>> HELLO. LET ME GET STARTED. SO THANKS FOR COMING. TODAY'S A STEM CELL SEMINAR SERIES. THIS IS MY GREAT PLEASURE TO INTRODUCE DR. KEN ZARET FROM THE UNIVERSITY OF PENNSYLVANIA SCHOOL OF MEDICINE. SO AS YOU KNOW, HE HAS BEEN A LEADING SCIENTIST DEVELOPMENTAL BIOLOGIST AND WELL RESPECTED IN THE FIELD OF DEVELOPMENTAL BIOLOGIES ESPECIALLY IN THE PANCREAS DEVELOPMENT. LET ME BRIEFLY INTRODUCE HIS ACADEMIC HISTORIES MUCH HIS PH.D. DEGREE IN BIOLOGY AT THE UNIVERSITY OF ROCHESTER. AFTER HIS POST DOCTOR TRAINING -- LAB AND HE STARTED HIS OWN LAB IN BROWN UNIVERSITY SINCE 1986. THEN HE MOVED TO THE CANCER CENTER IN PHILADELPHIA FROM 1999 TO 2009. SO SINCE 2009, HE'S THE PROFESSOR IN THE DEPARTMENT OF THE CELL AND DEVELOPMENTAL BIOLOGY AT THE UNIVERSITY OF PENNSYLVANIA SCHOOL OF MEDICINE. AND HE'S ALSO THE ASSOCIATE DIRECTOR OF THE INSTITUTE FOR THE DEGENERATION MEDICINE AND ALSO CODIRECTOR OF THE PROGRAM IN [INDISCERNIBLE] AND THEN HIS LAB HAS BEEN USING SUCH MAPPING AND MOUSE GENETICS AND STUDY THE MECHANISMS IN LIVER AND PANCREAS EXAMINATIONS [INDISCERNIBLE] HIS LAB ALSO STUDIES [INDISCERNIBLE] AND ALSO CHROMATIN MODIFICATIONS IN THE LIVER AND PANCREAS EXAMINATIONS. I BELIEVE HE IS GOING TO TALK ABOUT SUCH THINGS. SO I'LL FINISH UP MY INTRODUCE AND THEN PLEASE WELCOME DR. KEN ZARET. [APPLAUSE] >> THANKS VERY MUCH. IS THIS ON? THANKS VERY MUCH, I APPRECIATE THE INVITATION. AND JUST WANT TO SAY IF ANYBODY HAS A QUESTION OR A COMMENT WHILE I'M TALKING, PLEASE SHOUT OUT AND INTERRUPT AND MAKE IT MORE OF A DIALOGUE, MAKE IT MORE INTERESTING PERHAPS. AND THANKS AGAIN. SO I'M GOING TO TALK ABOUT HOW CELLS ARE PROGRAMMED IN THE EMBRYO AND HOW WE CAN REPROGRAM SULFATES AS WILL AND ASK A QUESTION WHEN DO WE KNOW ENOUGH ABOUT THE BASIC MECHANISMS BY WHICH CELLS ARE PROGRAMMED TO BEGIN TO PREDICT HOW TO MORE EFFICIENTLY PROGRAM CELLS FOR BIOMEDICAL PURPOSES. NOW, WHAT I'LL TALK ABOUT A BIT IS SOME OF THE HISTORY OVER MY LAB AND OTHERS IN UNDERSTANDING HOW TRANSCRIPTION FACTORS PROGRAM CHROMATIN IN A STEP WIDE FASHION BRING A PLURIPOTENT CELL TO A MORE DIFFERENTIATING CELL SUCH AS ENDO DETERMINE AND A DIFFERENTIATED CELL BUT ALSO DISCUSS ISSUES OF HOW YOU CONVERT ONE CELL TYPE TO ANOTHER. SUCH AS GOING FROM A PANCREATIC CELL TO A LIVER CELL OR VICE VERSA OR IF A FIBROBLAST. FOR A LONG TIME PEOPLE LIKE ME IN THE FIELD AND MANY OTHERS STUDIED TRANSCRIPTION FACTORS INVOLVED IN THIS PROCESS. NOW WE'RE INVESTIGATING HOW CHROMATIN MODIFIERS ENZYMES THAT CO-VIOLENT REMODIFY CHROMATIN AND SO FORTH PLAY A ROLE IN THESE PROCESSES AND I'LL DESCRIBE THAT TODAY. IN THE FUTURE I THINK WE'LL HAVE A BETTER SENSE OF HOW MODULATORY RNAs PLAY INTO THIS AS WELL. SO A WHILE BACK MY LAB WAS STUDYING EARLY LIVER DEVELOPMENT AND PROGENITORS TO THE LIVER BEFORE LIVER-SPECIFIC GENES WERE ACTIVATED AND DISCOVERED WHAT WE CALLED PIONEER FACTORS. SO THESE ARE TRANSCRIPTION FACTORS THAT ARE ERROR THE FIRST -- THAT ARE EITHER THE FIRST OR AMONG THE FIRST ARE TRANSCRIPTIONALLY SILENT AT THE TIME OF INITIAL BINDING. AND AS I'LL DESCRIBE IN A MINUTE, THESE PROTEINS HAVE SPECIAL BIOCHEMICAL FEATURES THAT DISTINGUISH THEM FROM OTHER TRANSCRIPTION FACTORS AND MAKE SENSE TO REGARD TO THEIR INITIAL BINDING OF CHROMATIN THAT'S NOT YET BEEN PROGRAMMED FOR EXPRESSION. NOW, A QUESTION IS, HOW CAREFULLY ARE THESE EARLY AND LATER NETWORKS THAT ARE INVOLVED IN ESTABLISHING A DIFFERENTIATED CELL ALSO GOING TO BE INVOLVED IN CELL REPROGRAMMING. AND SO THERE'S SOME WORK THAT'S COME OUT IN THIS AREA. AND JOHNATHAN SLACK AND DAVID -- AND HIS COLLEAGUES HAVE SHOWN A NUMBER OF YEARS AGO NOW THAT YOU CAN REPROGRAM A PANCREATIC -- TYPE CELL TO A HEPATIC-LIKE CELL USING TRANSCRIPTION FACTORS THAT BYPASS THIS EARLY STEP AND REPRESENTED A LATER STEP OF DIFFERENTIATION OF THE LIVER. SO THE LIVER AND PANCREAS HAVE A COMMON EARLY STEP BUT YOU DIDN'T NEED TO THE TO TRANSDIFFERENTIATE BETWEEN THE TWO. BY CONTRAST, EXPERIMENTS FROM SEVERAL LABS INCLUDING ONE EXEMPLIFIED AND ASKED WHICH OF THESE ARE NECESSARY FOR A FIBROBLAST CELL REACHING BACK FURTHER IF YOU WILL TO A LIVER CELL AND FOUND INEVITABLY YOU NEEDED ONE OR MORE OF THESE PIONEER FACTORS THAT WERE USEFUL EARLY IN NORMAL LIVER PROGRAMMING. WHAT I WOULD LIKE TO DO TODAY IS TAKE A STEP BACK FROM THIS AND ASK ABOUT CELL REPROGRAMMING AND WHAT WE'VE LEARNED FROM THESE CONCEPTS TO APPLY TO OTHER IS ASPECTS OF CELL REPROGRAMMING AND OTHER TRANSCRIPTION FACTORS AND ASK WHICH MAY WORK AS PIONEERS, WHICH MAY NOT. BUT ALSO ASK A VERY IMPORTANT QUESTION AND THAT IS ABOUT THE RESTRICTION OF CELL FATE AND PREVENTING CELL REPROGRAMMING. SO WHAT DO I MEAN BY THAT? SO CONSIDER FOR A MOMENT THE ASTOUNDING DISCOVERY FROM YAMANAKA'S LABORATORY THAT JUST ONE THE NOBEL PRIZE THAT FOUR TRANSCRIPTION FACTORS -- ARE SUFFICIENT TO ON A TOPIC EXPRESSION CONVERT A FIBROBLAST CELL TO A PLURALLY POTENT CELL TO MAKE A PLURIPOTENT STEM CELL. THAT'S FANTASTIC. THE FACTS ARE THAT ONLY ONE IN A THOUSAND OR 10,000 OF THESE IN A TYPICAL ASSAY USING HUMAN CELLS ACTUALLY FUNCTIONALLY CONVERT AND IT TAKES ABOUT A MONTH FOR THAT TO HAPPEN. BY CONTRAST, THE OTHER NOBEL LAUREATE IN THIS FIELD JOHN -- A NUMBER OF YEARS AGO SHOWED IF YOU TOOK A NUCLEUS FROM A SKIN CELL AND TRANSPLANTED IT INTO AN EGG THAT YOU COULD GENERATE PLURIPOTENT CELLS WITHIN A COUPLE DAYS. SO THERE'S A GAP. AND SO THE QUESTION THAT I WANT TO POSE IS THAT USING THE CONCEPT OF TRANSCRIPTION FACTORS TO REPROGRAM CELLS, WHAT IS IT THAT'S RESTRICTING THEIR ACTIVITY. HOW CAN WE BEGIN TO GET AT THE MOLECULAR COMPONENTS OF A CELL THAT PREVENTS CELLULAR REPROGRAM. AND THEN USING THAT TO