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In the LAPD’s Open-Unsolved Unit, not many murder victims die a decade after the crime. So when a man succumbs to complications from being shot by a stray bullet ten years earlier, Bosch catches a case in which the body is still fresh, but any other clues are virtually nonexistent. Even a veteran cop would find this one tough going, but Bosch’s new partner, Detective Lucia Soto, has no homicide experience. A young star in the department, Soto has been assigned to Bosch so that he can pass on to her his hard-won expertise.
Now Bosch and Soto are tasked with solving a murder that turns out to be highly charged and politically sensitive. Beginning with the bullet that has been lodged for years in the victim’s spine, they must pull new leads from years-old evidence, and these soon reveal that the shooting was anything but random.
As their investigation picks up speed, it leads to another unsolved case with even greater stakes: the deaths of several children in a fire that occurred twenty years ago. But when their work starts to threaten careers and lives, Bosch and Soto must decide whether it is worth risking everything to find the truth, or if it’s safer to let some secrets stay buried.
Identical twins, Maggie and Clara Ryder, both grew up with Leo Maddox, billionaire playboy, apparent alcoholic, and heir to his grandfather’s world-famous Maddox hotel empire. Their roles were cemented long ago: Maggie, as his best friend and Clara as his childhood nemesis.
But when a simple twist of fate changes everything and both girls start to fall for the lean, mean, ridiculously sexy and seemingly spoiled Leo… which girl will win his heart?
Throw in a little mischief, drama, and one smoking-hot bartender and lines are sure to blur. But one thing’s for certain, neither good-girl Maggie nor rebellious Clara will be satisfied until they each figure out where they belong.
*NEW ADULT ROMANCE. Standalone. Recommended for readers 17+
*LEO MADDOX ~the alternative version to this novel releases on Nov. 25th
Local cop Stevie Taylor and police chief Zane Duncan have startling new proof that a deadly street drug is expanding its poisonous grip on their small town: a skull found on the banks of the Rogue River has two bullet holes in the back. Still caught up in the excitement of their new relationship, Stevie and Zane are determined to track down the drug’s source before more lives in Solitude, Oregon, are lost.
Then one death strikes especially close to home, leading to heartache and confusion for Stevie. As the drug distributors strike hard and the body count in Solitude grows higher, she and Zane must hunt down the deadly killer—but are they the predators, or the prey?
A premier author-signing event, don’t miss the experience!
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The Novel Experience Event is the best parts of other more expensive events (signings, parties, and craziness) without the prices. TNEE offers general attendance to readers for FREE with an option to purchase VIP tickets for a very affordable price.
At The Novel Experience, ALL AUTHORS, traditionally published, self/indie published and small press will be signing alongside each other without the politics and issues that have come to rule the signing events of other conventions. With the world of publishing changing so rapidly, events are lagging behind. TNEE is the answer to the need that has recently cropped up.
Readers want what they want – BOOKS and we want to give it to them. Think of TNEE as a one stop shop! Readers will be able to get their reading fix and still be able to party with the authors and publishers they have come to love, in the genres they love.
TNEE includes a VIP preview for all VIP readers to get books signed earlier than the regular signing and exclusive events! Saturdays feature a massive signing and then another night of parties and merriment.
KEEP READING FOR THE LIST OF
FABULOUS AUTHORS SIGNING AT TNEE
AND REGISTRATION INFO!
Being a teenager is tough, especially when you have to pretend to be something you’re not, and you’re in love with someone you shouldn’t. 115 years ago, a small planet called Arcadia was invaded by a vicious alien race and nearly destroyed. Cut off from their resources, the Arcadians turned to Earth for help. A group of Arcadian explorers discovered a Prophecy that claimed their salvation lay in the hands of two children from Earth. To ensure their safety, the Arcadian Council sent their most gifted youngsters to Earth to act as protectors. Samantha is one of them.
To succeed in her mission she must learn to control her Arcadian powers and keep her true identity from her best friend, and the girl she swore to protect, Alexia. But Samantha will soon realize that nothing is as it seems. Someone is trying to prevent the Prophecy from taking place and the prophecy boy hasn’t been found yet. There is also a new drug circulating at school that is turning students into freakishly strong menaces.
To make matters worse, distractions keep getting in her way. Such as her love/hate relationship with her “cousin” Matthew. Or her confused feelings toward popular and mysterious Julian. She wants nothing more than to be free to live her life. But the survival of Arcadia depends on her and her friends. Free will is not an option.
*Note from the author: Due to strong language and mature content this book is recommended to readers 17+.
Copywriter by day, nerd by night, Hailey Holloway is living the post college dream. Her life may not be flashy and exciting but it’s enough for her. After a late night at work, Hailey stumbles upon two dragons brawling in the middle of Philadelphia. In an act of kindness and lapse of sanity, she comforts the dragon who was left behind to die. In doing so she inadvertently invited the soul of Dacea into herself for safe keeping. Hailey just wants to live her life as simply and quietly as possible but with the voice of a disembodied dragon in her head, she has little hope of achieving her goal.
Dacea will do anything to be free of Hailey, a human who is moments from checking herself into a psychiatric ward. He is grateful to be alive, but at what cost to his sanity? The dragon world is in upheaval. Green is at war with Red, and he is of no use to anyone while stuck with Hailey.
The solution to their problem is not without its risks. But just how far will Hailey go to be free of the dragon?

domain: library


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Local cricketers will soon be able to sharpen their skills in safer and more modern facilities .
CS Energy is proud to sponsor the annual Jules Armstrong NAIDOC Week Encouragement Award.
Engineering graduate puts his studies into practice at CS Energy’s major overhaul of Wivenhoe Power Station.
Major shipment arrives in Townsville bound for Kennedy Energy Park near Hughenden.
CS Energy's Wivenhoe Power Station supports Lowood locals in need.
Callide B Power Station has generated more than 150,000 gigawatt hours of electricity since its opening back in 1988.
Wivenhoe Power Station is undergoing a $11.8 million tune-up to keep it operating in peak condition and ensure it meets its 100-year design life.
CS Energy was proud to support the inaugural Big Skies 2018 festival on Queensland's Western Downs.
Kogan Creek Power Station welcomed four new apprentices and trainees recently and is
equipping them with the skills to succeed in Australia’s energy industry.
Four massive bulk head gates were reinstalled at Wivenhoe Power Station recently in a
meticulously planned operation involving two cranes and a convoy of semi-trailers.


text:
Separate names with a comma.
Discussion in 'Tennessee Titans and NFL Talk' started by Scarecrow, Jan 19, 2014.
They gave us a decent QB in Matt Hasselbeck
Hasselbeck was like Steve young compared to fitz...
Sucks to see Horse face win again, Gonna never hear the end of it here in East TN if he wins the damned super bowl.
Peyton should choke as usual.
Either of these defenses may be able to do the job, anyway.
Not this time.
you don't deserve to call yourself a titans fan. All you are is a worthless bandwagon fan who pretends to only do it to try and annoy us but you probably cheer for the broncos and tell your friends that you're a broncos fan any team that gets the best player you're a fan of eh? big heat fan too? you're a jackass and i don't care about the language or if this comment gets deleted and i get blocked because that is the only way to really describe people like you. They are a disgrace to our world I wish there was a way to spit on a comment such as the ones you have provided in this post.
yeah... hope you're here after the SB.
PLEASE PLEASE!!! Comeon Richard!!!
I can't stand fans like that...just don't ever go into east TN...they're the worst!
My pick at the beginning of the post season was Seattle over Denver. I'm sticking to that. AFC was very weak. NE have no offensive weapons for Brady.
Seattle D should be able to slow down Manning, and they'll do enough on offense to win.

domain: sports


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High-school history text divides Cambodians
A new textbook on the history of the Khmer Rouge—the Cambodian Communist faction which murdered an estimated 1.7 million people during its four-year reign of terror in the late 1970s—cannot be used as a stand-alone reader for high-school students, says the government in Phnom Penh.
Instead, the book will be assigned as a supplementary source and used by the Cambodian Ministry of Education to produce a less controversial work on the same topic.
A History of Democratic Kampuchea, written by 26-year-old Khamboly Dy, provides a warts-and-all assessment of the rise and fall of the Pol Pot regime. It was published by the Documentation Center of Cambodia, an independent organization that specializes in Khmer Rouge history, with funding from the Soros Foundation and the National Endowment for Democracy.
The government-appointed committee that reviewed the text criticized it for identifying specific members of the Khmer Rouge and focusing too much on the post-1979 Cambodian civil war. Explaining the reason for his veto, one member of the committee argued that history “should be kept for at least 60 years before starting to discuss it.”
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- Nelson Mandela Dead: Icon of Anti-Apartheid Movement Dies at 95
- George H.W. Bush Given Lyndon B. Johnson Foundation Award
- Bruce Springsteen's 'Born To Run' manuscript could fetch $100,000 at NY auction
- Hospital Donates Records of Jacqueline Kennedy Onassis to JFK Library
- Australia’s Eureka Flag Finds a New Patch

