visit milton nsw

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An elegant country escape awaits in Milton. Enjoy artistic offerings, colourful shopping and gourmet experiences in this fashionable town that boasts a picture-postcard scene of beautiful green hills and heritage streetscapes. In the distance, spot Pigeon House Mountain, a haven for bushwalkers, offering spectacular views of the Shoalhaven region.

Food & wine indulgences

Thanks to an abundance of fresh produce, fine wines, bakeries, country pubs and quality restaurants , Milton is known as a foodie destination on the NSW South Coast . Enjoy a beautiful breakfast at Milk Haus , then browse artisan breads and gourmet pies at Flour Water Salt  or The Heritage Bakery .

At Cupitt's Winery , you can taste wines at the cellar door, cheeses from the fromagerie and craft beers from the microbrewery. Enjoy lunch on the lawn or in Cupitt's Estate Restaurant . For dinner, book a table at Small Town Food and Wine  for a culinary treat, or Harvest Bar for tapas and live music on weekends. Check out the events calendar to see what’s on.

Cupitt's Winery , Ulladulla

Antiques & artistic endeavours

For antiques and arts, stop by Nulladolla Pottery , Millhouse Gallery ,   Gallery Alchemy   and Turnbull Bros Antiques, Milton’s oldest antique shop. Head along to the Milton Village Showground Markets , a bustling produce and craft market held on the first Saturday of every month. The historic Milton Theatre   hosts concerts and performances.

Gallery Alchemy - Credit: Ola Moszumanska

Scenic drives & outdoor adventure

For a scenic drive, venture out to the hinterland village of Woodstock, positioned on the western edge of Burrill Lake. Indulge in handmade chocolates at Woodstock Chocolate Company or enjoy a pick-your-own experience at  Woodstock Flower Farm . Continue further on and you will find the turn off to  Pigeon House Mountain , one of Shoalhaven's most iconic day walks.

Pigeon House Mountain (Didthul) , Morton National Park

Getting there 

Located in the Shoalhaven region on the NSW South Coast , Milton is a three-hour drive from Sydney and 2.5 hours from Canberra. Nearby, you’ll find Lake Conjola ,  and the seaside towns of Mollymook and Ulladulla . Make a road trip of your journey there by driving along the stunning Grand Pacific Drive .

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Plan your trip, explore jervis bay & shoalhaven.

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Shoalhaven | south coast nsw, milton nsw in the shoalhaven is a historic village perfect for a weekend getaway or a longer stay. enjoy unique shopping, hatted restaurants and stunning country scenes..

Milton NSW in the southern Shoalhaven is a trendy, historic village. Renowned cafes and restaurants mix with quirky arts and designer boutique stores to give a uniquely Milton experience – which has visitors coming back year after year. Milton offers a great range of quality accommodation which is ideal for a quick getaway with friends or a longer stay with the family, exploring all the region has to offer. The town was established in 1860 and much of its character is derived from the many historic buildings here, so much so that the entire town has now been classified by the National Trust as an historic village. The monthly village art and craft markets reflect the many skilled artists and designers living in the area and are a great way to spend the day after a tasty cafe breakfast. 

WHAT TO DO IN Milton

Venture out to Woodstock in the hinterland, just a short drive from Milton and positioned on the western edge of Burrill Lake. Milk Haus, a wholefoods cafe resides in the old cheese factory here and has a solid reputation built on using sustainable practices in delivering delicious and wholesome food. For those looking for some action, Skirmish Ulladulla is a little further down the road in Woodstock. Grab a bunch of mates and have a ball shooting each other in one of the six skirmish playing fields available. If you continue through Woodstock you will see the turn off to  Pigeon House Mountain , one of our most iconic day walks. Hotels, motels and self-contained options are available in Milton, Mollymook and Ulladulla.

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This Village In NSW Is Full Of Old World Charm With Antique Stores And 19th-Century Cottages 

Step back in time in the historic village of Milton which offers an unforgettable experience to history buffs with heritage architecture and beautiful landscapes.

The South Coast shines as a favourite destination for NSW residents thanks to its many wonderful secrets which include towns and villages that look straight out of a storybook . If you’ve ever dreamt of having your own cottage in a quaint little town, we may have found the perfect spot for you. The historic village of Milton comes with heritage buildings, boutiques, cafes and plenty of 1860s charm . It may be small, but it packs a punch. Its proximity to Mollymook means you get a lovely blend of beach and country charm. The two go hand in hand so we suggest making a pitstop at Mollymood if you’re driving out. Here’s how to spend a weekend in the area.

Milton, NSW

Milton has retained its old-world charm through its heritage buildings , churches and cottages that offer a glimpse into its historic past. It’s also enveloped in lush, rolling hills and cattle grazing the sweeping green fields. It’s as quaint as it sounds and it’s wonderfully walkable so the best way to enjoy the countryside is to put yourself in the heart of the town, mingle with friendly locals and enjoy some alone time.

Things to do

Milton has gained some love for its art galleries including Milton Art Gallery , Van Rensburg Galleries , and Milton Timber Gallery , and its boutique stores but it’s also home to cosy cafes and the award-winning Heritage Bakery which serves delicious pies and pastries. Other eateries to add to your list include Cupitt’s Winery which includes a brewery and restaurant and comes with accomodation and the charming garden-to-plate eatery Milk Haus which is perched on the outskirts of town.

Its heritage buildings include the Milton Court House, the intimate Milton Theatre where visitors can catch a variety of live performances and shows and some vintage churches. For your shopping needs, head to Turnbull Bros Antiques to snag a bargain or plan your trip on a weekend for the Milton Village Showground Market , which is held on the first Saturday of the month and features over 100 diverse stalls across retro and vintage goods, fashion, local produce, jewellery and excellent street food.

If you’re feeling extra adventurous, go on a lantern-lit ghost tour of the historic town.

Once you’ve had your fill of the countryside, make your way to the neighbouring Mollymook which offers great surf beaches, a golf course and an equally good food scene.

Getting there

The town of Milton is located 6km northwest of Ulladulla on the Princes Highway and is about a 3-hour drive from Sydney and a 2.5-hour drive from Canberra. The easiest and fastest way to get there is to drive but you can also catch the train from Central to Kiama , then take a connecting train from Kiama to Bombaderry Station. Plan your travel at Transport NSW .

If you’re visiting the South Coast , stay a while and explore its many natural wonders including striking rock formations , coastal walks and bioluminescent beaches .

Can’t get enough of small-town getaways? Plan a road trip to New England High Country for autumn foliage and incredible views .

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South Coast Experiences

How to spend a foodie weekend in beautiful Milton NSW

The perfect itinerary for a weekend getaway from Sydney

• Kate Mather
• 18 October 2020

The historic town of Milton has started making a name for itself in recent years. Less than three hours drive from Sydney, and just under two from Canberra – but a decade away from the bustle of the city – this quiet country village is the perfect choice for people seeking respite from the cities rush. 

Aside from the idyllic pace of life, Milton holds another secret lure: the food. This is a place where farm-to-table really means farm-to-table. Stroll between the country pubs, stop in at the local winery, and finish your day at one of Milton’s abundant restaurants – each one serving up fresh, delicious produce from the local land and sea. 

Here’s our SCx itinerary on how to spend the perfect weekend in Milton…

THE ROUTE FROM SYDNEY

How to get to milton.

1. Head out early from Sydney on the Princess Highway. Before you reach Gerringong, you’ll zip past the lovely Woolshed Kitchen – a great breakfast spot with delicious treats like warm-baked croissants, eggs benedict and char-grilled sourdough. The restaurant is right off the highway, with chic, contemporary interiors that signal you’re well on your way to the laid-back South Coast.

2. Continue down the highway until you get to the Gerringong turnoff. From here, it’s the scenic route all the way to Milton . Turnoff towards  Gerringong and continue on towards Gerroa, then all the way down past Shoalhaven Heads and into Nowra. This is a very pretty coastal drive – the ocean twinkling to your left and coastal bush land to your right. Factor in time for photo stops. 

3. As you continue into Nowra, you’ll cross over the Nowra river. Stop and pick up supplies in the town as you stretch your legs. The Nowra fresh fish and meat market has lots of local treats, but there’s also major supermarkets and other large retailers for all the essentials. From here, it’s about another 45 minutes on to Milton. 

ARRIVING IN MILTON

Get your bearings.

Milton is more of a village than a town. It’s full of country charm, with 19th century heritage streets and picturesque pastoral landscapes in every direction. Creativity exudes here, from the local artisans, producers and craftsmen who call Milton home. It’s a great place to window shop, with arts and crafts shops scattered across the village. But the best thing to do is eat, whether from the local farm shop or at one of the chic cool restaurants in town.

TIME FOR LUNCH

Where to eat.

1. Get lunch at Milk Haus Milton

Located in a converted cheese factory just outside Milton, Milk Haus is a wholefoods cafe specialises in honest, simple food using local produce. While the key message here may be simplicity, the menu is exciting and showcases some of the South Coast’s best suppliers. The founders farm as nature intended. What they can’t grow enough of in the garden, they source locally using a network of farmers, growers and fishermen. They aim to minimise wastage, feeding both the compost and the chooks in the process.

2. Pack a picnic from the local farm shop

Milton Farm Shop is a lovely little shop at the heart of town, selling in-season, organically-grown, and locally sourced produce from within a 100km radius of Milton. At SCx, we especially recommend you pick up some delicious Milton Mushrooms , which have all kinds of medicinal health benefits. Both Mick Ryan Park and Milton Memorial Park are small but pretty places to find a bench or patch of grass to eat your picnic. 

AFTERNOON ACTIVITIES

1. Explore the local beaches

After a long and lazy lunch, there’s nothing better than a walk on the beach. Jump in the car and head south – exploring the inlets and shores that most tourists will never see, including Pebbly Beach  (home to a local population of kangaroos) and the golden  Merry Beach,  just past Kioloa. If you’d prefer to stay closer to town, Mollymook  and  Narrawallee Beaches  are both great for surfing, while swimming in the tidal rock pool at  Bogey Hole  is a safe option for families. For surf lessons or wetsuit and board hire, check out  Mollymook Beach Surf School . Group lessons start from $50 per person, while boards are $20 for two hours or $50 for the day.

2. Get crafty

In Milton, you can take part in beautiful floristry workshops at Gypsy Carme n – mastering the art of flower arrangement or creating a luxe boho feature for your home. Clare is the passionate owner and creative director, who creates floral arrangements for all kinds of events. She’s an excellent teacher and will have you walking away with beautiful, memorable creations that represent your time on the South Coast.

3. Embrace your wellbeing

Although you’re only a few hours from Sydney, spending even just a weekend in the rolling countryside of New South Wales does wonders for your mental wellbeing. For the full ‘wellness’ experience, grab some time with Tracey Gunn , who runs healing food workshops and yoga classes in Milton. She can assist with on-one-on health sessions,  private yoga classes, and cooking classes in fermentation and raw food. Tracey is also the founder of Delicious Vibrant Beauty , a natural organic skincare company that makes products perfect for refreshing the skin after a little too long on the beach.

4. Stop for a drink

The foodie scene is strong in Milton – but the drinks scene is catching up quickly. Head for a tasting at local estate Cupitt’s Winery , offering one of the best cellar doors in the Shoalhaven region. The wine is delicious, only matched by their hearty cheese boards which feature cheese made onsite. You could also head to the newly-refurbished Milton Hotel , a great place for a cold afternoon beer sat on the outside deck. Check out the seasonal beers from their microbrewery before tucking into some locally cured meats on their charcuterie platter. Or for something a little stronger, try the cocktails at Harvest Bar Milto n ; drinking a Dark & Stormy overlooking the dark (and sometimes stormy) countryside of the South Coast is a great way to end a day in Milton.

TIME FOR DINNER

In Milton and the surrounding towns, there are an abundance of restaurants for dinner. From the super casual (a mobile wood-fired pizza cafe in town called Italian Guild ) to the super fancy ( Rick Stein at Bannisters ; the eponymous seafood master’s first restaurant outside of the UK, which helped put Mollymook on the map) you’re spoilt for choice when it comes to dining options. Here are a couple of our favourites…

1. Small Town in Milton

Small Town is a paired-back, neighbourhood bistro from the owners of the much-heralded (and sadly missed, if you ask many of Milton’s inhabitants) St Isidore – a coastal restaurant that closed in 2019. The owners wanted to switch their focus to delicious wine and simple food, although their menu is no less adventurous: you’ll find dishes like grilled octopus, nduja and lemon, and roast half chook, eggplant, bois boudran  and flat bread. There’s also a fantastic list of aperitifs to start the meal, and a great wine list.

2. The Ruse in Ulluadulla

If you want to head out of time, we recommend The Ruse in the coastal town of Ulluadulla, a 15 minute drive away. This causal bar and restaurant overlooks the twinkling Ulladulla Harbour, with a menu inspired by Central America. They’re always busy, so it’s best to book ahead if possible, though they do try and save space for walk-ins. Ordering a few of their creative cocktails is a great way to end an evening on the South Coast.