THEN IF WE UNDER WHAT'S RESTRICTING, CAN WE DOWN REGULATE THOSE COMPONENTS ENHANCE REPROGRAMMING AND FINALLY UNDERSTAND HOW THESE RESTRICTIVE COMPONENTS THAT PREVENT AN EXPERIMENTAL SITUATION SUCH AS REPROGRAMMING, HOW DO THEY ACTUALLY PLAY INTO HOW A CELL INITIALLY GETS DIFFERENCE SHAIFTD IN THE FIRST PLACE AND HOW WE SEPARATE CELL LINEAGES. A PICTURE VIEW OF ALL OF THIS IS WE'RE HAVING A TRANSITION IN MY LAB. FOR MANY YEARS WE FOCUSED ON POSITIVE REGULATION. WHAT IS IT THAT PROMOTES CELL DIFFERENTIATION FORWARD. AND NOW WE'RE LEARNING THAT UNDERSTANDING HOW CELLS LAY DOWN ROADBLOCKS TO PREVENT A CELL FATE FROM FORMING AND FORCING OTHERS MAY IN FACT BE JUST AS IMPORTANT. SO TO DO THIS, WE STARTED WITH A POST DOC IN MY LAB NAMED -- A TERRIFIC GUY AND A BIOINFOMATTIST. I'M GOING TO TALK ABOUT THAT BUT WHAT I WOULD LIKE TO DO FIRST IS GIVE YOU A LITTLE BIT OF BACKGROUND WHAT WE KNOW ABOUT THE BIOCHEMISTRY AND THE BIOMECHANISM BY WHICH BIOFACTORS WORK AND IN THAT FROM TIME TO TIME WORK I'LL COME BACK TO THIS QUESTION ON HOW THESE FACTORS REPROGRAM FIBROBLAST CELLS. WE GOT INTO THIS QUITE A FEW YEARS AGO PLAYING A GAME CALLED WHOSE ON FIRST. WE'RE LOOKING ON A LIVER SPECIFIC REGULATORY ELEMENT FOR THE ALBUMIN GENE -- LOOKING AT HEPATOCYTES AND EARLIER CELLS AND FINALLY ENDODETERMINE CELLS. AND THE -- FAMILY THAT WERE INVOLVED IN ESTABLISHING COMPETENCE FOR GENES TO BE ACTIVATED UPON -- SIGNALING. AND -- LABS HAVE DONE SOME NICE GENETICS TO SHOW THAT IN FACT THESE FACTORS ARE NECESSARY AT THIS EARLY STAGE TO ALLOW THE ENDO DETERMINE TO INDUCE LIVER. FOR A VARIETY OF REASONS THE FOX FACTORS APPEAR TO BE SLIGHTLY UP STREAM OF THE GADDA FACTORS WE FOCUSED ON THOSE. FOX TRANSCRIPTION FACTORS HAVE A CENTRAL CONSERVED DNA BINDING DOMAIN. THEY ALSO HAVE CONVENTIONAL TRANSCRIPTION FACTOR -- AND IN THE HUMAN THERE'S 43 DIFFERENT FOX GENES, EACH ENCODED BY SEPARATE LOCI AND THE FOX A CLUSTERS OF FOX A, 2 AND 3 IS FOR GUT TISSUE DEVELOPMENT. IN ANY CASE THE DNA BINDING DOMAIN WAS CRYSTALLIZED BY STEVE BURLY'S LAB AND FOUND TO HAVE A HELIX MOW TESTIFY THAT PROFESSION TRUDES INTO THE MAJOR GROOVE AS WELL AS TWO WINGS OF POLYPEPTIDE CHAINS THAT MAKE MINOR GROUP CONTACTS. THE IMPORTANT THING IS THIS STRUCTURE THE DNA BINDING DOMAIN ALLOWS THE PROTEIN TO BIND ONE FACE OF THE DNA HELIX WITH SPECIFIC SEQUENCE RECOGNITION ALLOWING THE OTHER FACE OF THE HELIX TO BIND OTHER STUFF, IF YOU WILL. NAMELY PERHAPS CHROMATIN OR NUCLEOSO MOW COMPONENTS SUCH AS HISTONES. THE LAB FOUND THAT HISTONES WHICH FINDS TWO NOOK SOLES AND HELPS COMPACT CHROMATIN CONTAINS THE SAME OVERALL STRUCTURE AS THE FOX TRANSCRIPTION FACTORS DO. AND THEN -- A POST DOC IN MY LAB FOUND THE PURIFIED FOX A PROTEIN CAN BIND TO ITS TARGET SITE ON A NOOK N U C LEOCELL. THE DNA IS WRAPPED NEARLY TWICE AROUND THE HISTONES TO MAKE NOOK SO MANY CORE PARTICLES. THESE ARE ARRANGED IN A PARTICULAR SPACIAL REGISTER THAT ALLOWS THEM TO FORM HIGHER ORDER STRUCTURES THAT CAN BE FURTHER BOUND BY LINK OR HISTONE TO COMPACT THE CHROMATIN AND KEEP IT QUIET OR CO-REGULATORY COMPLEXES THAT MIGHT HELP OPEN IT. ALL THESE ALLOW TWO METERS OF DNA TO SQUISH INTO EVERY NUCLEUS IN YOUR CELLS. IN ANY CASE THERE ARE HISTONE MODIFICATIONS THAT COVALENTLY OCCUR MOSTLY ON THE GLOB LEARN DOMAIN IN THE CENTER OF THE NOOK SO MANY. LISA IN MY LAB INVESTIGATING HOW THESE DPAWKS FAKERS MIGHT WORK CREATED NOOK SO MANY TEMPLATES WHERE THEY ASSEMBLED CHROMATIN IN VITRO WITH N LABEL DNA AND WITH A FOX A BINDING SEQUENCE IN THE CENTER OF IT AND THEN COMPACTED THE STRUCTURES WITH LINK OR HIS TOMORROW AND FOUND THAT THEY WERE RESISTENT TO NUCLEASE SUCH AS DNA. BUT WHEN YOU ADD PURIFIED FOX PROTEIN THAT CREATED A HYPER SENSITIVE SITE IN THE MIDDLE OF THE ARRAY RIGHT WHERE THE FOX BINDING SEQUENCES ARE. AND THE FOX BINDING SEQUENCE DEPENDENT. SO NOTABLY, THIS ACTIVITY OF CREATING A HYPER SENSITIVE SITE COULD OCCUR ON CELL CONTINUE THAT WAS ASSEMBLED FROM HISTONES THAT WERE ISOLATED FROM LIVER BUT ALSO HISTONES WE PREPARED FROM E COLI WERE COMMON IN HISTONE AND STILL CREATED THIS HYPER SENSITIVE SITE SHOWING THAT YOU DIDN'T NEED A HISTONE MODIFICATION FOR THESE FACTORS TO BIND IN OPEN CHROMATIN. THE TERMINAL DOMAIN INDEPENDENTLY HAD THE ABILITY TO BIND TO THE HISTONE OCULAR COMPONENTS AND WERE RESISTENT TO .6 MOLAR SALE. IF YOU TERMINATE THAT YOU NO LONGER HAD SUFFICIENT CHROMATIN OPENING BY THE TRANSCRIPTION FACTOR. SO PUTTING ALL THIS DATA TOGETHER IT'S BEEN CONSISTENT WITH THIS MODEL HERE. THE FACTOR CAN BIND TO ITS TARGET SITE ON A NUCLEOSOME AND IT CAN LEAH CROSS THE GLOBULAR DOMAIN A PORTION, A C TERMINAL PORTION OF THE PROTEIN. NOW KEEP THIS IN MIND ALONG WITH THE PREDICTED HIGHER ORDER STRUCTURE OF THE NUCLEOSOME ARRAY AS DETERMINED BY TIM RICHMOND'S LAB. WHAT REALLY STABILIZES THIS STRUCTURE IS ARE THE NUCLEOSOMAL ACTIONS THAT OCCUR. WHAT WE SUGGEST IS BY ESSENTIALLY STICKING A FINGER, IF YOU WILL, BETWEEN THE NUCLEOSOMES AS THEY WOULD OCCUR IN THE STRUCTURE, YOU ESSENTIALLY CAUSE ONE NUCLEOSOME TO BE EXPOSED. THIS WOULD ALLOW NOT JUST NUCLEASES TO GAIN ACCESS IF YOU WILL BUT OTHER TRACK SCRIPTION FACTORS AND OPEN UP THE CHROMATIN. OKAY. SO PUTTING ALL OF THIS TOGETHER. WE BASICALLY HAVE COINED THE TERM PIONEER TRANSCRIPTION FACTORS FOR PROTEIN THAT HAVE THE BIOCHEMICAL CAPACITY ON THEIR OWN TO BIND THEIR TARGET SITE ON A NUCLEOSOME AND WHERE WE'VE OBSERVED THAT IN DEVELOPMENT, THEIR BINDING PRECEDES THE BINDING OF OTHER FACTORS. IN THE TIME SINCE THIS MANY OTHER LABS NOW HAVE SEEN PIONEER TYPE ACTIVITIES EVEN IN DIFFERENTIATED CELLS WHERE FOX FACTOR CELLS OR OTHER KINDS OF FACTORS