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STARmeterSEE RANK
Up 60,654 this week
Jorge Van Rankin
Jorge Van Rankin was born on June 5, 1964 in Mexico City, Distrito Federal, Mexico as Jorge Gabriel Van Rankin Arellano. He is an actor and writer, known for ¡Qué madre, tan padre! (2006), Serafín: La película (2001) and Big Brother VIP: México (2002). See full bio »
More at IMDbPro » Represent Jorge Van Rankin? Add or change photos


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Yes I do not trade on Alpha and volume for at least one house position is off. Seems odd to me as I always thought stockwatch was bang on. I no longer subscribe since I do not daytrade as much anymore and my brokerage has almost all the features I used to pay for. I will admit that since pp announcement has come out I have traded opl a few times (token rebellion as I still think we would of been better served had pp been north of a buck). Made a grand total of 450$ ish flipping pennies woopie do (my core actually increased by 500 shares lol... some rebel) ZNN and HVU were better flips. Too much rhum I digress gn and gl. A drunken ty to fairchild and oogee and the dutchman for all their work.
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Monday, November 12, 2012
NEW WILMINGTON, Pa. - The Presidents' Athletic Conference (PAC) coaches voted five Westminster College football players to the 2012 All-PAC Football Teams and an additional seven Titans received honorable mention recognition.
Saturday, November 10, 2012
BEAVER FALLS, Pa. - The Westminster College football team lost its season finale against host Geneva, 33-14, on Saturday in Presidents' Athletic Conference (PAC) action. The Titans finish the season 3-6 overall and 2-6 in the conference. Geneva improved to 3-7 overall and 3-5 in conference play.
Saturday, November 3, 2012
NEW WILMINGTON, Pa. - The Westminster College football team lost 16-10 to Thiel at Harold Burry Stadium on Saturday for its Senior Day. The Titans dropped to 3-5 overall and 2-5 in the Presidents' Athletic Conference (PAC), while the Tomcats improved to 3-6 overall and 3-4 in the conference.
Saturday, September 22, 2012
NEW WILMINGTON, Pa. - Titan junior Tyler Banks (Meadville, PA / Meadville) rushed for a career-high 144 yards in Westminster's 17-6 win over the visiting Saint Vincent College under the lights of Harold Burry Stadium on Saturday. The Titans improved to 2-1 overall and 1-1 in the Presidents' Athletic Conference (PAC), while the Bearcats dropped to 0-4 overall and 0-2 in the conference.
Displaying 1-4 of 6 total records


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See All Contributors »
How long before the Big 12 Network for mens basketball goes HD?
Any guesses?
Too long.
Edit: Better question, how long after their first HD broadcast do they actually broadcast an ISU game in HD?
thats the only question i care about
Hooray for Metaphors!
I'm sure the "great" minds who run the Big XII will just start up a network for the South schools.
"The Constituiton...is a mere thing of wax in the hands of the judiciary, which they may twist and shape into any form they please."- - -Thomas Jefferson, Sept. 6, 1819.
how long before Mediacom gets the big 12 network? took 2 years to get the big 10.
Looks like they are using camera phones right now!
"Explode and tear this whole town apart
Take a knife and cut this pain from my heartFind somebody itching for something to start"
"The Promised Land" Bruce Springsteen
Originally Posted by ajk4st8
How long before the Big 12 Network for mens basketball goes HD?
Any guesses?
The Big 12 Network is just the name for ESPN+.
There's no actual Big 12 Network like the Big 10 Network.
Originally Posted by CrossCyed
The Big 12 Network is just the name for ESPN+.
There's no actual Big 12 Network like the Big 10 Network.
They said throughout the day that it was the inaugural weekend of the Big 12 Network, so I think it is something different than has been there the previous years; but you're correct, it is nothing like what the Big 10 has.
Originally Posted by Drive4cy
Well, great...the year the world ends. Figures.
Originally Posted by mustangcy
Well, great...the year the world ends. Figures.
Not till Dec 21 on Mayan calendar (12/21/12) and the 6000 year Jewish calendar and the 7000 year Mormon calendar.
Last edited by Wesley; 01-10-2009 at 07:03 PM.
Let Prohm's Posse Ride: Georges, Georgios, 3sus, Hallice, Deonte, Abdel, Monte, Matt, and McKaT.
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Stars Photos
Photos: Lane MacDermid throws punches; Brenden Dillon all smiles; Stars too much for Flames
Photo: Tom Fox/Staff Photographer
Dallas Stars center Cody Eakin (20) keeps the puck from Calgary Flames left wing Curtis Glencross (20) in the second period.
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The band turns it up for a return to Sacramento Institution Old Ironsides tonight. In April, we hit the hills of El Dorado for our debut at Kinnee O'Reilly's megapub. May sees the band open for legendary Real McKenzies at Sacramento Uberclub Ace of Spades. Then it's on to RENO June 1 and 2 for our 3rd year in a row gigging after Young Dubliners at Reno Sands' Eurofest. Plus, there are a couple little local gigs TBA. Keep your eyes and ears open.


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Originally posted by WarriorOfLight96
reply to post by wrkn4livn
Most of the orbs people catch are nothing more than dust or a fly flying around, but I did see a little light in the dark one time and didn't come from a light source and it had an orange glow but in the middle of the room and than faded. Yes if you open communication than you open the door and it`s what you let out that follows you and will bring an unbalance in your life and with it chaos.
Originally posted by VegHead
reply to post by wrkn4livn
Interesting questions. I don't know anything about #1. I don't understand 'the other side' at all, which is why I personally will never do ghost investigations or conduct EVP sessions. I don't want to open the door to something that I don't understand. Ok, yeah... I'm chicken. That's another way to put it.
With #2, your thought process with the idea of dust getting kicked up is just what I was thinking. But unless your guests are literally showing up with a lot of travel dust on their clothes, it doesn't seem to fully account for the big increase in orbs with their arrival. I assume you clean your house pretty well before company comes over, so one would think there would be dust kicked up (if there was going to be) in the "before" pictures you took. You've done a good job of trying to debunk them yourself.
Would you be open to sharing a few orb pictures? You can block out any family member faces to try to keep it anonymous.


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Their self-proclaimed title of "Ambassador's of Health - Affordably"', sums up their goals. However, the roots of this family operation reach deeper, as they've expanded the operation nurtured by this family's planning and dedication.
Leading the way in promoting natural, organic and 'raw' food sources, the entire Hamilton family is devoted to providing affordable, wholesome produce locally. Careful attention is paid, in every step of growing fruits and vegetables on their farm, to insure purity, flavor and vigor in the their crops. From the selection of organic seeds, and absolute avoidance of pesticides and chemicals, to conservation of the soil, care is taken in every step to harvest the most healthy and sustainable produce, Supporting local 'community oriented' farms not only helps protect our environment, by reducing greenhouse gases and carbon footprints for food delivery, but the food is fresher, healthier, and better tasting.
Now this incredible food source is available for delivery in Fresno and it's surrounding areas. Whole Farms Inc, of central California, is expanding their Organic Food Delivery service in within the local communities of Fresno, Madera, Clovis and Kerman. Their goal is to provide delicious affordable, delicious produce, conveniently delivered to you. Recently Whole Farms has formed a co-operative with local farmers, to deliver a wider variety of wholesome nutrition including local honey, raw milk, nuts and more.
While not off 'manning the farm' with family members, Ariana can be found at the family's newly opened 'Revive' Cafe, in Fresno's historic Fulton Mall. The cafe focuses on natural and vegan foods. Everything is prepared with organically grown and all-natural ingredients. The Revive Cafe offers a wonderfully tasty assortment of juices, smoothies, appetizers, entrees and deserts.
Do you know your local farmer? Whole Farms are eager to meet new customers & discuss health foods, and natural products. Feel free to drop by the Revive Cafe to chat with Ariana, or contact them (at http://www.OrganicFoodsDelivery.com) if you're interested in the delivery of whole foods and natural products. They have an abundance of information on healthy dieting, and ecological living to share.
# # #
Whole Farms Inc
Ambassador's of Health - Affordably
Address: 1807 Broadway Fresno, California
Telephone: 559-284-4803
Email: email@example.com
Follow us on www.Facebook.com/


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Press Release Distribution
March 26, 2012 News
3 years ago, 10515 views, By GingerNewYork-TV
"In the world of music there are those that play the drums and there are artists that earned the title percussionist...Dan Britt being the latter." -Ginger Broderick Host/Executive Producer GingerNewYork-TV
3 years ago, 10097 views, By Ranjan Sahu
Do you want to track a cell phone number but don't know how to do it? Without the right tools, it's near impossible to find who a cell phone number belongs to, because most of them aren't listed in public directories.
