Singer and songwriter Judy Collins asked the question rhetorically: Who knows where the time goes? But the issue raised by the refrain hits close to home for today's entrepreneurs.
But the problem arises not because these leaders are bad planners. It's because they are great entrepreneurs. People who are talented at juggling lots of ideas and problems tend to see interruptions as just another thing to juggle.
Those entrepreneurs who are essential to the smooth workings of a business often believe that work would grind to a halt if they didn't make themselves available for interruptions. As a result they tend to see interruptions as something that they should accommodate, no matter when and where. They think, It will just take a minute, then I'll get back to my own priorities.
But interruptions are not benign and don't just take a minute. If entrepreneurs don't guard against them, interruptions can wipe out three to five hours of a workday, every day.
Consider one of the most common interruptions that entrepreneurs deal with on a daily basis: unplanned questions from key employees.
Imagine an entrepreneur immersed in deep concentration on a priority, say, fine-tuning a big proposal due that very day whose acceptance could be pivotal for the company's third and fourth quarters. An interruption pops up, just a quick one: The head of marketing wants to share some prime results from the last campaign. And why not? She deserves the recognition.
So the entrepreneur does a quick review of the numbers, dispenses compliments and makes a note to follow up with congratulations to the team the next day and investigate further exactly what worked.
Now he can go back to his proposal. That only took five minutes. It's no problem, right? Wrong. Just as the marketing chief had walked in, the entrepreneur had just concluded that his proposal provided several different descriptions of the solution, and he wanted to highlight the best one and ask for the others to conform. Now he must go back and reread those sections and decide which one was the keeper.
He isn't irritated with the marketing chief but finds himself irritated with the situation. He doesn't like doing the same thing twice and his opinion about the keeper had been fresher and better before. Now he has lost the context so he is not sure. But this proposal is too important to guess about, so he restarts reviewing the proposal from the beginning, but with less energy and enjoyment than he brought to the task the first time.
A glance at the clock confirms that all this rereading has used up the time he wanted to spend on the all-important pricing section to reconfirm some of the underlying assumptions. He runs a couple of calculations. In the end he realizes that he has to trust his sales manager's numbers, so he hands it off for finalizing just in time to meet the deadline.
On the way home, he figures out that the proposal's pricing was wrong. He calls his sales manager, asks him to call the prospect and rescind the proposal. He and the sales manager spend a couple of hours reworking the numbers.
He's late to dinner, the kids are in bed, the sales manager is embarrassed and the prospect is unimpressed.
So tolerating interruptions is not necessarily the hallmark of a successful entrepreneur. Simultaneously handling multiple demands is not good for the health of the company — only for its demise. To be successful entrepreneurs must learn how to handle those who would interrupt them and not allow their time to be stolen. This should tackled not by discourteously dismissing an interrupter but by learning how to make it in that employee's interest to let the entrepreneur work uninterrupted when necessary.


As well as fascinating desert-adapted wildlife, it is the rugged beauty of Damaraland that entices travellers to this remote wilderness. Colossal sandstone cliffs, towering mountains and ancient geological formations make up one of Namibia's most dramatic landscapes.
Unfolding between the Skeleton Coast and Etosha, the geography of Damaraland is simply breath-taking. Casting a shadow over the desert highlands and rocky plains are some of the highest mountains in the country.
The ruggedness of this remote wilderness has produced some remarkable wildlife, including some of the last free-roaming desert-adapted elephants in the world. Desert-adapted rhino are also present and often seen traversing the sand dunes. Add to the list thriving populations of Hartmann's zebra, gemsbok, springbok, Damara dik-dik and kudu, and you have an unforgettable wildlife destination.
When you're not tracking wildlife in the north, venture to the south of Damaraland to discover a wealth of San artwork and geological features. Twyfelfontein is particularly impressive, with hundreds of rock engravings depicted on the sandstone cliffs that date back to 3300 BC. The 200 million-year-old Petrified Forest – a collection of fossilised tree trunks on a sea of sandstone – is also well worth a visit.
As one of Namibia's least populated regions, you can enjoy the delights of Damaraland with few others. Get in touch with one of our Africa specialists to start putting together your luxury tailor-made trip.


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An Abbey Theatre and Landmark Productions co-production with Octopus Theatricals, Ireland and USA
Theatre for One (and a Little One)
Dates: 29 – 31 December
11am – 1.30pm, every 30 minutes (recommended for ages 4 – 7)
3pm – 5.30pm, every 30 minutes (recommended for ages 7 – 10)
Tickets: Free, but ticketed
Running Time: Approx. five mins
Please note: If you do not have a ticket, you will not gain access. Please do not come to the performance if you do not have an assigned ticket.
Given the audience capacity, we anticipate significant demand for this production. If you cannot make it to your allotted performance time, please contact box office by phone or email to relinquish your ticket so it can be transferred to another audience member.
One ticket allows you to see one play. To see multiple plays, you need to book multiple tickets.
Four leading Irish writers present original, five minute plays, performed by one actor for one young audience member and their lucky guest.
Theatre for One is a very special, state-of-the-art performance space – and this Christmas, for the very first time, we are opening it up to younger audience members.
Conceived and created by the Olivier award-winning designer Christine Jones and designed by LOT-EK architects, one lucky young audience member at a time (accompanied by a grown-up – if they choose) will be able to enter the specially-designed booth, and experience the magic of a brand new play, performed just for them.
Two of the plays are written for children aged 4 – 7, and two for those aged 7 – 10.
Produced by Landmark Productions and Octopus Theatricals, in co-production with the Abbey Theatre, it features short plays for young audiences by four of Ireland's leading writers – Roddy Doyle, Sonya Kelly, Louis Lovett and Pauline McLynn – and promises a unique theatrical thrill for audience members young and old.
Supported by Accenture and Dublin City Council.
#T41andaLittleOne
Online Programme
Download the Theatre for One (and a Little One) – Online Programme
Audience members will be required to follow all HSE safety guidelines. Mandatory guidelines will be included within your ticket. See here for more information on safely visiting the Abbey Theatre.
Cast: Lorcan Cranitch
Cast: Eleanor Methven
Cast: Rory Nolan
Cast: Caoimhe O'Malley
Created by: Christine Jones
Directed by: Eoghan Carrick and Veronica Coburn
Costume Design: Ciara Fleming
Sound Design: Ivan Birthistle
Lighting Design: Colm Robinson
Writer: Roddy Doyle
Writer: Sonya Kelly
Writer: Louis Lovett
Writer: Pauline McLynn
Theatre for One

redpajama_set_name: RedPajamaCommonCrawl


to spend a few days on vacation? There are many places to stay in Sesimbra Municipality. Travelers from every corner of the Iberian Peninsula and other countries come to enjoy the beach, monuments, historical heritage, and beautiful nature of this Portuguese town in the region of Setubal.
Visitors who have the opportunity to stay in Sesimbra Municipality lodging can experience the tranquility of a city on the Atlantic Ocean and close contact with nature. Houses are abundant throughout Setubal and are a cheaper option than the hotels in Sesimbra Municipality. Keep in mind that the city is a few kilometers from Lisbon, the capital of Portugal.
If you're unsure of where to stay in Sesimbra Municipality, search Minube to find the accommodation that suits your needs. Search a variety of Sesimbra Municipality hotel deals at the best prices and in the most recommended areas of the city, especially near the beach area. Book now and come enjoy one of the best cities in the Iberian Peninsula!


On April 4, 2008, the European Union granted the Melton Mowbray pork pie the long-coveted Protected Geographical Indication status. For the last 14 months, only pork pies made within a designated zone around the modest town of Melton Mowbray may be packaged and sold as Melton Mowbray pork pies. These pork pies are distinguished by their uncured, greyish meat and scalloped, hand-raised crust (prepared according to a specific recipe, naturally). The melifluous town name, by the way, is half Anglo-Saxon (Melton means middle village or manor) and half pure Norman conquest - Mowbray comes from the family name of the lords of the manor. (Mowbray is itself a corruption of the French Manche.) Stilton cheese and the phrase "paint the town red" also originate from humble Melton Mowbray.
And now to the pie itself. Our particular pie was Brockleby's Melton Mowbray, the award-winning organic Melton Mowbray pork pie. The world's only organic Metlon Mowbray pork pie, apparently! Brockeby's is found in Hathern, a village of about 1800, located to the west of Melton Mowbray.
Our pie was a gorgeous, dense construction, its delicate pink interior cushioned in just the right amount of aspic and nestled inside a perfect golden-brown crust. We split the pie into sixths and ate it with apples and cheese, Stilton and a sharp Cheddar. The flavors were excellent compliments, but the pie was more than tasty enough to be eaten on its own. We washed down our bites with a delicious Badger First Gold ale.
All in all, an excellent summer's dinner with a superior pork pie.