3. …Or bring the restaurant to you

Milton is not just great for eating out: eating in is just as fun. Citrus Catering can organise dinner party menus at your accommodation, with a delicious four-course meal prepared, cooked and served to you by their professional team – ideal for gatherings of up to 20 guests. For something a little more casual, Dinner Pal is a takeaway service serving the Milton area, deliver cold-packed meal kits for you to prepare in your accommodation’s kitchen. They can also organise beautiful weekend hampers that can be waiting for you on arrival.

TIME FOR BED

Where to stay.

If you’re staying in Milton, it’s only right you choose accommodation that matches the town: something rustic and charming, surrounded by beautiful countryside and fresh coastal air…

1. The Old Schoolhouse

The Old Schoolhouse is set on 2.5 acres of tranquil lawn and orchard, just outside Milton. It offers sweeping with views in all directions – perfect to soak up the region’s country charm. While the schoolhouse itself is long gone, the historic Schoolmaster’s residence remains. The main homestead is the heart of this rambling property, with two separate accommodation choices beyond the house. It has all the trappings of a rural property – with a productive vegetable garden and orchard, and chooks that wander throughout the grounds. It even has a full-sized cricket pitch. While the property retains the charm of a colonial homestead, the interiors have been updated to incorporate the best of modern style. Guests are free to wander the gardens and orchard, picking fruit straight from the tree and collecting fresh-laid eggs for their breakfast.

2. Woodstock Homestead

Woodstock is a luxurious Rural Retreat only Minutes from the beach. Looking for a summer or winter getaway? Family holiday or friend’s getaway? Kids, No Kids, Fur baby’s? No matter what your reason Woodstock Homestead is the perfect place.

WE CAN HELP YOU PLAN

At South Coast Experiences, we’re here to help you plan the perfect trip to the South Coast NSW – free of charge – so please speak to us if there’s anything we can do to make your planning easier.  See our full range of Milton Experiences here >

We also help organise accommodation for groups of any size, so talk to us about what you need.  See our South Coast accommodation here >

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The delightful township of Milton is popular for its scenic rural coastal setting, its living history, rich cultural life, delightful range of colourful shops, fascinating galleries and lively food places.

Milton's magnificent location ensures something for everyone whether you'd like to spend time on the pristine beaches, adventure in the mountainous forested hinterland, muck around in boats,or any more of myriad other activities only minutes away.

The welcoming friendliness of the locals is wholeheartedly country and an atmosphere of creativity and personality is evident in the diverse artistry and craftsmanship on show at venues throughout the township.

The beautiful Narrawallee and Mollymook beaches are only a few minutes from Milton. Visit the fishing fleet in the picturesque Ulladulla harbour between the mountains and the sea.

Milton is also a good base from which to discover the magnificent wilderness and escarpment country of the Morton National Park and the Upper Clyde River. Escorted tours to this spectacular region are available or you can drive to places like Pigeon House Mountain for spectacular views over the coast and the rugged eastern escarpment. Climbing Pigeon House earns a badge of honour.

For details of tours, accommodation, businesses and services available from Milton, please visit Ulladulla District OnLine .

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Things to do in and around Milton

The Old Schoolhouse Milton is the perfect place to discover all the astonishing things that Milton has to offer. Set amongst stunning dairy country but only minutes away from Narrawallee and Mollymook Beach, Milton has become a centre for wonderful food and wine and a vibrant arts community.

Milton’s main street offers a range of casual eating.

Pilgrims vegetarian cafe is a well known stop in the middle of town. Wander up the hill and discover Flour Water Salt Bakery offering great coffee and delicious food.

Our favourite for an evening drink or dinner The Guild offers the best wood fired pizzas and Italian on the South Coast.

The Milton Hotel

As with all small townships, the local pub is the centre of the social scene and The Milton Hotel is no different. Whether it’s meeting up with friends for a drink, enjoying a delicious bistro dinner or listening to some of Australia’s best live music, The Milton Hotel warmly welcomes locals and visitors alike.

Harvest Bar

Harvest Bar is the perfect place to enjoy a glass of wine, beer or cocktail accompanied by delicious tapas.

Small Town Milton

Brought to you by the people of famed St Isidores, this cosy 20-seater  neighbourhood bistro is serving some of the finest food on the NSW South Coast.  With a reputation for creating memorable dishes using produce from local and Australian producers it is a must when visiting Milton.

Cupitts Winery

Born of the desire to provide something very special the Cupitts Winery family remains dedicated to providing the best wine and food experience on the NSW South Coast. The restaurant produces amazing food using fresh, local ingredients or you can just sit on the deck looking out across the vineyard and enjoy wood fired pizza with a wine or craft beer or enjoy a wine tasting or meal in the one hatted restaurant.

Milk Haus was dreamed up by passionate people, who have an appetite for cooking and love of the earth, to create a cafe that looks after you and nature.

Situated in the old Cheese Factory at 170 Woodstock Road, Milk Haus aims to deliver fresh, honest, simple fare; free of toxins, preservatives and anything artificial. Good food that makes you feels energised and alive.

Gwylo at Mollymook

Chef Matt Upson made the decision to close Tallwood and has reopened with a new look and a menu of  “Asian Fusion’ dishes with an Asian street food flavour.  At Gwylo you can sit at the bar, watch the woks fire and charcoal smoke or get comfy in a booth with friends and share a flavour-packed tasting menu.

The Milton Produce Markets

A small group of local growers sell their produce every Saturday morning at the Milton Showground.

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An insider’s guide to Milton, NSW

ACS1017_milton_main_street

It may be renowned for its historic streetscape, bucolic, evergreen setting and close proximity to the coast, but it’s the supportive community, local attractions, restaurants and thriving boutique hotels that make the small town of Milton, in the Shoalhaven region of the NSW South Coast, so special.

In fact, it’s estimated by locals that as many as two new families are moving to Milton each week, ensuring the town’s vibrant mix of food, retail, education and medical services continues to develop and evolve.

Fertile farmland surrounds Milton on the NSW South Coast.

The Budawang tribe are the original inhabitants of this part of NSW, and were sighted by Captain Cook on his voyage in 1770.

The first European settlers arrived in Milton in 1828, establishing a cedar-cutting industry that drew more farmers and saw Milton become the commercial centre of the district by 1875.

Today, the main street is a charming mix of old and new, and the town is framed by rolling pastures, the majesty of Pigeon House Mountain and the Budawang Range, and the occasional glimpse of sparkling surf.

Milton’s main street.

What to do in Milton, NSW

With mild temperatures year-round, there’s plenty to enjoy.

Beautiful bushwalks, delicious eateries, places for an afternoon tipple, cooking classes, as well as spots for yoga and meditation – there’s no shortage.

For more information, visit Milton NSW .

Milton restaurants and eateries

MILK HAUS, WOODSTOCK

A five-minute drive from Milton’s town centre you’ll find Milk Haus, in Woodstock. They serve breakfast and lunch in a stylishly converted cheese factory, taking produce from their flourishing kitchen garden.

For more information, visit Milk Haus .

FLOUR WATER SALT, MILTON

Stop in for bread, pastries and flavoured milks – a contemporary small-town bakery.

For more information, visit Flour Water Salt .

A local bar and restaurant serving up tapas, experimental cocktails and live music.

Follow them on Instagram @harvestmilton .

THE MILTON HOTEL

Beautifully restored and with views out over the coastline, The Milton Hotel is worth a visit for their great menu and to try home-brewed Dangerous Ales.

For more information, visit The Milton Hotel .

CUPITT’S WINERY

Located in nearby Ulludulla , this boutique winery also boasts a fromagerie, restaurant, brewery and beautiful accommodation.

For more information, visit Cupitt’s Winery .

This bakery, which prioritises the use of simple ingredients, is a cult favourite.

Milton attractions

MILTON THEATRE AND OLD TOWN HALL

Standing side by side, these historic buildings (the hall is now the local library) are a charming highlight along Milton’s main street.

For more information, visit Milton Theatre and Old Town Hall.

COLLERS BEACH

Nearby Collers Beach is a beautiful spot to watch the surfers. Plus, it’s dog friendly!

For more information, visit shoalhaven.com

The best shops in Milton

MILTON VILLAGE MARKETS

Food, clothes, bric-a-brac and more; held the first Saturday of each month.

For more information, visit Milton Village Markets .

The best of modern surf culture.

For more information, visit Akwa Surf .

Milton accommodation: where to stay

BANNISTERS HOTEL, MOLLYMOOK

Luxe accommodation plus day spa and acclaimed restaurant Rick Stein at Bannisters.

Bannisters Hotel .

THE OLD SCHOOLHOUSE, MILTON

The two renovated cottages on this two-hectare property are small but blissful. The entire property has recently undergone a stylish renovation, completed by interior designer Christina Prescott and will truly feel like a home away from home.

The Old Schoolhouse .

CLIFF HOUSE, MOLLYMOOK BEACH

When you envision an ideal beach house, uninterrupted ocean views are a must. This is exactly what Cliff House on Mollymook beach delivers in spades.

For more information, visit Cliff House on Airbnb .

See some of our favourite stays in Milton here .

The penthouse suites at Bannisters in Mollymook Beach were designed by Collette Dinnigan, fashion designer and former local.

Milton real estate

With its idyllic surrounds, café scene and close-knit local community, Milton’s “got everything”, says Ben Pryde, principal and director of the local Raine and Horne. He adds that about 65 per cent of buyers in the area hail from Sydney.

A tightly held market, Milton offers a mix of character homes and new estates — the median price for homes is $1.075 million — as well as premium acreage surrounding the village.

“There are also places on acreage further out, more in the bush, for those looking for solitude or escapism,” says Ben.

Milton has two primary schools, Milton Public School and St Mary’s Star of the Sea, and a handful of preschools and early learning centres. Ulladulla Public School and Ulladulla High School are also close by, as are TAFE campuses in Ulladulla, Nowra and Moruya. The University of Wollongong is two hours north, with hundreds of course options available.

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‘We’re in a good paddock’: a visitor’s guide to Mollymook and Milton, Shoalhaven’s surf-and-turf twins

Endless beaches, lush farmland and the ripple effect of a certain seafood chef from Cornwall are just part of the community-minded NSW region’s story

“Y ou’re not considered a local in Milton unless you’re in a band, raise chickens and tend your own veggie patch,” laughs Katie Stidwill, co-owner of wholefoods cafe Milk Haus, set inside a converted cheese factory, surrounded by dairy farms. A surfboard roof rack on your car is also a must.

Set between emerald hills and the aquamarine sea, three hours’ drive south of Sydney, Mollymook and Milton are the gorgeous, surf-and-turf fraternal twins of Shoalhaven. Alongside their sensible sibling, the fishing port and service centre of Ulladulla, they offer white-sand beaches, massive waves, lush farmland, tranquil lakes, national park wilderness and very impressive golf-courses, if you’re that way inclined.

Renowned local big-wave surfer Brett Burcher, who’s also a teacher at the Milton public school, sums it all up nicely when he says: “we’re in a good paddock down here.”

Thanks to the ripple effect of a certain seafood chef from Cornwall, the food and drink scene is also on par with an inner-city neighbourhood. Rick Stein may have kickstarted the gourmet ecosystem a dozen years ago (several of his former chefs now run their own restaurants), yet the region has always brimmed with finelocal meat, eggs, dairy, produce and, of course, seafood.

While Mollymook and Milton may be enviable holiday destinations, they’re also vibrant and viable year-round communities that not only are home to farmers and fishers, tradies and alternative lifestylers but also, in a post-Covid world, to digital commuters, tree- and sea-changers.

This shift is not without complexity. Like many coastal areas in New South Wales, the last two years have seen a rise in homelessness as the area’s property prices spike, while tourism dollars, and their associated jobs, plummeted.

Monica Mudge, a yoga teacher and founder of the not-for-profit Treading Lightly , remains optimistic. “Everybody’s part of the story,” she says. “We all have something to learn from each other. That’s what resilience looks like.”

After mobilising everyone to provide food hampers and clothing for those displaced during the 2019/20 bushfires, the Treading Lightly team have now created a whole pantry of multigenerational environmental and community initiatives.

The beaches go on for ever hereabouts. Mollymook’s 2km crescent (with lifeguards) is a great place to start. At mid-tide, swim at the Bogey Hole natural rock pool, accessible by walking around Mollymook’s southern headland or driving through the Beachside golf course. Narrawallee, Rennies and Racecourse beaches are other less-crowded gems. Burrill Lake enters the sea in a swirl of turquoise waters and shifting sands at Dolphin Point.

Take surfing lessons with world champion surfer Pam Burridge , who specialises in teaching women. Go kayaking or standup paddle-boarding at Narrawallee Inlet and Burrill Lake to see black swans, pelicans and sea eagles. Walking on Water will set you up at the best spots depending on tides and wind. They also do surf lessons.

From May to November watch whales breaching offshore from the Ulladulla Lighthouse or South Pacific Heathland preserve.

Follow the Aboriginal and interpretive signs on the new Giriwa walking track along Burrill Lake or take the short but challenging trek up Pigeon House Mountain in Morton national park.