3 years ago, 8018 views, By Owner
Check it out folks, no hype.. no salesman crud about some shotty program that ultimately makes you lose money.. I am talking about earning a full time income from two hours of work in your spare time.
3 years ago, 5690 views, By Howard E. Haller, Ph.D.-Intrapreneurship Institute
Intrapreneurship Institute Case Study by Howard Edward Haller, Ph.D. A classic example of a successful Intrapreneurship program which created many new 3M products. This is the Story of 3M's profitable product line "Post-it Notes" (TM).
3 years ago, 4366 views, By Jimmy Swaggart Ministries
Join Jimmy Swaggart Ministries for Easter Campmeeting - April 4-8, 2012 in Baton Rouge, Louisiana. Come and experience the difference!
3 years ago, 4020 views, By C. Harrell
With Reebok, you can get into shape for the summer. Make good on those New Years resolution with the REEBOK TrainTone Slimm Athletic shoe in Black/Silver.
3 years ago, 3800 views, By Courtney Scott
Griffin cases for the Apple iPad and iPhone are the case that I personally use. Whereas I don't own an iPhone, I do have an iPad as well as a 11 month old and 6 year old. My Griffin Survivor case has been safe-guarding my iPad 2 ever since Christmas.
3 years ago, 3514 views, By Vision For Equality
PA Representatives Gene DiGirolamo and Thomas Murt will be joining SAFA (Self Advocates and Family Alliance) for their Press Conference on March 29, 2012 at 9:00am in the Capitol Rotunda in Harrisburg, PA.
3 years ago, 3332 views, By Athena Marketing
Lothar C. Birkenfeld continues his career in the kitchen and bath cabinetry manufacturing industry as the President and CEO of Alno USA Corporation, a subsidiary of Alno AG.
3 years ago, 3037 views, By Ralph Lendez
YHVH Entertainment officially began the pre-production process of the play Burley Man in Los Angeles, CA.
3 years ago, 3012 views, By Ella Bee Media Group
Loyalty Jewelry will join Shecky's Girls Night Out to offer shoppers the latest from its 2012 spring and summer collection.
3 years ago, 2772 views, By East Africa Business Summit
The Innaugural East Africa Business Summit and Expo SA, aimed at highlighting the investment opportunities in East African region will be held at the Hilton Hotel, Sandton on the 20th July 2012 with over 300 business people and potential investors
3 years ago, 2711 views, By Barbara Bogo
Oldcastle Precast provided structural precast concrete building components for the new development at Forest Houses in New York. It is part of the largest municipal housing plan in the nation
3 years ago, 2707 views, By Bob Hawkinson
Hawkinson Nissan wins Nissan 1st Award of Excellence for 2012!
3 years ago, 2667 views, By Darren Spencer
Tulum, the sacred Mayan ruin which is perfectly located on the cliff overlooking the turquoise waters of the Caribbean Sea.
3 years ago, 2487 views, By Mark Wilson
New housing initiative partnership Southeastern Housing Foundation inc, announces partnership with Peabody Chase NY llc
3 years ago, 2432 views, By Monica Williams
American Idol star, Melinda Doolittle to embrace the cover of the May 2012 Issue of Social Diary Daily magazine that will benefit a health charity for kids.
3 years ago, 2410 views, By Dennis Nazelli
Detroit beauty salon and day spa is making eyes more beautiful and alluring with semi-permanent eyelash extensions.
3 years ago, 2389 views, By Eaton Smart Hong Kong
In the "Sustainable Meeting Package" of Eaton Smart Hong Kong, guests are encouraged to dine Sustainably and Fairly
3 years ago, 2388 views, By Innovative Medical Products
Single-patient-use, foam support is guaranteed sterile, helping reduce Healthcare Associated Infections (HAI) at the surgical site
3 years ago, 2384 views, By Jonathan Confeld
Young entrepreneurs Brenton Hayden (CEO & Founder of Renters Warehouse) and Ryan Marvin (Co-Owner of Renters Warehouse & part of the Marvin Window & Door family) purchased the brand & select assets such as trademarks, websites, & proprietary software
3 years ago, 2310 views, By Atul Malikram
The 2012 edition of extraaa innings T20 will once again see the 'fiery 4' anchors who dazzled the show in the last season led by the veterans Samir Kochhar and Gaurav Kapur coupled with the vivacious duo, Shibani Dandekar and Archana Vijaya.
3 years ago, 2307 views, By SGS Consumer Testing Services
A revised European standard EN 1930:2011 was published by the European Committee for Standardization (CEN) in December 2011.
3 years ago, 2290 views, By PJMC Limited
New consultancy service to optimise new or existing installations of interaction recording and quality management solutions to meet business requirements, reduce costs and deliver an improved customer service.
3 years ago, 2270 views, By Vigorous PR
Rachel'z Supports Tuscaloosa Native, Iris Hill's Appearance on NBC's 'Escape Routes'
3 years ago, 2244 views, By Danielle Aychouh
TNT Express Middle East has further strengthened its commercial and business development portfolio with the appointment of Sanae Murschel as the new commercial and business solutions director for the Middle East.
3 years ago, 2231 views, By Genietrack
Mobile family safety service that helps parents keep an eye on their kids without unnecessary calling or texting
3 years ago, 2180 views, By Sara Pierre - Communication Manager
Stimulated Emission Depletion Microscopy (or STED nanoscopy) offers a technological breakthrough in the environment of cell imaging by drastically increasing the resolution over traditional confocal microscopy.
3 years ago, 2168 views, By Inside2Outside Ltd
The Pastures Primary School in Narborough, Leicestershire recently installed one of the countries first thin-film solar tensile PV canopies. Designed to provide considerable energy savings whilst maximising usable outdoor space for schools.
Rare, masterfully carved rhinoceros horn libation cup hits $318,600 at Elite Decorative Arts auction
3 years ago, 2145 views, By Ken Hall
A rare, masterfully carved Chinese rhinoceros horn libation cup from the 17th or 18th century, just four inches tall and fitted to a reticulated teakwood base, sold for $318,600 at a sale of Asian antiques held March 17-18 by Elite Decorative Arts.
3 years ago, 2142 views, By Donald MacNamara
The most experienced home loan modification expert in the United States, Michael Fisher, has just released The Complete Loan Modification System, a step by step guide that virtually assures the success of your mortgage modification.
3 years ago, 2141 views, By LookeeTV, Inc.
LookeeTV Launches HD Internet TV Streaming Box to Extend Product Family - Award-winning LookeeTV Internet TV & Radio Players Available in HD Streaming Set Top Box Model
3 years ago, 2103 views, By M. Petrillo
Baskets-n-Beyond™ , located in Dumont, NJ, partners with several Bergen County Synagogues to offer Bimah Baskets to their members having Bar/Bat Mitzvah's. Baskets-n-Beyond™ first introduced Bimah Baskets to the Bergen County area in 1993.
3 years ago, 2082 views, By NETIA
NETIA, a leading software manufacturer of media asset management and radio automation solutions, today announced significant enhancements to its Radio-AssistTM 8 range of digital audio automation software.
3 years ago, 2065 views, By Jerry Milani
Successful entrepreneur John Macaluso has been elevated to CEO of Wizard World, Inc., and will lead one of North America's most diverse pop culture events brands.
3 years ago, 2038 views, By NLM Promotions
While 2011 was a very tough year for Brian Loftin and his #23 NASCAR Whelen Southern Modified Tour Race team, that is all behind them now and the entire team has a renewed spirit. The team is ready to win races and more importantly championships!
3 years ago, 2037 views, By KuicK Research
Rising investment from public and private sector entities along with government focus on market liberalization to drive power sector installed capacity.
3 years ago, 1951 views, By Polished For Prom
Polished For Prom is partnering with College is Cool to help seniors enjoy the prom of a lifetime through their 2012 prom sponsorships.
3 years ago, 1951 views, By Kevin Wong
Asian Fashion and Asian dress has been talked about a lot nowadays due to the great designs and fashion trends which are being introduced by the Asian designers. Today when we see the designs and trends which has been brought about by these Asian...
3 years ago, 1945 views, By Primal Youth
Primal Battle of the Bands is the bands competition of the year, and we're back in 2012 with a bang!