domain: history


Google Video Store Now Open
It’s now divided up into three categories.
+ Google Video Store
Also browsable at: http://video.google.com/videostore
+ Popular Video
+ Random Picks
Material from the Google Video Store can also be browsed by genre or show name. At the moment, three main categories are listed:
+ NBA Basketball Games
+ Music Videos
as well as the titles to purchase and download episodes of the following programs:
+ Brady Bunch
+ Charlie Rose
Talk about from one extreme to the other. (-:
+ Ed Sullivan Show
+ Have Gun Will Travel
+ I Love Lucy
+ Star Trek Deep Space Nine
+ Star Trek Voyager
+ Survivor Guatemala
+ Twilight Zone
You can also browse these CBS programs here.
A quick look at some of the CBS programs available shows that 15 episodes of I Love Lucy are currently available to purchase for $1.99. Three episodes of Star Trek Voyager ($1.99) and 5 episodes of The Twilight Zone (1.99).
Btw, you can view a 30 second clip (no Google Video Player required) before making a decision if you want to purchase or rent the material. After you make your first purchase, you’ll be prompted to download the video player (if required) if it’s not already on your system. Remember, material that’s not copy-protected can be viewed on any media player that can handle .avi files. They can also be downloaded to your iPod or PSP.
A new section Google Video FAQ offer info and assistance in downloading material and explanations of a buying a “Day Pass” for a video (24 hour viewing period) and purchasing a video. You can also read about how to tell if if a video is copy-protected?
Also, a simple Google Video search results pages now allows you to not only view your results in a grid or list, but also quickly limit your results to free video or video for sale by just clicking. You can also click to sort by the length of the material (Long – Medium – Short). Results can be also be sorted by relevance, date uploaded, and title.
As Danny pointed out the other day, at the moment Google Video content for purchase or rental is only available to those with credit cards with a US address. The Google Video Player is only available for Windows 2000 and Windows XP machines. Mac and Linux versions are in the works. After downloading the Google Video Player I was prompted with a box (here’s a screen cap) that was pre-checked or pre-ticked to change the default search provider on IE to Google. Is this something new that Google is doing when downloading software?
Finally, to learn more about the new store, take a look at Marketwatch.com video interview or read a pseudo transcript of an interview with Jennifer Feikin, the director of Google Video that I posted earlier today.
Want to discuss? Check out the thread: Google Opens Video Store, in the SEW Forums.
Postscript 1: A bit confused. In our chat with Google’s Peter Chane last week, we were told that Charlie Rose interviews would not be copy-protected and could be viewed on a myriad of devices. I just purchased and downloaded a Charlie Rose interview and was told that it DOES require the Google Video Player. Here’s a screen cap of the page that appeared after I paid to download the program. Google’s Jennifer Feikin also uses Charlie Rose as an example of material that would not be copy-protected in the interview she gave to Marketwatch.com.
Update: Well, I went and downloaded the file again and now get several download options (screen cap). In fact, the show can be downloaded to an iPod or PSP as an MP4 format file. Even more confusing is the Windows/Mac option that downloads the file in the Google .gvp format and requires the Google Video Player. Why is this confusing? Because there is no Google Video Player for the Mac as of today. In fact, Google’s documentation makes that point very clear.
Let me end this update by saying that I’ve now downloaded the video after paying $.99, installed the player, and still can’t watch the video. Why? It seems that at the moment (2 am EST) I’m unable get my Google account to authenticate so I’m unable to view the show. I keep receiving an error message. I’m told to go to video.google.com/support but I can’t find anything about authentication problems. Yes, I know, Google Video is a beta. (-:
Postscript 2: It looks as if the Google Video Player problem/authorization issue was on my end, some sort of compatibilty issue with my system. I dowloaded the player and video on another computer and watched my video. If you’re having issues, try doing the same thing (assuming you have more than one computer).
Postscript 3: If you like to read the small print, you’ll notice on the Terms of Service page that payments for Google Video are being handled by the Google Payment Corporation, a company Google set-up last year. The privacy page was unavailable when I checked the link a few minutes ago.
Postscript 4: If you’re a Rocky and Bullwinkle fan (I am) you’ll be happy to learn that the video store is home to 8 downloadable episodes.
Postscript 5: To get the Google Video Player, you need to search for any file, then click download, then a window should prompt to install it. No luck? Here’s a direct link to the file.
Just off the phone with Google. Some answers to my questions.
1) Look for the privacy link to work soon.
2) Why the Windows/Mac option? This appears because the Charlie Rose program is not copy-protected. A Mac user would be able to downoad an .AVI file that would play on their machine. The .avi file topic is discussed (it could be a bit more clear) on a video search help page. Google said they would try to make this more understandable on both the video download page and on the help page.
3) As of now, the first time you download from Google Video it will download a video player to your computer. That’s why I was told I needed the player. Google said they are looking into how this is worded and might make a tweak or two.


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I love my apothecary jar-so easy to switch out the contents each season. The little jack o’lanterns inside are actually froma string of dollar store lights I purchased years ago. The ghost and pumpkin votive holders as well as the “Happy Halloween” blocks were purchased last year for 50% off from Target in November. The BOO sign is from this year’s Target Dollar Spot, it was originally hot pink but I gave it a coat of Oil Rubbed Bronze spray paint. (And that pedestal it’s on-an IKEA candlestick holder and upside down baby plate!) And last but not least, the black cheesecloth and orange spiders are dollar store finds!
I love decorating for Halloween now that the boys are old enough to get excited! Have you put your Halloween decor up yet?

domain: design


Today is opening day for the almost NHL-sized rink situated in the west parking area of the train station. Warming cabins are accessible and It’s open from 10 a.m. to 9 p.m every day. Shinny hockey is allowed and Indoor washrooms located inside the Train Station are available from 9 a.m. to 5 p.m.
This rink joins the Fenlands Meadow Rink to become the 2nd outdoor Town of Banff-sponsored rink to open for the season.
- Use at your own risk.
- This rink is not supervised.
- Please respect the rink and all other users.
- The Town of Banff is not responsible for any loss, damage or injury resulting from use of this facility.
- In case of emergency, Call 9-1-1 . The skating rink is located at 327 Railway Avenue.
- To report damages or if you have concerns, please call the Town of Banff at 403.762.1240.


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Dick Baranzini talks to the Star Valley Town Council about thebenefits of a photo system to enforce town speed limits.
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Vice President Joe Biden said Wednesday that he will not run for president.
Biden delivered his statement at the White House Rose Garden with President Barack Obama in attendance.
“As my family and I have worked through the grieving process, I’ve said all along what I’ve said time and again to others, that it may very well be that that process, by the time we get through it, closes the window on mounting a realistic campaign for president,” Biden said. “I’ve concluded it has closed.”
This certainly makes Hillary’s life a lot easier. It also puts additional pressure on Obama to sandbag the FBI investigation into Hillary’s mishandling of top secret information and influence peddling while Secretary of State. If she flames out based on her inherent criminality, the Democrats will be stuck with Bernie Sanders. And no conservative political blog should be allowed to have that much fun.


The DC-19 Stealth Carbine was designed specifically for use by clone troopers designated Shadow Troopers. The weapon uses a special blend of Tibanna Gas to achieve an invisible blaster bolt without the tell-tale red, green or blue contrail. Painstaking efforts were also made in dampening the noise of the blast. As such, the Shadow Troopers proved able in assassination and other covert duties, allowing some respite for Republic commandos and ARC Troopers. The weapon would later be studied and serve as much of the basis for the Xerrol Nightstinger- another sniper weapon with an invisible blaster bolt.
Due to the invisibility and nigh-silence of the blaster bolts fired by the DC-19, the -10 penalty when using the Snipe feature of the Stealth skill is negated. The DC-19 uses a standard Power Pack, but must be reloaded after 10 shots. However, the Stealth Carbine uses a specialize gas canister (Each costing 500 credits, and weighs 0.25 kg) which must be replaced every 500 shots.
Weapon Type: Rifles
Stun Setting: Yes
Rate of Fire: Single-Shot ,Autofire
Weight: 5 Kilograms


