>> GOOD AFTERNOON. I HOPE EVERYONE HERE IS ENJOYING THE SEQUESTER. KAREN AGREED TO REDUCE HER TALK BY FIVE SLIDES. [LAUGHTER] WELL, 5 PERCENT, WHICH IS WHAT WE WANT. SO IT'S MY PLEASURE TO INTRODUCE KAREN ADELMAN, WHO IS A SENIOR INVESTIGATOR AT NIH S. SHE DID HER UNDERGRADUATE TRAINING FROM BUFFALO AND FROM BUFFALO TO UNIVERSITY OF PARIS, WHERE SHE GOT HER PH. D AND THEN CAME BACK TO THE UNITED STATES AND DID HER POST DOC AT CORNELL BEFORE BEING RECRUITED AS A TENURED TRACK INVESTIGATOR. HER WORK IS FOCUSED ON THIS INTERESTING INTERPLAY BETWEEN THE ENVIRONMENT, TRANSCRIPTION REGULATION AND SHE'S GOING TO TELL US TODAY ABOUT POTENTIALIATING SIGNAL RESPONSIVE TRANSCRIPTION, A DYNAMIC DANCE BETWEEN R FLA 2 AND CHROMATIN. KAREN? >> SO THANK YOU, FOR THE INVITATION TO PARTICIPATE IN THIS REALLY FABULOUS LECTURE SERIES, AND FOR THE OPPORTUNITY TO COME UP TO BIG NIH AND TELL YOU A LITTLE BIT ABOUT THE STORIES THAT WE'VE BEEN DEVELOPING IN MY LAB. SO WHAT I AM GOING TO TELL YOU ABOUT TODAY IS THE EFFORT IN MY LAB TO UNDERSTAND HOW GENE EXPRESSION IS REGULATED IN RESPONSE TO EXTRA CELLULAR ENVIRONMENTAL QUEUE, AND THE ROLE THAT CONTROLLING THE ELONGATION -- THE ROLE THAT THAT REALLY PLAYS IN THIS PROCESS, SO I AM GOING TO START BY BASICALLY DEFINING WHAT I MEAN WHEN I SAY "PAUSED RNA POLYMERASE" AND ABOUT THE ROLE OF THE REGULATION AND WHAT ROLE IS PLAYED WITHIN CELLS. AND THEN I AM GOING TO END WITH SOME UNPUBLISHED DATA FROM MY LAB, WHERE I'LL DESCRIBE A RELATIONSHIP SURPRISING FUNCTION OF POLYMERASE. SO THE OVERARCHING QUESTION IS HOW GENE EXPRESSION IS REGULATED BY ENVIRONMENTAL CUES. AND SO WE ALL KNOW THAT SIGNALS FROM THE ENVIRONMENT CAN BE RECOGNIZED BY SENSORS, EITHER AT THE CELL SURFACE AND THAT THESE SIGNALS CAN BE TRANSDUCED BY THESE CASCADES BUT CONVERGE ON TRANSCRIPTION FACTORS THAT GOES INTO THE NUKE LOUIS AND TURNS GENES ON OR OFF. AND SO ULTIMATELY WEG FROM SIGNAL TO ALTER GENE EXPRESSION. WE GO. BUT WHAT'S REALLY IMPORTANT FOR US TO UNDERSTAND BETTER IS WHY A SIGNAL DOESN'T ALWAYS LEAD TO THE EXACT SAME KINDS OF EXPRESSION FROM EVERY GENE THAT IT TARGETS. SO WE KNOW THAT THERE CAN BE VERY DIFFERENT CONTENTICS AND MAG ATTITUDES OF GENE EXPRESSION THAT ALL COME FROM A SINGLE COMMON SIGNAL. AND WE ALSO KNOW THAT THE SIGNAL RESPONSIVENESS CAN BE CELL-TYPE DEPENDENT SO THAT A SIGNAL CAN GIVE VERY DIFFERENT RESPONSES IN DIFFERENT CELL TYPES, YOU EVEN WHEN THEY POSSESS THE SAME GENOME. AND WHAT THAT TELLS IS