3 years ago, 1943 views, By Stereolabs
New products include mobile analyzer, multi-camera controller and 3D content verification software
3 years ago, 1921 views, By Jeff Geoghan
Central Pennsylvania Real Estate Company's Record Growth Honored by National Brand
3 years ago, 1919 views, By Blueprint LSAT Preparation
Premier LSAT company Blueprint LSAT Preparation expands to six new locations: Atlanta, Chicago, Dallas, Houston, Miami, and Seattle. Spring deal of $500 off and a 5 point practice exam guarantee! (restrictions apply).*
3 years ago, 1906 views, By Joe Moore CEO
In the aftermath of Google's changes to the algorithms used to index websites, business owners have seen their rankings drop and revenues decrease.
3 years ago, 1901 views, By Jetset Magazine
This jewelry is a little bit glam, a little bit rock 'n' roll. There's a lot to be said for the style impact demure femininity can make. But then again, isn't it fun to let your rocker side show through every once in a while, too?
3 years ago, 1893 views, By Melon Communications
Leamington Spa, United Kingdom. 16 March 2012. Leading IT security specialists, the Nettitude Group (www.nettitude.com) announce that they have become an ISO 14001 company.
3 years ago, 1876 views, By Glamour Destination Management
Their love for the community the environment and the work that they do are the binding agents that brought Jamaica's newest Eco- Tourism attraction and project, Glamour Destination Management together.
3 years ago, 1867 views, By Jothy Hughes- N O W! Communications
Everyday people are now able to build prosthetic hands and give a child the opportunity to grasp or clutch an object, after they have lost their hand in one of the 100 million active landmines, globally.
3 years ago, 1838 views, By Jesus Perez
Elemental Gold has signed option agreements with Dirae Exploration for the exploration of copper, gold and molybdenum porphyry projects in the Yauri mineral field located in south Peru.
3 years ago, 1816 views, By Michelle Silva
He reiterates the confidence of international capital markets in the UAE


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Photos: Being a Cowboys Cheerleader ... it's more than pom poms and hot pants
Photo: G.J. McCarthy / Staff Photographer
Focused on the routine during an October 2010 game against Tennessee.
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The Magazine
Mugged by Reality
Albert Shanker and the fall of American liberalism.
Sep 10, 2007, Vol. 12, No. 48 • By FRED SIEGEL
Kahlenberg, himself a thoughtful social democrat, best known for arguing on behalf of income-based affirmative action, wants to revive the "tough liberalism" once represented by Shanker. But this is unlikely. Social democracy, with its admirable solidarity-based instincts, was a product of the relatively closed economies that defined advanced societies between the end of the first period of globalization and the onset of the second. But as power has shifted, to some degree, from national governments to world markets, social democracy has languished, even in its European homeland. It's doubtful that we will see Albert Shanker's like again.
Fred Siegel, professor of history at Cooper Union, is the author, most recently, of Prince of the City: Giuliani, New York and the Genius of American Life.


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Mio ALPHA 2
Mio FUSE
Mio LINK
The holidays are notable time to back off from structured training and forgo working out. Maintaining and not gaining weight over the holidays can be tricky. The fire that once stoked the metabolic flame making you a lean, mean, calorie-burning machine, dims and the body shifts to energy storage.
While it's natural for the body to swing in winter hibernation mode, we also don't have to be like a squirrel storing all of its nuts during holiday festivities. And you certainly shouldn't go "starving in the dessert" either, to coin a phrase used by Registered Dietitian, Jae Berman, just to avoid the pleasures of holiday feasts.
Be the strategist, not the survivor with these strategies to prevent weight gain this Thaksgviking.
Strategy #1
Plan your wardrobe in advance. Wear an outfit that fits comfortably and that isn't too loose (you should still fit into it after dinner). Avoid wearing anything that is too tight, as well. Let ...
The post How to Prevent Weight Gain Over Thanksgiving appeared first on Mio Global.
Let's face it – injury happens and in the worst form: stress fractures, broken collarbones, muscle strains, ligament sprains, and even skin wounds, leaving the athlete sidelined for days to weeks, or even months. Left behind are the hopes and aspirations for a fruitful season.
The number one goal for any professional or amateur athlete when rehabbing an injury is optimizing the healing process for a speedy "return to play". Caring for an injury typically involves two processes: immobilization and rehabilitation. This is where nutrition plays an integral role in the healing process.
The first defensive strategy is Immobilizing the injured or inflamed site (i.e., foot in a boot or arm in a sling) and to reducing the risk of infection to open wounds (i.e., refraining from swimming in open water or in the pool). Depending on the severity of the injury, an athlete's ability to train is compromised. The reduction of ...
The post 6 Things Every Athlete Should Know About Nutrition for Injuries appeared first on Mio Global.
As a runner, one of the most difficult challenges we face during a race is our ability to maintain a steady pace from start to finish. We can usually start out strong but will often fade in the final miles. I have seen a handful of motivation posters over the years that all infer a similar message — the race is won by the runner who slows down the least. The key to unlocking race success can be summarized in three main concepts: 1) building the effort during long workouts, 2) proper pacing, and 3) creating mental toughness. Let's take a closer look at each of these and how we can incorporate them into specific workouts to help us build run endurance and avoid fading in our next big race.
Build the Intensity of Your Long Run
Intensity can be measured by pace ...
The post How to Avoid the Fade & Boost Your Run Endurance appeared first on Mio Global.
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We are Mio Athletes, performance athletes with an obsessive attitude toward fitness and precision training. Our training may differ, but our bond runs deep beneath the surface–straight to the heart. We train to master our performance. These are our stories.
Word on The Streets


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I don't know about you guys, but when we travel every inch counts! With 4 kids 10 and under space fills up quick, so something like the Keeper Waterproof Roof Top Cargo Bag is perfect in helping free up much needed space in our van. Plus, this product has an average of 4.5 stars out of 502 customer reviews! (we might just have to grab one of these ourselves )
Right now this is marked down 42% off retail to $43.99! (Ret. $76.39) Select super-saver shipping to get this item shipped free or get free 2-day shipping with Amazon Prime.
For the size of this cargo bag (15 cubic feet) along with the quality and price, I don't think you can go wrong with this option.
Click to see this deal on Amazon!
Here are some of the product's features:
- Waterproof rooftop cargo bag with 15 cubic feet of space
- Increases vehicle cargo space
- Waterproof design protects cargo against road grit, sun, wind and rain
- Soft sides flex to fit odd-shaped loads
- Folds compact for easy storage
NOTE: The prices on Amazon can vary from one minute to the next. I can make no guarantees that the price will be the same as the one above when you click on the Amazon link. Let me know in the comments section below if the price changes and I will get the post updated as soon as possible.
More great Amazon Deals!


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- Presentation
- edu_shrew2012_activities_pack.pdf
- (1 MB, Adobe Acrobat)
The Taming of the Shrew 2012 Teacher Pack
The activities in this pack are inspired by The Taming of the Shrew by William Shakespeare. They can be used either as stand-alone practical approaches to this play or as supporting activities for students seeing the production. They have been designed with KS3 to KS5 students in mind, but can be adapted for other age groups. Some are best suited to an open space such as a hall, but many can be used in a classroom.
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1 Review
Many thanks


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A man laughing (me, actually)
My laugh, recorded in home.
Me pressing the space bar
Pressing spacebar on my Logitech K120 Recorded using Zoom H1 You are free to use it anywhere and mix it ...
Typing or keyboarding on a computer keyboard.
What I imagine a heart failure might sound like. At least in a drama. editing and mixing of sounds under ...
Jim, according to technology, this person is dead. 1200Hz Sine made in Audacity. On FLAC and OGG Vorbis audio file ...
HP Printer making some noises.
my printer printing
My printer. Recorded with a Shure SM58 microphone and Focusrite Saffire 6 USB audio interface.
Office fax-printer beeps. HP LaserJet 1536dnf MFP. Loud background noise is ventilation. XY-Stereo
My printer doing some printing.
The obligatory printer sample. Fairly modern. Recorded with a Zoom H2.
A cymbal roll created using my Technics SX-KN720. Great with a long reverb.
Orchestral Effect ArmsProducts.tk


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IUCN in West Africa- Challenges and Opportunities
Refereed StatusNon-Refereed
MetadataShow full item record
The West African region covered by IUCN-BRAO comprises 17 countries: Cape Verde, Senegal, Mauritania, the Gambia, Guinea, Guinea Bissau, Mali, Sierra Leone, Liberia, Côted'Ivoire, Ghana, Burkina Faso, Niger, Togo, Benin, Nigeria, and Chad. It extends over a surface area of 7,500,000 sq. km with a population of nearly 250 million inhabitants, or an average density of 33 inhabitants per sq. km. With the exception of Chad and Mauritania (members of the Permanent Inter-States Committee for Drought Control in the Sahel1), IUCN-BRAO's target region corresponds to the ECOWAS (Economic Community of West African States) space. Eight of the fifteen ECOWAS member states --the CFA franc zone-- form an economic and monetary space: the West African Economic and Monetary Union (UEMOA)......