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EPOS Developer Portal
The New EPOS Developer Portal – a Simple, Scalable and Secure Way to Access All Digital Solutions Assets
EPOS Media Alert EPOS Developer Portal.pdf
Global PR Manager, Enterprise
Yasmin Gouhari
yagu@eposaudio.com
Copenhagen, Denmark – November 3, 2021: EPOS, the premium audio solutions brand, is excited to reveal the new EPOS Developer Portal. This digital platform provides access to EPOS APIs and SDKs, enabling partners and customer development teams to integrate and deploy EPOS devices into their voice and video solutions to ensure a superior user experience and achieve higher productivity.
Designed for easy accessibility to EPOS APIs and SDKs
The EPOS Developer Portal was created to facilitate access to EPOS digital solutions assets in one centralized platform. Technical partners can access here a variety of APIs and SDKs and work into their own solutions to take advantage of device management capabilities, interoperability or call control.
Designed to help users stay up to date with the latest software releases for our digital solutions assets, the Developer Portal features a built-in lightweight support system where you can get all your technical questions answered.
''We are thrilled to introduce the EPOS Developer Portal, a one-stop platform that supports longtime collaboration with our partners. This new program will improve the overall experience and enable organizations to work and communicate in a more productive way using EPOS audio and video solutions", Theis Mørk, VP Product Management.
Click here to find further information about the EPOS Developer Portal.
Established upon the former successful joint venture between audio specialist Sennheiser and the world-leading hearing technology group Demant, EPOS designs, manufactures and sells pioneering high-end audio and video solutions for business professionals and serious gamers.
Crafted with advanced engineering and pioneering technologies, EPOS offers uncompromised audio and video connectivity that enable business professionals to better communicate and collaborate – anytime, anywhere and on any device. EPOS audio is reliable, easy-to-use and designed for ultimate comfort.
Owned by the Demant Group and with headquarters in Copenhagen, Denmark, EPOS builds on more than 115 years' audio expertise and operates in a global market with offices and partners in more than 60 countries.
Alongside own-branded premium audio and video solutions, EPOS sells co-branded EPOS I SENNHEISER products under a trade license agreement with Sennheiser.
Poor Audio Causes our Brains to Work 35% Harder to Interpret Information, EPOS Study Finds
EPOS audio solutions built on EPOS BrainAdaptTM technologies can improve performance and reduce cognitive load
Copenhagen, Denmark – November 3, 2022 – Global audio and video brand EPOS has today launched findings examining the impact poor audio quality is having on the productivity levels of remote workers. The research found that poor audio quality causes our brains to work harder to process information. EPOS also found that the brain undergoes significant strain when struggling to process digital audio in comparison to sounds in a physical environment.
EPOS launches cashback campaign to enable businesses to invest in quality hybrid work solutions
EPOS incentives businesses to scale up hybrid work and optimize collaboration
Copenhagen, Denmark – 11 October 2022 - Global audio brand EPOS has today launched a cashback campaign to enable businesses to invest in technology solutions that stimulate collaboration and increase productivity without scrimping on quality, durability, or performance. The campaign is available to enterprise-end customers. The campaign will run in markets including Austria, Belgium, Germany, France, Ireland, Luxembourg, Netherlands, and the United Kingdom. From October 1st through to December 31st, 2022, business purchases of select products will be eligible for a fixed cashback payment for every purchased unit1.


Community-based and professional organizations play a crucial role in the iterative relationship between research and action. Below are a selected group of public health, research, and social justice organizations committed to racial health equity.
This list is provided as a courtesy. Being listed does not necessarily indicate endorsement or partnership.
The ACE Minority Affairs Committee promotes diversity in the field of Epidemiology and hosts an annual workshop on health disparities.
The nation's leading agency dedicated to monitoring and addressing health issues in the country. The CDC also provides helpful information and statistics about all major diseases and conditions, and the vaccinations needed when traveling internationally.
An APHA caucus concerned about social inequalities in health. Our purpose is to spur new connections among the many of us involved in different areas of public health, who are working on diverse public health issues (whether as researchers, practitioners, teachers, activists, or all of the above), and live scattered across diverse regions of the United States and other countries.
Nonprofit, independent professional association that represents a diverse membership of nearly 4,000 health education professionals and students in the United States and 25 international countries.SOPHE members work in schools, universities, voluntary organizations, health care settings, worksites, and in local, state and federal government agencies.
HCAP's main purpose is to study and improve health in underserved communities with community based organizations, academia, public health practitioners, and policy makers in a collaborative and co-learning process that stresses systems development, community capacity building, and balancing research and action.
Highlander is the anchor of organizing in the U.S. South. Rosa Parks, Rev. Martin Luther King, Jr., Septima Clark and many others trained and conducted workshops here.
Founded in 1970 to lend a hand to black leaders as the traveled the uncharted road from civil rights activisim to the political establishment. Its most prominent founders were Dr. Kenneth B. Clark, a renowned social psychologist, and Louis E. Martin, the legendary news paper editor who had become a key presidential adviser on issues affecting black America.
SAAPHI consists of researchers, academics, professionals and students who share and interest and passion in improving the health of African American communities.
The African American Policy Forum (AAPF) is an innovative think tank that connects academics, activists and policy-makers to promote efforts to dismantle structural inequality. We utilize new ideas and innovative perspectives to transform public discourse and policy.
The BCFBSM was founded in the mid 1980's after LAPD announced a serial murderer was operating in South LA. LAPD waited until close to a dozen women were killed before announcing anything to the public. Since then, the BC has campaigned to bring attention to these murders, to work for justice and accountability by law enforcement, city officials and the media.
An expanding social justice movement born in response to the perceived impunity with which police and others kill members of the African American community.
The City Project believes that all people should have access to healthy, livable communities. Our multicultural, Latino-led team works with diverse allies to ensure equal access to (1) healthy green land use through planning by and for the community; (2) climate justice; (3) physical education and schools of hope as centers of their communities; (4) health equity and wellness; and (5) economic vitality for all.
EmbraceRace is a multiracial community of parents, teachers, experts, and other caring adults who support each other to meet the challenges that race poses to our children, families, and communities.
A collection of people from all faiths, backgrounds, ethnicity, gender, socioeconomic status, joined together in mutual hope and admiration for essential fairness and compassion among society as a whole.
The organization's website. This grassroots organization challenges corporate agribusiness' adverse effects on the health, social, labor and environmental well-being of North Carolina communities. The organization educates the public to raise awareness about key issues, trains students and advocates, engages in political action and conducts empirical research.
A National Call for Moral Revival is uniting tens of thousands of people across the country to challenge the evils of systemic racism, poverty, the war economy, ecological devastation and the nation's distorted morality.
SPLC monitors hate groups and hate crimes across the nation, and it conducts workshops to raise awareness about these issues. It has also assisted persons interested in making a transition out of white supremacist groups.
Begun in 1962 by Cesar Chavez, Dolores Huerta, Gilbert Padilla and other early organizers, the United Farm Workers of America is the nation's first enduring and largest farm workers union. The UFW continues organizing in major agricultural sectors, chiefly in California.
The BRITE Center's mission is to support the innovative use of research, science and policy development to help eliminate disparities in physical and mental health for communities that are traditionally underserved by academic research.
The California Center for Population Research (CCPR) was established in 1998 and has since, been a leading research center for research and training in demography. CCPR is comprised of over 90 active faculty researchers from an array of academic disciplines, such as epidemiology, public policy, economics, sociology, and public welfare.
Center for Critical Race Studies at UCLA engages scholars and community members in interdisciplinary research that expands our understanding of crucial educational and social inequality issues. Centering the role of race, racism, and their intersectionality with other forms of discrimination such as sexism, classism, homophobia, and ableism, is key to the CCRS mission.
The Center for Culture, Trauma, and Mental Health Disparities is a multi-ethnic and multi-disciplinary group promoting interdisciplinary research examining the prevalence and impact of traumatic experiences on PTSD, depression and concomitant cognitive/emotional, behavioral, psychological and biological processes in ethnic minority populations.
UCLA Law's Critical Race Studies (CRS) program is the premier institutional setting for the study of the intersection between race and the law. Anchored by renowned scholars whose research represents the cutting edge of critical race theory in legal scholarship and related disciplines, our CRS program has no parallel in American legal education.
The mission of the UCLA Institute of American Cultures (IAC) is to advance our understanding of the new social and cultural realities in America. The unparalleled population shifts that have occurred in recent decades have transformed our sociocultural landscape, expanding both intra-group diversity as well as opportunities for intersectional exchanges.
The Institute on Inequality and Democracy advances radical democracy in an unequal world through research, critical thought, and alliances with social movements and racial justice activism. We analyze and transform the divides and dispossessions of our times, in the university and in our cities, across global South and global North.
The Center is committed to working with community, academic, and government parters to design, implement, and disseminate relevant research pertaining to racism and health disparities. If you would like to partner with us, please click the button below.