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The H3K27me3-demethylase KDM6A is suppressed in breast cancer stem-like cells, and enables the resolution of bivalency during the mesenchymal-epithelial transition
Metrics: PDF 2010 views | HTML 4454 views | ?
Joseph H. Taube1,3,4,*, Nathalie Sphyris1,*, Kelsey S. Johnson3, Keighley N. Reisenauer3, Taylor A. Nesbit3, Robiya Joseph1, Geraldine V. Vijay1, Tapasree R. Sarkar1,5, Neeraja A. Bhangre1, Joon Jin Song6, Jeffrey T. Chang7,8, Min Gyu Lee9,10, Rama Soundararajan1 and Sendurai A. Mani1,2
1Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
2Metastasis Research Center, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
3Department of Biology, Baylor University, Waco, Texas, USA
4Institute of Biomedical Sciences, Baylor University, Waco, Texas, USA
5Department of Integrative Bioscience, Texas A & M University, College Station, Texas, USA
6Depatment of Statistical Science, Baylor University, Waco, Texas, USA
7Center for Clinical and Translational Sciences, The University of Texas Health Science Center at Houston, Texas, USA
8Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Texas, USA
9Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
10Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
Sendurai A. Mani, email: [email protected]
Joseph H. Taube, email: [email protected]
Keywords: epithelial-mesenchymal transition, mesenchymal-epithelial transition, KDM6A, bivalent genes, GSK-J4
Received: July 20, 2016 Accepted: April 26, 2017 Published: July 10, 2017
The deposition of the activating H3K4me3 and repressive H3K27me3 histone modifications within the same promoter, forming a so-called bivalent domain, maintains gene expression in a repressed but transcription-ready state. We recently reported a significantly increased incidence of bivalency following an epithelial-mesenchymal transition (EMT), a process associated with the initiation of the metastatic cascade. The reverse process, known as the mesenchymal-epithelial transition (MET), is necessary for efficient colonization. Here, we identify numerous genes associated with differentiation, proliferation and intercellular adhesion that are repressed through the acquisition of bivalency during EMT, and re-expressed following MET. The majority of EMT-associated bivalent domains arise through H3K27me3 deposition at H3K4me3-marked promoters. Accordingly, we show that the expression of the H3K27me3-demethylase KDM6A is reduced in cells that have undergone EMT, stem-like subpopulations of mammary cell lines and stem cell-enriched triple-negative breast cancers. Importantly, KDM6A levels are restored following MET, concomitant with CDH1/E-cadherin reactivation through H3K27me3 removal. Moreover, inhibition of KDM6A, using the H3K27me3-demethylase inhibitor GSK-J4, prevents the re-expression of bivalent genes during MET. Our findings implicate KDM6A in the resolution of bivalency accompanying MET, and suggest KDM6A inhibition as a viable strategy to suppress metastasis formation in breast cancer.
The epithelial-mesenchymal transition (EMT) and the reverse process, the mesenchymal-epithelial transition (MET), are important cellular reprogramming events that endow tumor cells with the traits required to traverse the multiple steps of the metastatic cascade. The EMT program, which can be triggered by exposure to transforming growth factor beta (TGFB), converts polarized epithelial carcinoma cells into intrinsically motile and invasive spindle-shaped mesenchymal counterparts [1–3]. Additionally, EMT imparts cancer stem cell (CSC) characteristics, including intrinsic resistance to anoikis and genotoxic stresses as well as tumor-initiating capabilities [4, 5]. Consequently, EMT facilitates the initial steps of the metastatic cascade by promoting tumor cell detachment, invasion, and dissemination, whereas MET is evoked to restore the proliferative potential required for efficient colonization and formation of epithelial outgrowths at the distant site [6, 7].
The induction of EMT is accompanied by the downregulation of epithelial markers—most notably E-cadherin, the gatekeeper of the epithelial state (encoded by CDH1)—and the de novo expression of mesenchymal-associated genes . The extensive changes in gene expression accompanying EMT/MET, coupled with the dynamic and reversible nature of the transitions between the epithelial and mesenchymal phenotypic states, suggest the involvement of epigenetic regulatory mechanisms in these processes [8–10]. Moreover, recent studies have begun to unravel the complexity of the epigenetic mechanisms that regulate stemness and the transition from a pluripotent to a differentiated state.
Post-translational modifications of histones are amongst the most extensively studied epigenetic mechanisms that can fundamentally alter gene expression. Indeed, the existence of a complex histone code has been proposed to explain how distinct combinations of histone modifications may converge to alter the transcriptional output of the underlying chromatin . In particular, trimethylation of histone H3 at lysine 4 (H3K4me3) and lysine 27 (H3K27me3) has been associated with gene activation and silencing respectively [12–16]. The coexistence of these two conflicting activating and repressive marks within the same promoter, forming a so-called bivalent domain, was first described in human and mouse embryonic stem (ES) cells . In ES cells, bivalent domains are prevalent in the promoters of differentiation-control genes and serve to maintain these genes in a silent but transcription-ready state, poised for lineage-specific upregulation or downregulation [17, 18]. Differentiation of ES cells into distinct lineages entails the resolution of bivalency by the removal of either the activating H3K4me3 mark, resulting in developmental silencing, or the repressive H3K27me3 mark, leading to gene activation [17, 18].
The bivalent chromatin configuration is also important in the context of CSC plasticity. In the plastic non-CSC subpopulations of human breast tumors, the promoter of ZEB1—a key EMT-inducing transcription factor—is bivalent, and resolves to an active H3K4me3-monovalent state, following exposure to TGFB, eliciting the induction of EMT and conversion to a CSC state . Therefore, the resolution of bivalency is emerging as a critical epigenetic mechanism underpinning the switch between stem-like and differentiated cell states both during embryonic development and cancer progression.
We previously used genome-wide chromatinimmunoprecipitation followed by high-throughput sequencing (ChIP-Seq) to profile the patterns of H3K4me3 and H3K27me3 in immortalized human mammary epithelial cells (HMLE), and their counterparts induced to undergo EMT through ectopic expression of the EMT-inducing transcription factor Twist (HMLE-Twist) . In addition to the extensive switching of monovalent H3K4me3 and H3K27me3 marks throughout the genome, we observed a significant enrichment of bivalent genes in mesenchymal HMLE-Twist cells relative to vector-transduced epithelial HMLE counterparts . Here, we have focused on the subset of premarked monovalent H3K4me3-promoters, rendered bivalent and silenced through the addition of H3K27me3, that can be dynamically reactivated through subsequent H3K27me3 removal. Indeed, we found that modulation of H3K27me3 content is the predominant means of regulating gene expression during the transition from an epithelial to a mesenchymal state. The corollary of this observation is that the removal of the H3K27me3 mark from bivalent promoters may be a major route to the resolution of bivalency towards gene activation during EMT-reversal/MET.
To date, only two related H3K27me3-demethylases have been identified: lysine (K)-specific demethylase 6A (KDM6A)—also known as ubiquitously-transcribed X chromosome tetratricopeptide repeat protein (UTX1)—and KDM6B, also known as Jumonji-domain containing 3 (JMJD3) [21, 22]. Both KDM6A and KDM6B have been implicated in a wide range of differentiation processes as well as in cancer progression, but their respective transcriptional outputs are likely to be highly context-dependent [21, 23–25]. In fact, whereas KDM6B has been shown to promote EMT by removing the repressive H3K27me3 mark from the SNAIL (SNAI1) promoter , KDM6A has been implicated in the resolution of bivalency during retinoic acid-induced differentiation of mouse ES cells . We therefore reasoned that KDM6A is a more likely candidate to demethylate H3K27me3 marks and drive the resolution of bivalency during MET.
Herein, we found that KDM6A is differentially expressed between stem and non-stem populations of breast immortalized and malignant cell lines, and that its protein levels decline during TGFB-induced EMT, with levels restored following TGFB-withdrawal and EMT-reversal/MET. In addition, we demonstrate that a significant proportion of EMT-associated bivalent genes encode effectors of cell differentiation, proliferation, phenotypic commitment and intercellular adhesions that are consequently silenced in the EMT/stem-like state. We further demonstrate that KDM6A plays a critical role in the MET-associated resolution and reactivation of these bivalent genes. These observations are consistent with a role for KDM6A in the resolution and activation of numerous bivalent genes, involved in proliferation and differentiation, to facilitate colonization during the latter stages of metastasis.
Genes rendered bivalent following EMT are predominantly associated with cell-cell adhesion, proliferation and developmental specification
We previously used genome-wide ChIP-Seq to profile the patterns of H3K4me3 and H3K27me3 in vector-transduced epithelial HMLE cells (hereafter HMLE-vector) versus mesenchymal HMLE-Twist cells, and detected a striking 2.7-fold increase in the number of bivalent genes in HMLE-Twist cells, compared to their epithelial counterparts . Since genes may become bivalent by trimethylation of H3K4 at premarked H3K27me3 promoters, trimethylation of H3K27 at H3K4me3-monovalent promoters, or by the deposition of methyl groups at both sites, we classified the bivalent genes in HMLE-Twist cells into 4 groups, based on their pre-existing histone modifications in HMLE-vector cells: I) premarked with H3K4me3 in HMLE-vector cells that gain H3K27me3, II) premarked with H3K27me3 in HMLE-vector cells that gain H3K4me3, III) unmarked in HMLE-vector cells that acquire both H3K4me3 and H3K27me3, and IV) bivalent in HMLE-vector cells with no further change in status (Supplementary Table 1). Indeed, we found that the addition of H3K27me3 to premarked monovalent H3K4me3-promoters is the predominant modification contributing to the establishment of bivalency during the transition from an epithelial to a mesenchymal state, accounting for the de novo formation of 47% of bivalent domains (Supplementary Table 1) .
In order to understand which biological processes may be regulated through the establishment of bivalency following EMT, we determined the enrichment for specific gene ontology terms in each category through gene ontology analysis. Strikingly, all 4 categories of bivalent genes are enriched for genes regulating development, cell fate specification and differentiation (Figure 1A, green bars). Examples of genes in these categories include transcription factors and signaling molecules such as SOX4 and SOX9 in Group I, WNT1 and WNT2 in Group II, BMP4 and NGFR in Group III, and PITX1 and FGF9 in Group IV. Notably, the subset of genes that acquires bivalent status through the addition of H3K27me3 following EMT (Group I) is particularly enriched for genes involved in cell-cell adhesion and cytoskeletal architecture (Figure 1A, blue bars), consistent with the reduction of intercellular adhesions and the acquisition of intrinsic motility following EMT. Examples of Group I genes involved in the regulation of actin cytoskeletal integrity and cell-cell adhesion include genes encoding the actin-binding protein COBLL1, the desmosome components DSG2 and DSP, and the constituent of adherens junctions CADM1. The finding that the combination of histone modifications, deposited at promoters rendering them bivalent, can classify the underlying genes into functionally distinct categories suggests that histone modifiers are selectively deployed to silence coordinated gene networks during EMT.