THERE IS SOMETHING LAID ON TOP OF THAT THAT OFTEN EFFECTS A CHROMATIN ENVIRONMENT AROUND A TARGET GENE THAT INFLUENCES THE WAY IT'S ACTIVATED INTO RESPONSE TO A SIGNAL. AND IT'S IMPORTANT TO UNDERSTAND ALL ASPECTS OF THIS FROM THE SIGNAL TO THE GENE EXPRESSION BECAUSE THIS GENE EXPRESSION REALLY PLAYS A MAJOR ROLE IN THE IDEOLOGY OF A NUMBER OF DIFFERENT KINDS OF DISEASES. SO AS I WAS SAID IN MY INTRODUCTION, AN ASPECT OF SIGNAL RESPONSIVE GENE EXPRESSION THAT MY LAB REALLY FOCUSES A LOT OF ENERGY ON IS TRYING TO UNDERSTAND HOW TRANSCRIPTION REGULATION IS INFLUENCED BY THE CHROMATIN STRUCTURE. AND SO WE KNOW THAT CHROMATIN CAN BE A BARRIER TO ALL DNA-DEPENDENT PROCESSES. AND WE KNOW THAT REGULATING THE CHROMATIN STCTURE AROUND A PROMOTER CAN EFFECT HOW RESPONSIVE A GENE MAY BE, BUT WE DON'T REALLY FULLY UNDERSTAND HOW THAT REGULATION TAKES PLACE AND WHEN IT TAKES PLACE. AND THE EFFORTS THAT I AM GOING TO BE FOCUSING ON IS A PATROL THAT WE FOUND FOR THE TRANSCRIPTION MACHINERY ITSELF IN REGULATING THE EPIGENOME UNDER POORN A NUMBER OF THESE RESPONSIVE PROMOTEERS. SO THE STARTING POINT FOR ALL THIS IS REALLY THE FINDING THAT IN HIGHER -- GENE EXPRESSION IS COMPLICATED THAN THOSE SORT OF TEXTBOOK MODELS THAT I LEARNED BACK WHEN I WAS IN SCHOOL THAT ARE BASED LARGELY ON SOMETHING LIKE YEAST AS A MODEL SYSTEM. AND FOFOR A LONG TIME THE MODEL WENT SOMETHING LIKE THIS WHERE YOU WOULD HAVE NUCLEARSOME POSITIONED OVER THE TRANSCRIPTION START SITE AND TURNING A GENE ON WOULD INVOLVE BRING INNING A CHROMATIN REMODELER, MOVING CHROMOSOME OUT OF THE WAY THAT COULD THEN BRING IN THE TRANSCRIPTION MACHINERY AND FROM THAT POINT ON IT WAS GENERALLY ASSUMED THAT THIS POLYMERASE WOULD SORT OF PROCEED ALMOST AUTOMATICALLY INTO PRODUCTIVE ELONGATION. SO THE STORY THAT I AM GOING TO TELL YOU ABOUT TODAY IS WHERE WE FIND THE POLYMERASE HAS TO OVERCOME A NUMBER OF DIFFERENT OBSTACLES ON ITS PATH AND WHERE REGULATION OF PROMOTER RELEASE OR ESCAPE OF THIS EARLY ELONGATION COMPLEX DOWNSTREAM INTO THE GENE CAN BE THE KEY REGULATED STEP DETERMINING GENE EXPRESSION. NOW, THE IDEA THAT POLYMERASE RELEASE FROM THE PROMOTER CAN BE IMPORTANT ISN'T REALLY A NEW IDEA. IT WAS DESCRIBED IN THE MID 80'S BY JOHN LIST, WITH WHOM I DID MY POST DOC WHEN HE WAS STUDYING THE PROMOTEERS. AND WHO WHAT HE FOUND WAS NOT THAT THE POLY MARRIASE WAS RECRUITED TO THE PROMOTER OF THE GENES TRIER PRIOR TO HEAT INDUCTION. AND BEAUTIFUL WORK FROM KARL'S LAB, MUCH OF