Check out the self-guided history trail of Milton, stopping along the way to browse its antiques, homewares and clothing shops. In Ulladulla, source handmade clothing, jewellery, pottery and skincare from Slow: A Handmade Collective of local artisans.

A little bit of everything

A collective sense of wellbeing permeates Cupitt’s Estate with its views over the vineyards to Burrill Lake, whether it’s couples enjoying wine and cheese tastings in the historic cellar door, guys doing a beer flight on the deck, diners scanning through the farm-to-table menu in the arched restaurant, or young families eating pizza on the terrace as they listen to live music on Sunday afternoons.

There are also five new and sleek accommodation pods here – though their designer bathrooms and decks do not come cheap, with midweek prices starting from $550 a night.

Owner Rosie Cupitt is also the founder of the region’s Slow Food Convivium, and has worked with a local Budawang elder of the Yuin nation, Uncle Noel Butler, to add local Indigenous foods to Slow Food’s international Ark of Taste .

Where to eat

Dog-friendly, indoor-outdoor community hub the Milton Hotel has fabulous updated pub grub, craft brews from in-house Dangerous Ales brewery, local live music, and even a great children’s playground.

Bannisters Rooftop Bar and Grill is the casual alter ego to Rick Stein at Mollymook with local seafood from Lucky’s and other delicacies.

Mollymook’s moody Gwylo is Matt Upson’s pumping, neon-lit, Asian fusion street-food restaurant, with killer cocktails.

Alex Delly and Jo Thomas cook mostly with local ingredients at their bijou Small Town Food and Wine in Milton. Check out their next-door providore and Lagom sourdough bakery at Burrill Lake.

Two-storey Ruse , with floor-to-ceiling windows overlooking Ulladulla Harbour, is the place to indulge in tequila cocktails, tacos, bright salads and bar snacks.

Milk Haus provides splendid farm-to-table cafe fare with just-picked produce from its kitchen garden. Pick up whimsical flowers from Fred in the pop-up out the back.

Where to sleep

Location, location, location. With absolute water frontage as Lake Burrill enters the ocean, Dolphin Point Holiday Park has powered sites for caravans and simple cottages, with rates starting at $45 a night.

For beach-house rentals, check out the options at Riparide , which offers insider tips on what to do while you’re here. On Dolphin Point headland, the Nest is an affordable base for two couples and four kids.

Summer is getting booked up, but autumn is still balmy with warm water and fewer crowds – and there’s wonderful whale watching during the cooler months.

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South Coast NSW

The town of Milton, 6km northwest of Ulladulla on the Princes Hwy, is a cheerful caricature of its 19th-century history. European settlers flocked to the area in the 1820s, lured by logging, and a township was founded some 40 years later. A courthouse, a theatre and dozens of other heritage buildings preserve Milton's lost-in-time feel. Nowadays craft shops, boutiques and cafes have earned the town a spot on tourist itineraries.

Milton has several good places to eat, including an award-winning bakery and numerous daytime cafes.

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Out of Town Guide: Milton

Ashiki Life and Style Boutique

Bunyas Organics and Antiques

Milton is a three-hour drive south of Sydney. The town is easily missed, but it’s home to cafes, restaurants, boutique shops and art galleries, so passing it by would be a shame. Its focus on local is something the town prides itself on; top chefs design their menus around local produce, and bookstores showcase local writers.

EAT & DRINK

Pilgrims The older sibling to Pilgrim’s Cronulla , Pilgrims was the first to bring vegetarian fare to the town. The venue transforms from a trendy cafe serving great coffee and burgers by day, into a bustling Mexican restaurant at night. First timers should try the Bliss Burger. It’s a mixed-grain patty served in a wholemeal bun with fried onion, avocado, cheese, tabouli, sprouts and a satay sauce. The dinner menu features fresh Mexican comida and homemade sangria.

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8/9 Princes Highway (02) 4455 3421 Pilgrimsmenu.com

St. Isidore The lush surroundings and rolling hills visible from St. Isidore’s is where many of the ingredients that end up on your plate at this restaurant come from. Chef Alex Del’s focus is on sourcing the freshest, locally grown produce, including seafood. Leave room for dessert: pannacotta with rhubarb, rose geranium and pistachio is made with the next-door neighbour’s honey.

89 Croobyar Road (02) 4455 7261 Stisidore.com

Milk Haus Take a short drive out of town to Woodstock to visit this sustainable wholefoods cafe in an old cheese factory, The chefs are also gardeners at Milk Haus – they tend to the farm and weed the vegetable garden between service. The menu changes every month, reflecting what is ripe for the picking.

170 Woodstock Road (02) 4455 7293 milkhaus.com

Harvest Bar Harvest Bar filled a hole in the local drinking scene when it opened in 2014. It serves some of the region’s best wines and craft beers alongside a solid food menu featuring options such as sliders and a roast-pumpkin board. Harvest Bar is open Tuesday to Sunday from 3pm.

4/23 Wason Street (02) 4455 6269 harvestbarmilton.com

Cupitt’s Winery A little further South, towards Ulladulla, is Cupitt’s Winery. It straddles the glorious space between a winery, brewery, fine-dining restaurant, boutique hotel and to top it all off – it makes its own cheese. Its website lists the regular events it holds, such as its Long Table Lunches.

58 Washburton Road (02) 4455 7888 Cupitt.com

Milton Village Markets The Milton Village Markets are held on the first Saturday of every month. Buy the region’s best local produce and see some of the town’s artistic talent. The markets take place along the footpath next to the Princes Highway on Milton’s main drag.

Morton National Park Morton National Park is perfect for a day trip. It’s a track that caters to all bushwalking abilities, and has sweeping views of the South Coast. The Mount Bushwalker and Pigeon House Mountain tracks are a short drive from the heart of Milton. The Pigeon House Mountain track involves some steep climbs and navigating ladders, but the vistas are worth the effort. Maps of these tracks can be found on the NSW National Parks website .

Milton Theatre Check out Milton Theatre’s facebook page for any upcoming events. The small theatre attracts some great musical talent from the east coast.

69 Princes Highway (02) 4454 3636

Cuppitt’s Cottage Staying in one of these immaculately renovated, 100-year-old cottages on the grounds of Cuppitt’s winery means you can roll out of bed and into the vineyard. The family-run hotel is just five minutes from Ulladulla, Milton and Mollymook Beach. The rooms sleep up to four people. Each has its own stone fireplace, full kitchen, is furnished with antique furniture and has views of the Budawang Ranges and Burrill Lake. Owners Rosie and Griff Cuppitt will be more than happy to show you around the grounds. Ask them for a tour of their microbrewery, and sample some of their craft brews.

Bannisters By the Sea Bannisters is owned and run by Rick Stein and his wife, Sarah. The coastal boutique hotel is just a five-minute drive from Milton and has 32 rooms, including spa rooms, luxury suites and penthouses. Each has its own private balcony overlooking the ocean. Try booking one of the Collette Dinnigan-designed penthouse suites decorated with Australian photography and trinkets sourced from markets in France. The restaurant, Rick Stein at Bannisters, is the celebrity chef’s first restaurant in Australia and features local fish and shellfish.

191 Mitchell Parade, Mollymook (02) 4455 3044 bannisters.com

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Milton, NSW

Attractive, artistic and fashionable town on the ridge inland from the seaside towns of Ulladulla and Mollymook.

There was a time when no one mentioned Milton without twinning it with Ulladulla. The two towns were so close - one on the ridge, one beside the sea - that they had become Milton-Ulladulla in the minds of most visitors. Today Milton has acquired a separate identity. It has attracted artists and alternative lifestyle folk who don't want to live by the sea but want to live in a relatively inexpensive town with interesting inhabitants and an easy proximity to Mollymook, one of the best beaches on the South Coast. Milton's economy was originally driven by fishing, dairying and timber. Today it has become a chic village characterised by gift shops, good restaurants and cafes and well-preserved historic buildings. Unfortunately, like Berry further north, it has not been by-passed and consequently all traffic travelling through the area drives down the main street.

Milton lies 220 km south of Sydney via the Princes Highway.

Origin of Name

No one is entirely sure why Milton was named. There is a theory that it is a simple colonial corruption of the English Milltown. Alternatively it is argued that it was named after the great 17th-century English poet, John Milton. It was either named by the town's first postmaster, George Knight, or by John Booth who created a private township on part of his 80 acre (32 ha) farm. There is a rather romantic story that Booth saw a copy of Milton's Paradise Lost  in his library and was suitably inspired.

Things to See and Do

Exploring Historic Milton The most sensible way to explore historic Milton is to get Your Walking Guide to Historic Milton compiled by Sue Curran ,  a single A5 brochure which lists a total 25 places of interest all of which are, with only three exceptions, either on the Princes Highway or on Wasson Street. It is available from the Visitor Information Centre in Ulladulla. Some of the town's most interesting buildings include:

(No.1) The Milton Library - which was built by public subscription as the local School of Arts in 1871. It is a handsome building on the Princes Highway beside the Milton Theatre.

(No.5) Times Past Bed and Breakfast - a charming cottage which was built around 1890. It was originally the homestead of the dairy farm which supplied the town's milk.

(No.6) Wesleyan Methodist Manse - standing at the top of the main street on the corner of Thomas Street it was a gift to the church from ex-convict Henry Claydon. The church later sold the building as they thought it too ostentatious. The triangular plot to the west of the house contains some graves, one of which belongs to Joseph Whatman, the first settler on the site of Milton. The rear balcony was used for binocular surveillance of the coast during World War II.

(No.9) The Court House and Old Police Station - both date from 1877 and are an impressive part of the townscape. If you look carefully at the Court House you will notice that it is somewhat unusual being asymmetrical rather than symmetrical.

(No.10) The Post Office - was built in 1880 and extended in 1894 and 1904. It dominates the townscape. The first post office was established in the town in 1859.

(No.11)  The Settlement - was the centre of the town from earliest times. It was originally established as the Post Office Stores in 1860. The stores were purchased by John Kendall in 1874 and run by his son-in-law, Henry Carrington Blackburn. After Kendall died Blackburn demolished the original store and built 'The Popular' Store (1898) which, at the time, was one of the largest stores on the south coast. The Settlement Courtyard, with its specialty shops, now occupies the space where the original storeroom and stock feed barn were located.

(No.12) Victorian National Bank - built in 1870 and now a cafe named Coastal Indulgence it is a particularly elegant building. Behind it is the Village Green courtyard complete with an old pump and a number of specialty shops.

(No.13) Anglican Church of St Peter and St Paul - designed in a Gothic style it was completed in 1860, although it was not consecrated for many years owing to unpaid debts. The foundation stone was laid by John Kendall. Many believe that the Chinese Elm Tree in the grounds is the oldest in Australia, being planted by Alice Kendall when she returned from missionary work in China in 1920.

(No. 15) The Cottage -  'The Cottage', currently the Milton Medical Centre, was built in the 1870s. It is a typical Victorian villa with high ceilings and a simple graciousness. There is an old pump and a well behind the building.

(No. 16 & 17) Melrose and the Lighthouse Keeper's Cottage -  Now known as the Milton Lighthouse Medical Practice, this old lighthouse keeper's cottage is well hidden by a hedge. When the lighthouse was mechanised in the 1920s the cottage was dismantled and removed by bullock cart from Warden Head. 'Melrose', on the opposite corner, was originally a farm belonging to Walter and Amelia Alice Kendall.

(No. 18) Heritage Bakery - On the corner of the Princes Highway and Croobyar Road is the Heritage Bakery, an elegant two-storey structure. Built around 1887 by William Riley and his six sons it  was originally a substantial general store. The balcony, which gives the building much of its contemporary charm, was added at a later date.

(No. 19) Uniting Church - the present Uniting Church was built in 1872 as the local Congregational Church. Gothic in its architectural style it is decorated with quoins, cathedral glass windows and facings of hewn stone. The manse was built 1875 and was used as a school from 1899-1908.

St Mary's Star of the Sea Catholic Church Located at 41 Corks Lane, St Mary's Star of the Sea Catholic Church stands on a hill overlooking the northern entrance to Milton. It was opened in 1891 although, at the time, no foundation stone or memorial plaque was included on the building. There is very little information about the church apart from a weather vane tht was established in memory of Alf Martin who died in 1980 and a Lone pine in the grounds which was donated at Anthony Gallaher in 2006.

Other Attractions in the Area

Lake Conjola and Fishermans Paradise Lake Conjola is a large lake which lies to the north of Milton. It is thought the name comes from a Yuin Aboriginal word 'Kongoola' which was the name of a fish which could be found in the lake. The lake is a classic South Coast "get away from it all" retreat for holidaymakers, sea changers and retirees whose idea of heaven involves boating (there are boat ramps at the two towns - Lake Conjola and the amusingly named Fishermans Paradise), sailing, water skiing and safe swimming.  Bream, flathead, whiting, tailor, and blackfish can all be found in the lake. Access to the town of Lake Conjola is via Lake Conjola Entrance Road which heads east from the Princes Highway 6 km north of Milton and access to Fishermans Paradise is a few kilometres further north on Alma Road.