Document Languageen
Spatial CoverageWest African region
- Miscellaneous [667]


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A growing body of evidence has shown that Krüppel-like transcription factors play a crucial role in maintaining embryonic stem cell (ESC) pluripotency and in governing ESC fate decisions. Krüppel-like factor 5 (Klf5) appears to play a critical role in these processes, but detailed knowledge of the molecular mechanisms of this function is still not completely addressed.
By combining genome-wide chromatin immunoprecipitation and microarray analysis, we have identified 161 putative primary targets of Klf5 in ESCs. We address three main points: (1) the relevance of the pathways governed by Klf5, demonstrating that suppression or constitutive expression of single Klf5 targets robustly affect the ESC undifferentiated phenotype; (2) the specificity of Klf5 compared to factors belonging to the same family, demonstrating that many Klf5 targets are not regulated by Klf2 and Klf4; and (3) the specificity of Klf5 function in ESCs, demonstrated by the significant differences between Klf5 targets in ESCs compared to adult cells, such as keratinocytes.
Taken together, these results, through the definition of a detailed list of Klf5 transcriptional targets in mouse ESCs, support the important and specific functional role of Klf5 in the maintenance of the undifferentiated ESC phenotype.
Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation embryos; they are amenable to manipulation, enrichment and expansion and retain the developmental potency of embryonic founder cells, being able to differentiate into cells and tissues of all three germ layers both in vitro and in vivo [1,2]. For these characteristics, ESCs represent an invaluable source of different cell types, thus opening up new possibilities for cell therapy. The understanding of the transcriptional regulatory networks that operate in ESCs is fundamental to unravel the molecular basis of pluripotency, self-renewal and reprogramming. The complexity of this regulatory network was highlighted by the identification of hundreds of genes, targets of the ESC master genes Oct3/4, Nanog and Sox2 [3,4]. In addition to these master regulatory factors, several other transcription factors play important roles in the control of this regulatory network [5-9], such as Krüppel-like factors (Klfs).
Klfs belong to the Sp1 family of transcription factors with over 20 members [10,11]. Three members of this family (Klf2, Klf4 and Klf5) are expressed in undifferentiated mouse ESCs and downregulated during early stages of differentiation [12]. Klf4 is one of the original "Yamanaka factors" required to reprogram somatic cells to a pluripotent state [13], and Klf2 and Klf5 are able to substitute for Klf4 function in the reprogramming, including cocktail [14]. It has been proposed that Klf2, Klf4 and Klf5 have redundant functions because the impairment in the ESC undifferentiated state was only observed following knockdown (KD) of all these three genes [15]. Nevertheless, there is evidence that Klf5 has unique functions: We have recently shown that KD of Klf5 induces ESC differentiation, whereas its ectopic expression is able to maintain ESC pluripotency in absence of leukemia inhibitory factor (LIF) [16] and Klf5-knockout (KO) mice show developmental defects at the blastocyst stage [17], thus suggesting a specific requirement of this Klf in early embryogenesis and in turn a high hierarchical role in the transcriptional network. Opposite to other transcription factors regulating ESC pluripotency and the first step of embryo development, Klfs are also expressed in adult terminally differentiated cells [18]. Klf5 is expressed in skin, intestinal crypts, stomach, lung, testis, uterus, heart and kidney [19-21]. We systematically explored Klf5-specific targets in mouse ESCs by matching gene expression profiling with chromatin immunoprecipitation coupled with parallel short tag-based sequencing (ChIP-seq) that identifies all Klf5 genome binding sites. Here we report that Klf5 regulates at least 313 genes functioning as a repressor or activator. A total of 161 of 313 genes bear at least one binding site for Klf5 within 100 kb from the gene boundaries, indicating that they can be considered Klf5 primary targets. We first demonstrated that suppression or ectopic expression of some Klf5 targets strongly affects the ESC undifferentiated state, thus further supporting a high hierarchical role of this transcription factor in ESCs. Then we addressed the specificity of Klf5 function in ESCs by demonstrating that Klf2 and Klf4 have nonredundant roles and that Klf5 targets in ESCs are different from the gene targets of this transcription factor in adult differentiated cells.
Identification of Klf5 direct targets
To identify genes directly regulated by Klf5, we first analyzed the transcriptome changes upon Klf5 KD in ESCs by microarray analysis. To minimize indirect effects due to phenotypic changes induced by Klf5 KD, we performed a time course analysis both by qPCR and by Western blot analysis, which showed that Klf5 expression is significantly decreased already 12 hr after siRNA transfection (Figure 1a). On the contrary, the expression of stemness genes such as Oct3/4 and Nanog, previously demonstrated to be modified by Klf5 KD [16], is still unaffected at 12 hr (Figure 1b). Therefore, it is possible to capture early effects on transcriptome following Klf5 KD at 12 hr after transfection.
Figure 1. Identification of Klf5 targets by microarray analysis. (a) qPCR and western blot analysis of Klf5 expression following transfection of siKlf5 and siNS at different time points (12, 24 and 48 hr after siRNA transfection). (b) Expression level of stemness markers was measured 12 and 24 hr after transfection by qPCR and Western blot analysis. All qPCR data are expressed as fold changes relative to mock-transfected cells. Error bars represent standard errors (SD) of triplicates. *P < 0.01. (c and d) Expression of 53 Klf5-target genes measured by real-time PCR in undifferentiated and 4-day (4 d) and 7-day (7 d) differentiated ESCs. Both Klf5-induced genes (c) and Klf5-repressed genes (d) are reported. The results are reported as fold changes relative to undifferentiated cells.
Statistically significant probes (FDR < 0.1) were classified as upregulated and downregulated following Klf5 KD by using as cut-off > 1.25- and < 0.75-fold changes, respectively. On the basis of these constraints, we identified 239 upregulated and 74 downregulated genes (Additional file 1).
Additional file 1. Additional Table 1. Gene expression profile by microarray analysis upon Klf5 KD in ESCs. Fold change is calculated by comparing data from siKlf5 cells over the control (siNS). Cut-off >1.25- and <0.75-fold changes were used. Probes with FDR <0.1 were selected.
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These predicted genes encode a set of proteins involved mainly in development and differentiation (Additional file 2), in agreement with the Klf5 role in ESCs and embryonic development [16,17]. Interestingly, 52 genes controlled by Klf5 encode regulators of transcription, indicating that Klf5 regulates many transcription factors, which in turn regulate their downstream targets. Moreover, among the genes regulated by Klf5, there are factors that have already demonstrated a role in ESC pluripotency, such as Tcl1 [8,22], BMP4 [23], Nr0b1 [24] and CD9 [25]. We also compared our results with available data [15,17,26,27], and we found that 86 of 96 transcripts showed the same behavior upon Klf5 KD and upon triple KD of Klf2, Klf4 and Klf5 [15] (Additional file 3). Moreover, we found that 75% of the transcripts showed the same trend in our analysis and in Klf4 KD cells [26] (Additional file 3). Instead, no significant correlation was found when our data were compared with the data of LIF deprivation or expression of STAT3 dominant-negative form [27] (Additional file 3).
Additional file 2. Additional Figure 1. GO analysis of Klf5 targets and Klf expression profile during ESC differentiation. (a) Gene ontology (GO) annotation of the selected probe sets according to DAVID "Biological Process Classification" tool (Laboratory of Immunopathogenesis and Bioinformatics, Clinical Services Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD, USA). (b) Expression levels of Klf2, Klf4 and Klf5 were measured by qPCR in undifferentiated (t0) and 4-day (4d) and 7-day (7d) differentiated ESCs. The data are represented as fold changes relative to undifferentiated cells.
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Additional file 3. Additional Table 2. Microarray data were compared with published results. Column siKlf5/siNS is referred to our microarray data (see also Additional Table 1). Differences with published data are highlighted in red. dw: downregulated gene, up: upregulated gene; na: data not available.
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It is reasonable to assume that Klf5-regulated genes, identified by gene expression profiling, include both direct and indirect transcriptional targets. To distinguish between direct target genes and those indirectly regulated through alterations in transcriptional networks governed by Klf5, we employed genome-wide chromatin immunoprecipitation (ChIP). We introduced a FLAG-tagged Klf5 in ESCs and generated two independent pools of stable clones. The levels of Klf5 were assessed by Western blot analysis with anti-Klf5 and anti-FLAG antibody to detect both endogenous and exogenous Klf5 (Additional file 4). Chromatin from these cells was immunoprecipitated with anti-FLAG antibody, and purified DNA fragments were analyzed by direct high-throughput sequencing. We identified 6480 putative binding sites, and among these 5820 had an FDR lower than 5% (Additional file 5). ChIP-qPCR validations were carried out on 15 Klf5-binding sites with different numbers of ChIP tag counts within the defined overlap region (Additional file 4). Twelve of 15 target loci subjected to validation showed a significant enrichment over three independent control regions (Additional file 4).