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A revealing memoir by a witness to some of the most important events of recent U.S. history.
Hardback$22.48Indie
John Kerry tells the story of his remarkable American life—from son of a diplomat to decorated Vietnam veteran, five-term United States senator, 2004 Democratic presidential nominee, and Secretary of State for four years—a revealing memoir by a witness to some of the most important events of our recent history.
Every Day Is Extra is John Kerry's candid personal story. A Yale graduate, Kerry enlisted in the US Navy in 1966, and served in Vietnam. He returned home highly decorated but disillusioned, and testified powerfully before Congress as a young veteran opposed to the war.
John Kerry was a visiting distinguished statesman at the Carnegie Endowment for International Peace, where he focused on conflict resolution and global environmental challenges.
Kerry served as a prosecutor in Massachusetts, then as lieutenant governor, and was elected to the Senate in 1984, eventually serving five terms. In 2004 he was the Democratic presidential nominee and came within one state—Ohio—of winning. Kerry returned to the Senate, chaired the important Foreign Relations Committee, and succeeded Hillary Clinton as Secretary of State in 2013. In that position he tried to find peace in the Middle East; dealt with the Syrian civil war while combatting ISIS; and negotiated the Iran nuclear deal and the Paris climate agreement.
Every Day Is Extra is Kerry's passionate, insightful, sometimes funny, always moving account of his life. Kerry tells wonderful stories about colleagues Ted Kennedy and John McCain, as well as President Obama and other major figures. He writes movingly of recovering his faith while in the Senate, and deplores the hyper-partisanship that has infected Washington.
Few books convey as convincingly as this one the life of public service like that which John Kerry has lived for fifty years. Every Day Is Extra shows Kerry for the dedicated, witty, and authentic man that he is, and provides forceful testimony for the importance of diplomacy and American leadership to address the increasingly complex challenges of a more globalized world.


The first song of the new GB Soundtrack to hit the web is now the first song to release a music video (well, first new song - gotta love Debarge!). With plenty of GB action, the video for Good Girls shows off Elle King's proton-weilding skills - check it out!
Good Girls single available now on iTunes and Amazon.
GB2016 OST available from tomorrow on iTunes and Amazon.
When ghoulish-goings on start happening at the Manchester Printworks, Mad Mancunian Mr. Deansgate and resident Ghost Walker Flecky Bennett call in the professionals!
Members of our GMGB and YGB divisions team up to do battle with a couple of troublesome spooks and save the day!
Looks like another addition off the upcoming soundtrack has been leaked online!
ZAYN's offering, which is called wHo will be the fifth song on the trackless and is more of a smooth groove compared to the earlier-dropped tracks.
Have a listen, but be warned - this is a leaked audio and the end of the track is a little.... skippy.
The UKGB Hit London For New Film Celebrations!
The UK Ghostbusters & friends hit the town yesterday to celebrate the UK release of the new Ghostbusters film - and boy did we have a blast!
After getting a call about somethin' weird in Waterloo, we rolled out to find an old friend causing havoc! Everyone's favourite Marshmallow Man decided to make his big break in the station - right through the floor (Courtesy of Wild Creations)! On top of that, there is evidence of a local Slimer causing mischief, and a cool Forbidden Planet pop-up store selling all things Ghostbusters!
It seems forever ago when we placed our pre-order for this, and so you can imagine the excitement seeing the tired, disheveled postman hand over a slightly buffed Amazon box! After almost removing the arm of said postman, and squiggling some sort of electric signature on his pad, the door was barely shut when the package was ripped open.
And it's beautiful - you can't hep but stare at the cover for a while! Luckily the awaiting pages do not drop the bar and look just as awesome!
Happy 64th Birthday Dan Aykroyd!
The UK Ghostbusters wish a HUGE Happy Birthday to Dr. Ray Stantz himself, Dan Aykroyd.
We salute you, sir! Hope to see you again real soon!


Campus Classics first attended summer conventions and regional meetings throughout the country to market its sportswear and product line. Its look was unique and the higher quality immediately recognized. The demand for its product in this setting was realized.
Through the mid 1990's and long before the Internet, targeted mailings and salesmen crisscrossed the country selling sportswear in chapter houses in roughly 25 states! Although, this approach was not entirely unique at the time; the desire to bring these items to market in a "licensed" environment with the approval of each headquarters was.
As "Greek's" themselves, our founders knew disregarding trademarks owned by these organizations was not right or consistent with their beliefs or ideals. Until Greek Licensing evolved in the late 1990's, the greater majority of these companies weren't paying the same licensing fees Campus Classics had for almost ten years.
Twenty-five years later Campus Classics and its mission have evolved, adding in-house embroidery, screen-printing, laser engraving, talented graphic designers, and a deep "on-hand" inventory, shelved and ready to ship per group.
We remain a dynamic, passionate and family-owned sportswear and gift item provider to the customers we serve. We are also committed to "building the brand" and enhancing the image, identity and initiatives of the client groups we have partnered with these past twenty years.
We work diligently manufacturing quality merchandise in an ethical and charitable environment and know our staff and service will exceed your expectations!


Space Available: 2,100 to 30,000 sq. ft.
Class A office space in the heart of downtown Burlington. This 8-story building is one of the most prominent buildings in the city as viewed from Lake Champlain. Partial and full floor spaces available, from 2,100-30,000 sq. ft., some with lake and mountain views. Significant improvements are planned, including a new building entry, updated lobby with a café, fitness room, bike storage, and more. Join an impressive group of tenants, including Goldman Sachs, Willis Towers Watson, and Gannett/Burlington Free Press. Rent: Starting at $15/ sq. ft.+ fixed NNN.


We are still thinking about Narrowboat Bob, ideal in so many ways but do we really want to spend 6.5k over our top budget? We are also a bit hazy on the finer details of this boat, as we viewed so many other boats on the same day. We know we felt really positive about her, but we can't recall exactly why she felt right.
Bob is a two hour drive away, and as we have no plans the next day we decide that we should do yet another road trip. There is one marina that we didn't get to see on our last trip, so we figure we can call into see them too, and Michael checks to see if any other boats have been listed anywhere that might also fit our requirements. He finds one; it is another private sale, and another one that has been owned by the same people from new and used for continuous cruising. We arrange to see it at 9am the next morning.
When we arrive we spend over an hour chatting to the owners and looking over this beautiful boat, we'll call her C. The owners tell us it was their intention to live on this boat for the rest of their days, and that they have only changed their plans due to medical reasons. The love that they had shown the boat was clear, she was immaculate and clearly well loved. There are so many little details and they take such pride when taking about her. The engine is so clean, and there are so many little details; this couple really have thought of everything. And there are records and photos going back to her purchase, with the original contract to get her built as well as owners manuals for every piece of equipment on board. It is all looking really positive until we mention that we would need to get a survey done, they seem a bit taken aback and explain that it would be difficult to arrange and that they must to sell by the end of the month to complete on a house purchase. This boat is currently their home, and it is not convenient to get it pulled out of the water in the time frame available. They seem to reluctantly accept it, if we can arrange everything and it is not to far for them to travel.
When we leave I point the car in the direction of our next destination and Michael hits the phone to first find a marina that can accommodate the pull out, and then a surveyor that is free on the same day. Miraculously, after just 45 minutes of calling he has pulled it all together for four days time, it is an easy day's cruise for them, and we even confirm that there are no stoppages on that stretch of canal. So we offer the full asking price and pass on the details for the survey. We get a message back saying that they are currently grocery shopping and will contact us back this afternoon. All we can do is wait and hope.
We pass the time by viewing Bob again, and we remember why we liked her so much; she has more character than I previously gave her credit for, and, looking with more critical eye, we can see that she really would make a fine home. We take some more advice on the lack of central heating and conclude that, in the short-term, it is something we can live without, and can be installed at any stage in the future, for a not inconsiderable cost. The only drawback is the asking price, we don't really feel comfortable spending quite that much.
Still no news about C, so we head off to the brokerage that we didn't make it to on Saturday. This is a place we really wanted to visit. They make really good videos of each boat on their books and for the last six months we have watched every one they have released, it was a really good way of learning about the different types of boats available for sale while we were living in the States. When we arrive they are really welcoming and happy to chat and Michael spends a good five minutes bonding with the huge bear of a dog that is hanging around the office. Like many other places we have visited in the last week they also advise that they are low in stock and waiting for more boats to come in. There was one possibility, but on viewing she wasn't as nice as Bob and she was 5k more.
Heading off again -- this time to meet some friends for dinner -- and trying not to stress too much about the call that hasn't yet come. Then, finally, a call, and it wasn't the news we were hoping for. They had received another offer for C that matched ours, however the other offer came from a couple that didn't require a survey; we still had first refusal, but they now wouldn't move the boat to the marina to get pulled out of the water. I felt sick. I really wanted this boat. The next hour was horrible, as we debated our options and went round in circles. She was almost perfect and a such a good price, she had been priced to sell and really was a bargain. My instinct told me that it would be fine; they came across as a really genuine couple, their story stood up, there was evidence from the state of the boat that she was well looked after and in great condition. But what if there was a minor issue? How would we feel if we found out down the line that there was a problem, surely we could just fix it? Ok, we would have to spend a bit of extra money, but I felt if we took our time and sold this boat on, we could actually make some money. But, then, what if there was an issue that was catastrophic? Michael kept mentioning galvanic corrosion; this boat had been moored for the last two years next to other boats with mains hookups, so it was a possibility, it was really unlikely, but what if? We could buy her and get a survey done after the fact, but what if we pulled her out of the water and there was a 1mm section of hull? What if she sank on the way to get the survey? We could lose 40k overnight, and then what would we do?
I do believe that this couple were completely genuine, and that the boat would pass the survey with flying colours, but it would also be the perfect scenario for a con. At the end of the day we just couldn't take the risk, and I know we did the right thing for us. The couple that bought her are braver than we are; I am sure it will work out perfectly for them, and despite the loss I feel I am happy for them. At the same time, it hurts; I wanted that boat and it hurts to lose out. It hurts to write about too, and it will until we find our boat and understand why C wasn't the boat for us after all.
And so to the second rejection. We don't want to rebound to Bob, she is a very different boat and so we sleep on it. We realise that the one thing holding us back from her is the cost; she is advertised at over 16k more than C, and we are just not comfortable spending so much. We decide to put in a cheeky offer, not so cheeky to be insulting, but cheeky enough that we are pretty sure it will be rejected. Its just it is all we are comfortable spending, knowing how much we would then have to spend on top of that to get her how we want her. And the offer is rejected. We draw a line under Bob and walk away.