Pointers Gap 1.4 km north of Lake Conjola Entrance Road on the Princes Highway, and heading west, is a scenic drive on Pointers Gap Road which leads up to Pointers Gap Lookout which is on the edge of the  McDonald State Forest at the top of the escarpment. It offers panoramic views of Lake Conjola and the beaches south of Burrill Lake. The whole area, particularly the heathland, is particularly beautiful when the wildflowers are blooming in the spring and summer.

Mollymook and Rick Stein at Bannisters Five kilometres from Milton, via Matron Porter Road, is one of the best restaurants on the NSW South Coast - Rick Stein at Bannisters. It specialises in seafood and offers a reasonably priced menu (compared to Sydney prices) which ranges from French through Indian and Malaysian to Modern Australian with heavy hints of Asia. Also Mollymook (see https://www.aussietowns.com.au/town/mollymook-nsw ) has one of the best beaches on the South Coast - arguably on the whole of the east coast.

* Prior to European settlement the area was occupied, for an estimated 20,000 years, by the Yuin Aborigines.

* Captain Cook sailed up the coast  in 1770 and noted, at Bawley Point, south of Milton, indigenous people who 'appeared to be of a black or very dark colour'. He named Pigeon House Mountain on April 21, 1770 recording in his journal what he described as  'a remarkable peaked hill, which resembled a square dove-house, with a dome at the top, and which for that reason I called the Pigeon House'.

* In 1827 the first land grant was issued to Reverend Thomas Kendall (1778-1832) who settled north of the present township of Milton. Kendall ran cattle and felled timber using ticket-of-leave men for labour. He called his property 'Kendall Dale'.

* In 1839 Kendall's grandson Henry Kendall, who became one of Australia's most famous poets, was born on the estate.

* Other grants were issued in the 1830s and the site for a village was surveyed in 1837.

* The town's first name was 'The Settlement'. This 'settlement' grew on what is the site of present-day Milton. The area around The Settlement was soon occupied by farmers.

* In 1859 John Booth (he was a business partner of the department store owner, Anthony Hordern) purchased the 80 acre (32 ha) Myrtle Farm for £240 and subdivided it into 62 allotments. He developed a private town which was probably named Milton by George Knight, the local postmaster. That same year the foundation stone was laid at the Anglican Church of St Peter and St Paul.

* During the 1860s the district developed a reputation for mixed farming with dairying, wheat-growing (it was short lived and was destroyed when 'wheat rust' hit the south coast), pig-rearing, honey and vegetable-cultivation. A tannery was established at Millards Creek and silica and quartzite were mined and loaded on a wharf at Bannister Point, north of Mollymook.

* In 1862, as a result of a public subscription, the first volume of Henry Kendall's poetry, Poems and Songs , was published.

* The Congregational Church (now the Uniting Church) was erected in 1872.

* The town's first public school (there had been private schools since 1851) was opened in 1878.

Visitor Information

The nearest Visitor Information Centre is at Ulladulla. It is located opposite the harbour at the Civic Centre, Princes Highway Ulladulla 2539, tel: 02 4455 1269.

Useful Websites

Surprisingly Milton lacks a good, comprehensive local website. The best available is the Ulladulla website with the Milton entry. Check out http://www.ulladulla.info/milton

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What a fabulous website thank you Bruce. I was born/bred in Milton and my memory of the Victorian National Bank throughout the 1940’s-50’s-60’s is that of the Commercial Baking Company of Sydney. My sister worked there for many years. Loved your talk this morning at Wollongong U3A!

Thanks Gwenda. Glad you like the site. It really is a labour of love.

I am wondering what colour the outside walls of the Lighthouse cottage were painted before it was moved to town and now the Lighthouse Medical Practice building.

I discovered that John Booth, the founder of Milton, was my great great grandfather on my mother’s side of the family. He sounds a most interesting person. One of his daughters married Samuel Hordern owner of the now defunct Hordern’s Department Store in Sydney. I was in Australia in 2013 and did not know about Milton – very frustrating as I live in Scotland and can’t pop back to find out more.

• 12 Slaughterhouse Road Milton NSW 2538
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SeaChange Parks Milton

When it comes to gorgeous scenery, peaceful coastal surrounds, and postcard moments, you’ll find it here.

SeaChange Parks Milton is situated on 17 acres of manicured lush green parkland located a short distance from the historic township of Milton. A short drive will also get you to the famous Mollymook Beach and the township of Ulladulla.

Looking for a lakeside escape? Check out our sister park SeaChange Parks Lake Conjola .

WELCOME TO OUR holiday park

For the perfect south coast getaway offering value accommodation and extensive facilities set on a backdrop of green surrounds and fresh seaside air. We are ideally located close to beaches, wineries and wildlife, so come stay with us and enjoy what the location has to offer.

With a range of accommodation options available we can cater to all needs. Whether you need to park up a van, pitch a tent or find a bit of comfort in our range of cabins and on-site vans, we have the option for you!

Book with us today for your next adventure.

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Monday – Wednesday 09.00am – 04.00pm

Thursday – Friday 09.00am – 05.00pm

Saturday 10.00am – 02.00pm

Sunday CLOSED

Please keep in mind that our business hours may vary due to NSW Health requirements for Covid19.

Please contact us for up to date information.

JOIN US TODAY!

Ready to discover your next amazing adventure.

Do you have questions? Contact or visit us.

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Milton, NSW

Welcome to Petana!

Book your stay today for a rustic farm experience. 

Accommodation

Petana is an immersive farm stay, with all the best the country has to offer. Only three hours south of Sydney, it's the perfect getaway for families, friends and romantic weekends.

Take some time out to explore this beautiful stretch of coastal country. From the mountains to the sea, Shoalhaven offers a plethora of sights and activities for everyone.  

The Grounds

There are many nooks and crannies of the farm to explore, many leaves to be overturned and hidden gems to be discovered.

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Future Jobs and Investment Authorities

The NSW Government is committed to supporting communities reliant on the coal industry to secure their long-term economic future as the global demand for coal declines over time.

New South Wales has a long history of coal mining and coal-fired power generation, with most of this concentrated in the Hunter, Central West, Illawarra and North West regions. These regions contribute billions of dollars to the NSW economy and tens of thousands of jobs. The local economies of these regions are highly dependent on the coal sector.

The NSW Government has committed to establishing Future Jobs and Investment Authorities. The Future Jobs and Investment Authorities will be made up of local representatives in each coal-reliant region to advise the Government on regional priorities and investment opportunities. The Future Jobs and Investment Authorities will help deliver investment in new industries to support the creation of jobs to deliver the best possible economic outcome for these communities. They will coordinate across all levels of Government to ensure investment and action is timed in the right way to support the best outcomes for coal-reliant communities.

Driving investment to create jobs will be the key performance indicator for the Future Jobs and Investment Authorities, which will be measured by:

• job creation, particularly creation of similar or higher value jobs that provide continued direct and indirect economic benefits
• productive investment in each region through the growth of engine industries and activation of new industries
• coordination across all levels of Government and with local communities to maximise opportunities and deliver effective outcomes.

Action to date

To support the establishment of the Future Jobs and Investment Authorities, the NSW Government has:

• committed $5.2 million to support the establishment of the authorities to these four regions to grow future jobs and industries
• held four stakeholder roundtables in the Hunter (8 August 2023), Central West (10 November 2023), Illawarra (27 March 2024) and the North West (10 April 2024) regions to gain community and industry insights on regional priorities
• developed an Issues Paper to go out to public consultation on the proposed model and functions of the new Authorities. 

Have your say

The NSW Government has developed an Issues Paper with a proposed approach to the establishment of the Authorities including:

• a proposed structure with clear roles and responsibilities of the Authorities
• how the Authorities will work with local, state and Australian government entities, industry and communities
• the funding model for the Authorities
• what legislation will need to change
• how we will measure success.

We want to hear from you. Please click on the link below to Have Your Say.

Consultation opens 28 May 2024 and closes 12 July 2024 .

Royalties for Rejuvenation

The NSW Government sets aside at least $25 million each year from mining royalties to support coal mining communities in New South Wales through the Royalties for Rejuvenation Fund. The funding will ensure that coal mining communities can make targeted investments towards strategic planning, workforce development programs, constructing enabling infrastructure, and establishing new industries and employment opportunities.

As part of the Royalties for Rejuvenation Fund, the NSW Government established Regional Expert Panels to provide regionally specific advice to ensure decision-making is aligned with the needs and demands of the local area.

The Government will consider the Royalties for Rejuvenation Fund and Royalties for Rejuvenation Expert Panels established under the NSW Mining Act 1992 and Mining Regulation 2016 as part of the public consultation on the Future Jobs and Investment Authorities Issues Paper.

For more information on the Royalties for Rejuvenation Fund and Expert Panels, visit Royalties for Rejuvenation Fund and Royalties for Rejuvenation Fund resources and news .

To read media releases and documents on Future Jobs and Investment Authorities visit:

News and resources

In-person requirements blocking elderly and severely disabled from receiving photo IDs from Service NSW

Service New South Wales has refused to budge on a requirement to have people front up to get photo identification, despite pleas for an alternative option for elderly people or those with severe physical impairments.

Those denied a NSW Photo Card because they are unable to go to attend a Service NSW centre in person have been unable to make changes to their banking, switch phone plans or buy some items online.

Karen Carty said she felt like she was losing her independence and freedom of choice.

Ms Carty has primary lymphoedema, a degenerative disease that has left her with mobility issues, and needs full-time care.

"I've been bed-bound for more than five years. It's not so straightforward," she said.

She first contacted Service NSW in July 2022 because her driver's licence was about to expire.

Unable to renew her licence because she could no longer physically drive, Ms Carty applied for a NSW Photo Card, an alternative proof of identity.

Ms Carty asked if her old driver's licence photo could be used or if she could get an exemption with a note from her doctor.

She said Service NSW refused both and said she must attend in person to have her photo taken.

Now living in an aged care facility in Villawood, Ms Carty no longer receives utility bills addressed to her, further limiting the ways she can make up 100 points of identification.

This has stopped her from changing to a cheaper phone plan, having a new phone delivered, and changing her financial plan, which was recommended by her bank.

"I recently realised, if I were to pass away, my children could get my money more easily than I can, which is crazy," Ms Carty said.

"People absolutely rely on online for shopping and making changes. That's how we live our life but if you're medically housebound, there's just nothing you can do."

Alternative option needed

Getting nowhere with her application, Ms Carty started a petition calling for Service NSW to implement a remote service for individuals physically unable to visit the centre in person.

The petition currently has more than 700 signatures.

Struggling to access Service NSW centres was a common issue, according to physiotherapist Anne Bryant, who made home visits to clients, including Ms Carty.

"There are a lot of people out there who are slipping through the system," Ms Bryant said.

The physiotherapist said an alternative option should be available for people who were housebound or had limited mobility, such as people who used a wheelchair for short trips but could not sit for long.

"We want to have our ID secure of course,"  Ms Bryant said.

"Surely there is something that we can do for people who can't really get in there."

A Transport for NSW spokesperson said in some limited cases other options could be arranged but Ms Carty said she was repeatedly told she must come into the office.

A spokesperson said the requirement to take photographs on site was for security reasons.

"Without these controls, the risk of identity fraud increases."

Aging parents at risk

Some elderly people have been hitting the same obstacle.

Brett Mann has been trying to help his mother, Gladys, get a form of photo ID.

The 88-year-old is showing early signs of dementia and relies on her children to handle her finances.

Her bank informed Mr Mann in December 2022 that he needed to provide 100 points of ID for his mother due to anti-money laundering laws.

Mr Mann's mother no longer has a passport or a driver's licence and, like Ms Carty, lives in an aged care facility without any utility bills addressed to her.

When he tried to organise a NSW Photo Card he was told about the requirement to get a photo at a centre.

"She's extremely frail," Mr Mann said. "It's very hard to do anything because of her mobility challenges."

Mr Mann told Service NSW that if she was to be put into a wheelchair to attend the venue, the stress could result in a medical episode.

"They said ,'Oh, well we don't want that', but then they never actually came up with an alternative solution," he said.

Ms Mann's bank accounts were frozen for nine months as Mr Mann tried to find a solution.

While Service NSW refused to budge, Mr Mann eventually came to an agreement with the bank to forgo the proof of identity requirements.

"That doesn't mean that there aren't various limitations," he said.

"Given that we don't actually have 100 points of ID, I can't open other additional investments or accounts, all those sort of things with different financial institutions."

Mr Mann said he had many friends with aging parents who were at risk of being in a similar situation.

"I think in this day and age, they need to actually have some sort of process to look after the elderly or people with disabilities," he said.

"To give them some sort of option other than that they need to come into the office."

No plan for remote service

Transport for NSW said that while it had no plan to implement a remote service, it was able to assist customers on a case by case basis.

Ms Carty and Mr Mann both say this has never been offered during their dealings with Service NSW and they are still waiting on finding an alternative way to get photo ID.

Transport for NSW was not able to say how many people had been offered alternatives to resolve the issue.

"We understand that some people may have difficulty accessing a photo card," a spokesperson said.