Additional file 4. Additional Figure 2. ChIP-seq validation by ChIP-qPCR. (a) Expression level of FLAG-Klf5 stable clone pools used to prepare chromatin for ChIP-seq experiment. Western blot was stained with an anti-FLAG and anti-Klf5. (b) List of peaks validated by ChIP-qPCR. Peaks with different numbers of tags were chosen. Peak location are indicated (chr, chromosome). (c) ChIP-seq validation was performed by ChIP-qPCR using anti-FLAG antibody and IgG, as control, with extracts derived from FLAG-Klf5 and Mock transfected ESCs. The data are expressed as the amount of precipitated DNA calculated relative to the total input chromatin. Samples from 1 to 15 correspond to regions close to the following genes: Agap1, Lamc2, Fcgr3, 170009P17Rik, Tgfβ2, Smx16, Nlgn1, Epha2 (upstream region), Epha2 (downstream region), Igfbp7, Serpine1, Cyp2s1, 4930467E23Rik, AC152164, Inpp4b, respectively. Three different control regions were chosen (samples 16, 17 and 18): chr1:10573933-10573984, chr1:71481391-71481461, chr3:12034661-12034625, respectively, where no significant peaks were found. Bars represent SD of triplicates.
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Additional file 5. Additional Table 3. Results of ChIP-seq analysis. The putative binding sites of Klf5 are reported with relative number of tags for each peak and FDR. Match with gene microarray data is shown. The distance from the 5' and 3' boundaries of the Klf5-regulated genes is indicated.
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Next, we analyzed Klf5-ChIP target sequences with the motif-finding CisFinder software to search for over-represented motifs. The three best results were represented by CG-rich elements (Additional file 6), in agreement with previous reports [28]. Moreover, all three motifs contained the CTGC sequence, suggesting a putative binding site for Klf5.
Additional file 6. Additional Figure 3. Klf5 binding motifs identified with CisFinder via 200-bp sequences centered at binding peaks (E-score > 22).
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To identify primary targets of Klf5 among the genes regulated upon Klf5 KD, we matched microarray and ChIP-seq data, and we found that the relationship between target promoter occupancy and gene expression analysis is excellent. In fact, we found 161 of 313 genes whose expression is regulated by Klf5, showing a Klf5 binding site within 100 kb from the gene boundaries (Table 1, Additional file 5). To validate microarray data among these 161 Klf5 putative primary targets, we selected 60 genes where Klf5 binding sites are at various distances from gene boundaries. qPCR analysis of these genes performed upon Klf5 KD revealed that 53 (>88%) of 60 showed the same trend observed in microarray analysis (Additional file 7).
Table 1. List of Klf5-target genes obtained by matching microarray and ChIP-seq data. Klf5-repressed and activated genes bearing a binding site for Klf5 within 100 kb from gene boundaries are reported.
Additional file 7. Additional Figure 4. qPCR validation of microarray data. Sixty Klf5 target genes were analyzed by qPCR to confirm the microarray data. Probe set of both downregulated (a) and upregulated (b) genes upon Klf5 KD is shown. Black bars represent not validated probes. The data are expressed as fold change relative to siNS transfected cells. Validated probes showed a P < 0.01.
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To gain insights into the primary targets of Klf5, we analyzed the expression pattern of the 53 validated genes (Figures 1c and 1d), during neural differentiation of ESCs. Klf5, such as the other two ESC-specific Klfs, is highly expressed in undifferentiated ESCs, and its expression dramatically decreases when differentiation occurs (Additional file 2). We reasoned that genes activated by Klf5 are expected to show the same expression profile of Klf5, i.e., expressed in undifferentiated ESCs and downregulated when differentiation occurs. On the contrary, the genes that are negatively controlled by Klf5 are expected to show an opposite expression profile, i.e., not expressed in undifferentiated ESCs and upregulated when differentiation occurs. As expected, almost all the genes activated by Klf5 are expressed in ESCs and repressed during differentiation (Figure 1). On the other hand, almost all the genes that are inactive or repressed in ESCs, but are expressed during differentiation, appear to be negatively regulated by Klf5 (Figure 1d).
Suppression of Klf5 target genes impairs ESC undifferentiated state
The first point we asked is that of the relevance of Klf5 direct targets in the maintenance of ESC phenotype. Among the putative Klf5 direct targets, we decided to start by analyzing 23 genes that were selected on the basis of validation by qPCR (Additional file 7) and their expression profile during ESC differentiation (Figure 1). To this aim, we selected pools of stably transfected ESC clones, where the expression of these 23 genes was knocked down by specific shRNAs. A reduction of at least 20% in transcription levels was observed after selection (Additional file 8). The stably transfected cells were plated at clonal density and further grown for 7 days. The resulting colonies were stained for alkaline phosphatase (AP) activity (Figure 2a) and the percentage of undifferentiated and differentiated colonies was calculated. KD of Klf5 (positive control) resulted in at least 40% decrease of AP-positive colonies compared to control shRNA transfected cells (Figure 2b). Knockdown of 8 of 23 target genes analyzed resulted in a significant decrease of AP-positive colonies (Figure 2b). To confirm that these decreases in the number of AP-positive colonies were due to an impairment of ESC undifferentiated state, we measured the level of stemness markers in cells KD for these eight genes soon after selection. Expression of both Oct3/4 and Nanog was significantly reduced compared to control after knockdown of all these genes (Figure 2c). The observed decrease in Oct3/4 and Nanog expression corresponded to a significant increase of early differentiation markers (Additional file 9). These results were confirmed by further independent shRNAs (Additional file 8). Taken together, these results suggest that Klf5 controls the expression of at least 8 of 23 genes required to maintain the undifferentiated state of ESCs.
Additional file 8. Additional Figure 5. KD of a subset of Klf5-target genes. (a) ESCs were stably transfected with shRNA plasmids for selected Klf5-target genes or with control shRNA (shGFP) and KD was verified by qPCR. The results are represented as fold changes relative to shGFP-transfected cells. SD of triplicates is reported. (b) Percentage of undifferentiated (blue) and differentiated (red) colonies observed by AP staining upon KD of eight Klf5-target genes with a second independent shRNA. *P < 0.01. (c) Expression levels of Oct3/4 and Nanog upon KD of eight Klf5-target genes with a second independent shRNA. The data are represented as fold changes relative to shGFP-transfected cells.
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Figure 2. Effects of KD of Klf5-target genes in ESCs. (a) ESCs stably transfected with shRNA plasmids were grown for 7 days at clonal density (50-100 cells/cm2) and then stained for alkaline phosphatase (AP) activity. The results of shGFP-transfected cells (negative control) and shHck-transfected cells are shown as examples. Scale bars, 1 cm. (b) Histogram showing the percentages of undifferentiated (blue) and differentiated (red) colonies observed by AP staining after knockdown of Klf5 response genes. shKlf5-transfected cells are the positive control, and shGFP-transfected cells are the negative control. Bars represent SD of triplicates. *P < 0.01. (c) qPCR analysis of stemness markers after KD of Klf5 target genes that induced a significant decrease in AP-positive colonies. Efemp1 KD cells were used as negative controls. The data are represented as fold changes relative to shGFP-transfected cells. Error bars represent SD of triplicates. Differences are significant with P < 0.01.
Additional file 9. Additional Figure 6. Expression of early differentiation markers of endoderm (Sox17), mesoderm (Brachyury) and ectoderm (Fgf5) upon KD of eight Klf5-target genes.