CAN BE BOUND TO CHROMATIN AT A SILENT TEAM AND ALLOW A NUCLEAR RECEPTOR, OR MOAN RECEPTOR TO BIND THE CHROMATIN THERE OR ACTIVATE TRANSCRIPTION. NOW JERRY WORKMAN'S LAB IN THE 90'S SHOWED THAT IN FACT MANY TRANSCRIPTION FACTORS OR MOST OF THEM LACK THE INHERENT ABILITY TO BIND A TARGET SITE IN THE NUCLEOSOME WHICH YOU COULD SEE INHERENT COOPERATIVE EFFECTS WHEN YOU HAD MULTIPLE FACTORS BINDING TOGETHER, YOU COULD OVERCOME THE ABILITY OF HISTONES TO PREVENT THEIR BINDING AND YOU COULD NOW GET CHROMATIN BINDING. YOU HAD SIX BINDING SITES FOR VARIOUS FACTORS COMING ON THERE. THIS WOULD REQUIRE SIMULTANEOUS BINDING WITH THE OTHER FACTORS. SO WHAT'S THE UTILITY OF THIS KIND OF SYSTEM? WELL IF YOU HAVE THESE PIONEER FACTORS AS I NOTED, THEY COULD HAVE AN ACTIVE ROLE IN TERM OF PHYSICALLY OPENING THE CHROMATIN AND ALLOWING OTHER EVENT TO OCCUR THERE. BUT THERE COULD ALSO BE A PASSIVE ROLE WHICH IS TO SPEED INDUCTION. THAT IS IF YOU HAVE THE PRIOR BINDING OF A COUPLE FACTORS BUT NOT ALL OF THEM NEEDED TO STIMULATE TRANSCRIPTION, THAT BY HAVING PREBINDING OF A FEW, THEN IF YOU BRING IN A COUPLE MORE, YOU CAN MORE RAPIDLY JUST BRING IN A FEW RATHER THAN ALL OF THESE AND TURN ON A GENE. OKAY. SO WE WANTED TO BRING THESE PRINCIPLES TO BEAR NOT JUST IN TERMS OF CELL PROGRAMMING AS I'VE BEEN TALKING ABOUT BUT CELL REPROGRAMMING. AND ONCE AGAIN, INVESTIGATING THE SYSTEM THAT YAMANAKA HAD NICELY DEFINED WHERE YOU TOPICALLY EXPRESS THESE FACTORS IN FIBROBLASTS AND ULTIMATELY GENERATE PLURIPOTENT STEM CELLS. SO THE QUESTIONS WE WANTED TO ASK WERE FIRST, DO THESE FACTORS ACT AS PIONEERS. DO ANY OF THEM, DO ALL OF THEM. ANALOGOUS TO FOX. IF YOU JUST LOOK AT HOW THESE FACTORS CAN BIND TO DNA, WE'VE GOT SOME CLUES. SO THESE CAN BIND DNA LEAVING A FREE DOMAIN ON THE OTHER SIDE. THAT WOULD ALLOW THE FACTORS TO BIND NUCLEOSOMAL DNA OR NAIVE CHROMATIN, IF YOU WILL. THE SAME FOR TAIL F4. BY CONTRAST C -- AND ALSO INVESTIGATE CHROMATIN STATES THAT WOULD AFFECT BINDING AND TRY TO UNDERSTAND WHAT IT IS THAT MAKES IT SO DIFFICULT TO CREATE IPS CELLS. LET'S A LOT OF WORK IN THIS SYSTEM THAT'S GONE ON BEFORE WE GOT THERE FOR SURE. SO RICK YOUNG'S LAB WAS AMONG THE FIRST AND LENOIR BOYER IN HIS LAB TO MATCH WHERE THESE TRANSCRIPTION FACTORS OCCUR IN THE PLURIPOTENT CELLS. VARIOUS OTHER LABS HAVE MAPPED PLURALLY POTENT CELLS FOR THE BINDING EVENTS OF THE FOX -- LAB STUDY IN THE MOUSE CELLS THAT WERE PARTIALLY PROGRAMMED AFTER SEVERAL WEEKS OF ININDUCING THIS PROCESS ARE WOULD THESE FACTORS WERE BINDING AND FOUND MANY SIMILARITIES TO THAT SCENE IN EMBRYONIC STEM CELLS WITH SOME DIFFERENCES. WE LOOKED AT THE VERY EARLIEST EVENT. WE FOUND OUT USING A LENGTHY VIRUS SYSTEM TO CONDITION AND ACTIVATE THESE FACTORS BY WESTERN BLOTTING AND SO FORTH THAT THE FACTORS WERE INDUCED IN THESE CELLS BY 48 HOURS. 48 HOURS ALSO IS THE TIME, IS A TIME PRIOR TO WHEN YOU WOULD TRANSFER THE FIBROBLAST TO EMBRYONIC STEM CELL CULTURE CONDESIRE. THEY'RE STILL BEING GROWN AS FIBROBLASTS. WE'RE REALLY LOOKING AT THE INITIAL MASSES OF THESE FACTORS ON A GENOME. I WOULD LIKE TO POINT OUT THAT IN ALL OF THESE REPROGRAMMING TYPE EXPERIMENTS, YOU VERY TYPICALLY GET A PROFOUND AMOUNT OF APOPTOSIS OBSERVED IN THE CELL CULTURE. SO WHAT'S UNDERLYING THAT? IN ANY CASE UNDER THE CONDITIONS THAT WE'RE USING, WE'RE ABLE TO GENERATE IPS CELLS THAT WERE ALKALINE PHOSPHATE POSITIVE SHOWN HERE AND EXPRESS THE KNOWN PLURALLY POTENCY FACTORS AND THEY CAN DIFFERENTIATE INTO MULTIPLE GERMAN LAYERS WITH A BODY ASSAY AND SO FORTH. SO WE DID CHROMATIN IMMUNOPRECIPITATION SO BASICALLY YOU PREPARE CHROMATIN THAT'S BEEN CROSS LINKED THE TRANSCRIPTION FACTORS TO THE GENOME AND CHROMATIN AND WE DID EXPENSIVE -- AS SEEN HERE. WE DID EIGHT REPLICATES BIOLOGICAL REPLICATES OF THESE CHROMATIN IP'S AND THEN PULLED DOWN FOR EACH OF THESE, THE FOUR FACTORS AND MAPPED WHERE THEY GO IN THE GENOME. USING HIGH FREQUENCY TECHNOLOGY. AND WE HAD VERY CLEAN ANTIBODIES FOR THIS STUDY. SO I'M GOING TO SKIP A LOT OF THE DETAILS AND BASICALLY SUMMARIZE THAT WE SAW MANY BINDING EVENTS WHEN YOU FIRST EXPRESS THESE FACTORS IN THE GENOME. SO EXPRESSED, THEY BIND TO MANY MANY SITES AS SEEN HERE. AND THE BINDING IS LARGELY DRIVEN BY WHERE THERE'S A MOTIVE UNDERNEATH THE PEAK THAT YOU WOULD ASSIGN. AND WHAT I WOULD LIKE TO POINT OUT IS THAT WHAT REALLY STOOD OUT FROM THE VERY BEGINNING IS IF YOU LOOK AT ONE OF THESE FACTORS BINDING EVENTS SUCH AS ACT4 YOU CAN SEE THE OTHER FACTORS COINCIDE AT MANY OF THE OCT4 BINDING EVENTS AND THE SAME FOR ALL THESE OTHERS. THE MAIN POINT IS THEY'RE GOING TOGETHER TO A LOT OF SITES. NOW, WHERE ARE THEY GOING? THEY'RE GOING TO VARIOUS GENES INITIALLY AND THEY'RE GOING TOGETHER TO GENES INVOLVED IN. PROMOTE REPROGRAMMING SUCH AS THIS MIR CLUSTER DISCOVERED BY ED MORE SEE AT U PENN AND AT THE -- THEY TARGETED GENE THAT NEED TO BE DOWN REGULATED FOR THE EPITHELIAL TRANSITION TO GENERATE THE ES-TYPE MORPHOLOGY FROM THE FIBROBLAST. THEY DON'T BIND EVERYWHERE TOGETHER. WE FOUND THAT AT THE E CAT GENE IT'S KLF BINDING THERE AND THAT AGREED BEAUTIFULLY WITH THIS PAPER SHOWING THE KLF WAS UNIQUELY IMPORTANT FOR DOWN REGULATING -- I'M SORRY, UP REGULATING. SO WE ALSO SAW MANY GENES THAT WERE PRO APOPTOTIC BEING BOUND SUCH AS P53. IF YOU DO A GENE ONCOLOGY ANALYSIS, THERE ARE MANY HUNDREDS OF GENES THAT HAVE BEEN CATEGORIZED AS BEING INVOLVED IN PROGRAM CELL DEATH THAT ARE INITIALLY BOUND BY THESE FACTORS. SO IF I COULD EDITORIALIZE FOR A MOMENT, I THINK IT'S MAYBE POSSIBLE THAT THE GENES THAT SENSITIZE THE CELL TO A NEED FOR APOPTOSIS ARE IN SOME SORT OF SPECIAL OPEN CHROMATIN