"However, many of these people are also particularly vulnerable to identity fraud, which is why it is important that correct process is followed."

An authorised representative could apply for a renewal or replacement for the NSW Photo Card, if the cardholder was unable to attend a service centre due to medical reasons. To prove this, the applicant would have to provide a medical certificate.

This option, however, is not available to those applying for the NSW Photo Card for the first time.

Since being contacted by ABC Radio Sydney , Transport for NSW has offered to help both Ms Carty and Mr Mann with their case.

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• Digital Health Challenge winners visit eHealth NSW office

26 April 2024

eHealth NSW hosted 10 students from Loreto Normanhurst School for a special event at the Chatswood office on 11 March 2024. These students were winners and finalists of the Digital Health Challenge, presented by eHealth NSW in collaboration with the Association of Independent Schools (AIS) NSW late last year.

Students had the opportunity to pitch their ideas to the eHealth NSW Executive Management Team (EMT), demonstrating their application of digital health solutions.

The challenge, held as part of the Partnering with Schools program, was designed to inspire creative problem-solving among students, while introducing them to new health and digital literacy concepts.

Ideas pitched included a leave support app for staff on maternity leave (inTouch) and a platform that translates resources into multiple languages (ALBUT), with students impressing the EMT with their research and presentation skills.

The winning idea was Digital Health Drive, an internet-enabled van that visits rural areas to support people with internet access so they can learn and navigate digital health solutions.

Tenielle Davies, a teacher at Loreto Normanhurst said the challenge sparked the curiosity of students to further engage with NSW Health resources that many did not know existed.

“Students had the opportunity to use innovative problem solving as well as gain a greater perspective of career opportunities in health,” she said.

During their visit to our office, students had the opportunity to learn more about eHealth NSW, its staff and programs, like Telestoke and the organisation’s role in keeping the health system safe from cyber-attacks.

eHealth NSW has been partnering with the Association of Independent Schools (AIS) NSW for over two years to encourage students to engage with STEM (Science, Technology, Engineering and Mathematics) learning and introduce them to a possible future career in digital health.

The Partnering with Schools program provides a platform for eHealth NSW to connect with students, schools and their broader communities to understand their health system needs to inform the planning, design and delivery of digital solutions.

The program is an opportunity to give back to the community. It’s a way for us to share our knowledge of the health system and digital health technologies. This can contribute to building greater health literacy in the community.

The program also showcases how digital technologies can impact individual health and wellbeing as well as health information now and into the future.

If you want to learn more about the program, please email the eHealth NSW Consumer Engagement team .

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• Published: 23 May 2024

Identification of the RSX interactome in a marsupial shows functional coherence with the Xist interactome during X inactivation

• Kim L. McIntyre 1   na1 ,
• Shafagh A. Waters 2   na1 ,
• Ling Zhong 3 ,
• Gene Hart-Smith 4 ,
• Mark Raftery 3 ,
• Zahra A. Chew 5 ,
• Hardip R. Patel 5 ,
• Jennifer A. Marshall Graves 6 &
• Paul D. Waters   ORCID: orcid.org/0000-0002-4689-8747 1  

Genome Biology volume  25 , Article number:  134 ( 2024 ) Cite this article

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The marsupial specific RSX lncRNA is the functional analogue of the eutherian specific XIST , which coordinates X chromosome inactivation. We characterized the RSX interactome in a marsupial representative (the opossum Monodelphis domestica ), identifying 135 proteins, of which 54 had orthologues in the XIST interactome. Both interactomes were enriched for biological pathways related to RNA processing, regulation of translation, and epigenetic transcriptional silencing. This represents a remarkable example showcasing the functional coherence of independently evolved lncRNAs in distantly related mammalian lineages.

The sex chromosomes of therian mammals (marsupials and eutherians) share a common ancestry [ 1 ], having evolved from a pair of autosomes [ 2 ] after the divergence of therian and monotreme mammals approximately 187 mya [ 3 ]. X chromosome inactivation (XCI) occurs in both groups of mammals, implying an ancient origin. However, XCI in eutherians and marsupials involve molecular mechanisms that are remarkably different.

XCI in therian mammals silences transcription of one of the two X chromosomes in female somatic cells [ 4 ]. It is established in the early embryo and maintained through subsequent cell divisions and serves as an important model for epigenetic silencing due to its unparalleled scale and stability. Long-noncoding RNAs (lncRNAs) have emerged as common regulators in therian XCI. Eutherian XCI is mediated by a lncRNA called XIST [ 5 ]. Its mouse orthologue, Xist [ 6 ], shares ~ 67% sequence conservation with human XIST. This includes a series of tandem repeats (A to F), of which only repeat A is well conserved across all eutheria [ 7 ].

The protein interactome of Xist has been investigated in mouse cell lines using techniques involving chromatin isolation by RNA precipitation with mass spectrometry (ChIRP-MS) and its variations [ 8 , 9 , 10 , 11 , 12 ]. These investigations have identified 494 proteins in total, with only 6 proteins (Hnrnpm, Hnrnpu, Myef2, Raly, RBM15, Spen) common to all studies [ 8 , 9 , 10 , 12 ]. An alternative technique, RNA immunoprecipitation (RIP) combined with deep sequencing, identified epigenetic regulators in the human XIST interactome that were not identified in the mouse studies: EZH2 and SUZ12, subunits of polycomb repressive complex 2 (PRC2), and CHD4, a subunit of the NuRD histone deacetylase complex [ 13 , 14 ].

Marsupials lack an XIST gene; instead, ancient protein-coding genes have been retained at the loci homologous to those from which XIST and neighbouring genes evolved in eutherians [ 15 , 16 , 17 ]. In marsupials, XCI is mediated by a lncRNA called RSX [ 18 ] that is derived from a non-homologous and physically distinct region of the X chromosome. RSX is 27 kb in Monodelphis domestica (grey short-tailed opossum) [ 18 ] and 30 kb in koala ( Phascolarctus cinereus ) [ 19 ], longer than the 15 kb mouse Xist [ 6 ] and the 17 kb human XIST [ 20 ]. Although lacking linear sequence homology, a k-mer analysis classified two major groupings of repeat domains that are shared between Xist and RSX ( RSX repeat 1 with Xist repeats B, C and XIST repeat D, and RSX repeats 2, 3 and 4 with Xist repeats A and E). Each of these domains is enriched for specific protein binding motifs [ 21 ]. Therefore, although RSX and Xist share no sequence homology they could be functional analogues.

Xist and RSX are both nuclear transcripts that are spliced, capped and polyadenylated in the manner of mRNAs, and are expressed only in female somatic cells, exclusively from the inactive X chromosome. In both cases, the clustered transcripts can be visualised using RNA fluorescence in-situ hybridisation (RNA FISH) as a distinctive cloud-like signal accumulated on the inactive X chromosome [ 18 , 20 ]. Induction of RSX expression from an autosomal transgene in mouse silences transcription in cis [ 18 ]. This indicates a silencing capacity similar to that of Xist [ 22 ], although marsupial XCI is ‘leakier’ or more incomplete than the XIST -driven process in eutherians [ 23 ], perhaps due to the evolution of two different lncRNAs in different ancestral genomic contexts.

Here, we investigate the protein interactome of RSX in a marsupial, Monodelphis , and compare it with the Xist protein interactome. We consider the molecular mechanisms underlying the convergent evolution of XCI in therian mammals to enhance understanding of the evolution of the adaptations for balancing gene expression between the sexes. Our findings show that RSX interactors significantly overlap with Xist interactors, falling within the same protein–protein association network related to RNA splicing and processing, translation regulation and ribosome biogenesis, and epigenetic transcriptional silencing. This highlights the remarkable functional coherence of these non-homologous and independently evolved lncRNAs. We identified overlap between the Xist and RSX protein interactomes, both of which are enriched for functions associated with post-transcriptional regulation of gene expression. Post-transcriptional regulation has been shown to contribute to the balancing of expression of X-borne genes between the sexes in eutherians [ 24 , 25 , 26 , 27 ], although the underlying mechanisms are unknown.

Identification and validation of RSX interactors and comparison with Xist interactome

To investigate the protein interactome of RSX we used ChIRP-MS to capture proteins associated with RSX using six biotinylated oligonucleotides complementary to different RSX regions (Additional file 1 ). Cell lysates were prepared from female Monodelphis fibroblast cells that were either UV crosslinked, formaldehyde crosslinked, or uncrosslinked. We identified 131 proteins that were associated with RSX using alternate criteria of presence/absence and greater than two-fold enrichment versus a control, either absence of oligonucleotides or scrambled oligonucleotides (Fig.  1 A, Additional file 2 ).

RSX and Xist interactomes share common orthologous proteins and protein–protein association networks with distinctive functional enrichments. A Overview of ChIRP-MS workflow. * Two proteins were identified by a single pulldown from a UV crosslinked sample. ** includes 4 additional proteins identified using RIP-qPCR. Graphic created using BioRender.com. B Protein–protein interactions of the RSX and Xist interactomes based on experimentally determined interactions, co-expression, and curated database annotations for human orthologs (STRING database v11.5) [ 28 ]. Each node represents an interactome protein, each edge represents an annotated protein–protein interaction of minimum confidence 0.4. Interaction networks were visualised using Cytoscape (v3.8.2) [ 29 ], omitting proteins with no annotated interactions. Nodes were clustered based on connectivity (number and weight of edges) using the GLay Cytoscape plugin [ 30 ] with default settings. Intercluster edges to minor clusters (4–10) omitted for clarity. # denotes mean intracluster node degree (21). C Key functional and structural enrichments of each major protein interaction cluster. GSEA was conducted using gProfiler2 [ 31 ] with multiple testing correction based on false discovery rate. D Enrichment of RSX (fold change relative to Igg controls) by immunoprecipitation of protein targets from female Monodelphis fibroblast cell lysates, followed by quantitative PCR using RSX -specific primers. Enrichment (30-fold) was also detected for HNRNPK, as previously published [ 21 ]

We validated two RSX interactors using RNA immunoprecipitation (RIP) followed by quantitative PCR (qPCR). The RIP targets were chosen for their potential role in XCI. In eutherian models, SUZ12/EZH2 (core components of PRC2), HDAC2, HNRNPK and CTCF have roles in eutherian XCI. MBD2 + 3, MBD4, and MECP2 bind methylated DNA and are involved in chromatin remodelling and gene regulation. The histone marks H3K9me3 and H3K27me3 are known to accumulate on the inactive X in marsupials [ 32 , 33 ]. PCAF is an acetyltransferase, so served as a negative control (Fig.  1 D). Target proteins were immunoprecipitated from female Monodelphis fibroblast cell lysates, followed by qPCR using RSX -specific primers [ 21 ]. Enrichment of RSX (relative to IgG controls) of greater than tenfold was detected for seven targets. These included HNRNPK [ 21 ] and SFPQ, which were identified by the ChIRP-MS. The RIP-qPCR also identified four additional RSX interactors not detected by ChIRP-MS: EZH2 (PRC2 catalytic subunit), HDAC2 (a histone deacetylase), MBD4 (a methyl-CpG binding domain protein), and MECP2 (a methyl-CpG-binding protein).

These proteins were included in the RSX interactome, bringing the total to 135 proteins. We considered whether proteins of the RSX interactome had orthologues in the Xist interactome. Of the 135 RSX -interactors, 81 did not have orthologues in the Xist interactome, so were specific to the RSX interactome. The remaining 54 RSX -interactors had orthologues that were identified in the Xist interactome (which comprises 497 proteins in total). Therefore, we identified a substantial cohort of proteins that interact with both RSX and Xist , despite the lack of homology in the primary sequence of these two lncRNAs. We also considered the extent to which the two interactomes might include different proteins from common functional pathways, potentially providing insights into how therian XCI evolved to be mediated by different lncRNAs in marsupials and eutherians.

Network analysis of the RSX and Xist interactomes reveals functional similarities

Gene set enrichment analysis (GSEA) [ 34 ] of each of the RSX and Xist interactomes identified that over 90% of the 136 ontology terms enriched for the RSX interactome were also enriched for the Xist interactome ( p  < 1 × 10 −3 ) (Additional file 3 : Tab 5), suggesting functional similarities between the two interactomes.

We queried the protein–protein interactions within the combined RSX and Xist interactomes using the STRING database (v11.5), with experimental findings, co-expression data, and evidence from curated databases as interaction sources [ 28 ]. Of the 578 proteins in the combined interactomes, 516 proteins had at least one interaction (confidence score > 0.4) and formed a network with 8721 edges (a mean of 15.1 edges per node). This was significantly higher than the 3633 edges expected for a random set of 578 proteins selected from the same proteome ( p  < 1 × 10 −16 ). Clustering of the interaction network partitioned it into three larger clusters and five small clusters (Fig.  1 B, Additional file 4 : Tab 1). The key functional enrichments of each of the three major clusters were determined using GSEA. The three large clusters were individually enriched for functions including mRNA binding, translation (and regulation of translation), and nitrogen compound catabolic process (Fig.  1 C, Additional file 3 : Tab 4). In addition to these common terms, the clusters had distinctive functional enrichments, including ribosomal biogenesis in cluster 1, RNA splicing and processing in cluster 2, and chromatin modification and epigenetic silencing in cluster 3 (Fig.  1 C, Additional file 3 : Tabs 1–3, with Column E in each case listing the interactome proteins underlying each enriched ontology term, Additional file 5 : Fig. S1).