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Ectopic expression of Serpine1 impairs ESC undifferentiated state
As shown above, we found many genes whose expression in ESCs seems to be negatively regulated by Klf5. Two of them, Serpine1 and Runx1, were selected for functional studies. Their Klf5-dependent regulation is confirmed by the observation that they are expressed at very low levels in undifferentiated ESCs. Furthermore, these genes that seem to be negatively regulated by Klf5 in undifferentiated ESCs showed a strong induction upon ESC differentiation when Klf5 disappeared (Figure 1d) [16]. Thus, we investigated their effect on stemness by forced expression in undifferentiated ESCs. We transfected ESCs with expression vectors bearing FLAG-tagged Serpine1 or Runx1 (Figure 3a); these cells, grown at clonal density for 7 days, were stained to assay AP activity. Alkaline phosphatase staining showed that forced expression of FLAG-Serpine1 is able to induce a significant decrease in the number of undifferentiated colonies, whereas FLAG-Runx1-transfected cells did not show a significant decrease in AP expression (Figure 3b). To further study the effect of forced expression of Serpine1 and Runx1, we measured the expression level of Oct3/4 and Nanog. As shown in Figure 3c, the expression of Oct3/4 was impaired upon Serpine1 constitutive expression in ESCs, and also Nanog showed a moderate but significant decrease, whereas no significant changes were detectable in FLAG-Runx1-expressing cells. This decrease of Oct3/4 and Nanog levels corresponded to a significant increase of early differentiation markers (Figure 3d) even in the presence of LIF, indicating that Serpine1 alone is able to impair the undifferentiated state by inducing an uncontrolled differentiation.
Figure 3. Ectopic expression of Serpine1 impairs ESC undifferentiated state. (a) ESCs were transfected with empty vector (mock) or FLAG-tagged Runx1 and Serpine1 expression vector. Western blot stained with anti-FLAG antibody show the expression level of FLAG-Serpine1 and FLAG-Runx1 24 hr after transfection. (b) ESCs transfected with empty vector, FLAG-Serpine1 or FLAG-Runx1 were grown at clonal density (50-100 cells/cm2) for 7 days and then stained for alkaline phosphatase activity (scale bars, 1 cm). The percentage of undifferentiated (blue) and differentiated (red) colonies is reported. Bars represent SD of triplicates. *P < 0.05. (c) Expression level of stemness markers was measured 48 hr after transfection by qPCR. The data are expressed as fold changes relative to mock-transfected cells. Error bars represent SD of triplicates. *P < 0.01. (d) Expression levels of early differentiation markers of ectoderm (Fgf5), endoderm (Sox17) and mesoderm (T, also known as brachyury) were measured 48 hr after transfection by qPCR. Error bars represent SD of triplicates. *P < 0.01.
ESC-specific targets of Klf5
Next, we asked the question of the specificity of Klf5. Previous works demonstrated that Klf5 is highly expressed in skin [19] and that alterations in Klf5 expression level may affect the epidermis-differentiated phenotype [29], suggesting that Klf5 could regulate the regenerative potential of stem cells in the epidermis. We analyzed expression changes of the 53 qPCR-validated Klf5 targets upon Klf5 KD in primary keratinocytes (Additional file 10). In fact, we found that the transcripts of six genes expressed in ESCs were not detectable in primary keratinocytes, both in Klf5 KD and control cells, whereas there were 21 of 47 genes similarly regulated by Klf5 in primary keratinocytes and ESCs. On the other hand, 14 of 47 genes showed opposite changes in ESCs versus keratinocytes, while 12 of 47 genes were regulated by Klf5 only in ESCs (Figure 4). Although the extent of Klf5 suppression is different in ESCs versus keratinocytes, the comparison of expression profiles of these two cells indicated a cell type-specific gene regulation by Klf5.
Additional file 10. Additional Figure 7. Klf5 KD in primary keratinocytes and Klf2 and Klf4 KD in ESCs. (a) Klf5 or NS siRNA were transfected in primary keratinocytes and Klf5 expression level was measured 12 hr after transfection by Western blot with anti-Klf5 antibody. (b) Expression levels of Klf2, Klf4 and Klf5 were measured by qPCR in ESCs 12 hours after siRNA transfection. The results are represented as fold changes. Bars represent SD of triplicates. P < 0.01.
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Figure 4. ESC-specific targets of Klf5. qPCR analysis of expression changes of Klf5 target genes upon Klf5 KD in primary keratinocytes compared to results obtained in ESCs. Klf5 or NS siRNA were transfected in both primary keratinocytes, and ESCs and gene expression levels were measured 12 hr after transfection. Probe sets of both genes induced (a) and repressed (b) by Klf5 in ESCs were analyzed. Arrows indicate genes regulated only in ESCs; arrowheads indicate genes showing an opposite change upon Klf5 KD in ESCs and primary keratinocytes. All data are expressed as fold changes relative to siNS transfected cells. Error bars represent SD of triplicates.
A further aspect of the specificity of the Klf5-based regulation concerns the possibility that Klf2, Klf4 and Klf5 have redundant functions by binding and thus regulating common targets in ESCs [15]. To address this point, we investigated gene expression changes of the 53 qPCR-validated Klf5 targets, following KD of each single Klf. We transfected Klf2 or Klf4 siRNA in ESCs, and 12 hr after transfection we found that the level of Klf2 and Klf4 were significantly reduced, although with different extent of KD (Additional file 10). In these conditions, we found that only a low percentage (<10%) of examined genes showed the same trend upon KD of all these Klfs (Figure 5). The same low percentage was observed by comparing Klf5- versus Klf4-regulated genes. Instead, we found that the genes that showed the same behavior upon Klf2 and Klf5 KD represent about 40%. Finally, we observed that about 45% of the genes show a Klf5-specific gene regulation, different from that dependent on Klf2 or Klf4 KD (Figure 5), indicating that some redundancy could exist in the genes controlled by Klf5 and Klf2, rather than Klf4.
Figure 5. Klf5-specific targets. Comparison of gene expression changes following KD of Klf2, Klf4 or Klf5 in ESCs. Klf2, Klf4, Klf5 or NS siRNA were transfected in ESCs. RNA samples were collected 12 hr after transfection and subjected to qPCR. (a) Klf5-activated genes. (b) Klf5-repressed genes. Arrowheads indicate the expression changes induced by Klf5 only. All data are expressed as fold changes relative to siNS transfected cells. Error bars represent SD of triplicates.
In the past 3 years, several reports have shown Klf5 to be an important player regulating ESC self-renewal, pluripotency and embryonic development with a unique requirement of Klf5 in inner cell mass (ICM) establishment compared to the other ESC-specific Klfs [16,17,30-32]. Although the pivotal role of Klf5 in ESC self-renewal and ICM development has been at least in part elucidated, its mechanism of action and the genes that Klf5 controls are still not completely understood. In the present study, we analyzed Klf5 regulatory targets in ESCs by comparing microarray and ChIP-seq analysis. We identified 74 Klf5 target genes whose expression is activated by Klf5 and 239 whose expression is repressed. Among them, we found 161 genes bearing Klf5 binding sites within 100 kb from the gene boundaries and that can be considered putative primary targets of Klf5.
We have shown that Klf5 controls genes that play a crucial role in ESCs such as Tcl1 [8,22], BMP4 [23] and Nr0b1 [24], and thus it may be required in ESCs to maintain pluripotency by activating expression of these self-renewal promoting genes and by simultaneously inhibiting expression of differentiation promoting genes such as TGFβ2 [33], Otx2, Pitx2 [8] and GDNF [34]. To further support the role of Klf5 in the control of regulators of the ESC phenotype, we have explored the effects of the suppression of 23 genes target of Klf5. Among these 23 genes, we identified 8 genes whose KD induces the loss of ESC undifferentiated state even in the presence of LIF. The eight identified genes encode cell adhesion molecules (Bcam, Perp), two protein kinases (Hck, Dgka), a growth factor binding protein (Igfbp3), an endoplasmic reticulum protein (Niban) and two proteins with still unknown function (1600029D21Rik, E130012A19Rik). Such a variety of molecules indicates that the ability of Klf5 to maintain the ESC undifferentiated state results from the concomitant regulation of a wide range of cellular activities. These eight genes showed the same trend of Klf5 expression during ESC differentiation and, moreover, a different transcriptional control by Klf2, Klf4 and Klf5 with Igfb3, Niban and Perp responding only to Klf5. Among these proteins, Hck has been shown to be involved in gp130-mediated signaling of LIF, since introduction of constitutively activated Hck alleviates the requirement of ESCs for LIF to remain undifferentiated [35]. Our data suggest that Klf5 is required for proper control of the expression of ESC-specific genes as well as genes whose transcription seems to be induced upon ESC differentiation. Among the genes suppressed by Klf5 in undifferentiated ESCs, we found Serpine1, also known as plasminogen activator inhibitor-1 (PAI-1). Serpine1 is a downstream target of TGFβ1 and is induced upon ESC differentiation by TGFβ1 stimulation [36]. We have found that ectopic expression of Serpine1 resulted in an impairment of the ESC phenotype by promoting the appearance of differentiation markers even in the presence of LIF. This phenotype mimics the effect of Klf5 KD as well as of Klf5 KO ESCs [16,17]. Although a redundant function was suggested for Klf2, Klf4 and Klf5 [15], we have shown that Serpine1 is regulated specifically by Klf5 and not by Klf2 and Klf4 suppression. This specific control by Klf5 could explain the inability of Klf2 and Klf4 to compensate for the loss of Klf5 function in ICM establishment.