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Toronto police arrest four suspects in connection with teen's alleged abduction in March
Published May 14, 2020 Updated May 14, 2020
Toronto police say they have arrested four suspects and are seeking at least one more in the case of a 14-year-old boy who was allegedly abducted over his stepbrother's criminal activity.
Police say the arrests were made earlier this week and the suspects – all men between the ages of 25 and 38 – face a total of 26 charges combined.
The charges include kidnapping for ransom, forcible confinement, conspiracy to commit an indictable offence, and uttering threats.
Toronto police Chief Mark Saunders says officers are still seeking a fifth man, and there may be more arrests and charges as the investigation continues.
The teenage victim was nabbed off the street in early March in response to a multimillion-dollar cocaine heist the previous summer.
The ordeal lasted 36 hours and triggered an Amber Alert until the boy was found, dishevelled but otherwise healthy, in Brampton, Ont.
Police say the teen was not involved in any criminal activity.
The boy went missing around 8:30 a.m. on March 4, but his parents did not know their son had never made it to school because the school missed an absence-reporting deadline.
The police chief says the arrests were conducted during a series of searches that also led officers to seize a kilogram of cocaine, more than 31 kilograms of marijuana and thousands of dollars.
Saunders says another man arrested during the operation is not believed to be involved in the abduction but is facing several drug-related charges.


Sara, an apathetic teenager, gets art history tutoring from a woman named Helena. What neither of them know is that these simple art lessons will become a more personal journey for Sara to accept herself.
Notas Aparte | S1:E1 | Sara meets her new Art History tutor, Helena.
Notas Aparte | S1:E2 | Sara confronts herself.
Notas Aparte | S1:E3 | Sara and Helena discuss crushes and play The Lesbian Quiz.
Notas Aparte | S1:E4 | Helena and Sara exchange notes, and Helena's lessons continue.
Notas Aparte | S1:E6 | Sara tries to cheer up Helena.
Notas Aparte | S1:E7 | Helena teaches Sara how to flirt.


Inevitably the tragic death of Mikaeel Kular is prompting many nurseries to reflect on how they would respond to a child's questions in the event of a family member or friend dying.
The phasing of Universal Credit will have significant implications for the finances of families using nurseries and for nursery staff themselves.
Having been reception leader for the past two years, Leisa Towle is now about to embark on an exciting new role in this very large primary school.


NEW ORLEANS – The New Orleans Pelicans announced that after discussions with Dell Demps this morning, the team and Demps have decided to mutually part ways.
Danny Ferry will assume the team's interim general manager duties. Ferry most recently served as the Pelicans' Special Adviser for the last three seasons and has over 25 years of NBA experience as both a player and front office executive. His previous front office experience includes President of Basketball Operations and General Manager for the Atlanta Hawks (2012-15), Vice President of Basketball Operations for the San Antonio Spurs organization (2010-12), and General Manager of the Cleveland Cavaliers (2005-10).