Clustering and enrichment analyses were also conducted on the RSX and Xist interactomes separately using the same approach. Each interactome had four major clusters, with GSEA enrichments reflecting those of the combined interactome analysis, subject to division of cluster 2 in the RSX interactome, and division of cluster 1 in the Xist interactome (Additional file 5 : Fig. S2).

RSX -specific interactome proteins were of interest in unravelling the differences between eutherian and marsupial XCI. GSEA of the 81 RSX -specific proteins identified enrichments for spliceosomal complexes, ribosomal subunits, cytosolic translation, nucleosome binding and chromatin organisation (Additional file 3 : Tab 6) in proportions similar to those of the overall RSX interactome, other than perhaps for nucleosome binding which predominantly involves RSX -specific proteins. Apart from this, RSX -specific proteins did not appear to have gross unique function compared to the full RSX interactome.

Collectively, the clustering and GSEA enrichment analyses revealed an overlap between the RSX and Xist interactomes. This encompassed common proteins and also interactions with different proteins in common molecular pathways, providing insights into the functions modulated by RSX and Xist.

Functional analysis of HNRNPK in Monodelphis XCI

We focused on the functional role of HNRNPK, which was identified in our RSX interactome and is also an Xist -interacting protein. HNRNPK is important in recruiting polycomb repressive complex 1 (PRC1), a significant part of the epigenetic silencing machinery, during eutherian XCI [ 11 , 35 ]. In the combined RSX/Xist interactome network it was in cluster 2, which was enriched for functions in RNA splicing and processing (Fig.  1 B). We depleted HNRNPK expression in a female Monodelphis fibroblast cell line using RNA interference (RNAi), adapting eutherian-based construct design and delivery for our non-traditional model organism. We assessed the effect on XCI using RNA FISH, which allowed us to determine the transcriptional status of MSN, an X-borne gene that is usually silenced on the inactive X chromosome, which should have monoallelic expression. In control nuclei (transfected with an empty RNAi vector) biallelic expression of MSN (indicating transcription from both X chromosomes) was detected in only 18% of cells ( n  = 286; Additional file 5 : Figs. S3 and S4). Knockdown efficiency was assayed by measuring transcript abundance using RT-qPCR. The knockdown effect on protein abundance may differ due to variations in post-transcriptional processing.

Following HNRNPK knockdown (by ~ 24–35%) biallelic expression of MSN increased from 18% in control cells to 39% in cells with depleted HNRNPK expression ( n  = 159; p  = 1.0 × 10 −11 chi-squared test goodness of fit test) (Additional file 5 : Figs. S3 and S4). Increased biallelic expression of MSN signified reactivation of transcription from the silenced allele on the inactive X chromosome. This outcome was observed across two independent experiments, and provides evidence that HNRNPK plays a role in maintenance of transcriptional silencing on the inactive X chromosome in Monodelphis .

Functional analysis of CKAP4 in Monodelphis XCI

CKAP4 has not been identified as an Xist interactor, and has no predicted interactions with any protein in either the Xist or RSX interactomes (Additional file 4 : Tab 3). In the RSX interactome, CKAP4 was unexpectedly the protein with the highest fold-change (20 ×) enrichment relative to controls in the native (uncrosslinked) ChIRP-MS (Additional file 2 : Tab 2). Therefore, we used RNAi to suppress CKAP4 in female Monodelphis fibroblasts by ~ 53–55%. We observed an increase in biallelic expression of MSN from 18 to 26% ( n  = 165; p  = 8.7 × 10 −3 Chi Squared Test Goodness of Fit Test). This suggests that CKAP4 plays a role in maintenance of Monodelphis XCI.

Interestingly, despite the absence of CKAP4 from the GSEA analysis of the combined interactome network, the rough endoplasmic reticulum (where CKAP4 is usually localised) was significantly enriched in cluster 1 ( p  = 1.1 × 10 −6 ), along with three other associated terms ( p  < 9.8 × 10 −4 ) (Additional file 3 : Tab 1). This finding aligns with the functional enrichment of ribosomal and translation-associated machinery observed in the same cluster.

The role of CKAP4 in marsupial XCI prompted a comparative analysis of its protein sequences across a broad phylogenetic spectrum, including eutherians (mouse, human, and hyrax — an afrotherian), monotremes (platypus and echidna), and eight marsupial species. This comparative sequence analysis (Fig.  2 A), unveiled a large expansion of a glutamine (Q)-rich repeat at the N-terminus in the Monodelphis CKAP4, which contrasted eutherians, monotremes and most other marsupials.

CKAP4 has a glutamine-rich repeat expansion in monodelphis. RSX and Xist interactomes are enriched for proteins with IDRs. A Protein sequence alignments of representative mammalian CKAP4. An expansion of a glutamine (Q) rich repeat was observed at the N-terminus in Monodelphis and yellow footed antechinus. Inset shows a subregion of the repeat expansion. B AlphaFold predicted structure of Monodelphis CKAP4, with the Q rich repeats highlighted in black. C Alignment of predicted CKAP4 structures for human (blue), mouse (red), hyrax (orange) and Monodelphis (green and black). Sequence independent RMSD values (for all atoms with outlier rejection) of Monodelphis CKAP4 to the eutherian orthologues were 24.5 Å (human), 24.5 Å (mouse), and 13.7 Å (hyrax). D Median protein IDR scores for the RSX and Xist interactomes represented as violin plots (depicting density distribution) overlayed with boxplots depicting the median for all proteins of the RSX and Xist interactomes (mouse orthologs), and randomly sampled proteins of a subset of the mouse proteome comprising only proteins within the gene ontology terms enriched in clusters 1, 2 and 3 (20 × sets of 200 proteins). Statistical significance assessed using Dunn’s test (with Holm adjustment) for pairwise comparisons, following Kruskal–Wallis test

We employed AlphaFold to predict the tertiary structures of CKAP4 in Monodelphis alongside three representative eutherian species: human, mouse, and hyrax (Fig.  2 B and C). The structural predictions highlight a distinctive helical conformation within the Poly Q-rich N-terminus of Monodelphis CKAP4 (2B). Such structural motifs are known for their stability and propensity to engage in functional interactions with RNA and proteins [ 36 ], and provides a molecular mechanism by which Monodelphis CKAP4 could interact with RSX .

Enrichment of proteins with intrinsically disordered regions (IDRs) in the RSX interactome

Recent studies have revealed that the Xist compartment is founded on an assembly of dynamic RNP complexes comprising Xist RNA in association with the intrinsically disordered regions (IDRs) of Xist -interacting proteins, such as SPEN, PTBP1, MATR3, CELF1, and CIZ1 [ 37 , 38 , 39 , 40 ]. Of these, only PTBP1 was identified in the RSX interactome, so we considered whether other proteins with IDRs might also be present in the RSX interactome.

We assessed the proportion of proteins enriched for IDRs in each interactome with IUPred2A [ 41 ], which calculates a disorder score for each residue using amino acid composition and energy estimation. Disorder scores above 0.5 (range 0 to 1) correspond to disordered residues. We calculated the IDR score for each protein as the median disorder score of its residues. We found that RSX interactome proteins had higher median IDR scores than the Xist interactome proteins ( p  = 2.5 × 10 −9 ). Both interactomes had higher median IDR scores ( p  = 1.4 × 10 −27 for RSX and p  = 2.7 × 10 −16 for Xist ) than a randomly sampled group of 200 proteins from a subset of the reference proteome (Fig.  2 D). The background proteome subset comprised all proteins within the ontology terms (Additional file 3 , Tabs 1–3, column A) enriched ( p  < 1 × 10 −3 ) in one or more of the combined interactome clusters 1, 2 and 3. Proteins common to both the RSX and Xist interactomes had higher median IDR scores than proteins that interact exclusively with either RSX or Xist ( p  = 2.5 × 10 −2 cf RSX , p  = 1.4 × 10 −10 cf Xist ) (Additional file 5 : Fig. S5A).

IDRs have important roles in supporting protein–protein interactions, protein-RNA interactions, and the formation of phase-separated condensates that form nuclear subcompartments [ 42 , 43 , 44 ]. Our finding suggests that an enrichment for proteins with IDRs may play a role in the formation of RSX -associated RNP complexes, aiding subcellular organisation, as has been identified for Xist -associated RNPs.

This research provides a novel insight into the complex protein interactions of RSX , a lncRNA in marsupials with a role similar to the eutherian Xist in epigenetically silencing the inactive X chromosome. We found that that the RSX interactome has functional enrichments analogous to Xist that underscore their functional similarities. We also showed that alleles on the inactive X chromosome were partially reactivated following the partial depletion of HNRNPK and CKAP4, two proteins in the RSX interactome, indicating a role for each in marsupial XCI maintenance.

Of note was the glutamine (Q)-rich repeat at the N-terminus of CKAP4. Poly-Q motifs play a pivotal role in modulating protein–protein interactions, often leading to the formation of aggregates with distinct biological consequences [ 36 ]. The structure of the Poly-Q rich repeat in Monodelphis CKAP4 suggest a novel change that might underpin its different function when compared to the eutherian counterpart. The helical conformation of Monodelphis CKAP4 N-terminus could enhance affinity for RNA and/or proteins, which could enable its binding with RSX . Interestingly, the Poly-Q motif expansion is not common to all marsupials, appearing to be specific to Monodelphis and a species of antechinus, suggesting linage specific adaptation.

LncRNAs provide an important organising mechanism in epigenetic regulation, including in the recruitment and sequestration of RNA splicing and processing factors. Many of these proteins are multifunctional, often with distinct nuclear and cytoplasmic functions. This interaction of lncRNAs with multifunctional proteins can provide an efficient mechanism by which lncRNAs can impact diverse molecular networks. Of the 54 proteins identified in common in the RSX and Xist interactomes, 43 form part of interactome network cluster 2, which features proteins involved in RNA splicing and processing.

The 54 proteins common to both interactomes were enriched for intrinsically disordered regions (IDRs), a characteristic identified in each interactome individually. These IDRs could potentially contribute to epigenetic silencing by facilitating protein–protein interactions, as proteins enriched in IDRs are characterised by their flexible and adaptable binding with multiple partners. This binding plasticity may contribute to the dynamic regulation of gene expression on the inactive X chromosome, potentially including alternate silencing and escape from silencing, depending on specific cellular contexts. Further, this plasticity might be important for the observed ‘leakiness’ of XCI observed in marsupials as partial or full expression from the inactive X chromosome [ 45 , 46 ].

Analysis of interactome network clusters 1 and 3 provided further insight into the mechanisms by which RSX and Xist might regulate gene expression. GSEA of each of these clusters indicated a functional coherence: despite a relatively small overlap in interactomes, they had different protein interactors involved in shared pathways. Cluster 1 was functionally enriched for ribosomal biogenesis, rRNA processing and regulation of translation. For RSX , this is consistent with the nucleolar association of the inactive X in marsupials. Cluster 3 contained proteins typically associated with XCI, including those involved in epigenetic regulation of transcriptional silencing, histone modifications and heterochromatin. These proteins include SPEN, which was identified in all of the Xist ChiRP-MS studies but was absent from the RSX interactome. SPEN is required for upregulation of Xist during XCI initiation [ 47 ], but becomes less important during the maintenance stage [ 48 ]. Therefore, it was not unexpected that SPEN was absent from the RSX interactome in our fibroblast model, which represents XCI maintenance.

Enrichment of functions associated with post-transcriptional regulation is a fascinating aspect of the Xist and RSX interactomes. Post-transcriptional regulation of X-borne gene expression has been identified in eutherians and Monodelphis by comparing gene expression in the transcriptome and translatome (based on ribosomal occupancy) [ 24 ]. Balancing of sex chromosome-borne gene expression between sexes in the proteome has also been identified in the more distantly related platypus ( Ornithorhynchus anatinus ) and chicken ( Gallus gallus ). This highlights the possibility of post-transcriptional regulation being an ancestral strategy for fine tuning the expression of sex chromosome genes in both sexes [ 49 ]. Our understanding of the evolution of the therian sex chromosomes suggests that silencing of X-borne genes would have evolved as the X and Y chromosomes diverged, perhaps initially involving other noncoding RNAs and only localised silencing before the Y chromosome was as degraded as it currently is. The emergence of independent chromosome wide regulation of XCI by XIST and RSX would then have coordinated presumably more efficient silencing, balancing the expression of X-borne genes between the sexes as degeneration of the Y chromosome progressed.

Conclusions

This work highlights a striking example of convergent evolution of lncRNA protein interactome evolution that achieves XCI in diverse mammalian clades. The independently evolved XIST and RSX recruit similar molecular pathways to repress the activity of almost an entire chromosome. These molecular pathways are associated with epigenetic transcriptional silencing, which typifies XCI, in addition to post-transcriptional regulation of gene expression, notably RNA splicing and processing, translation regulation and ribosome biogenesis. The functional coherence between the RSX and Xist interactomes, and the prevalence in both interactomes of proteins enriched for IDRs, adds a novel and critical dimension to our understanding of lncRNA mediated epigenetic regulation.