THAT WE HAVE NOT FIGURED OUT. IF THERE'S AN ECTOPIC EXPRESSION OF TRANSCRIPTION FACTORS THAT YOU GET RID OF THOSE CELLS INSTEAD OF REPROGRAMMING THE CELL IN A TISSUE WHICH COULD RESULT IN A DELETERIOUS METAPLASIA. IN ANY CASE, IF WE PUT A LOT OF THIS DATA, THESE DATA TOGETHER, THE BINDING EVENTS WHERE EACH OF THE FACTORS OCCUR WITH RESPECT TO THE TRANSCRIPTION START SITE AND LOOK AT FROM 5KB UP STREAM TO THE START SITE MINUS 50KB TO 5KB FROM MINUS 500KB TO 50KB. IT'S CLEAR THAT THERE'S EXTENSIVE AND PREDOMINANT BINDING TO DISTAL ELEMENTS IN THE GENOME AS OPPOSED TO TRANSCRIPTION START SITES AND PROMOTERS. BY CONTRAST EMBRYONIC STEM CELLS THERE'S A LOT OF BINDING PROMOTERS PARTICULARLY BY C MYC. IN FACT RICK YOUNG'S LAB WAS FIRST TO POINT OUT THAT C MYC FORMS A DIFFERENT NETWORK IN PLURALLY POTENT CELLS THAN IT DOES FOR OCT AND KLF WHICH ARE DISTAL ELEMENTS IN THE CELLS. INITIAL BINDING EVENTS IN THE GENOME ARE IN FACT QUITE DIFFERENT THAN THEY ARE IN STABLE EMBRYONIC STEM CELLS. IF WE COMPARE THE BINDING EVENTS TO FUNCTIONAL ENHANCERS THAT HAVE BEEN IDENTIFIED AS PART OF THIS VISTA PROJECT. IN FACT THEY'RE EXTENSIVELY BOUND. IT'S NOT JUST RANDOM ELEMENTS WHERE THEY'RE BOUND, WE FOUND UP TO ABOUT 60% OF THE SITES WHERE THEY'RE BOUNDS IN THESE HUMAN CELLS ARE CONSERVED IN THE MOUSE. AGAIN THEY BIND TO A LOT OF FUNCTIONAL ENHANCERS SO THESE ARE NOT JUST NON-SPECIFIC EVENTS, BINDING WOULD APPEAR TO BE CONSERVED FUNCTIONAL DISTAL ENHANCER ELEMENTS. NOW WHAT ABOUT GENE EXPRESSION CHANGES? WELL, IF WE FOCUS ON THE BINDING EVENTS THAT OCCUR AT TRANSCRIPTION START SITES OR NEAR THEM AND HOW THEY MAY OR MAY NOT CORRELATE WITH GENE EXPRESSION CHANGES. ALEX-LAB RECENTLY PUBLISHED TRANSCRIPTIONAL CHANGES THAT OCCUR DURING REPROGRAMMING. IF YOU TAKE THE FINE BLASTER GENE EXPRESSION PROFILE AS SHOWN HERE TO GENES THAT ARE OFF VERSUS GENES THAT ARE ON IN MORE HIGHLY ON, THE GENE THAT COME UP DURING EARLY STAGES OF REPROGRAMMING ARE PREDOMINANTLY THE GENES THAT ARE ALREADY ON. IN OTHER WORDS YOU JUST BOOSTING THE EXPRESSION OF GENES THAT WERE ALREADY OPEN FOR BUSINESS, IF YOU WILL. AND IF YOU LOOK AT WHERE THE OCT, SOX, YOU CAN SEE MYC AND KLF4 ARE TARGETED PROMOTERS ALREADY ACTIVE. ANOTHER WAY OF SAYING THIS IS THAT MANY, MOST OF THE GENES THAT ARE TARGETED BY THESE FACTORS EARLY AT ENHANCERS DO NOT AT 48 HOURS RESULT IN PROMOTER BINDING OR TRANSCRIPTIONAL CHANGES. IN OTHER WORDS MOST OF THESE GENES ARE STILL SILENT WHERE THEY'RE BOUND AT 48 HOURS. SO THIS COMES TO THE FIRST IMPEDIMENT, IF YOU WILL, TO THE REPROGRAMMING PROCESS, WHICH IS THAT MANY OR MOST OF THE PROMOTERS TO WHICH THESE FACTORS ARE BOUND ARE NOT RESPONDING AT THIS EARLY TIME POINT. SO THERE'S SOMETHING THAT'S GOT TO OVERCOME THE INABILITY OF PROMOTERS TO RESPOND. AND WE DON'T YET KNOW WHAT THAT IS. RIGHT. SO THE QUESTION IS, WHAT IS THE ROLE OF SO-CALLED VIE -- METHYLATION INDICATIVE OF AN ACTIVE POTENCY IF YOU WILL IN AN H3K27 TRY METHYLATION INDICATIVE OF INACTIVE OR BEING RESTRAINED. THAT WAS LARGELY CHARACTERIZED, THAT WAS INITIALLY CHARACTERIZED IN AN EMBRYONIC STEM CELLS AND WE'RE LOOKING HERE IN FIBROBLASTS. AND WE DID NOT SEE, WELL WE HAVEN'T CHARACTERIZED WHAT CHROMATIN CHANGES OCCUR IN RESPONSE TO THESE FACTORS BINDING BUT AS YOU'LL SEE IN A MOMENT, WE HAVE NO EVIDENCE FOR TARGETING, IF YOU WILL, TO ANY DOMAINS THAT MIGHT BE BINDING. SO OKAY. SO LET'S GET BACK TO THE BUSINESS AT HAND, THE ORIGINAL QUESTION. WHICH IS WHAT IS IT THAT MAY ALLOW THESE FACTORS TO BIND CHROMATIN OR CAN THEY BIND NEW CHROMATIN NAIVE CHROMATIN ON THEIR OWN AS ACTIVE PIONEERS. SO IT'S A FIRST STEP TOWARDS DOING THIS. WE DOWNLOADED SOME DATE THAT CAME OUT OF THE N CODE PRODUCT FOR RICHARD'S LAB AT THE UNIVERSITY OF WASHINGTON. THEY HAD DONE DNA SENSITIVITY ASSAYS ON THE SAME FIBROBLAST TUMOR FIBROBLAST CELLS WE WERE WORKING WITH FIBROBLAST. AND WE RANK ORDERED THE SENSITIVITY LEVELS OF ALL OF THE 188,000 BINDING EVENTS THAT WE MAPPED AT THESE BINDING SITES. BUT IN THE PREEXISTING BY BLASTS, IN OTHER WORDS BEFORE WE INDUCED THERE WERE ALL THESE SITES WHERE THE FACTORS WILL BIND WHERE WE INDUCE THEM, WHERE ARE THESE HYPER SENSITIVE SITES. SO IN FACT HYPER SENSITIVITY IS WAY OVER HERE AND MODERATE SENSITIVITY DNA GOES TO ABOUT HERE. AND THIS IS COMPLETELY CLOSED. THIS IS BASICALLY SILENT CHROMATIN. THE QUESTION IS WHERE DO THESE BINDING EVENTS MAP. AND THE ANSWER IS, FOR MYC, THERE'S A LOT OF BINDING TO ALREADY OPEN SITES OF THE CHROMATIN. FOR KLF, SOME OF THAT, THE ENCLOSED CHROMATIN AND OCT4 AND FOR THE MOST PART SOX 2 ARE BINDING OUT IN THE CLOSED CHROMATIN DOMAIN THAT ARE NOT HYPER SENSITIVE. IN OTHER WORDS YOU DON'T NEED A SITE TO BE HYPER SENSITIVE FOR THE FACTORS TO BIND. IMPORTANTLY, MANY OF THE GENES REQUIRED FOR CONVERSION TO PLURALLY POTENCY ARE OUT IN THIS REGION AS YOU MIGHT EXPENSE IF FIBROBLASTS ARE GOING TO KEEP THESE GENES OFF. WHAT ABOUT HISTONE MODIFICATIONS. SO WE DOWNLOADED MANY AND WE'VE LOOKED AT MORE AND THE ANSWER IS THE SAME. AND THAT IS THAT THERE ARE NO KNOWN HISTONE MODIFICATIONS THAT OCCUR OUT IN THIS CLOSED CHROMATIN REGION THAT STRONGLY OR THAT CORRELATES SIGNIFICANTLY WITH THE BINDING EVENTS FOR THESE FACTORS. AND WE LOOKED AT IT A WHOLE BUNCH OF WAYS. THERE ARE INDEED AS YOU MIGHT EXPECT AT THE OPEN CHROMATIN SITES MARK'S TYPICAL OF OPEN CELL CONTINUE PROMOTERS AND SO FORTH WHERE THE FACTORS WILL BIND. SO THEY'RE HAPPY TO BIND IF THERE'S AN OPEN SITE BUT THEY'RE BINDING PREDOMINANTLY OCT, SOX AND KLF AT SITES THAT ARE CLOSED. YOU CAN SAY WELL MAYBE THERE ARE COOPERATIVE EFFECTS BETWEEN THESE FACTOR AS I DESCRIBED EARLIER. WE