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Islet Studies
MLL3 and MLL4 Methyltransferases Bind to the MAFA and MAFB Transcription Factors to Regulate Islet β-Cell Function
David W. Scoville1,
Holly A. Cyphert2,
Lan Liao3,
Jianming Xu3,
Al Reynolds4,
Shuangli Guo2 and
Roland Stein1,2⇑
1Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN
2Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN
3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX
4Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN
Corresponding author: Roland Stein, roland.stein{at}vanderbilt.edu.
Diabetes 2015 Nov; 64(11): 3772-3783. https://doi.org/10.2337/db15-0281
Insulin produced by islet β-cells plays a critical role in glucose homeostasis, with type 1 and type 2 diabetes both resulting from inactivation and/or loss of this cell population. Islet-enriched transcription factors regulate β-cell formation and function, yet little is known about the molecules recruited to mediate control. An unbiased in-cell biochemical and mass spectrometry strategy was used to isolate MafA transcription factor–binding proteins. Among the many coregulators identified were all of the subunits of the mixed-lineage leukemia 3 (Mll3) and 4 (Mll4) complexes, with histone 3 lysine 4 methyltransferases strongly associated with gene activation. MafA was bound to the ∼1.5 MDa Mll3 and Mll4 complexes in size-fractionated β-cell extracts. Likewise, closely related human MAFB, which is important to β-cell formation and coproduced with MAFA in adult human islet β-cells, bound MLL3 and MLL4 complexes. Knockdown of NCOA6, a core subunit of these methyltransferases, reduced expression of a subset of MAFA and MAFB target genes in mouse and human β-cell lines. In contrast, a broader effect on MafA/MafB gene activation was observed in mice lacking NCoA6 in islet β-cells. We propose that MLL3 and MLL4 are broadly required for controlling MAFA and MAFB transactivation during development and postnatally.
Diabetes mellitus is a disease that affects the body's ability to maintain euglycemia, with type 1 characterized by a loss of insulin-producing islet β-cells and type 2 (T2DM) by peripheral insulin resistance and β-cell dysfunction. One proposed treatment for type 1 diabetes is to replace diseased β-cells with those generated from human embryonic stem cells (hESCs) or induced pluripotent stem cells (1). The principal limitation in producing functional β-cells has been directing the final postnatal maturation steps (2), which involves expression of proteins required for glucose sensitivity and insulin secretion (3).
Islet-enriched transcription factors are essential for embryonic formation and postnatal function of β-cells (4–6). For example, very early exocrine and endocrine pancreatic development is driven by Pdx-1 starting at embryonic day 8.5 (e8.5) in mice, with mice and humans both lacking a functional copy suffering from pancreatic agenesis (4,5). In contrast, Ngn3 expressed from e9.5 is only required in the formation of endocrine cell types (i.e., β, α [hormone glucagon producing], δ [somatostatin], ε [ghrelin], and pancreatic polypeptide) (6). MafA is expressed even later during development and only in β-cells (i.e., e13.5), contributing in postnatal maturation steps (7). Interestingly, the induction of glucose-sensitive insulin secretion in vivo from transplanted hESC-derived endocrine progenitors correlates with MAFA expression (8). Moreover, the production of only Pdx-1, Ngn3, and MafA is sufficient to reprogram mouse exocrine, intestinal, and liver cells into insulin+ β-like cells in vivo (9–11). Although these examples clearly illustrate the fundamental importance of islet-enriched activators to β-cells, the transcriptional mechanisms involved are not well defined.
Transcription factors primarily regulate gene activation by recruitment of coregulators, which often influence expression by directly binding to the basal transcriptional machinery and/or through epigenetic remodeling of the chromatin structure. These coregulators can have positive (coactivator) and negative (corepressor) actions on target gene transcription (12), thus conferring a second level of specificity to the transcriptional response. Coregulator recruitment is, in turn, controlled by the spatial and temporal expression patterns and posttranslational modifications of the transcription factor and/or coregulator. Unfortunately, little is known about the coregulators recruited by islet-enriched transcription factors. Although there are hundreds of known coregulators (http://www.nursa.org/), such knowledge is limited to candidate studies linking, for example, Pdx-1 to p300 (13), Set7/9 (14), HDAC1/2 (15), PCIF1 (16), and Bridge-1 (17). In contrast, MafA has only been linked to p/CAF (18).
In this study, we used an "in cell" reversible cross-link immunoprecipitation (Re-CLIP) and mass spectrometry (MS) approach to isolate coregulators of MafA from mouse β-cells. Notably, all nine subunits of the Mll3 and Mll4 histone 3 lysine 4 (H3K4) methyltransferase complexes were identified in the MafA immunoprecipitates, but none of the unique subunits of the other mammalian Mll complexes were detected (e.g., Menin, Mll1, Mll2 of Mll1/2 complexes; Set1A, Set1B, Wdr82 in Set1A/B complexes). (Mll3 and Mll4 will be referred to as Mll3/4 for simplicity.) These methyltransferases were also found to bind MAFB, a closely related transcription factor essential to mouse β-cell development and coexpressed with MAFA in adult human islets (19,20). Notably, mouse islet β-cell function is compromised in a heterozygous null mutant of NCoA6, a key subunit of Mll3/4 (21). Decreased NCOA6 levels reduced expression of a subset of MAFA- and MAFB-regulated genes in human and rodent β-cell lines, with evidence provided that this results from limiting gene transcriptional start site (TSS) H3K4 trimethylation. Interestingly, there is essentially complete overlap of MafA and Mll3/4 transcriptional control in islets isolated after embryonic β-cell–specific removal of NCoA6 or MafA in vivo. These results suggest that MLL3/4 coactivator recruitment by MAFA and MAFB is important to the formation and adult function of islet β-cells.
Cell Culture and Immunoblotting Analysis
Mouse βTC-3 cells were maintained in DMEM containing 25 mmol/L glucose, 10% FBS, and 100 units/mL penicillin/streptomycin (22). Human EndoC-βH1 (23) cells were grown in DMEM containing 5.6 mmol/L glucose, 2% BSA, 50 μmol/L 2-mercaptoethanol, 10 mmol/L nicotinamide, 5.5 μg/mL transferrin, 6.7 ng/mL selenite, 100 units/mL penicillin, and 100 units/mL streptomycin. βTC-3 cells were infected with an adenovirus overexpressing GFP alone or a Flag-tagged MafA (provided by Dr. Qiao Zhou, Harvard University) at a multiplicity of infection of 200 under conditions described previously (24). Nuclear extract was prepared 48 h postinfection as described below. Primary antibodies for immunoblotting (25) are listed in Supplementary Table 1. Horseradish peroxidase–conjugated anti-rabbit, anti-mouse, or anti-goat secondary antibody was used at 1:2000 (Promega). Immunoblots were quantitated with ImageJ software (National Institutes of Health).
Re-CLIP and Immunoprecipitation Assays
The Re-CLIP protocol was adapted from Smith et al. (26). Briefly, ∼109 βTC-3 cells were cross-linked using 0.1 mmol/L dithiobis (succinimidyl propionate) (DSP) in PBS for 30 min at 37°C. Cells were then harvested and nuclear extract was prepared by high salt (400 mmol/L NaCl) extraction (25). Magnetic protein G beads (Life Technologies) were covalently cross-linked with MafA antibody (20 μg) and were preincubated with an MafA-blocking peptide (32KKEPPEAERFC42, 100-fold excess antibody) or with PBS alone. Extract and beads were incubated for 3 h at 4°C, washed with radioimmunoprecipitation (RIPA) buffer (10 mmol/L Tris, pH = 8.0, 140 mmol/L NaCl, 0.5% Triton X-100, 1% sodium deoxycholate, 0.1% SDS), and eluted in RIPA supplemented with 200 mmol/L dithiothreitol (DTT). The eluted proteins were visualized by PAGE/silver staining, and protein identification was determined by liquid chromatography–tandem MS (LC/MS/MS) or Multidimensional Protein Identification Technology (MudPIT) analysis in the Vanderbilt University Proteomics Core. Immunoprecipitation experiments were performed with the antibodies listed in Supplementary Table 1 at 10 μg each, as described above for Re-CLIP, omitting the DSP step and substituting PBS for RIPA.
Sucrose Gradient Ultracentrifugation and Electrophoretic Mobility Shift Assays
Sucrose gradients were performed as described earlier (25). Briefly, βTC-3 nuclear extract (600–1000 μg) was collected by high salt extraction (25) and separated over a 5–35% sucrose gradient (4.5 mL total volume). Fractions (300 μL each, excluding the first 500 μL) were analyzed by immunoblotting, rat insulin II C1 element gel shift (25), and immunoprecipitation analysis. HeLa cells were transfected with expression plasmids encoding human MAFA and/or MAFB (25). Antibodies used for gel shift are listed in Supplementary Table 1.
Small Interfering RNA Treatment of β-Cell Lines
Knockdown in βTC-3 and EndoC-βH1 cells was achieved using ON-TARGETplus small interfering (si)RNAs of mouse MafA (#J-041353–09), mouse NCoA6 (#J-041129–6), human MAFA (#L-027343–01), human MAFB (#L-009018–00), and human NCOA6 (#L-019107–00). The targeting siRNA (0.5 nmol) or a nontargeting control (#D001810; GE Healthcare Dharmacon) was introduced into βTC-3 cells (4 × 106) using Buffer V (Lonza #VVCA-1003) with an Amaxa Nucleofector 2 (Program G-016; Lonza, Walkersville, MD). EndoC-βH1 cells (2 × 106) were transfected with siRNAs (50 pmol/L) using the Dharmafect #1 reagent (GE Healthcare Dharmacon #T-2001), following the manufacturer's protocol. Nuclear extract, RNA, and chromatin were collected 72 h after transfection.
Quantitative PCR and Qiagen RT2 Profiler PCR Array Analysis