IT DONE HERE, SHOWED THAT IN ADDITION TO THE POLYMERASE BEING PROMOTED, THAT NUCLEARASE WERE BEING MOVED OFF OF THE PROMOTER. WHAT I FOUND MOST AMAZING ABOUT THIS WAS THAT THE POLYMERASE ACTUALLY BEGAN TRANSCRIBING THE GENE. IT TRANSCRIBED ABOUT A 20 TO 60 TRANSCRIPT AND THEN PAUSED WITHIN THE PROMOTER PROXIMAL REGION. AND BEFORE HEAT STRESS, THIS POLYMERASE WOULD BE RELEASED ABOUT EVERY TEN NIENZ THE GENE TO GIVE YOU THIS LOW BASAL LEVEL OF GENE EXPRESSION. AND WHAT'S REALLY AMAZING IS DURING HEAT SHOCK, ONE OF THE THING THAT HAPPENS IS THIS PAUSE IS REALLY ALLEVIATED SUCH THAT NOW INSTEAD OF GETTING STUCK FOR ABOUT TEN MINUTES IN THE PROMOTER REGION BEFORE RELEASES INTO THE GENE, THE RECRUITED POLYMERASE WAS STOPPED FOR ABOUT FOUR SECONDS BEFORE THEY MOVED ON INTO PRODUCTIVE ELONGATION. SO BY CHANGING THE HALF LIFE OF THE POLYMERASE AND THE RESIDENCE TIME FROM TEN MINUTES TO FOUR SECONDS, THESE GENES COULD BE UPREGULATED NEARLY 1,000FOLD DURING JUST ABOUT A HALF-HOUR AFTER HEAT STRESS. AND SO I JUST WANT TO POINT OUT THAT TWO THINGS THAT ARE REALLY CRITICAL FOR THIS KIND OF RAPID GENE EXPRESSION. ONE IS THE PRESENCE OF THIS AND THE OTHER IS THE FACT THAT THIS IS A VERY NUKESOME DEPRIVED PROMOTER REGION. SO THIS HAD BEEN DESCRIBED AND AT A NUMBER OF OTHER INDUCIBLE GENE SYSTEMS. BY THE LAB SHOWN HERE. SO OTHER GENES THAT WERE REGULATED BY EARLY TRANSCRIPTION ELONGATION INCLUDED SOME VERY IMPORTANT GENE. BUT BEFORE YOU COULD REALLY BROADLY ASK WHERE IS IT DISTRIBUTED OVER A GENOME, THIS KIND OF REGULATORY MECHANISM SEEMED TO BE RELATIVELY RARE. SO THIS IS AN EXAMPLE OF THE KIND OF EXPERIMENT I DID AD NAUSEAM AS A POST DOC WHERE WE WERE USING PRECIPITATION AND ASKING WHAT IS THIS DISTRIBUTION, AT FOR EXAMPLE HERE ONE OF THE HEAT SHOCK GENES? SO I PULLED DOWN THE POLYMERASE AND ASKED WHAT DNA BY USING TILED ACROSS THE GENE. AND WHAT YOU CAN SEE CLEARLY HERE IS THAT THE START SITE GIVES YOU VERY GOOD SIGNAL, BUT THAT THAT POLY MARRIASE IS NOT RELEASED, IS NOT TRANSCRIBING EFFICIENTLY INTO THE REST OF THE GENE. SO WHEN I WAS STARTING MY LAB, WITH THE ADVENT OF CHIP-CHIP, IT OCCURRED TO ME THAT WE COULD USE A GENOMIC SORT AF APPROACH FOR ASKING DO OTHER GENES IN THE FLY JEROME EXHIBIT THIS AND WITH A REALLY TALENTED TECHNICIAN WHO HAD JOUND JOINED MY LAB, WE EMBARKED ON THIS GENOME-WIDE ASPECT OF DISTRIBUTION. AND WHEN WE GOT THE DATA, IT WAS REALLY, REALLY EXCITING, BECAUSE WHILE WE FOUND GENES THAT TOOK A LOOKED