PARTITION THE DATA TO JUST LOOK AT SITES THAT ARE BUMPED BY ONLY ONE OF THE FACTORS. AND IF YOU DO THAT, IT RESOLVES EVEN FURTHER. SO MYC IS JUST ABOUT EXCLUSIVELY CONFINED TO BINDING TO PREOPEN DOMAINS BY ITSELF. AND SOX 2 IS ESSENTIALLY GOING TO CLOSED CHROMATIN. THE SPIKES ON THE OTHER SIDE AND THE GAPS HERE ARE WHERE THEY REALLY LIKE TO BIND TOGETHER AT PARTICULAR SITES. SO PUTTING THIS TOGETHER, OOPS, LET ME GO BACK. IT INDICATES THAT THE SOX, OCT AND KLF FACTORS ARE PIONEERS AND THEY SEE MYC WITH THE DEPENDENT UPON THOSE FOR ENTERING CHROMATIN. SO WE WANTED TO TEST THAT DIRECTLY AND SET UP THE FOLLOWING EXPERIMENT. WHERE WE TOOK THE SAME HUMAN FIBROBLASTS AND NOW INDUCED EITHER MYC ALONE, FOCUS, OCT OR KLF ALONE. ONLY WHEN WE HAVE ALL FOUR OF THE FACTORS DO WE GET NICE -- BUT WE LOOKED AFTER JUST TWO DAYS. IF YOU CONSIDER THE FIBROBLASTS IN WHICH MYC HAS BEEN INDUCED AND LOOK AT THE SITES WHERE ALL FOUR OF THEM CAN BIND, IF YOU DON'T HAVE OCT, SOX AND KLF THERE, MYC DOES NOT BIND. BUT IF YOU HAVE OCT, SOX AND KLF THERE AS SHOWN HERE NOW YOU GET GOOD BINDING BY MYC. AND THERE'S NO NON-SPECIFIC EFFECT OF MYC ENTERING SITES WHERE JUST THESE HAVE BEEN MAPPED WHEN YOU ADD THE OCT, SOX AND KLF FACTORS. THIS SHOWS THESE FACTORS ACT AS PIONEERS FOR MYC. THESE HAVE BIONEER ACTIVITY AND THIS FACTOR DOES NOT NOT AND WAS DEPENDENT UPON THESE TO ENTER THESE CLOSED CHROMATIN SITES. YES? >> [INDISCERNIBLE] THERE ARE MAJOR DIFFERENCE AND I'M GOING TO TALK ABOUT THAT NEXT. GREAT QUESTION. THEY'RE OVERLAPPED WITH OCT, SOX AND KLF BINDING. IT'S A VERY STRIKING MARKED DIFFERENCE I'LL TALK ABOUT. THANKS. OKAY. SO TO SUMMARIZE, SOX, OCT AND KLF ARE NOT TARGETED BY A HISTONE MODIFICATION STATE OR CHROMATIN HYPER SENSITIVITY AND THEY ENABLE C MYC BINDING TO CHROMATIN. SO LOOKING AT THIS, WE CAN NOW TALK ABOUT SOME OF THE EVENTS THAT SEEM TO BE IMPEDIMENTS AND THAT IS YOU'VE GOT A LOT OF BINDING OF THESE FACTORS OUT TO DISTAL ELEMENTS. AND THERE'S MANY OF THE PROMOTERS AT THESE GENES ARE NOT YET ACTIVATED SO THAT'S KIND OF A BLOCK. WE KNOW THAT WE'RE ACTIVATING A LOT OF AGAINST THAT ARE PROAPOPTOTIC AND THAT'S A BLOCK. AND THEN AS I WAS JUST ASKED, IN FACT THERE'S NOT ONLY A CHAIN FROM MYC GOING TO PROMOTERS WHEN YOU GET PLURAL E POTENT CELLS BUT THE OCT, SOX AND KLF FACTORS CAN DISTRIBUTE TO OTHER GENES. I WOULD LIKE TO TURN THIS AROUND AND THINK ABOUT IT THE OTHER WAY WHICH IS IF WE KNOW IN ES CELLS THESE GUYS ARE GOING TO WIND UP AT THESE SITES. HOW COME WE SEE ABSOLUTELY ZERO BUT NOT JUST A SQUEAK BUT NADA, NOTHING OUT AT THESE DOMAINS. SO WHAT I'M ABOUT TO TELL YOU WAS DONE INDEPENDENTLY FROM A NEW STUDY THAT JUST CAME OUT IN RUDOLPH --'S LAB THAT BEARS UPON THIS QUESTION. SO THEY WERE NOTING AND INVESTIGATING THE POINT THAT WHEN YOU TOPICALLY INDUCE THESE FACTORS IN FIBROBLASTS, ONLY A FRACTION OF THE CELLS WIND UP BEING REPROGRAMMED. AND BY LOOKING AT THE RNA'S EXPRESSED IN INDIVIDUAL CELLS THAT DEPICT AT VARIOUS TIMES HERE, YOU BASICALLY FOUND THAT THERE WAS STOW CASTIC ACTIVATION OF GENE EXPRESSION. SOME CELLS, SOME OF THE TARGET GENES WOULD BE TURNED ON AND OTHERS EARLY ON, OTHERS WOULD BE TURNED ON AND SO FORTH. THERE WAS A SORT OF SCATTER SHOT EXPRESSION PATTERN AND THERE ARE RARE CELLS INDICATIVE OF TRUE PROGRAMMING. AT SOME POINT YOU ACTIVATED A CASCADE THAT WAS AS THEY CALLED DETERMINISTIC IN THE SENSE THAT ONCE THAT CASCADE GOT ACTIVATED, THEN ELICIT EVENTS THAT WERE SYNCHRONOUS IN THE CELLS AND LED TO FULL REPROGRAMMING. AT THE TOP OF THEIR CASCADE WAS SOX 2. IN THIS FUNCTIONAL CASCADE THAT THEY FOUND. ANY OTHER FACTORS WERE DOWN HERE. OKAY. SO WAY PRIOR TO THIS, THOUGH -- A POST DOC IN MY LAB WAS LOOKING AT HIS DATA. AND IT'S GREAT TO HAVE A BIOINFO MATTACIST AND A STATISTICIAN WORKING FOR YOU TO STAY GIVE US A PLOT, D VALUES AND FDRs AND ALL THAT STUFF. BEING BIOLOGISTS THERE'S NOTHING LIKE LOOKING AT THE DATA AND GETTING A SPREADSHEET, A SCREEN SHOT ON A BROWSER AND JUST LOOKING TO SEE WHAT DO THESE HISTONE MARKS LOOK LIKE, YOU KNOW. MAYBE WE'RE TOO OLD AND WE'RE STILL INTERESTED IN DOING THAT. AT ANY RATE, HE SAW SOMETHING REALLY AMAZING WHEN HE DID THAT AND STARTED ZOOMING OUT TO LOOK AT WHOLE CHUNKS OF THE CHROMOSOME. WHEN HE TOOK A CHROMOSOMAL VIEW OF THE BINDING EVENTS OF THESE FOUR FACTORS. SO THIS IS A WHOLE CHUNK OF HUMAN CHROMOSOME 3. I'M LOOKING AT ABOUT A QUARTER OF IT AT A TIME. THIS IS TEN MILLION BASE PAIRS GOING ACROSS. YOU SAW THESE HUGE GAPS WHERE THE FACTORS WERE NOT BOUND. NOW THIS IS INPUT NORMALIZE. INPUTS ARE FLAT ACROSS HERE. SO THESE LARGE GAPS, HERE'S HUMAN CHROMOSOME 20 WHERE THE FACTORS WERE NOT BOUND. GETTING TO YOUR QUESTION. BUT IF WE LOOKED IN EMBRYONIC STEM CELLS FROM PUBLISHED DATA, IN FACT AT MOST OF THESE SITES THEY GET RESOLVED. THEY'RE NICELY BOUND. SO WE CALL THESE DIFFERENTIALLY BOUND REGIONS. THIS REPRESENTS ANOTHER IMPEDIMENT TO CONVERSION AND GENE ACTIVATION. ACTIVATION OF THE PLURALLY POTENCY PROGRAM. HAVING DISCOVERED THESE WE DECIDED LET'S GET QUANTITATIVE. WE GOT GREG INVOLVED AND HE MADE THE FOLLOWING PLOT E FOUND THERE WERE THERE ARE HOLDS WE SET. THERE WERE 264 OF THESE IN THE HUMAN GENOME, AN AVERAGE OF TWO MILLION BASE PAIR CHUNKS THAT HAD SIGNIFICANTLY DIMINISHED BINDING OF ALL FOUR OF THE FACTORS WHERE THERE WAS INCREASE BINDING AT 2 MILLION BASE PAIRS OF FLANKING REGION ON EITHER SIDE. IT WAS FOR THE MOST PART FLAT ACROSS THOSE DOMAINS IN TERMS OF ALLOWING BINDING. WE ALSO LOOKED AT THESE REGIONS AS THEY WERE IN THE STUDY I TALKED ABOUT EARLIER FROM MEISNER'S LAB WHERE THEY LOOKED AT THE TEMPORAL INDUCTION OF GENE EXPRESSION