Figure 1: EMT-associated bivalent genes are enriched for regulators of proliferation, differentiation, and cell-cell adhesion, and are substantially repressed by addition of H3K27me3. Previously reported analyses of the epigenomes of HMLE-vector and HMLE-Twist cells, using ChIP-Seq for H3K27me3 and H3K4me3 and RNA-Seq, revealed that the number of bivalent genes (carrying both H3K4me3 and H3K27me3 marks within their promoters) is increased following Twist-induced EMT . In the present study, all bivalent genes in HMLE-Twist cells were divided into 4 Groups: Group I: premarked with H3K4me3 in HMLE-vector cells that gain H3K27me3; Group II: premarked with H3K27me3 in HMLE-vector cells that gain H3K4me3; Group III: unmarked in HMLE-vector cells that acquire both H3K4me3 and H3K27me3; and Group IV: bivalent in HMLE-vector cells with no further change in status. (A) The top biological process gene ontology (GO) terms for each bivalent gene group were determined using GOrilla. The most enriched GO terms in each category are shown with their corresponding p-values, calculated as the exact minimum hypergeometric score using the GOrilla web server . Green bars indicate categories relevant to developmental specification and blue bars indicate categories relevant to cell-cell adhesion and cytoskeletal architecture. Black bars indicate gene categories related to other biological processes. (B) The odds ratios of the overlap between the ES cell-derived and EMT-associated bivalent gene signatures were calculated, and are presented for each group of EMT-associated bivalent genes. (C) The percentage of bivalent genes in each group, exhibiting lower expression in HMLE-Twist cells compared to HMLE-vector cells, is graphed. Gene expression values are represented as Fragments Per Kilobase of transcript per Million mapped reads (FPKM), with data derived from RNA-Seq performed in . (D) The cumulative gene expression differences (measured in FPKM) between HMLE-Twist and HMLE-vector cells were determined, and the means graphed. Values above 0 indicate that the average change in expression is higher in HMLE-Twist cells, compared to HMLE-vector cells. Error bars indicate the standard error of the mean. The p-values were determined by comparison of ranks by the Mann-Whitney test for non-parametric data.
Since, in pluripotent ES cells, bivalent domains mark the promoters of genes involved in development and differentiation for silencing, we next examined the overlap between the sets of bivalent genes generated via EMT and genes identified as bivalent in ES cells. All 4 groups of bivalent genes, identified in our study, overlap at a statistically significant level (p<0.001 by Fisher’s exact test) with the ES cell-derived bivalent gene signature . In particular, the two groups of bivalent genes most associated with developmental specification (Group II, bivalent through acquisition of H3K4me3 and Group IV, pre-existing bivalent marks) show the highest odds ratios of overlap with the ES cell-derived bivalent gene signature  (Figure 1B), underscoring the similarities in the epigenetic regulation of the EMT and ES cell stemness modules. It is interesting to note that the Group IV bivalent promoters, unaffected by EMT, correspond most closely with those that are also bivalent in ES cells, supporting the notion that a subset of bivalent promoters are maintained in a ‘poised’ state despite a multitude of both differentiation and de-differentiation signals. Our results suggest that these constitutively bivalent Group IV genes encode products pertaining to a high degree of differentiation and specialized functions (e.g. LHX4, LHX9, LRRC6, PFKP, PPFIBP2, MEGF11, SEMA5B), or genetic programs that are suppressed under normal physiological conditions such as the response to genotoxic stresses and cell death pathways (e.g. TXNRD3, CERKL, SFXN1, PYCARD, PDRG1, NOC2L). Conversely, the genes in Group I show the least significant overlap with the ES cell-derived bivalent gene signature and encompass components of cell adhesion complexes and the cytoskeleton that play a prominent role in organizing cellular architecture and intrinsic motility. Therefore, we reasoned that these genes likely represent an EMT/MET-relevant structural gene module that regulates cell shape, cell-cell adhesion, and migration.
We next examined the differences in gene expression between each of the four groups of genes which became bivalent through distinct histone modification events during EMT. Consistent with our expectations, 69.4% (256/369) of the bivalent genes that gained the H3K27me3 mark were expressed at a lower level in their bivalent state in HMLE-Twist cells, compared to their monovalent H3K4me3 state in HMLE-vector cells (Figure 1C). In fact, further analysis demonstrates that the cumulative gene expression changes among genes in Group I are significantly more dramatic than the other groups (II-IV) and, overall, tend towards lower expression in cells induced to undergo EMT (HMLE-Twist) compared to epithelial counterparts (HMLE-vector), as might be expected due to the addition of the H3K27me3 repressive mark to the active H3K4me3 monovalent configuration, rendering the gene repressed/bivalent (Figure 1D). Given the significant differential expression of Group I genes between the epithelial and mesenchymal states, the relatively small overlap with the ES cell bivalent signature, and the enrichment of biological pathways involved in the regulation of cell adhesion and cell-cell communication in the Group I gene dataset, we next focused on the modulation of H3K27me3 as a major factor regulating the formation of bivalent domains and the silencing of gene expression following EMT.
The H3K27me3-demethylase KDM6A is dynamically regulated during EMT/MET and is differentially expressed in mesenchymal stem-like and non-stem cells
It is known that the resolution and activation of bivalent genes can promote differentiation in ES cells [28–30]. Given that MET at least partially restores differentiated epithelial characteristics to cells that have previously undergone EMT, and that our findings implicate the acquisition of H3K27me3 as a key modification leading to EMT-associated bivalency, we reasoned that an H3K27me3-demethylase activity might be suppressed during EMT, and reinstated during MET, thus enabling the resolution of bivalency. Therefore, we hypothesized that the activity and/or expression of the H3K27me3-demethylases KDM6A and KDM6B might be decreased following EMT, allowing the de novo accumulation of H3K27me3 and/or the retention of premarked H3K27me3 at bivalent domains.
We first investigated the expression of KDM6A in epithelial HMLE-vector cells in comparison with their mesenchymal counterparts overexpressing the EMT-inducing transcription factors Twist (HMLE-Twist) and Snail (HMLE-Snail). Using immunoblotting, we detected markedly reduced KDM6A protein levels in mesenchymal HMLE-Snail and HMLE-Twist cells compared to the epithelial HMLE-vector cells (Figure 2A). In addition, we observed intense immunostaining for KDM6A in the cytoplasm and nuclei of epithelial HMLE-vector cells, compared to the decreased KDM6A staining intensity of HMLE-Twist cells, most notably reduced in the nuclear compartment (Supplementary Figure 1A). We verified that HMLE-vector cells stained positive for E-cadherin, as expected, whereas HMLE-Twist cells were devoid of this epithelial marker, consistent with their mesenchymal phenotype (Supplementary Figure 1B). Interestingly, Twist1 has been shown to bind to a sequence 31 kb upstream of the Kdm6a gene in the context of mouse limb bud and endocardial cushion tissues . However, the functional relevance of Twist occupancy, at this site, to KDM6A gene expression has yet to be ascertained. Nevertheless, our data are consistent with the notion that Twist may repress KDM6A transcription.
Figure 2: KDM6A protein levels are decreased following EMT and upregulated during MET. (A) HMLE cells were transduced with either an empty vector (HMLE-Vector), or genes encoding the EMT-inducing transcription factors Snail (HMLE-Snail) or Twist (HMLE-Twist) . Whole-cell lysates were analyzed by immunoblotting for KDM6A. β-actin was used as a loading control. (B-F) MCF10A cells were treated with sterile 4 mM HCl containing 0.1% bovine serum albumin (Vehicle) or TGFB for 5 days (TGFB+). In parallel, MCF10A cells were exposed to TGFB for 5 days in order to induce EMT, and subsequently subjected to TGFB-withdrawal, for a further 5 days, to elicit MET (TGFB+/-). (B-C) MCF10A cells, treated as described, were fixed and immunostained for E-cadherin (B) and N-cadherin (C). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (D) MCF10A cells, treated as described, were analyzed by immunoblotting for KDM6B and vimentin (Vim). COX IV was used as a loading control. (E) MCF10A cells, treated as described, were fixed and immunostained for KDM6A. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (F) Cells were analyzed by immunoblotting for KDM6A, EZH2 and RING1A. β-actin was used as a loading control.
We also investigated the expression of KDM6A following TGFB-treatment of the E-cadherin+ve mouse mammary tumor cell line, 4T1 . As visualized by phase-contrast microscopy, treatment of 4T1 cells with TGFB over 5 days, elicited a marked reduction in cell-cell contacts and the acquisition of an elongated spindle-shaped morphology, consistent with the induction of EMT (Supplementary Figure 2A). Whereas KDM6A was readily detected in vehicle-treated 4T1 cells, we observed a dramatic reduction in KDM6A immunostaining following TGFB treatment, concurrent with the loss of the epithelial marker E-cadherin (Supplementary Figures 2B and 2C). These findings are consistent with an association of KDM6A expression with the epithelial phenotype, and its exclusion from the mesenchymal lineage. Moreover, these expression patterns suggest that KDM6A may function selectively/primarily in the epithelial setting, with the corollary that KDM6A depletion during EMT may account for the marked increase in bivalent domains detected in the genome of HMLE-Twist cells.
Since our findings indicate that KDM6A protein levels are depleted following EMT, we next sought to test whether KDM6A levels are reinstated during MET to facilitate the resolution of bivalency and the activation of genes involved in differentiation. For this, we utilized a physiologically-relevant model of TGFB-induced EMT in spontaneously immortalized mammary epithelial MCF10A cells, followed by EMT-reversal/MET elicited by TGFB-withdrawal. In this model, we observed diminished levels of membrane-localized E-cadherin in TGFB-treated MCF10A cells, compared to vehicle-treated epithelial counterparts, which exhibited a predominantly membranous expression pattern (Figure 2B). Moreover, TGFB treatment of MCF10A cells elicited an increase in the expression of the mesenchymal markers N-cadherin (Figure 2C) and vimentin (Figure 2D and Supplementary Figure 3). Conversely, TGFB-withdrawal was accompanied by restoration of E-cadherin to the cell membrane, and a decrease in the expression of vimentin and N-cadherin, consistent with EMT reversal/MET and re-differentiation towards an epithelial phenotype (Figures 2B-2D and Supplementary Figure 3). As expected, KDM6A protein levels decreased upon TGFB treatment (Figures 2E and 2F). Interestingly, upon TGFB-withdrawal/MET, KDM6A protein levels were restored to levels commensurate with those of vehicle-treated cells (Figures 2E and 2F). In contrast to our results for KDM6A, we observed increased expression of the related H3K27me3-demethylase KDM6B in TGFB-treated MCF-10A cells that have undergone EMT, relative to vehicle-treated epithelial counterparts (Figure 2D). This finding is consistent with a recent study showing that KDM6B expression is induced during EMT and, in fact, that KDM6B promotes EMT progression by de-repressing the SNAI1 promoter, through demethylation of the H3K27me3 mark, thus driving Snail expression . Overall, our data indicate that KDM6B and KDM6A protein levels are inversely regulated during EMT, commensurate with decreased E-cadherin protein levels and increased mesenchymal marker expression. Conversely, during TGFB-withdrawal/MET, KDM6A protein levels are restored to the levels of vehicle-treated cells, concurrent with a decrease in KDM6B levels.