SORT OF WHAT HAD BEEN DESCRIBED WHERE THE POLYMERASE HAD BEEN OR BE -- OR WAS DISTRIBUTED EVENLY OVER THE GENE UNIT, WE FOUND A NUMBER OF GENES WHERE THERE WAS THIS ACCUMULATION OF US POLYMERASE AT THE PROMOTER THAT WASN'T EFFICIENTLY MOVING INTO THE BODY OF THE GENE. AND WE DEVISED THIS SIMPLE METRIC FOUR IDENTIFYING HOW PAUSED OR HOW STUCK THESE PROMOTER-ASSOCIATED POLYMERASES WERE, WHICH WAS TO LOOK AT THE AVERAGE SIGNAL PROMOTER AND CALCULATE THAT HAS A RATIO OVER THE AVERAGE PROBE SIGNAL DOWNSTREAM WITHIN THE GENE AND WE CALL THAT THE PAUSING INDEX AND I'LL BE TALKING ABOUT THE PAUSING INDEX THROUGHOUT THE TALK AS A MEASURE OF HOW EFFICIENTLY POLYMERASE IS RELEASED DOWNSTREAM INTO THE GENE. AGAIN, THIS DOESN'T MEAN THAT THERE IS NO POLYMERASE IN THE GENE BODY. THESE ARE -- THIS IS ACTUALLY A VERY HIGHLY ACTIVE GENE. IT SIMPLY MEANS THAT THERE IS EVIDENCE OF A RATE LIMITING STEP THAT HAPPENS NOT AT BRINGING IT INTO THE PROMOTER BUT LICENSING IT TO GO INTO THE CHAIN. NOW, WITH THE IDENTIFIED GENES THAT HAD THE PROMOTER, THE THING THAT GOT US INTERESTED IN THIS IS AMONG THE 1,000 GENES, IT TURNED OUT THESE WERE REALLY DRAMATICALLY ENRICHED IN STIMULUS RESPONSIVE PATHWAYS AND THINGS LIKE DEVELOPMENT. AND SO THIS MADE US WONDER WHETHER OR NOT THE PARADIGM THAT HAD BEEN SET BY THE HEAT SHOCK GENES WAS GOING TO BE MUCH MORE BROADLY APPLICABLE THAN WE'D ORIGINALLY GUESSED. NOW, THE EXPERIMENTS THAT WERE DONE IN MY LAB WERE DONE IN THE CULTURE BUT WE'RE COLLABORATING TO DO VERY SIMILAR EXPERIMENTS IN THE EMBRYO AND THERE WE FOUND IN THIS BEAUTIFUL DEVELOPMENTAL CONTEXT THE VERY SAME PRINCIPLE, THAT DEVELOPMENTAL GENES WERE PRELOADED WITH POLYMERASE IN A MANNER WHERE NARELEASE OF THE POLYMERASE INTO THE GENE WAS THE REGULATORY STEP AND BETTER YET, THIS WAS NOT JUST A WACKY FLY THING BUT ACTUALLY WAS FOUND IN HUMAN CELLS. AND THIS STUDY WITH RICK YOUNG IN COLLABORATION WITH ROBERT, THEY WERE LOOKING AT EMBRYONIC STEM CELLS AND FOUND THAT THE RECRUIT TO JEANS EVEN FROM GENES THAT WEREN'T BEING TRANSCRIBED AND IT WAS RELEASED OF THIS RECRUITED POLYMERASE THAT WAS REGULATORY FOR GENE EXPRESSION. SO IN THE YEARS SINCE THESE PAPERS HAVE BEEN PUBLISHED, WE'VE COME TO APPRECIATE THAT THIS IS A BROADLY USED STRATEGY ACROSS METAZOA. AND PAUSING EXISTS AND HAS BEEN WELL-DESCRIBED NOW IN FLIES AND IN A NUMBER OF DIFFERENT MAMALLIAN SPECIES BUT PAUSING DOES NOT OCCUR IN THINGS LIKE YEAST, OR IN ARABADOB SIS. SO THIS SEEMS TO BE