IN THE FIRST FEW DAYS OF CONVERSION. AND THESE REGIONS BASICALLY ARE SILENT THROUGHOUT THAT INITIAL DOMAIN. THESE GENES ARE REFRACTORY TO FACTOR BINDING AND TRANSCRIPTIONAL ACTIVATION. WHEREAS IF YOU LOOK AT FLANKING DOMAINS YOU SEE THIS BUMP HERE IF YOU WILL AND TRANSCRIPTIONAL EXPRESSION. EVENTUALLY, THESE REGIONS DO EXHIBIT TRANSCRIPTIONAL ACTIVITY IN IPS AND IN ES. AND NOTABLY I SHOULD HAVE ON HERE SOX 2 FALL IN THESE DOMAINS. SO SOX 2, ONE OF THE GENES THAT -- LAB HAD FOUND WAS A LATE GENE TO GET ACTIVATED. WHEN IT FINALLY GOT ACTIVATED AND THE BALL WAS ROLLING TO PLURALLY POTENCY FALLS IN ONE OF THESE DOMAINS. I SHOULD POINT OUT THESE DOMAINS ARE GENERALLY ENRICHED FOR REPETITIVE ELEMENTS AND THEY'RE GENERALLY GENE POOR BUT THEY STILL HAVE LOTS OF GENES AND WE'LL TALK ABOUT THAT IN A MINUTE. SO IT WOULD SEEM THAT THESE ARE HETEROCHROMATIC REGIONS THAT MIGHT BE HUGE BLOCKS SHUT DOWN. THEY ARE DNA RESISTENT BUT AS I SAID THE PIONEER FACTORS COMBINING H RESISTENT CHROMATIN SO IT CAN'T BE JUST THAT. SO THESE DOMAINS HAVE ASSOCIATION WITH LAMIN WHICH -- LAB HAS SHOWN IS ASSOCIATED WITH BEGAN SILENCING. BUT IF YOU LOOK AT THE LAMIN RICH DOMAIN, THERE'S MANY MORE OF THEM THAN THERE ARE THESE DBRs. SO WE STARTED LOOKING AT HISTONE MODIFICATIONS AND WE MAP THOSE ACROSS THESE AND MANY MORE NOW THAT THE N CODE DATA CAME ON-LINE. MORE ACROSS THESE DOMAINS AND ONE OF THEM STOOD OUT AND THAT WAS HISTONE H3 LYSINE 9 TRY METHYLATION. IT'S REALLY HIGH OVER THESE DOMAINS COMPARED TO ALL OF THESE OTHER MODIFICATIONS. SO THAT WAS REALLY NEAT BECAUSE THE TRY METHYLATION HAS BEEN ASSOCIATED WITH HETEROCHROMATIN AND HETEROCHROMATIC FEATURES FOR A LONG TIME. MOST OF IT IS CENTROMERES AND TELOMERES AND HIGHLY REPEATED SEQUENCES OF THE GENOME AND THERE HASN'T BEEN AS MUCH FOCUS ON HOW THOSE DOMAINS MAY PLAY INTO DEVELOPMENTAL GENE REGULATION. SO TO TEST THE HYPOTHESES THAT THESE KINDS OF, THIS MODIFICATION COULD PLAY A ROLE IN RESTRICTING PIONEER FACTOR BINDING AND REPROGRAMMING, WE DEVELOPED A KNOCK DOWN PROTOCOL WHERE WE TOOK SIRNASE TO VARIOUS OF THE ENZYMES THAT ELICIT H3K9 TRY METHYLATION. TO GET STABLE KNOCK DOWN WE HAD TO DO TWO CYCLES OF KNOCKING DOWN TO FINALLY GET TO A POINT WHERE WE COULD OBSERVE LOWER LEVELS OF H3K9 TRY METHYLATION. AND THEN WE ALSO DID CHROMATIN IMMUNAL PRECIPITATION AT 12 SITES WITHIN THESE DBRs AND SHOWED THERE WAS A DEMINUTION OF RESIDENT K9 TRY METHYLATION WITHIN THE DBRs WHEN WE DID THAT. THEN WE ASKED IF NOW THESE FACTORS COULD HAVE BINDING IN THOSE DOMAINS, INCREASED BINDING. WHEN WE COMPARE THE NON-TARGETING SRNA TO THE VARIOUS COMBINATIONS OF TARGETED SRNAs WE SAW INCREASED BINDING OVER 18 SITES SELECTED AND PULLED HERE WITHIN THESE DIFFERENTIALLY DOWNED REGIONS. AND NO NON-SPECIFIC EFFECTS SUCH AS JUST BULKING UP SOX 2 FOR WHATEVER REASON AT FLANKING REGIONS. AND WE SAW SIMILAR RESULTS FOR OCT4. NOW FINALLY WE ASKED IF THIS HAD ANY EFFECT ON REPROGRAMMING. AND I REMEMBER IN 2011 GETTING THIS FRANTIC E-MAIL FROM MY POST DOC WHO SAID THAT NORMALLY WE DON'T SEE COLONIES COMING UP IN THIS REPROGRAMMING PROTOCOL UNTIL TWO WEEKS. WE FINALLY START TO SEE COLONY COME UP IN THE HUMAN SYSTEM BUT WITHIN SEVEN DAYS ARE HE HAD REALLY NICE COLONIES STARTING TO COME UP WHEN HE WAS KNOCKING DOWN VARIOUS OF THESE HISTONE LYSINE 9 METHYL TRANSFERASES. WE SAW INDUCTION OF THE REPROGRAMMING PROCESS AS WELL AS IF YOU STAIN COLONIES OUT BIONEERLY A MONTH AN INCREASED NUMBER OF REPROGRAMMED CELL COLONIES COMING UP. SO TAKING THESE DATA TOGETHER, IT INDICATES THAT WHILE PIONEER FACTORS HAVE SPECIAL PROPERTIES IN TERMS OF BEING ABLE TO BIND NAIVE CHROMATIN DOMAINS AND OPEN UP GENES AND REPROGRAM CELLS, YOU'RE NOT SUPER HEROES IN A SENSE THERE'S NOT CRYPTO NIGHT OUT THERE -- IN THE PROCESS OF GENERATING IPS CELLS YOU HAVE TO LOSE THE INHIBITORY DOMAIN, NOW ALLOW THE FACTORS TO BIND ELSEWHERE. HERE'S THE BLACK HERE THAT'S UNDER 2 MEGA BAIT REGIONS WHERE THEY ARE NOT BINDING, THESE ARE PEAKS. AND THEN THE SOX 2 LOCUS IS IMBEDDED DEEP WITHIN THIS DBR AS SEEN THERE. AND SO THAT FITS BEAUTIFULLY FROM THE DATA OF THE LAB THAT SOX 2 IS A LATE EVENT IN FINDING TO CRACK THAT OPEN SOMEHOW, YOU KNOW ELICIT MORE SYNCHRONOUS REPROGRAMMING WITHIN THE CELLS. TAKING IT ALTOGETHER WE KNOW INITIALLY THIS PIONEER FACTOR BINDING AT DISTAL ELEMENTS. THIS ENABLES MYC BINDING IN A WAY THAT IT ISN'T BINDING EARLY ON OR LATER IN REPROGRAMMED CELLS. THERE'S APOPTOSIS. YOU'VE GOT TO UNDERGO THE MA SECRETARY MULL EPITHELIAL TRANSITION AND THE DBRs. FINALLY WAY OUT HERE YOU CAN GET REPROGRAMMED CELLS. SO THESE DATA WERE JUST PUBLISHED A FEW WEEKS AGO. I'D LIKE TO TELL YOU IN THE LAST COUPLE MINUTES SOME OF THE THING WE'RE DOING NOW. SO IF YOU THINK ABOUT THE DBRs AND THE FIBROBLASTS, YOU KNOW, AGAIN THE FIBROBLASTS PREMIUMABLY DIDN'T EVOLVE SO THAT HUMANS LIKE US CAN'T REPROGRAM THEM. PRESUMABLY THESE DOMAINS ARE THERE FOR SOMETHING ELSE. AND SO WE ASKED WHAT KIND OF GENES FALL IN THESE DBRs AND FIBROBLASTS. THERE ARE ABOUT 530 OR SO GENES THAT FALL IN THERE. AND FULLY ONE THIRD OF THEM IN THE FIBROBLAST CELL HAVE TO DO WITH CELL ADHESION. THE FIBROBLASTS ARE MOTILE CELLS. THEY DON'T STICK TO THEIR NEIGHBORS LIKE A LIVER CELL WOULD. AND SO IT WAS REALLY INTERESTING TO SEE THAT THESE DBRs WOULD STAND GENES THAT YOU JUST PLAIN NEVER WANT TO BE ACTIVATING PRESUMABLY IN A FIBROBLAST. SO LET'S TAKE A LOOK AT ONE OF THESE. AGAIN A BROWSER VIEW. SO HERE'S WHERE THE DBR'S MAP OVER THIS DOMAIN. IN OTHER WORDS THIS WHOLE REGION IS GREATLY REDUCED FOR OCT, COX AND KLF BINDING WITH THE FIBROBLAST. THIS IS THE H3K9 TRY METHYLATION