RNA was collected from βTC-3 cells using the RNeasy kit (Qiagen). The Trizol reagent (Life Technologies) and the DNA-Free RNA Kit (Zymo Research) were used for EndoC-βH1 cells and mouse islets. The iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc.) was used for cDNA synthesis. The quantitative (q)PCR reactions were performed with the gene primers listed in Supplementary Table 2 on a LightCycler 480 II (Roche), and analyzed by the ΔΔCT method (27). Significance was calculated by comparing the ΔCT values in all but Fig. 7A and B, wherein each mutant was normalized to a control littermate. The Qiagen RT2 Profiler PCR array for human diabetes genes (Cat. No. 330231 PAHS-023ZA) was screened with 1 ng cDNA from EndoC-βH1 cells, following the manufacturer's guidelines.
Chromatin Immunoprecipitation
Chromatin was prepared from βTC-3 cells and EndoC-βH1 cells by cross-linking for 10 min with 1% formaldehyde, followed by lysis in buffer containing 1% NP-40. The centrifuged pelleted nuclei were lysed in SDS buffer (50 mmol/L Tris, 10 mmol/L EDTA, 1% SDS), and chromatin was sonicated using a Diagenode Bioruptor. DNA (10 μg) was incubated with 5 μg of antibody or species-matched IgG, and chromatin immunoprecipitation (ChIP) was performed as described previously (28). Antibodies are listed in Supplementary Table 1, and the qPCR primers used for the analysis are listed in Supplementary Table 2.
Mouse Lines, Intraperitoneal Glucose Tolerance Test, and Islet Isolation
Floxed (fl) MafA (19) or NCoA6 (29) mice were crossed with rat insulin II promoter-driven Cre (RIP-Cre) (30) transgenic mice to delete MafA (termed MafAΔβ) or NCoA6 (NCoA6Δβ) specifically in β-cells. All mice were maintained on a mixed background (C57BL/6J, I129, Balb/c, 129S6/SvEvTac, FVB/N), although MafAΔβ mice were mostly C57BL/6J. MafAΔβ, NCoA6Δβ, MafAfl/fl and/or NCoA6fl/fl mice were fasted for 6 h in the morning before the intraperitoneal glucose tolerance test. There was no change in total body weight in MafAΔβ or NCoA6Δβ mice (Supplementary Fig. 2A). Blood glucose levels were measured before (time 0) and then 15, 30, 60, and 120 min after an intraperitoneal injection of glucose (2 mg/g body weight) prepared in sterile PBS (20% w/v). Islets from 8-week-old mice were isolated using collagenase P (Roche) digestion and hand picking (31). The Vanderbilt University Institutional Care and Use Committee approved all of these studies.
Tissue Collection and Immunofluorescence
Pancreata were dissected and fixed in 4% paraformaldehyde in PBS for 4 h, followed by paraffin embedding (32). The entire e15.5 pancreas was serially sectioned (6 µm per section) and every 10th section analyzed by immunofluorescence. Three sections at least 120 μm apart were examined at 8 weeks for islet architecture and for Slc30a8 and Slc2a2 expression. The primary antibody list is provided in Supplementary Table 1. Species-matched secondary antibodies were used for immune detection at 1:1000 (Jackson ImmunoResearch Laboratories). Slides were mounted with Dapi Fluoromount-G (SouthernBiotech #0100-20) and images acquired on a Zeiss Axio Imager M2 widefield microscope with Apotome.
Glucose-Stimulated Insulin Secretion
EndoC-βH1 cells (2 × 106) were placed in medium containing low glucose (1.1 mmol/L) for 12–14 h. Low or high glucose (15.5 mmol/L) medium was then added for 1 h. Medium and cell lysates (lysis buffer: 20 mmol/L Tris, 1% Triton X-100, 10% glycerol, 137 mmol/L NaCl, 2 mmol/L EGTA, 1X protease inhibitor tablet [Roche Diagnostics]) were collected and analyzed for insulin content by the Vanderbilt Hormone Assay Core. Glucose-stimulated insulin secretion was performed with 8-week-old islets in the Vanderbilt Islet Procurement and Analysis Core. Briefly, islets were size matched, incubated overnight in 5.6 mmol/L glucose and then treated with 5.6 mmol/L or 16.7 mmol/L glucose for 1 h. The insulin content in the medium and cell lysate was analyzed. Insulin secretion capacity was calculated as media insulin over cell lysate insulin content. Glucose-stimulated levels are presented as the 15.5 mmol/L and 1.1 mmol/L values for EndoC-βH1 cells and 16.7 mmol/L glucose and 5.6 mmol/L glucose for isolated islets.
Mean differences were tested for significance using a Student two-tailed t test. The minimal level of statistical significance is listed in the figure legend.
Identification of MafA Proteins Using Re-CLIP
MafA interacting proteins were isolated from mouse βTC-3 cells using a combined Re-CLIP/MS strategy (Fig. 1A) (26). The Re-CLIP procedure was selected over other isolation methods (e.g., tandem affinity purification [33]), because the dimerization, DNA binding, and activation capacity of MafA is regulated by many phosphorylation events (25). Amine-reactive DSP was used to covalently cross-link MafA to neighboring proteins, and MafA antibody was used to precipitate bound proteins. The signal-to-noise of the procedure was improved by using the MafA antibody blocked by MafA peptide in the control immunoprecipitation (as opposed to IgG) and by washing precipitated proteins with RIPA buffer. Bound proteins were eluted from MafA by the addition of DTT to cleave the disulfide bond in the cross-linker. As expected, Pdx-1 (22) and p/CAF (18), known MafA-interacting proteins, were bound to MafA after Re-CLIP (Fig. 1B). Moreover, many proteins were selectively bound to MafA in the immunoprecipitation conducted in the absence of blocking peptide (Fig. 1C).
MafA interacting proteins in β-cells were identified by Re-CLIP/MS. A: Schematic of the Re-CLIP protocol used for isolating MafA interacting proteins from mouse βTC-3 cells. DSP was the cross-linker used in step 1 and DTT was used to elute MafA cross-linked proteins in step 5. B: Immunoprecipitations performed with βTC-3 cells using control IgG or MafA antibody in the absence (−) or presence (+) of DSP treatment. The precipitate was then immunoblotted with MafA, p/CAF, and Pdx-1 antibodies. The input lanes contain ∼1% of the nuclear extract; treatment with (+) DTT breaks the MafA-protein disulfide cross-link and returns MafA to its normal molecular weight. C: A representative image of proteins precipitated by MafA antibody preincubated with a blocking or scrambled control peptide after SDS-PAGE and silver staining. D: All of the proteins of the Mll4 complex were detected by Re-CLIP (IP); a representative MS result is shown. Mll3 was also present in the MudPIT analysis (Supplementary Table 3).
Numerous MafA bound proteins were detected upon MS analysis of the Re-CLIP products, with their activities consistent with a role in mediating MafA activity (Supplementary Table 3). It is notable that no other islet-enriched transcription factors or the p/CAF coactivator was identified by MS, presumably reflecting their relatively low abundance (e.g., Pdx-1, p/CAF) or inability to interact. This indicates that the results in Supplementary Table 3 represent only a portion of MafA-interacting proteins.
Our analysis focused on determining the significance of Mll3/4 coactivator binding to MafA because the evolutionary conserved COMPASS family of methyltransferases is strongly associated with gene activation due to their ability to mono-, di-, and trimethylate H3K4 within the enhancer and/or promoter region (34). MS identified all nine proteins of the ∼1.5 MDa Mll4 complex (Fig. 1D), and Mll3 was additionally detected by the more sensitive MudPIT analysis (Supplementary Table 3). However, none of the unique COMPASS subunits of mammalian Mll1/2 (i.e., Mll1, Mll2, menin, Hcfc1/2, Psip1/2) or Set1A/B (i.e., Set1A, Set1B, Cfp1, Wdr82, Bod1, Bod1l) were detected. The ability of MafA to bind to Mll3/4 was also independently demonstrated in βTC-3 cells producing an adenovirus-driven Flag-tagged MafA (Supplementary Fig. 1).
MafA Comigrates With the High–Molecular Weight Mll3/4 Complexes Upon Sucrose Density Gradient Separation
βTC-3 nuclear proteins were separated by size in a 5–35% sucrose gradient to determine if the mobility of the ∼90kDa MafA dimer was affected by interaction with Mll3/4. MafA was found in two distinct gradient size fractions. The faster mobility MafA, containing fractions 6–8, were surrounded by islet-enriched transcription factors of relatively low molecular weight (Fig. 2A) (Pdx-1, 31 kDa, fractions 3–5; Nkx6.1, 38 kDa, fractions 3–5; Hnf1α, 67 kDa, fractions 6–8; Nkx2.2, 30 kDa, fractions 3–5; Pax6, 50 kDa, fractions 6–8), whereas the high–molecular weight fraction 14 comigrated with the Rbbp5 (59 kDa), NCoA6 (220 kDa), Wdr5 (36 kDa), and Utx (154 kDa) subunits of the Mll3/4 complex. In contrast, few other islet-enriched transcription factors were found in this high–molecular weight fraction. Antibody supershift and competitor analysis illustrated that the binding properties of MafA in fractions 7 and 14 were identical (Fig. 2B). In addition, immunoprecipitation experiments conducted with antibodies to NCoA6 or MafA demonstrated that MafA interacted with several Mll3/4 components in fraction 14 (Fig. 2C). These results strongly indicate that a significant fraction of MafA is associated with the Mll3/4 complex in β-cells.
Sucrose gradient sedimentation reveals that MafA comigrates with the Mll3/4 complex. A: βTC-3 nuclear proteins were separated in a 5–35% sucrose gradient, and fractions were screened for MafA, Mll3/4 (i.e., Rbbp5, NCoA6, Wdr5, Utx), p/CAF, Pdx-1, Nkx6.1, Hnf1α, Nkx2.2, and Pax6 by immunoblotting. A significant portion of MafA was present in fraction 14, which contains Mll3/4 complexes. B: MafA2 in fractions 7 and 14 binds specifically to the rat insulin II C1 enhancer element. MafA in these fractions was supershifted by anti-MafA antibody, while only unlabeled C1 and not an insulin Pdx-1 binding element (Pdx-1 BS) competed for binding. C: Immunoprecipitations (IP) performed on fraction 14 with antibody to NCoA6 (left) and MafA (right), which were then probed with MafA, NCoA6, Rbbp5, or Ash2l antibodies. The input lane contained ∼5% of fraction 14.