TIED TO SORT OF A DIFFERENT LEVEL OF POTENTIALLY DEVELOPMENTAL SOPHISTICATION. NOW, IN ADDITION TO THE FIELD MOVING FORWARD, THE TECHNOLOGIES FOR STUDYING SOMETHING LIKE PAUSING HAS ALSO MOVED FORWARD A LOT. AND SO WE'VE FOFFED MOVED FROM DOING THINGS LIKE CHIP CHICHIP SO NOW YOU ARE IMMUNOPRECIPITATING THE DNA SO I GET A VERY BROAD PICTURE YOU HAVE THE GENOME AND HERE IS AN EXAMPLE OF A HIGHLY ACTIVE GENE WHERE YOU CAN SEE IT VASTLY EXCEEDS THE AMOUNT RELEASED INTO THE STREAM. AGAIN, CHIP REALLY ONLY TELLS YOU WHERE THE POLYMERASE IS. WHAT WE WANTED TO KNOW WAS WHERE EN COURAGED POLYMERASE WAS LOCATED, BECAUSE THAT'S REALLY THE HALLMARK OF THE PAUSED, TOO,. AND TWO ASSAYS HAVE BEEN DESCRIBED TO REALLY LOOK AT THE RNA COMPONENTS OF THESE COMPLEXES TO GET A HANDLE ON WHETHER WHERE THE ENGAGED AND ELONGATION CONFIDENT POLYMERASE IS. AND RONAN SEEING IS AN ASSAY ACROSS THE GENOME AND SOO HERE YOU CAN SEE THAT WHILE THE POLY MER E. P. A. S IS MOVING THROUGH THIS GENE, THERE IS A PILE-UP AS AN AT THE PROMOTER REGION, WHEREAS THE RNA JUST ISOLATES THE RNA ASSOCIATED WITH THE PAUSED POLYMERASE SO THAT YOU GET NUCLEOTIDE POLY MERATION OF WHERE IT'S PAUSING AND WHERE IT STARTED. SO PAUSING HAPPENS AND IF IT'S REALLY IMPORTANT, IT IS PROBABLY GOING TO BE REGULATED SO LET ME TELL YOU A LITTLE BIT ABOUT ITS REGULATION. SO ONE OF THE KEY REGULATORS AND THE THING THAT ESTABLISHES PAUSING IS CALLED NEGLECT ELONGATION FLEX. AND THIS REACTS TO THE PROTEIN AND THIS BIOCHEMICALLY CAN INHIBIT TRANSCRIPTION ELANGUAGEATION. IT EXISTS ONLY IN THOSE ORGANISMS WHERE I TOLD YOU THAT PAUSING EXISTS. SO THERE IS NO IN YEAST BUT IT SHOWS UP IN FLIES AND IS VERY HIGHLY CONSERVED INTO THE MAMMALS. SO IT IS A COMPLEX AND SO THAT WILL BECOME IMPORTANT WHEN I TALK ABOUT KNOCKING DOWN THE DIFFERENT SUBUNITS. THIS IS AGAIN BIOCHEMICALICALLY SORT OF THE ACTIVE PAUSE YOU HAVE PAUSING AND THIS IS WHAT SLOWS TRANSCRIPTION DOWN. SO GETTING RID OF IT IS CALLED POSITIVE TRANSCRIPTION ELONGATION FACTOR AND THIS STIMULATES A RELEASE OF THE COMPLEX AND ALLOWS THE POLYMERASE TO MOVE INTO THE GENE. SO I WANT TO POINT OUT THAT THERE IS A MYRIAD OF DIFFERENT WAYS OF RECRUITING IT TO THE PROMOTER AND IN SOME GENES IT COMES WITHIN SECONDS OF THE BEING RECRUIT SOD THAT PAUSING IS SHORT-LIVED AND REALLY TAKES ITS TIME GETTING TO THE PROMOTER AND THESE ARE THE GENES WHERE YOU SEE LONG-LIVED PAUSING AND CAN BE REGULATORY FOR GENE EXPRESSION. OKAY, SO WHEN WE FIRST