One potential explanation for the accumulation of H3K27me3 at bivalent genes could be an increase in the expression of components of the Polycomb Repressor Complex 2 (PRC2), which mediates methylation at H3K27 . To determine if this is the case, we used our dynamic EMT/MET model to profile the expression of EZH2, the histone methyltransferase that functions as the catalytic subunit of PRC2, and RING1A which acts as a transcriptional repressor within the PRC2 complex. Surprisingly, we found that the protein levels of both EZH2 and RING1A were decreased during TGFB-induced EMT in MCF10A cells (Figure 2F). This unexpected finding underscores the significance of the reduction in KDM6A expression for the establishment of bivalency during EMT. Thus, the accumulation of H3K27me3 at bivalent genes, during EMT, is not accomplished through the increased activity of a molecular writer driving increased deposition, but rather through the absence of KDM6A demethylase activity, evidenced by the persistence of pre-existing or newly-deposited marks at H3K4me3-premarked promoters.
We and others have shown that the stem cell-enriched fractions of established mammary cell lines exhibit mesenchymal characteristics [4, 5], suggesting that these stem-like cell subsets may also express lower levels of KDM6A, compared to more differentiated subpopulations. To test this, we used the lipophilic fluorescent membrane-intercalating dye, PKH26, as a functional means to enrich for mammary stem cells . PKH26 labels all cells evenly in the initial population, but is progressively diluted out from highly proliferative cells during cell division. Thus, PKH26 is selectively retained in the membranes of the relatively slow-cycling cells, a population that includes mammary stem cells, which can therefore be recovered using fluorescence-activated cell sorting (FACS) [34–36]. To quantify the differences in KDM6A expression between the stem-like and differentiated cell subsets, we sorted PKH26+ve and PKH26-ve/low MCF10A cells, 7 days following PKH26 addition. Given the small numbers of PKH26+ve stem-like cells compared to the bulk PKH26-ve/low populations, we centrifuged FACS-sorted PKH26-ve/low and PKH26+ve cell suspensions onto glass slides by cytospin and performed immunofluorescent staining for KDM6A (Figure 3A). To quantify our results, the intensity of KDM6A staining within a nucleus stained with 4', 6-diamidino-2-phenylindole (DAPI) was measured using Gen5 image analysis software. The mean intensity of KDM6A staining was significantly lower in the stem-like PKH26+ve MCF10A cell fraction compared to the PKH26-ve/low MCF10A subpopulation (Figure 3B). To expand our findings to additional cell lines, MCF7 and MDA-MB-231 cells were sorted for PKH26-ve/low and PKH26+ve cells, with MCF10A cells serving as a control. In order to better visualize the KDM6A staining pattern, we performed immunofluorescence using sorted cells deposited directly onto glass coverslips, without centrifugation. In line with our previous observations, KDM6A was noticeably absent from the nuclei of the stem-cell enriched PKH26+ve subpopulations from MCF10A cells as well as MCF7 and MDA-MB-231 breast malignant cell lines (Figure 3C). In contrast, we observed prominent nuclear KDM6A staining in the more differentiated PKH26-ve/low subpopulations (Figure 3C). Collectively, these results show that the slow-cycling, stem cell-enriched subpopulations of multiple mammary cell lines display significantly diminished KDM6A staining compared to the faster-cycling, differentiated subpopulations.
Figure 3: Stem cell-enriched subpopulations of mammary cell lines express lower KDM6A levels compared to non-stem counterparts. (A) MCF10A cells were labeled with PKH26 and sorted by FACS into PKH26-ve/low and PKH26+ve populations, and subsequently processed for KDM6A immunostaining. Images from random fields were captured using a Cytation 3 automated digital microscope. Representative images of KDM6A immunostaining (green) in PKH26-ve/low and PKH26+ve MCF10A cells, centrifuged onto slides by cytospin, are shown. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. The insets represent magnified images of selected areas. Scale bars, 20 μm. (B) Image analysis was performed to determine the relative intensity of the immunofluorescence signal within the nuclei of individual cells. The mean immunofluorescent intensity of KDM6A staining was plotted for the respective PKH26-ve/low and PKH26+ve subpopulations. n equals the number of nuclei analyzed for each MCF10A subfraction. P-values were calculated using an unpaired, two-tailed t-test. (C) Cells from the indicated cell lines were sorted into PKH26-ve/low and PKH26+ve subfractions by FACS. Immunofluorescent staining for KDM6A was performed on PKH26-ve/low and PKH26+ve cells that were seeded onto glass coverslips after sorting. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm.
KDM6A occupancy inversely correlates with H3K27me3 levels at the CDH1 locus
Given that the EMT and MET programs unfold over time, and are underpinned by gradual changes in gene expression with CDH1/E-cadherin repression widely regarded as the hallmark of EMT, we next undertook to conduct a timecourse analysis of the changes in H3K27me3 occupancy at the CDH1/E-cadherin promoter. For this, we transiently treated MCF10A cells with TGFB over 5 days, followed by TGFB-withdrawal for a further 5 days, and harvested samples for quantitative RT-PCR analysis and ChIP assays at appropriate timepoints. Consistent with the induction of EMT, TGFB treatment of MCF10A cells elicited a gradual decrease in the expression of the CDH1 gene. Conversely, TGFB-withdrawal was accompanied by a progressive increase in CDH1 transcript levels, signifying a return to an epithelial phenotype (Figure 4A). Accordingly, we found that H3K27me3 was enriched at the transcription start site of the CDH1/E-cadherin promoter following TGFB treatment, consistent with the notion that accumulation of H3K27me3 drives CDH1 repression during EMT. Following TGFB-withdrawal, the levels of H3K27me3 at the CDH1 transcription start site were diminished, thus establishing a transcription-permissive state (Figure 4B).
Figure 4: H3K27me3 deposition is reversible upon MET and revokes CDH1/E-cadherin silencing. MCF10A cells were treated with TGFB (5 ng/ml) for five days to induce EMT, followed by five days of culture without TGFB, eliciting MET. As a control, MCF10A cells were treated with vehicle during the first 5 days of the timecourse. (A) qRT-PCR was used to monitor CDH1/E-cadherin mRNA levels at the indicated timepoints. GAPDH was used as the reference gene to normalize the variability in template loading. (B) Chromatin immunoprecipitation-PCR (ChIP-PCR) analysis was done on the transcription start site of the CDH1 gene to determine the relative enrichment of H3K27me3 at this locus in MCF10A cells, harvested at the indicated timepoints. (C, D) ChIP-PCR analysis was used to determine the relative enrichment of H3K27me3 (C) and the KDM6A occupancy (D) at the CDH1 transcription start site, following immunoprecipitation of lysates from HMLE-vector and HMLE-Twist cells with the respective antibodies. For H3K27me3, values were normalized to immunoprecipitation of total H3. The background enrichment for a subtype-control IgG antibody was subtracted. P-values were calculated using an unpaired, two-tailed t-test.
Additionally, we sought to determine whether KDM6A directly regulates CDH1 expression and if selective enrichment of H3K27me3 at the CDH1 promoter is associated with CDH1/E-cadherin repression in the mesenchymal state. For this, we performed ChIP assays, using HMLE-vector and HMLE-Twist cells, which exhibit epithelial and mesenchymal characteristics respectively. As predicted, H3K27me3 was detected at 2.4-fold higher levels at the CDH1 promoter in HMLE-Twist cells, which do not express E-cadherin (Figure 4C), compared to HMLE-vector cells. On the other hand, KDM6A binding to the CDH1 promoter was detected to a much higher degree in HMLE-vector cells, suggesting a role for KDM6A in demethylating H3K27me3 to enhance/maintain basal E-cadherin expression levels (Figure 4D). Taken together, these data support a dynamic role for KDM6A in the removal of the H3K27me3 repressive mark to maintain the CDH1 promoter in a transcriptionally-active state, with the corollary that the loss of KDM6A activity, during EMT, will lead to accumulation of H3K27me3 and silencing of CDH1.
Inhibition of KDM6A activity during MET blocks reactivation of bivalent genes
In order to test whether KDM6A is involved in the resolution of bivalency and the reactivation of epithelial genes during EMT-reversal/MET, we exposed MCF10A cells to TGFB to elicit EMT, followed by TGFB-withdrawal to instigate MET. Furthermore, during TGFB-withdrawal, we treated the cells with either DMSO (as a vehicle control) or the H3K27me3-demethylase inhibitor GSK-J4 [37, 38] (Figure 5A). We confirmed the induction of EMT and MET by immunostaining for the intermediate filament protein vimentin, a well-established marker of mesenchymal cells. As expected, TGFB-induced EMT was accompanied by markedly increased immunostaining for vimentin, relative to vehicle-treated MCF10A cells, whereas TGFB-withdrawal, commensurate with MET, was evidenced by greatly diminished vimentin levels (Supplementary Figure 3). Interestingly, cells subjected to concurrent TGFB-withdrawal and GSK-J4 treatment exhibited intermediate levels of vimentin expression, suggesting that only a partial MET was achieved, consistent with a role for KDM6A activity in the restoration of the epithelial phenotype during MET progression (Supplementary Figure 3).
Figure 5: KDM6A inhibition by GSK-J4 blocks reactivation of bivalent genes. (A) RT-PCR for the indicated genes was performed on RNA extracted from MCF10A cells treated with vehicle or TGFB (2.5 ng/ml), and TGFB-treated MCF10A cells, subsequently subjected to TGFB-withdrawal in the presence of DMSO or GSK-J4, for the durations indicated. (B) Gene expression was compared between MCF10A cells treated with either vehicle or TGFB for 6 days. (C) Gene expression was compared between MCF10A cells treated with TGFB for 6 days, and cells treated with TGFB for 6 days, followed by TGFB-withdrawal for 10 days. (D) Gene expression was compared between MCF10A cells treated with TGFB for 6 days, and subsequently subjected to TGFB-withdrawal for 10 days, in the presence of either vehicle control (DMSO), or 50 nM GSK-J4. Expression was normalized to GAPDH. Data are presented as the mean and standard deviation of triplicate experiments. P-values were calculated by unpaired t-tests and are corrected for multiple comparisons by the Holm-Sidak method. ns=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p< 0.0001. (E) The table summarizes the expression changes for each gene across the different experimental conditions in panels B-D. Genes that exhibit significantly different expression in the direction indicated by each individual criterion are marked with “Yes”, while significant changes in the other direction or non-significant differences are marked with “No”. Genes marked with “Yes” for all three conditions are highlighted in yellow to indicate that they are suppressed following EMT, and that their re-expression following MET is sensitive to inhibition of H3K27me3-demethylase activity by GSK-J4.