A Subset of MafA-Regulated Genes Are Activated by the Mll3/4 Complexes in Mouse βTC-3 Cells
We next analyzed how Mll3/4 affected MafA activity by depleting MafA or NCoA6 from βTC-3 cells by siRNA-directed knockdown. The NCoA6 subunit lacks a DNA binding domain and is involved in recruiting Mll3/4 to a variety of transcription factors and nuclear receptors (35–38). Furthermore, Mll3 and Mll4 have redundant roles in the liver, and NCoA6 is required for activity of both methyltransferase complexes (36). Notably, mouse NCoA6+/− mutants have impaired glucose clearance (21), whereas overexpression of NCoA6 in rat INS-1 β-cells increases cell function (39). In addition, polymorphisms in NCOA6 are associated with decreased human β-cell function (39). Our objective was to determine how closely Mll3/4 is linked to MafA-mediated transcriptional control.
MafA and NCoA6 protein levels in βTC-3 cells were both effectively (>80%) and specifically depleted by siRNA treatment (Fig. 3A). The effect of these conditions on expression of β-cell genes bound by MafA and downregulated in pancreas-specific deletion MafAΔpanc mice was determined (40, 41) (Supplementary Table 4). As expected, the expression of most of these genes was decreased upon depletion of MafA (Fig. 3B; Slc2a2, Ins2, Lifr, Prss53, Atp2a2), but these conditions had no effect on NCoA6 levels. In contrast, only a small subset of these MafA-activated genes was affected in the NCoA6 knockdown (i.e., Slc2a2, Prss53). This outcome was considered likely because a number of other potential MafA coregulators were identified here and previously (e.g., p/CAF (18) (Supplementary Table 1), although it is possible that some fraction of Mll3/4 activity is independent of NCoA6. In addition, ChIP analysis showed that Mll3 and MafA were bound within the same control regions of the Slc2a2 and Prss53 genes (Fig. 3C). Moreover, H3K4 trimethylation and elongating carboxy terminal tail phosphorylated RNA polymerase II levels were significantly reduced within the TSS region of Prss53 upon knockdown of MafA or NCoA6, the regulatory pattern expected for MafA-recruited Mll3/4 activity (Fig. 3D and E).
Knockdown of NCoA6 reduces MafA target gene expression in βTC-3 cells. Control siRNA (Controlsi) or siRNA specific to MafA (MafAsi) and NCoA6 (NCoA6si-1 and -2) was introduced into βTC-3 cells. A: MafA and NCoA6 were immunoblotted after corresponding depletion, with NCoA6si-2 most effective at reducing protein levels and used in all subsequent experiments. Rbbp5 levels were unaffected by siRNA treatment and served as a loading control. Densitometric analysis indicated effective knockdown of MafA (MafAsi = 0.04 ± 0.02 standard deviation) and NCoA6 (NCoA6si-2 = 0.16 ± 0.12). n = 3; *P < 0.05. B: NCoA6 affects expression levels of a subset of MafA-regulated genes in βTC-3 cells. mRNA expression was determined by qRT-PCR, normalized to Gapdh expression (n = 3–6). C: ChIP analysis reveals that MafA and Mll3 are bound to the same region of Slc2a2 and Prss53. Fold enrichment was calculated using the percent input and normalized to background binding at the Albumin promoter (n = 4). D: H3K4 trimethylation levels were reduced near the TSS of Prss53 in MafAsi- and NCoA6si-treated βTC-3 cells in ChIP analysis. The percentage input was calculated and normalized to Controlsi treated (n = 3). E: MafA and NCoA6 knockdown in βTC-3 cells decreased phosphorylated RNA polymerase (Pol) II binding to the TSS region of Prss53 in ChIP assays (n = 3). All error bars indicate standard deviation. *P < 0.05.
Human MAFA and MAFB Interact With the MLL3/4 Complexes
Our next objective was to determine if human MAFA and closely related MAFB bind MLL3/4. MafB is produced in insulin+ cells only during mouse embryogenesis and plays an important role in β-cell formation (19). In contrast, human MAFB is expressed in developing and postnatal β-cells, being coproduced with MAFA in adult islet β-cells (20). These basic leucine-zipper proteins bind to DNA with identical specificity as homo- or heterodimers (32,42), as also observed in MAFA- and MAFB-transfected HeLa cell extracts (Fig. 4A). Gel shift analysis indicates that both MAFA/MAFB and MAFB2 complexes are also formed in nuclear extracts prepared from the human EndoC-βH1 β-cell line (Fig. 4B), although they were difficult to distinguish due to their similar size (MAFA at ∼45 vs. MAFB at ∼42 kDa). Supershift analysis with MAFA and MAFB antibodies provided further support for these protein associations (Fig. 4A: lanes 3–5; Fig. 4B: lane 3). Significantly, immunoprecipitation experiments demonstrated that MAFA and MAFB were both able to associate with multiple proteins of the MLL3/4 complex in EndoC-βH1 cells (NCOA6, UTX, RBBP5) (Fig. 4C), although whether this results from binding MAFB2 and/or MAFA/MAFB is unclear. In addition, MAFA and MAFB were able to interact with the islet-enriched PAX6 and ISL1 transcription factors as well as P/CAF; however, these likely represent relatively weak or indirect binding due to their absence from the Re-CLIP/MS analysis performed in mouse βTC-3 cells (Supplementary Table 1).
MAFA and MAFB associate with MLL3/4 in human EndoC-βH1 cells. A: MAFA2, MAFB2, and MAFA/B are produced in MAFA and MAFB transfected HeLa cells. Supershift (S.S.) reveals three distinct complexes, with anti-MAFA and anti-MAFB altering homo- and heterodimer formation. B: MAFB2 and MAFA/B appear to be the predominant gel shift complexes formed in EndoC-βH1 nuclear extracts. Note that complex formation is profoundly affected by the addition of anti-MAFB. The rat insulin II C1 and Pdx-1 binding element competitions illustrate the specificity of complex binding. C: Subunits of MLL3/4 were precipitated from EndoC-βH1 nuclear extracts with antibodies to MAFA and MAFB. Immunoblotting was performed for MLL3/4 subunits (NCOA6, RBBP5, UTX), islet-enriched transcription factors (PAX6, ISL1, HNF1α), and mouse MafA associated P/CAF (18). Representative blot is shown (n = 3).
MAFA, MAFB, and the MLL3/4 Complexes Are Necessary for Glucose-Responsive Insulin Secretion in EndoC-βH1 Cells
MAFA, MAFB, and NCOA6 were siRNA depleted from EndoC-βH1 cells to analyze their significance to human β-cell gene expression and function. Protein levels were reduced by ∼50–70% under these conditions (Fig. 5A). The effect on transcriptional control was determined on genes regulated by mouse MafA and a candidate set available on a commercial array composed of 84 genes associated with T2DM onset, development, and progression.
MAFA, MAFB, and MLL3/4 are required for glucose-stimulated insulin secretion in EndoC-βH1 cells. A: Protein levels of MAFA, MAFB, and NCOA6 were reduced upon targeted siRNA treatment. Densitometric analysis indicated effective knockdown of MAFA (0.44 ± 0.18), MAFB (0.53 ± 0.29), and NCOA6 (0.26 ± 0.05; n = 3). *P < 0.05. The effect of knockdown on β-cell mRNA levels was determined using bona fide MafA-activated genes of mouse β-cells (B) and by candidate gene screening using the Qiagen human diabetes RT2 profiler array (C). qPCR results were normalized to GAPDH expression. Error bars depict the SD (n = 4). D: MAFA, MAFB, and MLL3 bind within the same region of AGT. Fold enrichment was calculated from the percentage input normalized to GAPDH (n = 4). E: Glucose-stimulated insulin secretion was measured 1 h after glucose stimulation in cells treated with siRNA for 72 h. Results are presented as fold stimulation at 15.5 mmol/L vs. 1.1 mmol/L glucose; all measurements were normalized for insulin content (n = 4). Error bars indicate the SD. *P < 0.05.
INSULIN, G6PC2, PRSS53, and SLC2A1 were significantly affected by MAFA or MAFB depletion in EndoC-βH1 cells (Fig. 5B). However, their expression was unchanged by NCOA6 depletion, presumably due to differences in human MAFA/MAFB:MLL3/4 control in relation to rodents and/or the β-cell lines used in our analysis. Notably, the levels of several genes linked to T2DM and important to glucose-stimulated insulin secretion were reduced upon NCOA6 depletion (Fig. 5C), specifically, AMP-kinase subunit PRKAA1 (43), glucose-6-phosphate dehydrogenase (44), and angiotensin (AGT) (45). In addition, the expression of each was compromised in MAFB siRNA-treated EndoC-βH1 cells, whereas MAFA depletion only affected AGT. Moreover, MAFA, MAFB, and MLL3 were all corecruited to the same endogenous 5′-flanking control region of the AGT gene (Fig. 5D). Furthermore, glucose-stimulated insulin secretion in EndoC-βH1 cells was compromised upon knockdown of MAFA, MAFB, or NCOA6 (Fig. 5E). Collectively, these data support an important role for MLL3/4 in MAFA and MAFB activity in human β-cells.
Conditional Knockout of MafA or NCoA6 in Islet β-Cells In Vivo Decreases MafA Target Gene Expression and Glucose-Responsive Insulin Secretion
To evaluate the influence of MafA:Mll3/4 on islet β-cells in mice, we compared the phenotype of β-cell–specific loss of NCoA6 with that of MafA. Rat insulin II–driven Cre transgenic mice (30) were bred with MafAfl/fl (19) and NCoA6fl/fl (46) mice to remove these proteins specifically from insulin+ cells in MafAΔβ and NCoA6Δβ mice during development (Fig. 6A). Approximately 70% of mutant insulin+ cells lacked the MafA or NCoA6 proteins by e15.5 (Fig. 6B), a time at which MafB is still expressed in developing β-cells (47).
Islet architecture is unchanged in NCoA6Δβ mice. A: MafAΔβ and NCoA6Δβ mice were generated by breeding MafAfl/fl and NCoA6fl/fl mice with rat insulin II enhancer/promoter (RIP)-driven Cre transgenic mice. The MafA protein coding sequence is only present within exon 1 (19), while deletion of exon 8 causes a frame shift that prevents the production of NCoA6 (46). B: Representative images illustrate the effectiveness of MafA and NCoA6 removal from insulin+ cells at e15.5; ∼72% of insulin+ cells lack MafA or NCoA6. The blue arrows indicate insulin+ cells lacking NCoA6, and the yellow arrow indicates a cell still expressing NCoA6 (n = 3). C: Islet architecture is only distorted in MafAΔβ mice at 8 weeks.