MAPS SPANNING MOST OF THIS DOMAIN. WHAT'S REALLY NEAT IS THERE'S A WHOLE CLUSTER OF GENES CALLED DESMO COLLINS WHICH ARE -- LIKE GENES AND THOSE FALL UNDER THIS DOMAIN OF H3K9 TRY METHYLATION. SO WHEN THE N CODE DATA CAME ON-LINE WE DOWNLOADED IT AND THEY HAD THREE SAMPLES OF HUMAN LIVER. AND SO WE LOOKED AT ALL THREE OF THESE AND LO AND BEHOLD THE LIVER SPECIFIES AN H3K9 BLAST OVER THIS REGION. BUT IT TERMINATES INSTEAD OF HERE IT TERMINATES ON THIS SIDE OF THESE DESMO COLLIN GENES ALLOWING THESE GUYS TO BE EXPRESSED IN THE ADHERENT LIVER TYPE CELLS. SO WE'RE JUST BEGINNING TO THINK ABOUT THIS IN NEW WAYS BUT WHAT WE'D REALLY LIKE TO DO IS UNDERSTAND HOW THESE HETEROCHROMATIC DOMAINS NOW ARE SET DOWN IN DEVELOPMENT. HOW DO THEY REPRESENT BLOCKS FOR INTERCELLULAR CONVERSION. AND WE'VE BEEN TALKING NOW ABOUT A DIFFERENCE BETWEEN ORIGINALLY FIBER BLAST AND PLURALLY POTENT CELLS BUT NOW LOOKING BETWEEN FIBROBLASTS AND LIVER. IF YOU TAKE THESE 264DBRs THAT WE ORIGINALLY MAPPED IN THE FIBROBLASTS AND QUANTITATE K9 TRY METHYLATION IN EACH OF NO, SIR YOU CAN SEE DIMINISHED LEVELS IN LIVER. IT'S NOT JUST DIMINISHED LEVELS IN EACH OF THESE IT'S WHERE THE DOMAINS MATCH SO WE NEED ALGORITHMS FOR THAT. MOST OF THESE SIGNALS FOR H3K9 ARE LOST WHEN YOU FULLY REPROGRAM THE CELLS. AND WE WOULD LIKE TO NOW START LOOKING AT HOW THESE ARE LAID DOWN IN DIFFERENT LINEAGES BUT ALSO HOW OTHER TRANSCRIPTION FACTORS MAY OR MAY NOT BE RESISTENT TO BINDING THESE KINDS OF DOMAINS AND OTHER RESTRICTIVE DOMAIN. IN OTHER WORDS, WHAT'S THE RESTRICTION CODE OUT THERE. ARE THESE DOMAINS RESTRICTIVE TO ALL TYPES OF PIONEER FACTORS OR NOT. SO WE MOVED THE REGION ENCODED BY THIS DBROR THESE TWO FROM THE HUMAN OVER TO THE MOUSE GENOME AND LOOKED AT MOUSE FIBROBLASTS DATA THAT WAS ON-LINE. AND YOU CAN SEE THAT IN FACT, THERE'S AN INCREASE OF H3K9 TRY METHYLATION OVER THIS REGION GENERALLY IN THE MOUSE. SO IT SEEMS TO BE SOMEWHAT CONSERVED. WE THEN LOOKED IN LIVER CELLS AND LIVER KIND OF LOOKS DIFFERENT. SO IT'S A LOT FUZZIER, THERE'S A LOT MORE H3K9 ALL OVER BUT YOU CAN SEE THE DENSITY INCREASES RIGHT OVER THIS DOMAIN. FINALLY WE DID SOME LIVER CELLS FOR THE FOX A FACTOR IN MY LAB AND STRIKINGLY YOU CAN SEE THAT WHILE FOX A PEAK ARE NICE ON EITHER SIDE, THEY BASICALLY FLATTEN OUT OVER THIS DOMAIN. SO IT SUGGESTS THIS KIND OF HETEROCHROMATIN IS REFRACTORY TO VARIOUS KINDS OF SO-CALLED PIONEER FACTORS AND WE'D LIKE TO UNDERSTAND HOW THIS MODIFICATION AND OTHER MODIFICATIONS MAY OR MAY NOT UNIFORMLY BLOCK OR NOT BLOCK PIONEER FACTORS AND OTHER KINDS OF PROTEINS. SO WE CAN MODIFY THIS SLIDE NOW BY SAYING THAT THERE ARE RESTRICTIVE CHROMATIN FEATURES THAT BLOCK BOTH YOUR AVERAGE FACTOR AND A PIONEER FACTOR AND THAT THESE GUYS ARE NOT ABLE TO BIND ABSOLUTELY ANYWHERE. SO WE NOW BELIEVE THIS RESTRICTIVE MODEL, YOU CAN PREVENT THEM FROM BINDING BUT THEY DON'T NECESSARILY NEED A LICENSING MODEL WHICH IS SOMETHING ALREADY THERE FOR THEM TO BIND. AND IN TERMS OF NEXT STEPS FOR MY LAB, WE'RE VERY INTERESTED IN UNDERSTANDING HOW WE CAN TAKE THESE PRINCIPLES OF CHROMATIN RESTRICTION FEATURES TO ENHANCE REPROGRAMMING NOT FROM FIBROBLASTS TO PLURALLY POTENCY BUT CONVERTING RELATED CELL TYPES HOW CAN YOU GO FROM A LIVER CELL TO A BETA CELL AND SPECIFICALLY KNOCK DOWN THESE RESTRICTIVE FEATURES IN CHROMATIN FOR A LOCAL PERIOD OF TIME TO ALLOW THE FACTORS THAT ARE INHIBITED FROM BINDING TO THOSE TO GAIN ACCESS AND THEN MOVE THINGS ON STEP BY STEP. SO THAT'S WHAT WE'VE GOT AND THAT'S WHERE WE'RE GOING. IT'S KIND OF NEW DAYS AND IT'S VERY EXCITING FOR A LAB ACTUALLY TO BE PURSUING SOME OF THESE NEW DIRECTIONS. THE WORK I TALKED ABOUT WAS REALLY LED BY -- POST DOC PAR EXCELLENCE IN MY LAB -- SPECIALIST AND JASON WATSON MD PH.D. STUDENT. SO THANK YOU VERY MUCH FOR YOUR TIME. [APPLAUSE] >> [INDISCERNIBLE] >> GREAT QUESTION. SO THE QUESTION WAS WHAT'S KNOWN ABOUT THE REGULATION OF THE H3K9 TRY METHYL HISTONE METHYL TRANSFERASES DURING THE REPROGRAMMING PROCESSES. WHEN DO THEY START TO GO DOWN. DO THEY GO DOWN OR IS IT SIMPLY THAT THEY ARE NO LONGER TARGETED OR NOW YOU MORE DOMINANTLY TARGET SOMETHING THAT ERASES THAT MODIFICATION. SO WE DON'T KNOW THE ANSWER TO THAT BUT WE'RE TRYING TO SET UP SOME GENETIC WAYS OF LOOKING AT THAT BY MARKING PARTICULAR GENES IN FIBROBLASTS THAT WOULD REFLECT THE TIMING EVENTS FROM WHEN THAT WOULD OCCUR. AND THEN PERTURB THE CELLS. I DON'T HAVE A GOOD ANSWER BUT THAT'S EXACTLY ONE OF THE DIRECTIONS I THINK WOULD BE VERY IMPORTANT TO UNDERSTAND. SO IN TERMS OF THE GENES THAT CAME ON DURING THE INITIAL BINDING OF THE FACTORS, IT WAS GENES, 48 HOURS WHEN THESE FACTORS ARE FIRST EXPRESSED, IT WAS GENES ALREADY EXPRESSED IN FIBROBLASTS. SO THAT WAS ONE OF THE UNEXPECTED FEATURES OF THIS THAT WE WEREN'T LOOK OPENING UP A WHOLE LOT OF NEW GENES EARLY ON. THERE ARE SOME IMPEDIMENTS TO THAT. >> EVEN THE PIONEER EFFECT IT CANNOT BIND TO THE EDR BUT [INDISCERNIBLE] REPROGRAMMING. SO WHAT DO YOU THINK [INDISCERNIBLE] >> RIGHT. SO THIS IS RELATED TO THE OTHER QUESTION. I MEAN WE KNOW EVENTUALLY THE DBRs GO AWAY OR MOST OF THEM DO. THE ONE AT SOX DOES. SO THE ES CELLS ALL THOSE FACTORS ARE BINDING THOSE MEGA BASE REGIONS, NO PROBLEM. SO WE WOULD VERY MUCH LIKE TO KNOW WHAT IS IT THAT ERASES THOSE DOMAINS, IF YOU WILL OR PREVENTS THEM FROM FORMING. SO AGAIN WE'RE TAKING TWO WAYS TO LOOK AT THIS. ONE OF THESE IS TO SET UP A SCREEN IN FIBROBLASTS SO WE CAN DO SOME GENETICS WITH SRNA SCREENING AND SO FORTH AND DETECT WHAT IS IT THAT WILL PEAK, YOU KNOW, ALLOW THAT IMPEDIMENT TO GO AWAY. BUT THEN THE OTHER WAY IS LOOKING AT FORWARD HOW