We next used this model to measure the mRNA expression of a subset of bivalent genes in Groups I-IV. The expression of the majority of genes in Groups I-III was consistently downregulated to a statistically significant degree following TGFB-induced EMT in MCF10A cells (Group I: 15/22, Group II: 6/8, Group III: 5/8), while the expression of the majority of genes in Group IV remained unchanged, as would be predicted by their constitutively bivalent status (Figure 5B). Modest fluctuations observed in the expression of Group IV genes are potentially due to additional gene expression regulatory mechanisms. We next examined how expression of these select bivalent genes was altered during TGFB-withdrawal/MET. In Group I, the majority of genes that were downregulated during EMT were subsequently reactivated during MET (10/15) and this pattern was also seen for Group II (5/6), Group III (5/5) and Group IV (2/2) (Figure 5C). Remarkably, the MET-associated re-expression of nearly all the genes that displayed this pattern of regulation was sensitive to inhibition of KDM6A by GSK-J4 (Group I: 9/10, Group II: 5/5, Group III: 4/5, Group IV 1/2) (Figure 5D). While sensitivity to GSK-J4 was expected for genes in Groups I and III (Figure 5E), which exhibited a gain of H3K27me3 to become bivalent in our Twist-driven model of EMT, we did not expect to observe sensitivity to GSK-J4 in Group II genes, which gained H3K4me3 to become bivalent. As expected, the expression of Group IV genes, which retain a bivalent status throughout EMT and MET, remained largely unaltered, consistent with the notion that the establishment of bivalency at these loci is independent of KDM6A. Collectively, our data demonstrate that KDM6A-dependent H3K27me3 demethylation plays an important role in the re-expression of bivalent genes, involved in proliferation, differentiation and cell adhesion, during MET.
Expression of KDM6A and its target genes, which become bivalent during EMT, is lower in CSC-enriched, triple-negative breast cancers
Since we observed reduced KDM6A expression levels in cells that have undergone EMT and in stem cell-rich populations, we next sought to determine if breast cancer subtypes, enriched for EMT/CSC properties, also exhibit lower KDM6A levels. Breast cancers are markedly heterogeneous and can be classified into distinct subsets on the basis of their gene expression profiles or histopathological criteria. Differential gene expression profiling separates breast cancers into “intrinsic” molecular subtypes designated luminal A, luminal B, HER2-enriched and basal-like , which includes the majority of the recently characterized claudin-low tumors . Of these, the basal-like tumors are most enriched for mesenchymal and CSC features [41, 42]. Consistent with these characteristics, the basal-like tumors in The Cancer Genome Atlas (TCGA) dataset express lower KDM6A levels compared to the other subtypes examined (Figure 6A).
Figure 6: KDM6A and Group I bivalent genes are expressed at lower levels in basal-like and triple-negative breast cancers. Breast cancers from the TCGA datasets were categorized based on PAM50 classification (A) or ER-, PR- and HER2-status (B), and the expression of KDM6A was deduced from the available RNA-Seq data. P-values were calculated using Student’s two-tailed t-test. (C) The cumulative expression levels of all the Group I genes were compared between breast cancer samples, classified as triple-negative (TN) or non-triple negative (Non-TN). P-values were calculated using Student’s two-tailed t-test. (D) The cumulative expression levels of orthologous Group I genes from embryonic and adult mouse tissue samples were compared and plotted. Each dot represents a different timepoint during embryo development (Embryonic Stages) or tissue of origin (Adult Tissues). P-values were calculated using Student’s two-tailed t-test.
Based on immunohistochemical criteria—more often used clinically—breast cancers can be broadly classified into hormone-receptor positive luminal, HER2-expressing, and triple-negative subtypes. Triple-negative breast cancers (TNBCs), i.e. tumors lacking ER-, PR- or HER2-expression, are also enriched for EMT/CSC features when compared to ER-, PR- or HER2-positive breast cancers, consistent with the fact that the majority of basal tumors are also TNBCs . Importantly, KMD6A expression is lower in TNBCs compared to non-TNBCs (Figure 6B). Furthermore, we determined that the cumulative expression of the Group I genes, which become bivalent following EMT through addition/retention of H3K27me3, is also decreased in TNBCs compared to non-TNBCs (Figure 6C). Moreover, these Group I genes are suppressed in the embryonic stages of mouse development relative to their expression in a spectrum of adult/differentiated mouse tissues (Figure 6D). These data support our model that KDM6A expression/activity is a key enforcer of the epithelial phenotype and that loss of KDM6A expression correlates with EMT and the acquisition of stem-like properties during development and cancer progression.
The EMT and MET processes have been increasingly recognized as critical cellular reprogramming events facilitating carcinoma progression and metastasis [1–3]. The induction of EMT endows stationary carcinoma cells with the migratory and invasive potential, as well as the stemness attributes, required to navigate the sequential steps of the metastatic cascade and initiate micrometastases [4, 43–45]. Upon encountering the foreign microenvironment at the distant site, disseminated tumor cells undergo MET and revert to an epithelial-like proliferative phenotype, which is important for colonization and formation of overt epithelial metastatic outgrowths [6, 7, 46–49]. While key molecular effectors of the EMT/MET program have been deciphered, the changes in the chromatin architecture underlying EMT/MET, and the epigenetic regulators effecting these changes, are only just beginning to be explored. Herein, we identify increased H3K27me3 occupancy as the predominant modification leading to the establishment of bivalent domains and gene silencing following EMT. Most importantly, we establish a role for the H3K27me3-demethylase KDM6A in the resolution and activation of bivalent genes accompanying MET and the transition between the mesenchymal and epithelial states (Figure 7).
Figure 7: Schematic representation of the regulation of bivalency by KDM6A during EMT/MET. In mammary epithelial cells, KDM6A constitutively catalyzes the removal of di- and trimethyl groups from the H3K27me3 repressive mark of bivalent promoters (H3K4me3/H3K27me3), thus unmasking an active configuration (H3K4me3/H3K27me1). KDM6A target genes, associated with differentiation, proliferation and intercellular adhesion, are hence maintained in a transcriptionally active state reinforcing the epithelial phenotype. Following EMT and loss of KDM6A expression, cells accumulate H3K27me3 at KDM6A target promoters leading to the transcriptional repression of genes associated with differentiation, proliferation and intercellular adhesion, and to the acquisition of a mesenchymal/stem-like/slow-cycling phenotype. Conversely, KDM6A levels are restored during MET, concomitant with target gene activation/resolution of bivalency through H3K27me3 removal, and restoration of epithelial attributes. Inhibition of KDM6A, using the H3K27me3-demethylase inhibitor GSK-J4, prevents the re-expression of bivalent genes during MET. Non-TNBCs and TNBCs are enriched for cells in the “KDM6A high” and “KDM6A low” states respectively.
We previously reported a significantly increased incidence of bivalent genes in epithelial cells induced to undergo EMT via ectopic expression of Twist, relative to their vector-transduced epithelial counterparts . We made the striking observation that 47% of the bivalent domains, established de novo during EMT, arise through the accumulation of H3K27me3 at monovalent premarked H3K4me3-promoters . Through gene ontology analysis, we found herein that Group I—encompassing H3K4me3-premarked genes that accrue H3K27me3 following EMT—likely represents a coordinately-regulated EMT/MET-associated structural gene module that controls cellular architecture, inter-cellular adhesion and intrinsic motility, in addition to a gene module involved in the regulation of proliferation. Accordingly, we found that Group I genes are most markedly differentially expressed between the epithelial and mesenchymal states and, overall, tend towards lower expression in HMLE-Twist cells compared to HMLE-vector cells, consistent with the establishment of a repressed/bivalent configuration. With these considerations in mind, we focused on the modulation of H3K27me3 as a major factor regulating the formation of bivalent domains and the silencing of gene expression following EMT.
We reasoned that the observed accumulation of H3K27me3 marks following EMT might be attributed to an increased deposition of methyl groups, mediated by the PRC2 complex, and/or to a reduction in H3K27me3-demethylase activity. However, we found that the protein levels of both EZH2 and RING1A, two key components of the PRC2 complex , were downregulated during TGFB-induced EMT in MCF10A cells, suggesting that H3K27me3 accumulation is not primarily achieved through increased deposition of methyl groups. We also examined the expression of the two known H3K27me3-demethylases, KDM6A and KDM6B, and found that the induction of EMT is accompanied by markedly decreased KDM6A protein levels and, conversely, by elevated KDM6B expression. As the establishment of bivalency requires the accumulation of H3K27me3 marks, we conclude that the attenuation of KDM6A-associated H3K27me3-demethylase activity is the principal mechanism leading to increased H3K27me3-occupancy at H3K4me3-premarked promoters, underpinning the emergence of newly-formed bivalent domains following EMT.
The downregulation of CDH1/E-cadherin expression is considered a hallmark of EMT, and the enrichment of H3K27me3 at the CDH1 promoter has been shown to be a key factor—amongst other gene-repressive mechanisms—in silencing CDH1 expression in many tumor contexts [50–54]. Given that H3K27me3 occupancy can be modulated through enzymatic removal, we aimed to understand how EMT-associated CDH1 repression may be reversed during MET. First, using MCF10A cells, we showed that, while KDM6A protein levels decline during TGFB-induced EMT, its expression is restored following TGFB-withdrawal and EMT-reversal/MET. Our findings, herein, clearly show a progressive accumulation of H3K27me3 at the CDH1 promoter during TGFB-induced EMT, which correlates with a marked reduction in CDH1 transcript levels. Following TGFB-withdrawal and MET, the H3K27me3 mark is rapidly lost from the CDH1 promoter, preceding the upregulation of E-cadherin expression and restoration of the epithelial phenotype. These results are consistent with the notion that KDM6A enhances CDH1 transcription during MET, by demethylating the repressive H3K27me3 mark previously deposited at this locus during EMT. This is also supported by the recent observation that KDM6A is necessary for H3K27me3 demethylation at the CDH1 locus in colon cancer cells . Our results further suggest that KDM6A is constitutively present at the CDH1 locus in epithelial cells, thus regulating the basal levels of E-cadherin. Conversely, the KDM6A occupancy at the CDH1 promoter is diminished in mesenchymal cells and is inversely correlated with H3K27me3 levels.
We also aimed to understand how EMT-associated bivalent gene repression may be reversed during MET. Using the H3K27me3-demethylase inhibitor, GSK-J4, in our dynamic MCF10A EMT/MET model, we demonstrated that KDM6A plays a critical role in the MET-associated resolution and reactivation of bivalent genes. Significantly, our results indicate that GSK-J4 treatment, concurrent with TGFB-withdrawal/MET, prevents the activation of numerous bivalent genes implicated in proliferation, differentiation and cell-cell adhesion. Notably, bivalent genes that are repressed during EMT and de-repressed following MET in a GSK-J4 sensitive manner, include genes encoding regulators of cell growth and differentiation (CBX4, CCNA1, CDK4, FAM60A), components of desmosomes or microtubules functioning in the regulation of cell adhesion, polarity and migration (PKP2), and members of signal transduction pathways (FZD8, MAP3K9). Similarly, bivalent genes have been shown to be resolved by KDM6A during mouse ES cell differentiation . Furthermore, KDM6A-demethylase activity has been shown to regulate the reprogramming that underpins the derivation of induced pluripotent stem cells from somatic cells , a process wherein MET is considered a critical initiating event [57, 58]. Overall, these results suggest an important role for KDM6A in the reactivation of bivalent genes, associated with the establishment of a proliferative, differentiated, epithelial phenotype following the induction of MET.