Like the pancreas-specific Pdx1-Cre removal of MafA (MafAΔpanc) (41), MafAΔβ mice were euglycemic and had impaired islet architecture, where glucagon+ α-cells were no longer restricted to the outer mantle of the islet (Fig. 6C). Adult NCoA6Δβ mice were also euglycemic, but their islet architecture was unchanged. In addition, only MafAΔβ mice displayed the impaired whole-body glucose clearance observed in MafAΔpanc mice (41) (Supplementary Fig. 2B–E).
Strikingly, most of the genes regulated by MafA in 8-week-old MafAΔβ mouse islets were now also significantly compromised in 8-week-old NCoA6Δβ islets (Fig. 7A). In addition, glucose-stimulated insulin secretion was impaired in islets isolated from both MafAΔβ and NCoA6Δβ mice (Fig. 7C), as found upon MAFA, MAFB, or NCOA6 depletion in human EndoC-βH1 cells (Fig. 5D). The basal insulin secretion level of NCoA6Δβ islets was unusually high in relation to wild-type controls and MafAΔβ (Fig. 7C), a sign of β-cell immaturity (48). The more penetrant Mll3/4 control properties observed in NCoA6Δβ mice likely represent the combined actions of Mll3/4 on MafB activity during development and MafA postnatally.
Glucose-stimulated insulin secretion is impaired in MafAΔβ and NCoA6Δβ islets. Expression of all the direct targets of mouse MafA was decreased in 8-week-old MafAΔβ islets (A) but not those identified as human MAFA- and/or MAFB-regulated genes by Qiagen human diabetes RT2 profiler screening (B) (see Fig. 5C). Moreover, essentially all of these genes were also compromised in NCoA6Δβ islets. The error bars indicate the SEM (n = 3–5). Fold expression was normalized to control littermates. C: Isolated islets were incubated overnight in low glucose (5.6 mmol/L) and then treated with 5.6 mmol/L or stimulating 16.7 mmol/L glucose for 1 h. The data are presented as the percentage (%) of total insulin content secreted. The error bars represent the SEM. n for MafAfl/fl = 3, MafAΔβ = 4, NCoA6fl/fl = 3, NCoA6Δβ = 3. *P < 0.05.
Gene induction involves the recruitment of coregulators by enhancer-bound transcription factors such as MAFA and MAFB. These protein-bound effectors ultimately influence recruitment of the RNA polymerase II transcriptional machinery. We used an unbiased "in cell" chemical cross-linking, immunoprecipitation, and MS strategy to identify coregulators of the MafA transcription factor, an essential regulator of postnatal rodent islet β-cell proliferation and activity. Many distinct coregulator candidates were identified in our biochemical screen, and we focused on the Mll3/4 methyltransferase complex because of the established role of this coactivator in gene transcription. Our studies are consistent with a model where MLL3/4 recruitment by MAFA and MAFB is essential to islet β-cells.
MLL3 and MLL4 are members of the evolutionary-conserved COMPASS family of methyltransferases that are strongly associated with gene activation due to their ability to methylate H3K4 within enhancer and/or promoter regions. Strikingly, all of the proteins in the Mll3/4 complexes were identified in the MafA antibody precipitates by MS, and not the other mammalian Mll complexes (e.g., Mll1/2 complex or Set1A/B complex). MLL3/4 binding to MAFA was also confirmed by immunoprecipitation analysis in mouse and human β-cell lines (Figs. 2 and 4). Moreover, a significant fraction of MafA was bound to the ∼1.5 MDa Mll3/4 complex by sucrose gradient centrifugation (Fig. 2). Interestingly, the islet-enriched Nkx2.2 and Nkx6.1 transcription factors were also found in the same high–molecular weight fraction. Although the identity of these islet-enriched complexes are unknown, a very high–molecular weight complex would be predicted from the association in β-cells of Nkx2.2 with the histone deacetylase-1 corepressor, the Groucho-3 transcription factor repressor, and the DNA (cytosine-5)-methyltransferase 3A corepressor to prevent expression of the α-cell specification factor Arx (49). Likewise, Pax6 comigration with MafA:Mll3/4 could reflect binding to high–molecular weight proteins like p300 (50). However, Nkx2.2, Nkx6.1, and Pax6 were not detected by MafA antibody in Re-CLIP experiments, and no other islet-enriched transcription factors were either (Supplementary Table 3), strongly suggesting that these proteins are not directly, and certainly not abundantly, associated with MafA:Mll3/4. At least some interaction is likely with PAX6 and ISL1 in human β-cells based on the MAFA and MAFB antibody precipitation results in human EndoC-βH1 cells (Fig. 4C). Collectively, these data strongly implied that MafA recruitment of Mll3/4 was highly specific and important to β-cell activity.
Immunoprecipitation experiments conducted with the EndoC-βH1 cell line showed that human MAFA and MAFB are both capable of binding and recruiting the MLL3/4 complexes. However, reducing the protein levels of these transcription factors or the core NCOA6 subunit of the MLL3/4 complexes in human (Fig. 5) and mouse (Fig. 3) β-cell lines disclosed that the coactivator only influenced some MAFA/MAFB activated genes. For example, only the Slc2a2 and Prss53 genes were bound and coregulated by MafA and Mll3/4 among a variety of tested MafA-regulated genes in βTC-3 cells (Fig. 3). Importantly, and as expected, recruitment of Mll3/4 by MafA regulated H3K4 trimethylation levels of the Prss53 gene as well as RNA polymerase II engagement.
In contrast to what was found upon reduction of NCoA6 levels in β-cell lines, nearly all of the islet MafA target genes were dependent on NCoA6 in 8-week-old NCoA6Δβ islets (Fig. 7A). Because of the early action of Cre-recombinase in developing MafA+ and MafB+ NCoA6Δβ β-cells, this more penetrant phenotype is likely due to the activity of Mll3/4 on MafB-bound enhancers during development. Notably, this supposition is consistent with analysis of the MafB−/− (19) and MafAΔpanc (41) mutants, which established that many genes first regulated by MafB developmentally were subsequently influenced by MafA after birth (41). In NCoA6Δβ mice, the lack of Mll3/4 activity on MafA- and MafB-bound enhancers during development may explain why glucose-stimulated insulin secretion was differentially affected in NCoA6Δβ and MafAΔβ islets (Fig. 7C). The results also indicate that Mll3/4 is involved in some, but not all, MafA- and MafB-regulated gene expression. Thus, Mll3/4 was necessary for glucose-stimulated insulin secretion in both human and mouse β-cells but did not have the same influential effect on islet cell architecture as MafA (Fig. 7A).
Our understanding of the genes regulating glucose-stimulated insulin secretion in human β-cells is very limited. The results in EndoC-βH1 cells suggest that regulation by MAFA/B:MLL3/4 of the AMP-kinase subunit PRKAA1 (43), glucose-6-phosphate dehydrogenase (44), and angiotensin (45) expression may be important (Fig. 5C). For example, angiotensin appears to have a direct role in regulating β-cell activity (51). However, this candidate list certainly represents only a fraction of the gene products involved in β-cell function, and future efforts will be aimed at identifying more using ChIP-Seq and RNA-Seq. This analysis should also be conducted with human islets, because the transformed and proliferative nature of the EndoC-βH1 cell line could possibly influence how MAFA and MAFB regulate gene expression. It is also important to note how similar the phenotype of the MAFA, MAFB, and NCOA6 regulatory response was in our β-cell line and mouse studies, and yet different from the other islet-enriched transcription factors present in high–molecular weight complexes (Fig. 2). Thus, islet β-cell–specific deletion of Pax6, Nkx2.2, or Nkx6.1 produces a much more severe effect on cell identity and causes lethality soon after birth due to hyperglycemia (52–54).
In conclusion, our combined proteomic, cell line, and mouse strategies have led to the identification and characterization of MLL3/4 as a novel coactivator of the MAFA and MAFB transcription factors in islet β-cells. Future efforts will be aimed at determining how other isolated candidate coregulators affect MAFA and MAFB control, such as CoREST (LSD1, RCOR1, RCOR3, HDAC2) and histone deacetylase 6, both of which are known to play a major regulatory role in other cell contexts (55,56). It will also be of interest to determine if coregulator recruitment is influenced by homodimeric (i.e., MAFA2, MAFB2) or heteromeric (MAFA/MAFB) transcription factor composition and the physiological or pathophysiological state of the β-cell.
Acknowledgments. Adenoviral Flag-tagged MafA was kindly provided by Dr. Qiao Zhou, Harvard University. The authors thank Dr. Raphael Scharfmann, INSERM, Paris, France, and Dr. Philippe Ravassard, French National Centre for Scientific Research, Paris, France, for generously providing EndoC-βH1 cells.
Funding. This work was supported by grants from the National Institutes of Health (DK-090570 to R.S., F32-DK-102283-01 and T32-DK-007061 to H.A.C., and CA-112403 and DK-058242 to J.X.) and the Vanderbilt Diabetes Research and Training Center (DK-20593). Imaging was performed with National Institutes of Health support from the Vanderbilt University Medical Center Cell Imaging Shared Resource (CA-68485, DK-20593, DK-58404, HD-15052, DK-59637, and EY-08126) and Vanderbilt University Medical Center Islet Procurement and Analysis Core (DK-20593).
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. D.W.S. and H.A.C. researched data and wrote the manuscript. L.L. and J.X. provided animals. A.R. provided technical support. S.G. researched data. R.S. wrote the manuscript. R.S. is the guarantor of this work, and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0281/-/DC1.
Received February 27, 2015.
Accepted July 3, 2015.
© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
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