Klf5 has been described to play a role in various biological processes, such as the control of the stress response in the myocardium [37] and the induction of somatic cell reprogramming [14]. Furthermore, Klf5 has many roles in regulating cell cycle, development, proliferation, apoptosis and tumorigenesis [18]. Interestingly, Sur et al. [29] described a role of Klf5 in the regulation of the keratinocyte differentiation program and thus in the regenerative potential of stem cells in the epidermis. By investigating changes in expression profile of Klf5 target genes both in ESCs and primary keratinocytes, we have found that Klf5 may work as a repressor or activator in a context-specific manner. In fact, about half of the examined genes seem to be regulated by Klf5 in an ESC-specific manner, and in many cases the effect of Klf5 KD in ESCs and primary keratinocytes was opposite. This different regulation by Klf5 could be due to a different accessibility to transcriptional regulators as a consequence of chromatin folding and/or to the interaction with cell-specific transcriptional complexes.
Recently, Jiang et al. [15] proposed that Klf5 has a redundant function with Klf2 and Klf4 in ESCs. The same authors showed that there is a greater overlap between genomic binding sites of Klf2 and Klf4 than with Klf5. Accordingly, we found that about 45% of the examined genes are significantly regulated by Klf2 and Klf5. Interestingly, we also observed that almost half of the examined genes appear to be regulated specifically by Klf5. This can explain the unique requirement of Klf5 for derivation of ESCs and ICM proper development [17], in contrast to the dispensable role of Klf2 and Klf4 in these processes [31,32].
Numerous results indicate that Klf5 plays an important role in maintaining ESC pluripotency and in governing ESC fate decisions. In this work, we have explored the mechanisms through which this transcription factor regulates ESC functions. We have identified a likely complete set of genes, putative primary targets of Klf5 in ESCs, by comparing the early changes of the gene expression profile induced by Klf5 KD and the results of ChIP-seq analysis for Klf5 binding sites. About half of these genes seem to be regulated by Klf5 in an ESC-specific manner, and Klf5-dependent gene regulation in ESCs appears to be different from that based on Klf2 and Klf4, two other members of the Krüppel-like family, previously involved in ESC functions. Finally, we demonstrated that suppression or constitutive expression of Klf5 target genes clearly impair the ESC undifferentiated state. These results contribute to the understanding of the regulatory role of Klf5 in ESCs and suggest a high hierarchical role in these cells for this transcription factor.
Materials and methods
Plasmid construction
Serpine1 and Runx1 cDNA were derived from pSport vector (NIH Mammalian Gene Collection, Open Biosystems, National Institutes of Health, Bethesda, MD, USA) by PCR [16] with the following oligonucleotides:
HindIII-Serpine1: 5'-GATGACAAGCTTCAGATGTCTTCAGCCCTTGCTTGCCTCATCC-3'
NotI-Serpine1: 5'-GGCGATGAGCGGCCGCTCAAGGCTCCATCACTTGGCCCATGAAGAGG-3'
HindIII-Runx1: 5'-GATGACAAGCTTGCTTCAGACAGCATTTTTGAGTCATTTCCTTCATATCC-3'
NotI-Runx1: 5'-GGCGATGAGCGGCCGCTCAGTAGGGCCGCCACACGGCCTCCTCC-3'
Next, these cDNAs were cloned downstream FLAG-tag in the p-CBA-FLAG vector by using HindIII and NotI restriction sites.
Cell culture, transfection, differentiation and alkaline phosphatase staining
E14Tg2a (BayGenomics, San Francisco, CA, USA) mouse ESCs were maintained on feeder-free, gelatin-coated plates in the following medium: GMEM (Glasgow Minimum Essential Medium) (Sigma, St. Louis, MO, USA) supplemented with 2 mM glutamine, 100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1× nonessential amino acids (all from Invitrogen, Carlsbad, CA, USA), 0.1 mM β-mercaptoethanol (Sigma), 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA), and 103 U/ml leukemia inhibitory factor (LIF; Millipore, Billerica, MA, USA). Neural differentiation was induced as previously described [16]. Briefly, undifferentiated ESCs were trypsinized into a single-cell suspension and plated at low density (1-5 × 103 cells/cm2) on gelatin-coated dishes in the following medium: knockout Dulbecco's minimal essential medium supplemented with 10% knockout serum replacement (both from Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), 2 mM glutamine (Invitrogen), 100 U/mL penicillin/streptomycin (Invitrogen). Medium was changed on alternate days.
Primary mouse keratinocytes were isolated from 2-day-old Swiss ICR(CD-1) mice (Harlan Laboratories, Correzzana, Italy) and cultured as previously described [38]. Transfections were performed 5 days after plating.
Transfection of expression plasmids and shRNA plasmids (Open Biosystems; see Additional file 11) both in ESCs and in primary keratinocytes were performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. To generate the stable cell lines, E14Tg2a cells were transfected with shRNA plasmids and recombinant clones were selected with Puromycin (Sigma).
Additional file 11. Additional Table 4. Sequences of shRNAs.
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For alkaline phosphatase staining, ESCs were cultured at clonal density (30 cells/cm2). The cells were fixed in 10% cold neutral formalin buffer (10% formalin, 110 mM Na2HPO4, 30 mM NaH2PO4 H2O) for 15 min and then rinsed in distilled water for 15 min. The staining was obtained by incubation for 45 min at room temperature with the following staining solution: 0.1 M Tris·HCl, 0.01% naphthol AS MX-PO4 (Sigma), 0.4% N, N-dimethylformamide (Sigma), 0.06% red violet LB salt (Sigma).
RNA isolation and qPCR
RNA from ESCs and primary keratinocytes was isolated by using the Tri reagent (Sigma) and then reverse transcribed using MuMLV-RT (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions. Real-time RT-PCR was performed using PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA USA) and Power SYBR Green PCR Master Mix (Applied Biosystems). Gene-specific primers used for amplification are listed in Additional file 12.
Additional file 12. Additional Table 5. Primers used for qPCR.
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Microarrays and bioinformatics analysis
Total RNA from ESCs was isolated with Tri reagent (Sigma) and further purified with the Qiagen column kit (Qiagen, Milan, Italy). Then samples from three independent experiments were sent to Coriell Genotyping and Microarray Center (Coriell Institute for Medical Research, Camden, NJ, USA), where, after proper sample processing, cRNA were hybridized with the Affymetrix Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA, USA).
For bioinformatics analysis, raw probe intensities for each of the hybridized microarrays were normalized to gene expression levels using the dChip algorithm [39]. To identify genes significantly responding in the experiment, we computed the P values and false discovery rate (FDR). A total of 313 probes, corresponding to 313 different transcripts, have been identified (FDR < 0.1 corresponding to P < 0.005) that responded significantly.
Preparation of cell lysates and Western blot analysis
ESCs and primary keratinocytes were lysed in a buffer containing 1 mM EDTA, 50 mM Tris·HCl, pH 7.5, 70 mM NaCl, 1% Triton protease inhibitor cocktail (Sigma) and analyzed by Western blot analysis. The following primary antibodies were used: rabbit anti-Klf5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Nanog (Calbiochem, San Diego, CA, USA), mouse anti-Oct3/4 (Santa Cruz Biotechnology), mouse anti-GAPDH (Santa Cruz Biotechnology) and mouse anti-FLAG (Sigma). Antibody-protein complexes were detected by HRP-conjugated antibodies and ECL (both from Amersham Pharmacia, Milan, Italy).
Chromatin immunoprecipitation
For ChIP-seq analysis, ESCs stably transfected with FLAG-Klf5 were cross-linked with 1% formaldehyde for 10 min at room temperature, and formaldehyde was then inactivated by the addition of 125 mM glycine. Then the chromatin was sonicated to an average DNA fragment length of 200 to 500 bp. Soluble chromatin extracts were immunoprecipitated using the mouse monoclonal anti-FLAG (Sigma) or mouse IgG (Santa Cruz Biotechnology) as control. Then samples from two independent experiments were sent to the DNA sequencing service of EMBL (Heidelberg, Germany) and subjected to high-throughput sequencing with Illumina Genome Analyzer platform (Illumina, San Diego, CA, USA).
For ChIP-qPCR, samples were prepared as described above. Supernatant obtained without antibody was used as an input control. qPCR analyses were performed using the ABI PRISM 7900HT sequence detection system and SYBR Green PCR Master Mix (Applied Biosystems). Primers used for ChIP-qPCR are listed in Additional file 13.