Consistent with published reports, we found that KDM6B protein expression was increased during TGFB-induced EMT  and decreased following MET, in a manner inversely correlated with the levels of KDM6A. Since both KDM6A and KDM6B act by removing repressive H3K27me3 marks, and, as they exhibit differential expression patterns in the distinct settings of EMT and MET, it is unlikely that they target the same genes. This is supported by the inability of either demethylase to compensate for the loss of the other during embryonic development, which implies significant non-overlap in either their respective target genes, their tissue-specific expression patterns, or both . Importantly, although GSK-J4-mediated inhibition of KDM6B is likely to lead to the accumulation of H3K27me3 marks in a distinct set of target genes, the fact that KDM6B levels are decreased during TGFB-withdrawal/MET renders GSK-J4 inhibition of KDM6B redundant in respect to blocking the reactivation of bivalent genes during MET.
Remarkably, KDM6A protein levels are significantly decreased in the stem-like subpopulations of established mammary cell lines, compared to non-stem cell subsets, consistent with the links between stem-like attributes and the EMT phenotype [4, 5]. Taken together with the finding that the expression of KDM6A is restored in cells undergoing EMT-reversal/MET, we propose that KDM6A target gene occupancy is mostly associated with differentiation, and markedly diminished in the mesenchymal, stem-like state. This expression pattern is in concert with many recent findings, including an unbiased genetic screen that identified KDM6A as an EMT-suppressor in hepatocellular carcinoma cells [60–63]. In the present study, we also found that many Group I genes, rendered bivalent and silenced through retention of the H3K27me3 mark in the absence of KDM6A, are functionally involved in proliferation. Consistent with this, a recent study linked the high expression of KDM6A to the proliferative capacity of breast cancer cell lines . Taken together with the attenuated levels of KDM6A in stem-like cells, we suggest that low overall KDM6A expression and/or activity may be a contributing factor in the establishment of the relative quiescence manifested by normal and cancer stem cells [64, 65]. Moreover, we found that the expression of KMD6A, and its Group I target genes, is lower in CSC-enriched TNBCs compared to non-TNBCs, lending support to the potential clinical relevance of our findings.
Since tumor cells can disseminate early during tumor progression, the initial steps of metastatic dissemination, facilitated by EMT, may not represent amenable therapeutic targets for tumors with a propensity to metastasize. However, the reliance of colonization on the induction of MET at the distant sites may provide a potential therapeutic window for intervention. Our findings, that KDM6A activity is harnessed towards the resolution of bivalency and subsequent gene activation during MET, suggest that inhibiting KDM6A activity may be a viable therapeutic strategy to compromise the onset of MET-associated proliferation, differentiation, and colonization. Indeed, the small molecule inhibitor of H3K27me3-demethylase activity, GSK-J4, represents a promising drug candidate in this regard . In keeping with our finding that KDM6A maintains the expression of E-cadherin, a recent publication by Choi et al.  highlighted a role for KDM6A in suppressing the expression of the genes encoding the EMT-inducing transcription factors SNAIL, ZEB1, and ZEB2, thereby acting essentially as an EMT-suppressor. Intriguingly, however, this study showed that KDM6A elicits the repression of SNAIL, ZEB1, and ZEB2 in an H3K27me3-demethylase-independent manner, through its function as a scaffold protein, capable of recruiting LSD1, HDAC1, and DNMTs to promoters bound by MLL4 . Taken together, these findings present a compelling case for the development of small molecules that selectively block the KDM6A H3K27me3-demethylase activity, and/or modulate its scaffold function. We suggest that such molecules may comprise promising therapeutic candidates to suppress EMT or prevent MET-associated colonization by maintaining disseminated tumor cells in a dormant/quiescent state.
Overall, we demonstrate that KDM6A expression is decreased following EMT, and suppressed in stem-like subpopulations and TNBCs, with the corollary that genes involved in proliferation, cell adhesion, and differentiation are repressed through de novo deposition and/or retention of the H3K27me3 repressive mark. Significantly, KDM6A plays a critical role in the MET-associated resolution and reactivation of bivalent genes by removing H3K27me3 marks deposited during EMT. Taken together with a direct role for KDM6A in the maintenance of basal E-cadherin levels, these results suggest that KDM6A is a critical enforcer of the epithelial phenotype. These observations are consistent with a role for KDM6A in the resolution and activation of numerous bivalent genes, involved in proliferation and differentiation, to facilitate colonization during the latter stages of metastasis.
MATERIALS AND METHODS
Cell culture and drug treatments
Immortalized human mammary epithelial cells (HMLE), including cells expressing the empty vector (pWZL), Snail or Twist were maintained as in Mani et. al . MCF10A cells were cultured in 2D as a monolayer, as described in Debnath et al. . 4T1 cells were cultured as in Yang et al. . MCF7 and MDA-MB-231 cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum and penicillin/streptomycin. TGFB (R&D Biosystems, Minneapolis, MN, USA) was reconstituted in sterile 4 mM HCl containing 0.1% bovine serum albumin. TGFB was added to the culture medium at a concentration of 2.5 ng/ml unless otherwise indicated, with the reconstitution solution serving as a vehicle control. GSK-J4 (Selleckchem, Houston, TX, USA) was added to the culture medium at a concentration of 50 nM, with DMSO as a vehicle control.
Antibodies, immunoblotting, and immunofluorescence
Primary antibodies, used for immunoblotting and immunofluorescence, were raised against the following antigens: KDM6A (A302-374A; Bethyl Laboratories, Montgomery, TX, USA, and A302-374A; NBP1-80628, Novus Biologicals, Danvers, MA, USA), vimentin (10082-248; Proteintech, Rosemont, IL, USA), N-cadherin (D4R1H; Cell Signaling Technology, Danvers, MA, USA), E-cadherin (61081; BD Biosciences, San Jose, CA, USA), COX IV (926-42214; Licor, Lincoln, NE, USA), KDM6B (NBP1-06640; Novus Biologicals), EZH2 (5246; Cell Signaling, Danvers, MA, USA), RING1A (13069; Cell Signaling) and β-actin (ab8227; Abcam, Cambridge, MA, USA).
For immunoblotting, proteins were extracted by lysing cells in ice-cold radio-immunoprecipitation (RIPA) buffer containing protease and phosphatase inhibitors (Roche, Nutley, NJ, USA). Protein was quantified using the Bradford Assay (BioRad, Hercules, California, USA). Cell lysates (50 μg) were resolved using SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were probed with primary antibodies overnight at 4°C, followed by extensive washing with TBST, and incubation with secondary antibodies for 1 hr at room temperature. Chemiluminescent signals were detected with ECL™ prime (Thermo Fisher Scientific, Waltham, MA USA) using the Biorad ChemiDoc system.
Immunofluorescent staining was conducted as described in Mani et. al. , and images were acquired using an inverted Zeiss Axio Observer fluorescent microscope or an inverted Nikon Eclipse Ts2R fluorescent microscope.
PKH26 (Sigma-Aldrich, St. Louis, MO, USA) labeling was performed according to the manufacturer’s protocol. Label-retaining cells were sorted by FACS during which non-viable cells were excluded by propidium iodide staining. Sorted cells were then fixed in suspension for 10 minutes in 10% paraformaldehyde. Equal numbers of PKH26-ve/low and PKH26+ve cells were centrifuged onto slides by spinning for 2 minutes at 1500 rpm in a Cytospin 4 Cytocentrifuge (Thermo Fisher Scientific), or pelleted and resuspended in growth media and allowed to attach to coverslips. Cytospun cells were subsequently immunostained as described in Mani et. al. . Images were acquired using a Cytation™ 3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA), and analyzed for fluorescence intensity using Gen5 image analysis software (BioTek).
Quantitative reverse-transcription PCR and chromatin immunoprecipitation
Total RNA was isolated using Trizol (Ambion, Foster City, CA, USA) according to the manufacturer’s instructions. Relative quantification of the mRNA levels was performed using the comparative Ct method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene and with the formula 2−ΔΔCt. All quantitative reverse transcription-PCR (RT-PCR) experiments were run in triplicate and a mean value was used for the determination of mRNA levels.
To select bivalent genes for follow-up RT-PCR analysis, as presented in Figure 5, we first narrowed the sets of bivalent genes to those with appreciable levels of expression [Fragments Per Kilobase of transcript per Million mapped reads (FPKM)>10] in our RNA-Seq-derived dataset of gene expression in epithelial HMLE-vector cells. Next, we randomly selected 25 genes from Group I, and 10 each from Groups II, III and IV, and designed RT-PCR primers (Supplementary Table 2). Only those genes that were detectable by RT-PCR are presented in Figure 5.
Chromatin immunoprecipitation, followed by quantitative PCR analysis, was performed as described in Malouf et al. .
Statistical analyses of gene expression, gene ontology, and gene set overlap
RNA-Seq expression data were extracted from The Cancer Genome Atlas (TCGA) for breast cancer patients (BRCA). We categorized samples by PAM50 classification or by ER, PR and HER2 status. Samples with definitively negative values for ER, PR and HER2 were categorized as triple-negative (TN), while samples with a definitively positive value for ER, PR or HER2 were categorized as non-triple negative (Non-TN). We compared gene expression levels using a two-sided t-Test with unequal variance. The mouse tissue gene expression data were derived from Soundararajan et al. . Gene ontology analysis was performed by uploading selected gene name sets to the GOrilla web server  as the target set, using all of the gene IDs, from our previously reported ChIP-Seq data, as background . The association between two sets of genes was examined using Fisher’s exact test to find statistical significance. A small p-value means that the association (or overlap) is significant. Odds ratio was employed to measure the strength of association. Generally, an odds ratio greater than one indicates a strong association.
We acknowledge Dr. Huolin Tu and Dr. Jenaro Garcia-Huidobro at MD Anderson Cancer Center for invaluable discussion and technical help. We also thank Hunter Graham, Tom Niu, Tim Philip, and Collin Campion for providing technical assistance to support this work at Baylor.
This work was supported in part by the National Science Foundation Center for Theoretical Biological Physics (NSF PHY-1605817; SAM), the Temple Health & Biosciences District Research Award (JHT) and the Department of Biology and Molecular Bioscience Core facility at Baylor University. Flow cytometry was in part funded by the MD Anderson Cancer Center Support Grant from the National Cancer Institute (CA16672).
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
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domain: medical