Cell culture

Female Monodelphis fibroblasts were cultured at 35 °C with 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM), 10% v/v Newborn Calf Serum, 0–10% v/v AmnioMAX™-C100. Cells were passaged at 70–100% confluency using Trypsin–EDTA (0.25% w/v) (Thermo Fisher Scientific).

Cells were cultures on 15-cm plates to 70–80% confluence. Three plates (~ 6 µg total protein) were used for each pulldown. Cross-linking of samples occurred prior to cell harvest using either: (1) UV using Stratalinker UVP Crosslinker CL-1000 (200 mJ/cm 2 at 200 nm) on ice in 10 ml of phosphate buffered saline (PBS); or (2) 3% formaldehyde solution in PBS (30 min, RT) followed by quench in 0.125 M glycine for 5 min. ‘Native’ samples were not cross-linked. Cells were scraped from the plate into Eppendorf tubes and pelleted at 500 rcf for 5 min at 4 °C. Cell pellets were alternately flash-frozen and stored at – 80 °C, or proceeded directly. The cell pellet was resuspended (1 ml per plate of cells) in NP-40 buffer with Roche cOmplete™, EDTA-free Protease Inhibitor Cocktail (1 tablet per 7 ml of NP-40 buffer) and incubated for 15 min either: 1) at 4 °C on rotating platform; or) on ice, with vortexing for 5 s every 5 min. Cell lysates were sonicated using a Q700 sonicator (Qsonica) in a 4 °C water bath at amplitude 16 for 12 min pulsing 30 s on/off. Sonicated cell lysates were pelleted at 20,000 rcf for 5 min at 4 °C. Supernatant lysate was removed and assayed for total protein concentration using a Qubit™ fluorometer. Aliquots of 1.5 ml of supernatant (~ 3 µg total protein) were combined with 50 µl of prepared Dynabeads™ M-280 Streptavadin (beads). Beads were prepared in accordance with manufacturer’s guidelines with the following modifications: 100 µl of resuspended beads were used for each sample. After washing in 1 × Binding + Wash buffer (10 mM Tris–HCl (pH 7.5) 1 mM EDTA 2 M NaCl), beads were washed twice in Solution A (DEPC-treated 0.1 M NaOH DEPC-treated 0.05 M NaCl) followed by twice in Solution B (DEPC-treated 0.1 M NaCl), in each case vortexing for 5 s before magnetic capture for one min. Suspended beads were divided into 100 µl aliquots before final magnetic capture, followed by addition to each aliquot 200 µl of 2 × B + W buffer, 5 µl of 100 mM biotin-labelled oligonucleotide (omitted for control) and 195 µl of DNase-free H 2 O. Samples were incubated for 15 min at room temperature on slow rotating platform. Beads were magnetically captured for 3–5 min, before removal of clear supernatant. Beads were washed 3 times with a 1 × B + W buffer (200 µl) on rotation for 2 min with a one min magnetic capture. Beads were re-suspended in 100 µl of NP40 Buffer, before dividing between the two 1.5-ml aliquots of supernatant for each sample. Lysates were incubated with the pre-cleared beads on a rotating platform overnight at 4 °C. Beads were magnetically captured for one min at 4 °C, and supernatant removed. The beads for each sample were then washed with twice with 2 ml of NP40 Buffer (divided equally between the bead aliquots for the first wash before combining for the second wash), followed by twice for 15 min with 1 ml RIPA Buffer on rotating platform at 4 °C. After the final magnetic capture, the supernatant was removed. One hundred microliters of pre-warmed (65 °C) Elution Buffer (10 mM Tris–HCl, pH 6.0, 1 mM EDTA, 2.0M NaCl) was added to the supernatant before incubating for 20 at 65 °C with shaking. The supernatant was magnetically cleared of beads twice before assaying the protein concentration of the supernatant using a Qubit™ fluorometer, and submitting for LC/MS–MS. For validation, supernatant containing 20–60 μg of protein was dissolved in 1 × Laemmli Buffer (Bio-Rad), heated to 95 °C for 10 min to denature, and then size-separated on a 7.5% SDS-PAGE gel (Bio-Rad TGX) in 1 × Tris/Glycine/SDS (TGS) Buffer (Bio-Rad) at 160 V. Protein gels were washed in Milli-Q water three times for 5–10 min each before staining overnight in Commassie blue, washing in RO water three times and then excising protein bands.

Probe design

ChIRP-MS probes (Additional file 1 ) were designed using online tools [ 50 ]. Oligonucleotide probes were synthesised with 3′ Biotin-TEG, obtained from Integrated Data Technologies, Inc.

For each pull-down oligonucleotide probes were either pooled, or used individually. Probe 3 targeted the RSX Repeat 1. A probe with no homology to any sequence in the Monodelphis genome was used as an additional control to filter proteins identified in native (uncrosslinked) pull-downs.

Mass spectrometry

Samples were analysed at the Bioanalytical Mass Spectrometry Facility at the Mark Wainwright Analytical Centre (UNSW, Australia). Briefly, samples were firstly buffer exchanged to ammonium bicarbonate via 3 kDa spin cartridge. Samples were reduced (5 mM DTT, 37 °C, 30 min), alkylated (10 mM iodoacetamide, RT, 30 min), and incubated with trypsin at 37 °C for 18 h, at a 1:20 ratio (w/w). Samples were desalted with 200 µl C18 stage tip tips (Thermo Fisher Scientific). Eluted peptides from each clean-up were reconstituted in 10 µL 0.1% (v/v) formic acid and 0.05% (v/v) heptafluorobutyric acid in water. Digest peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex, Amsterdam, Netherlands). Samples (2.5 µl) were concentrated and desalted onto a micro C18 precolumn (300 µm × 5 mm, Dionex) with H2O:CH3CN (98:2, 0.05% TFA) at 15 µl/min. After a 4 min wash the pre-column was switched (Valco 10 port valve, Dionex) into line with a fritless nano column (75µ ×  ~ 10 cm) containing C18 media (1.9 µ, 120 Å, Dr Maisch, Ammerbuch-Entringen Germany) manufactured according to Gatlin [ 51 ]. Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 200 nl/min over 30 min. High voltage 2000 V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~ 0.5 cm from the heated capillary ( T  = 275 °C) of an Orbitrap Velos ETD (Thermo Electron, Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Orbitrap operated in data dependent acquisition mode (DDA).

A survey scan m/z 350–1750 was acquired in the Orbitrap (resolution = 30,000 at m/z 400, with an accumulation target value of 1,000,000 ions) with lockmass enabled. Up to the 10 most abundant ions (> 4000 counts) with charge states >  + 2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q  = 0.25 and activation time of 10 ms at a target value of 30,000 ions. M/z ratios selected for MS/ MS were dynamically excluded for 30 s.

LC–MS/MS spectra were analysed using the MaxQuant software suite (version 1.6.2.10.43) [ 52 ]. Sequence database searches were performed using Andromeda [ 53 ]. Label-free protein quantification was performed using the MaxLFQ algorithm [ 54 ]. Delayed normalizations were performed following sequence database searching of all samples with tolerances set to ± 4.5 ppm for precursor ions and ± 0.5 Da for peptide fragments. Additional search parameters were: carbamidomethyl (C) as a fixed modification; oxidation (M) and N-terminal protein acetylation as variable modifications; and enzyme specificity was trypsin with up to two missed cleavages. Peaks were searched against the reference genome for Monodelphis (Ensembl release 97). MaxLFQ analyses were performed using default parameters with “fast LFQ” enabled. Protein and peptide false discovery rate (FDR) thresholds were set at 1% and only non-contaminant proteins identified from ≥ 2 unique peptides were subjected to downstream analysis.

Protein groups files were imported into R Studio for analysis. Proteins were identified using a combination of (i) presence/absence analysis, to identify proteins detected in two or more pulldowns for native samples and a single pulldown for UV crosslinked samples and formaldehyde crosslinked; and (ii) intensity-based analysis, to identify proteins enriched more than threefold (log 2 ratio > 1.584963) relative to a control. For the fold-change analysis for formaldehyde crosslinking and UV crosslinking, proteins were selected on this basis alone. For the fold-change analysis for native (no crosslinking), proteins were subject to additional filtering: (i) proteins detected by more than 4 of 6 different ChIRP probe combinations (comprising 5 RSX probes individually + all RSX probes together); and (ii) t -test (with Benjamini–Hochberg correction for multiple testing), p value < 0.05.

RNA immunoprecipitation (RIP)

The RIP was performed on female M.domestica cells as described in [ 21 ] using the antibodies set out in Additional file 1 . Quantitative RT-qPCR was performed using RSX -specific primers as described in [ 21 ].

RNAi knockdown

The shRNA-expressing constructs were designed using a combination of online tools [ 55 , 56 , 57 ], and nucleotide BLAST of candidate sequences against the MonDom5 genome assembly (Ensembl release 84). For each target mRNA, candidate constructs were trialled for different regions of the mRNA to accommodate the possibility that binding may be impeded at certain sites by secondary structure or sequence variants. Constructs were cloned into a pCDNA3-U6M2 plasmid vector using BglII and KnpI restriction sites as described previously [ 58 ]. In summary, 2 ug of vector DNA was digested with KpnI-HF in 1 × CutSmart® buffer, then with BglII in 1 × NEB buffer 3.1. Each digest was incubated in total volume of 50 μl (including BSA 5 μl, 1 mg/ml) at 37 °C for one hour. The vector DNA was purified after each digest using the QIAquick® PCR Purification Kit. The vector was then 5′ dephosphorylated using Antarctic Phosphatase. The shRNA construct was prepared by 5′ phosphorylation of the oligonucleotides with T4 Polynucleotide Kinase, followed by annealing of the complementary oligonucleotides at 95 °C for 5 min, cooled to 25 °C over 1 h. The cut vector and shRNA construct were ligated with T4 DNA Ligase and transformed into competent Escherichia coli DH5α cells. Five microliters of vector (10–15 ng) was added to 50 μl cells, incubated on ice for one hour, heat-shocked at 42 °C, for 45 s, then incubated in 350 μl SOC medium at 37 °C for one hour. Competent DH5α cells were prepared by culturing in Luria Broth at 37 °C to optical density A 600 , then incubating on ice for 10 min, pelleting by centrifugation at 1520 rcf for 10 min at 4 °C, resuspending in Transformation buffer (6 mL), before storing at − 80 °C. Transformed DH5α cells were plated on ampicillin selective Luria Both agar. Gel electrophoresis of colony PCR product was used to screen for colonies cloned with the shRNA template. PCR reactions were performed with primers p008 and p080 using Taq 2 × Mastermix according to manufacturer’s instructions. PCR products of candidate clones were then sequenced using BigDye v.3.1 by the Ramaciotti Centre for Genomics (UNSW Sydney, Australia) to confirm cloning accuracy. Successful clones were cultured overnight at 37 °C in selective Luria Broth (ampicillin 100 μg/ml), and then extracted using the QIAGEN® Plasmid Midi Kit according to manufacturer’s instructions.

Transfection

The shRNA vector plasmids were introduced into the Monodelphis fibroblasts by transfection with Lipofectamine 3000. First transfection was carried out when cells were at 70–80% confluency. For transfection of cells on coverslips in 6-well plate, vector DNA (2.5 μg), Lipofectamine® 3000 reagent (6.5 μl) and P3000® reagent (5 μl) diluted in 250 μl Opti-MEM medium, added to 1.5 ml of cell culture medium. For transfection of cells in T25 flask, vector DNA (7.5 μg), Lipofectamine® 3000 reagent (18 μl) and P3000® reagent (15 μl) diluted in 375 μl Opti-MEM medium, added to 4 ml of cell culture medium. A second transfection was carried out ~ 24 h after initial transfection. For each transfection, incubations were conducted as per manufacturer’s instructions and cell culture media was replaced 6 h after transfection. Cells were harvested for RNA extraction, or progressed to RNA FISH, ~ 24 h after the second transfection.

RNA extraction

RNA was extracted from transfected cells using TRIzol® reagent (Invitrogen), 1.5 ml for T25, 500 µl for per well of 6-well plate, with incubation at room temperature for 5 min with mild agitation. Chloroform was added (0.2 ml chloroform per 1 ml of TRIzol® reagent), vortexed and incubated for 10–15 min at room temperature, before centrifuging at 10,000 rcf for 15 min at 4°. The aqueous (upper) phase was aspirated, and 1.5 × volume of 100% ethanol was slowly added and mixed. RNA was purified from the sample using RNeasy spin column kit (according to manufacturer’s instructions and on-column DNAse digestion using the RNase-free DNase set according to the manufacturer’s instructions. Final elution of RNA was in Ultra-Pure™ DEPC-treated water. RNA concentration was assayed using Qubit™ RNA Assay.

shRNA knockdown RT-qPCR

cDNA was prepared using Superscript™ IV First-Strand Synthesis System according to the manufacturer’s instructions, with 70–300 ng of total RNA as template and using oligo dT primers. RT-qPCR was conducted using the Viia7 Real-Time PCR System (Applied Biosystems) in technical triplicate using the KAPA SYBR® FAST qPCR Kit Mastermix (2 ×) Universal, using 0.5 μl of cDNA template and gene-specific primers (10 μM) (Additional file 1 ) in a 10-μl reaction. PCR was conducted at 95 °C for 20 s for enzyme activation, followed by 40 cycles of: 95 °C (1 s), 60 °C (20 s), then 60 °C to 99 °C melt curve analysis. Target expression level was calculated using the ΔΔCt method relative to the control cells transfected with an empty pCDNA3-U6M2 plasmid vector, and with normalisation with reference to GAPDH.