Additional file 13. Additional Table 6. Primers used for ChIP-qPCR.
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The amount of precipitated DNA was calculated relative to the total input chromatin and expressed as the fold enrichment relative to total input according to the following formula [40]: fold enrichment = 2{Delta}Ct × 10, where {Delta}Ct = Ct(input) - Ct(immunoprecipitation), where Ct refers to cycle threshold.
ChIP-seq bioinformatics analysis
More than 13 million sequences were produced and aligned to the mouse genome (version m37) masked for DNA repeat by using Bowtie tool version 0.9.9.2 (Center for Bioinformatics and Computational Biology, Institute for Advanced Computer Studies, University of Maryland, College Park, MD, USA) [41]. About 50% of sequences were univocally aligned, and the resulting coordinates were fed to MACS software version 1.3.5(Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, MA USA) [42] to detect genomic regions enriched for multiple overlapping DNA fragments (peaks) that we considered as putative binding sites. False discovery rate (FDR) was estimated by MACS, by comparing the peaks from anti-FLAG samples with those from control (anti-IgG ChIP) at the same P value cutoff. Motif analysis was performed by using CisFinder tool (Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging, NIH, Baltimore, MD, USA) [43] on 200-bp sequences centered at the expected binding site indicated by peak summit calculated using the MACS tool. Flanking sequences 1000 bp away from the peak summit have been used as control sequences.
(AP): Alkaline phosphatase; (BCAM): basal cell adhesion molecule; (BMP4): bone morphogenetic protein 4; (KLF): Krüppel-like factor; (CHIP): chromatin immunoprecipitation; (DGKα): diacylglycerol kinase α; (ESC): embryonic stem cell; (GDNF): glial cell-derived neurotrophic factor; (HCK): hemopoietic cell kinase; (ICM): inner cell mass; (IGFBP3): insulin-like growth factor binding protein 3; (KD): knockdown; (KO): knockout; (LIF): leukemia inhibitory factor; (NR0B1): nuclear receptor subfamily 0, group B, member 1; (OCT3/4): octamer-binding protein 3; (OTX2): orthodenticle homeobox 2; (PITX2): paired-like homeodomain 2; (RUNX1): runt-related transcription factor 1; (SHRNA): short hairpin RNA; (SIRNA): short interfering RNA; (Sox2): SRY-box containing gene 2; (TCL1): T-cell lymphoma breakpoint 1; (TGFβ2): transforming growth factor β2.
Authors' contributions
SP was responsible for the study's conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; LC was responsible for collection and assembly of data; CT was responsible for collection and assembly of data; FP was responsible for collection and assembly of data; SC was responsible for collection and assembly of data; LA was responsible for collection and assembly of data; DA was responsible for collection and assembly of data; VDS was responsible for data analysis and interpretation; LP was responsible for manuscript writing; TR was responsible for conception and design, data analysis and interpretation, and manuscript writing. All authors read and approved the final manuscript.
We thank Dr. Scott Frank for helpful discussion. This work was supported by grants from EC-Sirocco Consortium, Associazione italiana ricerca sul cancro (AIRC) and Italian Ministry of Health.
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Waffles the Corgi (Video)
This adorable puppy named Waffles loves showing off in front of the video camera. He sure likes to steal all of the attention, but this is because he is just too cute.
Corgi Picks Terrible Playmate for Fetch (Video)
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Icater.menuRead MoreiCater.menu is a full service event design, planning, production, and culinary company that elevates ordinary events to extra extraordinary experiences. We not only manage all the planning and details of your day, but we also create a tenacious look that reflects your vision. Our services are board which include event design, logistics, budget planning, management, rentals, staging, linens, lighting, flowers and more! We also assist with the selection of your collateral – invitations, place cards, programs and signage – to ensure that the overall look is carried through in all the details of the event. Our team brings over 10 years of creative innovation to custom decor and flowers as well as every other aspect of event production including venue selection, invitations, catering, entertainment and integrated branding.OnlineSpringfield, VA US
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County Limerick Self-Catering Holiday Cottages
Lots of holiday homes to rent overlooking the River Shannon in County Limerick
The River Shannon estuary is a popular place for weeks and weekends sailing, boating and angling. Holidaymakers love to be around water, when they're not in it, they like to view it, photograph it and stay nearby. There is a mass of holiday accommodation to rent near the Shannon estuary; holiday apartments, cottages and houses of all sizes. Limerick is a popular choice for self catering cottage breaks in Ireland and forms part of what is known as Ireland's 'Shannon region', named after the river Shannon. Limerick is a mostly rural region with a rich heritage and has lots to interest tourists. Where do you prefer to stay on holiday, near the coast or in the countryside?
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Visit Limerick on holiday
Even if you book a holiday cottage in the country, day trips to the city are possible. Limerick itself is an attractive city next to the river Shannon with a medieval old town area as well as the impressive King John's castle. You can take cruises on the River Shannon and enjoy relaxing and soaking up the scenery. County Limerick is well connected in terms of travel with Shannon International Airport being located not too far from Limerick, making County Limerick easily accessible by air for self catering cottage holidays.
Self catering cottages often appeal to people for a variety of reasons when taking a holiday in Ireland. Cottage stays can offer the advantage of a home from home environment and very often more space than would be available to you with a traditional hotel stay. Country cottage or self catering holidays very often offer the kind of flexibility that appeals to those with large or young families. Enjoy browsing through the cottages in the mid west of Ireland on Country Cottages Online. Don't forget to use our search assistant for specific types of cottages such as cottages with hot tubs or farmstays. We hope you find the right cottage in Limerick for you.
Please note we have no country cottages or cottages to rent in Limerick currently. We recommend that you take a look at cottage rentals in Ireland. Alternatively use our Search Assistant can help with your search for a suitable holiday cottage in Ireland.
Why not take a look at available Last Minute Cottages in Ireland?
Featured Holiday Cottages near County Limerick, Ireland :
River House Coastal Cottage, Sneem, County Kerry, South West , Sneem, Kerry, Ireland
6 bedroom Riverside Holiday House, South West Ireland.
With river views and a very comfortable high quality interior which includes granite work tops in the kitchen and a Jacuzzi bath, this is a luxury Irish holiday home and could be a great base for discovering some of the best of Kerry on a cottage holiday.
Sleeps from 1 to 12 people in 6 bedrooms
Number of bathrooms: 5
Pet Friendly: Yes
Quality Rating: (5)
01244 500 540
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Next availability: from 27 November 2015 to 19 December 2015; from 09 January 2016 to 25 March 2016; from 29 March 2016 to 30 April 2016;
Rannagh View Secluded Coastal Cottage for 9, Liscannor, Clare, Ireland
4 bedroom Coastal Holiday Cottage, Mid West Ireland.
Fantastic 4 bedroom Irish holiday home with country and sea views plus a modern and stylish interior, well placed for a seaside holiday and for exploring the Burren National Park.
Sleeps from 4 to 9 people in 4 bedrooms
Number of bathrooms: 4
Pet Friendly: No
Quality Rating: (5)
01244 500 540
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Next availability: from 02 April 2016 to 18 June 2016; from 02 July 2016 to 16 July 2016; from 06 August 2016 to 20 August 2016;
Donour Holiday Lodge, Fanore, Clare, Ireland
7 bedroom Holiday House, Ireland - County Clare.
Delightful large Irish holiday cottage with 7 bedrooms and lovely views over Burren National Park out to sea, just a walk away from the local blue flag award winning beach.
Special features: free internet access.
Sleeps from 6 to 16 people in 7 bedrooms
Number of bathrooms: 6
Pet Friendly: Yes
Quality Rating: (4)
01244 500 540
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more info >
Next availability: from 29 November 2015 to 23 December 2015; from 09 January 2016 to 13 February 2016; from 20 February 2016 to 19 March 2016;
St. Anthonys, Lauragh near Kenmare, Kerry, Ireland
3 bedroom Cottage, Beara Peninsula south west Ireland.
Picture-perfect Irish country cottage sleeping up to 4, conveniently located close to the coast on the Beara Peninsula and also near to beautiful Glanmore Lake.
Sleeps from 2 to 4 people in 3 bedrooms
Number of bathrooms: 1
Pet Friendly: Yes (1 dog welcome)
Rating: Not assessed
Lios Dana Lodge, Annascaul, Kerry, Ireland
8 bedroom cottage, overlooking Dingle Bay.
Fantastic large coastal cottage with impressive sea and beach views, located very close to Dingle Bay, ideal for large families, groups or special get togethers.
Special features: games room.
Sleeps from 6 to 16 people in 8 bedrooms
Number of bathrooms: 6
Pet Friendly: No
Rating: Not assessed