text:
When Your Editor Tells You There Are One Too Many Siblings
The further I get into the writing process of my novel, CHASING CRAZY, the further away I am from being finished.
Let that resonate. Feel free to read it again—I’ll wait.
I wrote down the first words for CHASING CRAZY on August 25, 2013. As I began writing, I questioned whether I wanted to keep this venture to myself or share my quest with everyone within earshot. Wouldn’t be great to just present a finished novel to the world, and have them go, “Oh my god, you wrote a novel?” But I decided to announce my writing intentions as a check and balance, believing that if I told the world that I was writing a novel, I better damn well do it.
So I did.
The downside to that logic is I had never written a novel and knew absolutely nothing about what goes into the process, thinking sweetly that my editor would look over my first draft and fix a few typos. I envisioned her saying, “It’s incredible, darling! This book will be a best seller!”
But the reality is a long road of new characters, deleted chapters and new directions. What started out as a memoir turned into a novel, and the reality is, a tight storyline means less characters. Translation—my editor informing me that, “You need to lose a sibling.” Which, if you have a sibling or two (or four), is the equivalent of being told that you need to cut off your arm. I balked at this suggestion for over a year as I rewrote and reworked the storyline.
But today, I killed a sibling. I didn’t actually kill the kid. I simply (literally) erased the poor child from the pages of my novel—nothing personal. The editor made me do it. With eyes now wide open, I continue to rework, delete and rewrite. CHASING CRAZY is not a great novel yet, but it will be.

domain: artistic


text:
The customs revenue collection in the Thiruvananthapuram Commissionerate has shown a growth of 55 per cent compared to the previous year, Customs Commissioner C.M. Murugan has said.
Addressing the Customs Day celebrations here on Tuesday, Mr. Murugan said the gross customs revenue collection from April 1, 2012 to January 31, 2013 was Rs.51 crore, as against the Rs.32 crore registered from April 1, 2011 to January 31, 2012.
The target for 2012-2013 was Rs.40 crore, which was achieved in November 2012 itself, he said.
Mr. Murugan pointed out that the main customs formations under the commissionerate were the Thiruvananthapuram International Airport, the Air Cargo complex and the two minor ports at Vizhinjam and Thangassery in Kollam district.
The growth of 55 per cent was possible with the help of the Airports Authority of India, the Kerala State Industrial Enterprises and other stakeholders, including the Central Industrial Security Force and the airline operators, he said.

domain: finance