ARE THOSE DOMAINS ESTABLISHED. I'M VERY EXCITED, IN FACT, ABOUT SOME NEW STUDIES WE'VE GOT WHERE WE'RE LOOKING AT EMBRYONIC DEVELOPMENT BECAUSE WE CAN NOW FACT SORT PANCREATIC PROGENITORS BASICALLY AT EVERY STAGE AND LIVER PROGENITORS AND HOW DO THESE DOMAINS GET SET UP. WHAT MOTIFS ARE ASSOCIATED WITH THE BOUNDARIES. AT FIRST WE THOUGHT IT WAS EXTEND OF F3K9 AND IT'S NOT. I SHOWED YOU SOME EVIDENCE. IT'S WHERE YOU SET THE BOUNDARIES TO THESE PATCHES. SO WHAT'S HAPPENING AT THESE BOUNDARIES I DON'T KNOW BUT THAT'S WHERE WE'RE GOING. >> HOW DO YOU THINK THAT THE TRANSCRIPTION FACTORS AVOID OPEN CHROMATIN. I GUESS THEIR BINDING SITES ARE PROBABLY THERE. >> YES. I MEAN FOR EXAMPLE SOX 2, WHY IS SOX 2 GOING SO MUCH TO CLOSED CHROMATIN. I MEAN, I CAN JUST SPECULATE THAT IT'S KD FOR NUCLEOSOMAL DNA MIGHT BE LOWER THAN THAT FOR FREE DNA OR SOMETHING. AND WE'VE ACTUALLY MADE ALL OF THESE FACTORS RECOMBINANTLY AND HOW THEY BIND TO NUCLEOSOMAL DNA. >> IS THERE -- IN THAT OPEN REGION. >> YES. THE ES CELLS ARE BINDING OPEN REGIONS. THEY WILL EVENTUALLY BE BOUND TO ACTIVE GENES SO THEY'RE BOUND TO ACTIVE ENHANCERS AND SO FORTH. IT'S NOT THAT THEY CAN'T DO THAT BUT IN THIS SITUATION, IT'S A GOOD QUESTION. I DON'T KNOW THE ANSWER. I HADN'T THOUGHT ABOUT IT THAT WAY. YES? >> [INDISCERNIBLE] >> GREAT QUESTION. SO WE'RE DOING THAT NOW FOR BOTH LIVER CELLS AND BETA CELLS. WE WANTED TO KNOW BY ECTOPIC EXPRESSION JUST EMPIRICALLY WHERE DO THOSE MAP, HOW WELL DO THEY COINCIDE WITH K9 TRY METHYL REGIONS AND HOW CAN WE USE THAT TO OUR ADVANTAGE TO TRANSDIFFERENTIATE CELLS. AND I'M SORRY, THE OTHER PART OF YOUR QUESTION? >> [INDISCERNIBLE] >> YES, RIGHT. HOW ARE THESE LAID DOWN. I THINK IT'S GOING TO BE MORE COMPLEX THAN THEY'RE JUST AT ONE SITE BECAUSE THEY'RE SPREAD OUT SO MUCH. I HAD LONG THOUGHT THEY WERE JUST WITH ARE REPETITIVE ELEMENTS AND I DIDN'T HAVE TO WORRY ABOUT THAT BUT MAYBE THERE'S SOMETHING COMPLICATED ABOUT HOW REPETITIVE ELEMENTS ARE REGULATED AND THAT'S ARE INTERDIGITATED WITH DEVELOPMENTAL REGULATIONS IN WAYS WE DON'T KNOW BUT IT OPENS UP A LOT OF THOSE QUESTIONS. >> A QUESTION ABOUT THE PROTEINS. YOU MENTION THAT NOW YOU'VE EXPRESSED SOME YEARS AGO WE WERE PLAYING WITH THE PCP, YES, MEMBRANE PERMEABLE TAG -- AND SOX 2. WE FOUND THAT WHEN WE ADDED THE TWO TOGETHER AND THEN ADDED THOSE DIRECTLY TO CELLS, WE DID MUCH BETTER. WE DIDN'T GET, YOU KNOW, OPTIMAL COLONIES BUT WE GOT COLONIES EXPRESSING THE STANDARD MARKERS. AND IT BECAME CLEAR THAT THE SOX 2 WHEN BOUND TO OCT4 STABILIZED IT BUT WE HAD NO IDEA WHETHER OR NOT THERE WAS A POSSIBILITY THAT THE TWO FORMED A COMPLEX BEFORE THEY BOUND AND ONE HELPED THE OTHER. ANY IDEA ABOUT THAT? >> WELL, IT'S AN INTERESTING POINT. WE DEFINITELY SEE THAT THEY CAN BIND INDEPENDENT OF ONE ANOTHER AT CHROMATIN SITES AND I SHOWED DATA FOR THAT. SO IT APPEARS THAT IT'S NOT NECESSARY FOR THEM TO BIND EVEN NAIVE CHROMATIN AS A PAIR. THAT BEING SAID, IT'S WELL ESTABLISHED FROM YOUR LAB AND VARIOUS OTHERS THAT THESE GUYS LIKE TO GO IN PAIRS. I CAN TELL YOU SOMETHING REALLY STRANGE AND AGAIN WE'RE JUST READING THE DATA HERE. THE FIRST THING WE LOOK FOR IS THE OCT SOX COMPOSITE MOTIF. FRANKLY IF WE DO DENO NO ANALYSIS IT'S NOT COMING UP. WE SAW THE SOX MOW FEE AND OCT MOTIF AND THEY'RE OCCURRING TOGETHER WITH AN A HUNDRED BASE PAIRS. SO REALLY CLOSE, NOT LIKE IT'S THE SAME GENE KBOA. I DON'T KNOW, I'M JUST READING IT OUT. SO SOMETHING THAT'S HAPPENING LATER ON I THINK IS A PREFERENCE FOR THE OCT SOX PAIRED MOTIF AS OPPOSED TO INDEPENDENT EVENTS OCCURRING. >> [INDISCERNIBLE] >> RIGHT. >> [INDISCERNIBLE] >> THAT'S ACTUALLY A GREAT QUESTION AND I HAVE TO CONFESS WE HAVEN'T THOUGHT ABOUT IT THAT WAY. THE POINT WAS OKAY, WE SHOW THAT THESE ALL FOUR OF THESE FACTORS ARE DEFICIENT IN BINDING THESE DBRs BUT IS ONE OF THEM A LITTLE BETTER THAN THE OTHER AND MAYBE AT THE END OF THE DAY THAT'S TELLING YOU SOMETHING ABOUT THE FIRST TO CRACK IN. SO YOU KNOW, I CAN JUST, YOU CAN LOOK AT THIS TOGETHER OR I CAN LOOK AT IT LATER. I DON'T, I HAVEN'T THOUGHT ABOUT THAT. I MEAN, TO THE FIRST APPROXIMATION THEY'RE ALL PRETTY DOWN THERE BUT MAYBE SOX 2 IS A LITTLE BETTER THAN THE OTHER GUYS. AND WE CAN LOOK AT THAT AFTERWARDS OR SOMETHING. IT'S A GREAT THOUGHT, THANK YOU. >> [INDISCERNIBLE] >> SO WHEN YOU MAKE BETA CELLS FROM EF CELLS OR WHATEVER YOU WANT FROM EF CELLS, DOES THE PATTERNING OF THESE DBRs FOLLOW WHAT YOU WOULD SEE IN VIVO. AND THE ANSWER TO THAT IS I HOPE TO TELL YOU IN NOT TOO MUCH TIME BECAUSE WE'RE STARTING TO LOOK, EVERYBODY'S BEEN FOCUSING ON K27 TRY METHYLATION STUFF AND I THINK THIS IS A NEW DIMENSION SO WE DON'T KNOW. >> [INDISCERNIBLE] >> LINK Electronics Inc. MODEL NUMBER: PDA-895 SOFTWARE VERSION: 1.0C WHEN GURDEN REPROGRAMMING IT TAKES TWO DAYS. SO WE KNOW YOU CAN DO THIS FAST. WHETHER WE'RE DOING IT CORRECTLY OR NOT, I CAN'T SAY. BUT THE GOAL I THINK IF WE UNDERSTOOD THE SYSTEM, WE SHOULD BE ABLE TO DO IT PRETTY QUICK. I DON'T THINK SPEED IS NECESSARILY AN INDICATOR OF NOT DOING IT RIGHT. THAT'S ALL I CAN SAY. >> [INDISCERNIBLE] >> THE MODEL FOR HOW IT CAN WORK IN TWO DAYS. WELL IT'S INTERESTING. YOU KNOW, I THINK THERE'S SOMETHING PHYSICAL GOING ON BECAUSE I'VE HEARD JOHN GERDEN TALK A BUNCH OF TIMES. HE SAYS THE FIRST THING THAT HAPPENED IS THE NUCLEUS INCREASES FOUR FOLD IN SIZE. AND H2AZ COMES IN AND FUNDAMENTAL CHROMATIN COMPONENTS ARE CHANGING. SO IT WOULDN'T SURPRISE ME IF REALLY SOME OF THE KEY STUFF IS INCREDIBLY BASIC ABOUT JUST OPENING UP THE CHROMATIN. AND WHAT A BETTER WAY TO DO IT THAN TO GET RID OF HETEROCHROMIN, THAT'S ALL I KNOW. >> THANK YOU.