RNA FISH probe preparation

RNA FISH probes were derived from BAC clones from the VMRC-18 BAC library (CHORI BACPAC Resources Centre (Oakland, CA)). Msn (BAC clone VM18-777F) was identified as a highly expressed X-linked gene based on RNA-seq transcriptome data (unpublished) from the same female Monodelphis fibroblasts, and mapping using Ensembl monDom5 assembly (release 84). The BAC clone containing RSX (VM18-839J22) was previously identified [ 18 ]. The BAC clones were acquired in E. coli DH10B, cultured in selective Luria Broth (chloramphenicol, 34 mg/ml) and extracted with the QIAGEN® Large Construct Kit. The BAC DNA was labelled by nick translation using DNase1, DNA Polymerase 1, fluorescent dUTP (0.03 mM Green 496 dUTP or Orange 552 dUTP) and nick translation buffer, with incubation at 15 °C for 1.5 h. Labelled probes were filtered through a sephadex column to remove unincorporated nucleotides. Probe size (~ 200–700 bp) was verified using gel electrophoresis (1% agarose). The labelled probes (100–200 ng per coverslip) were co-precipitated overnight at − 80 °C with Monodelphis C 0 t-1 DNA, and 100% ethanol v/v (3 × volume). After centrifugation (18,000 rcf, 4 °C, 30 min), the probe was washed twice in 70% ethanol, then air dried, dissolved in formamide (5 μl per coverslip, UNILAB), then denatured (75 °C, 7 min). The probe was then combined with hybridization buffer (5 μl per coverslip) and incubated on ice for 5 min, then at 37 °C for 20 min.

Sterilised coverslips were coated with gelatin before seeding in a 6-well plate with cells to density of ~ 70% confluence in overnight culture. Cells were washed with 1 × PBS before being permeabilized with Cytoskeletal buffer on ice for 5–7 min, then fixed in freshly made paraformaldehyde (3% w/v in 1 × PBS) for 10 min at room temperature, washed twice in 70% ethanol for 5 min, then dehydrated in an ethanol series (80%, 95%, 100% each for 3 min) before air drying. The prepared coverslip and probe (10 μl per coverslip) were hybridised on an RNase-free glass slide, sealed with vulcanised rubber, incubated overnight at 37 °C in a chamber, humidified with tissues soaked in 5 ml of 50% formamide/2 × SSC. Coverslips were then washed in a solution of formamide 50% v/v/2 × SSC (3 washes, each at 42 °C, 5 min), and then in 2 × SSC (3 washes, each at 42 °C, 5 min), air-dried and mounted. Coverslips were mounted with Prolong™ Diamond AntiFade Mountant with DAPI and sealed with clear nailpolish. Prepared slides were analysed using an Olympus BX53 microscope with proprietary cellSens software. Images were processed and compiled using Fiji (ImageJ) [ 59 ].

Interactome analysis

The Xist and RSX interactome analysis was conducted using the STRING database (v 11.5) [ 28 ] using human as the reference species. Human orthologues of Monodelphis genes were identified using Ensembl 97 BioMart [ 60 ]. For genes without a one-to-one orthologue, a human orthologue or equivalent was identified using reciprocal protein BLASTs [ 61 ]. There were two exceptions to this, where the MonDom5 gene did not have a human 1 to 1 orthologue (ENSMODG00000025105), or where the best reciprocal blast hit was already represented in the RSX interactome (ENSMODG00000013903). In these cases, the gene was excluded from downstream analysis. Evidence of interaction was based on experimentally determined interactions, curated database annotations, and experimentally determined co-expression. Minimum interaction confidence was set at 0.400 (calculated on a scale of 0 to 1). Interaction networks were visualised using Cytoscape (v 3.8.2) [ 29 ], omitting proteins with no interactions. Clusters were generated using the GLay Cytoscape plugin [ 30 ] with prefuse force directed layout. GSEA was conducted using gProfiler2 (v 0.2.2) [ 31 ] for annotations GO:MF, GO:CC, GO:BP (BioMart classes releases 2023–03-06) in R Studio with multiple testing correction based on false discovery rate and filtering for ontology term size < 1600.

Intrinsically disordered region analysis

The IDR content in interactome proteins was assessed using IUPred2A [ 41 ] using the idpr package (v 1.12.0) in R Studio using UniProt Accession ids (release 2023_02). This calculated a disorder propensity score for each residue based on parameters designed to detect long IDRs (at least 30 consecutive residues), with scores ranging from 0 to 1 and a score over 0.5 indicating a disordered residue. The median IDR score for each protein was calculated as the median of the per residue disorder scores.

Random sets (mouse and Monodelphis orthologs) were generated by randomly sampling (20 sets of 200 proteins each) from subsets of the UniProt (release 2023_02) proteomes UP000000589 (Mus musculus, Organism ID 10090) and UP000002280 (Monodelphis, Organism ID 13616), respectively, using the set.seed() function in R (version 4.1.3). The background proteome subset comprised all proteins within the ontology terms (Additional file 3 , Tabs 1–3, column A) enriched ( p  < 1 × 10 −3 ) in one or more of the combined interactome clusters 1, 2 and 3. Gene sets were obtained for each species from the GMT files for each of GO:BP, GO:MF, GO:CC [ 62 ]. Mouse orthologues of proteins in the RSX interactome were identified using Ensembl 97 BioMart [ 60 ].

For genes without an identified one-to-one orthologue, a mouse orthologue was identified using reciprocal best hit protein BLAST [ 61 ]. Three RSX interactors were not represented in the mouse orthologue set, either because no 1 to 1 orthologue was identified (ENSMODG00000006291, ENSMODG00000024476), or where the best reciprocal BLAST hit was already in the RSX interactome (ENSMODG00000008362). The statistical difference between groups was determined using the Kruskal-Wallace test followed by Dunn’s test (with Holm adjustment) for pairwise comparisons.

CKAP4 protein sequence alignment and structure prediction

We retrieved CKAP4 orthologues sequences from the NCBI Gene database [ 63 ] and Ensembl (v 111) [ 64 ]. The selected orthologues included representatives from marsupials, monotremes and eutherians (human, mouse) model organisms and an Afrotheria species (rock hyrax), aiming to encompass a broad phylogenetic spectrum. The specific protein accession numbers selected for this analysis were ENSP00000367265 (human); ENSMUSP00000050336 (mouse); ENSPCAP00000004540 (rock hydra); XP_020838076.1 (koala); XP_027728405.1 (wombat); XP_031793680.1 (Tasmanian devil); XP_036617112.1 (common brushtail possum); XP_043823845.1 (monito del monte); XP_044535072.1 (agile gracile opossum); XP_051817421.1 (yellow-footed antechinus); ENSMODP00000002342 (monodelphis); XP_028935129.1 (platypus); XP_038612756.1 (short-beaked echidna). Alignment was performed using the NCBI Multiple Sequence Alignment Viewer (v 1.25.0, COBALT) [ 65 ] with default settings.

For structural predictions, we used Colabfold v1.4.0 running on the Gadi supercomputer system at the National Computational Infrastructure (NCI), Canberra, Australia. This approach leverages the predictive power of AlphaFold2, incorporating both template-based and template-free modelling to predict protein structures with high accuracy. The FASTA sequence files for CKAP4 from human, mouse, hyrax, and Monodelphis were inputted for structural prediction. Default parameters were used for the database search. To ensure robustness of predictions, a recycle count of 3 was used, enhancing the iterative refinement of the predicted structures. Furthermore, we employed the –amber flag to incorporate molecular dynamics simulations for refining the predicted structures, and the –templates flag to utilise available structural templates that could guide the folding prediction.

Availability of data and materials

Mass spectrometry protein files and custom R scripts used to filter them are available via github.com [ 66 ]. RNA FISH images additional to Figures S3 and S4 are available via Figshare [ 67 ]. All other data are available in the manuscript or the supplementary materials.

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McIntyre KL, Waters SA, Zhong L, Hart-Smith G, Raftery M, Chew ZA, Patel HR, Marshall Graves JA, Waters PD. Identification of the RSX interactome in a marsupial shows functional coherence with the Xist interactome during X inactivation. Figshare.  https://doi.org/10.6084/m9.figshare.25807144 .

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Acknowledgements

Not applicable.

Peer review information

Andrew Cosgrove was the primary editor of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Review history

The review history is available as Additional file 6 .

P.D.W. and J.A.M. are supported by Australian Research Council Discovery Projects (DP170101147, DP180100931, DP210103512 and DP220101429). P.D.W. is supported by NHMRC Ideas Grants (2021172, 2027730). H.R.P. is supported by an NHMRC Ideas Grant (2021172). S.A.W. is supported by the UNSW Scientia program and an NHMRC Ideas Grant (1188987).

Author information

Kim L. McIntyre and Shafagh A. Waters contributed equally to this work.

Authors and Affiliations

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, 2052, Australia

Kim L. McIntyre & Paul D. Waters

School of Biomedical Sciences, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW, 2052, Australia

Shafagh A. Waters

Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW, 2052, Australia

Ling Zhong & Mark Raftery

Australian Proteome Analysis Facility, Macquarie University, Macquarie Park, NSW, Australia

Gene Hart-Smith

National Centre for Indigenous Genomics, Australian National University, Canberra, ACT, 2601, Australia

Zahra A. Chew & Hardip R. Patel

Department of Environment and Genetics, La Trobe University, Melbourne, VIC, 3086, Australia

Jennifer A. Marshall Graves

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Contributions

K.L.M. drafted the manuscript and figures, performed RNA FISH, and conceived and performed IDR and GSEA analysis. S.A.W. conceived and performed ChIRP and RIP-qPCR, and performed manuscript editing and figure preparation. L.Z. generated mass spectrometry data. G.H.S. performed mass spectrometry data analysis. M.R. was involved in mass spectrometry data collection and analysis. Z.A.C. was involved in protein structure prediction. H.R.H. was involved in protein structure prediction. J.A.M. commented on and edited the manuscript. P.D.W. conceived and oversaw the project, and assembly of the manuscript.

Corresponding author

Correspondence to Paul D. Waters .

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Supplementary Information

Additional file 1: table of reagents, resources and oligonucleotides., additional file 2: tab 1. rsx and xist interactomes. tab 2. chirp-ms results., 13059_2024_3280_moesm3_esm.xls.

Additional file 3: Tabs 1 - 3. GSEA analysis of combined interactomes network clusters 1 – 3, respectively. Column E (‘Genes’) lists the proteins in each enriched gene ontology term for the interactomes. Tab 4. GSEA terms enriched in all of combined interactomes network clusters 1-3. Tab 5. GSEA analysis of all proteins in each of RSX and Xist interactomes analysed separately (i.e., not combined) and including proteins with no predicted protein-protein interactions). Columns G and J (‘Genes.Rsx’ and ‘Genes.Xist’) list the interactome proteins underlying each enriched gene ontology term in the RSX and Xist interactomes, respectively. Tab 6. GSEA analysis of all proteins present in RSX interactome and absent from Xist interactome. Column E (‘Genes’) lists the interactome proteins in each enriched gene ontology term.

13059_2024_3280_MOESM4_ESM.xls

Additional file 4: Tab 1. Numbers of proteins and edges in STRING networks and each cluster of combined interactomes. Tab 2. Proteins in each cluster of combined interactomes. Tab 3. Proteins of combined interactomes with no predicted protein-protein interactions.

13059_2024_3280_MOESM5_ESM.pdf

Additional file 5: Five additional supplementary figures and legends: Figure S1. Graphical abstract. Figure S2. Protein-protein association networks of Xist and RSX interactomes. Figure S3. RNA FISH images using probes for RSX and X-borne gene, MSN, following RNAi knockdown of HNRNPK and CKAP4. Figure S4. Additional RNA FISH images (control and HNRNPK RNAi knockdown). Figure S5. Median protein IDR scores for RSX and Xist interactomes.

Additional file 6: Review history.

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McIntyre, K.L., Waters, S.A., Zhong, L. et al. Identification of the RSX interactome in a marsupial shows functional coherence with the Xist interactome during X inactivation. Genome Biol 25 , 134 (2024). https://doi.org/10.1186/s13059-024-03280-0

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Received : 02 November 2023

Accepted : 14 May 2024

Published : 23 May 2024

DOI : https://doi.org/10.1186/s13059-024-03280-0

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