DISCOVERED THAT PAUSING WAS HAPPENING, WE WANTED TO GET A HANDLE ON WHAT GENES AT WHICH THIS IS REALLY AN IMPORTANT PART OF THE REGULATION. AND SO WE DECIDED FIRST APPROXIMATION TO KNOCK OUT -- TWO OF THE UNITS B AND E, AND WE STARTED WITH THIS AND I WANT TO POINT OUT THAT WHEN WE KNOCK OUT THE CENTRAL SUBUNIT B IT'S KIND OF CUTE BECAUSE IT DESTABILIZES THE ENTIRE COMPLEX AND ALL OF THE SUBUNITS ARE ACTUALLY DEGRADE.^ AND SO WE'RE ABLE TO REALLY KNOCK DOWN THE ENTIRETY OF THE COMPLEX. OKAY, SO THIS WORK AND A LOT OF THE WORK WE ARE TALKING ABOUT TODAY WAS DONE BY DAN GILCHRIST AND SO THE FIRST THING HE DID WAS KNOCK DOWN AND DO A STUDY. OKAY, SO WHEN WE GOT THE DATA BACK, WE FOUND THAT JUST LIKE THE GENES THAT WE DESCRIBED AS HAVING PAWINGS, THE TARGET GENES WERE ENRICHED IN RESPONSES TO STIMULUS. AND HERE I'M JUST SHOWING YOU A COUPLE OF DIFFERENT SKGZ THAT THIS FALLS INTO. AND WE'RE HAPPY TO SEE THAT THE GENES WERE ON THIS LIST AND SO WE FOUND THIS KIND OF MATCHED THE MODEL THAT WE HAD IN OUR HUT WHERE WE WOULD KNOCK DOWN MOUSE AND THE POLYMERASE WOULD GO INTO THE GENE AND THEN NEW POLYMERASE WOULD COME TO THE PROMOTER AND IT WOULDN'T GET STRUCK STUCK AND TRANSCRIPT LEVELS WOULD GO UP. BUT TRANSCRIPT LEVELS WENT UP ABOUT THREE SDPOILD ALREADY TOLD YOU THAT WHEN THIS GENE IS ACTUALLY INDUCED, TRANSCRIPT LEVELS GO UP ABOUT 1,000FOLD. SO WHAT THIS TOLD US IS THAT PAUSING, RELIEVING PAUSING WASN'T THE SAME AS GENE ACTIVATION. AND IN FACT, IT SUGGESTED THAT THE MAIN ROLE OF PAUSING MAY NOT BE TO REPRESS BASAL TRANSCRIPTION BUT IT MAY BE DOING SOMETHING ELSE. THIS IS ONE OF THESE AHA MOMENTS THAT I HAD PASS A NEW INVESTIGATOR, WHERE I THOUGHT YOU KNOW, MY POST DOC ADVISOR USED TO SAY SOMETHING ABOUT THIS AND MAYBE I SHOULD THINK ABOUT THIS SDMOUCHLT IT TURNS OUT THAT JOHN HAS BEEN SAYING FOR A LONG TIME THAT IN HIS VIEW, PAUSING WASN'T THERE TO REPRESS BASAL TRANSCRIPTION. IT WAS THERE TO SET UP THE ACTIVATED STATE. IT WAS THERE SO THAT AT ANY TIME IF THESE POOR LITTLE FLIES WERE EXPRESSED OUT, POLYMERASE WOULD BE WAITING AT THESE OPEN PROMOTEERS FROMMED TO GO. AND SO THAT GAVE US THIS IDEA THAT PAUSING MAY ACTUALLY BE USEFUL FOR MAINTAINING GENE ACTIVITY AND SETTING UP FOR GENE ACTIVATION. AND IT REALLY HELPED US UNDERSTAND THE KIND OF CURIOUS PART OF THIS RESULT, WHICH WAS THAT WHERE THE GENES WERE UPREGULATED, THE VAST MAJORITY OF GENES WERE DOWNREGULATED UPON LOSS OF A NEGATIVE SFOOFRJT THAT'S NOT WHAT YOU'D EXPECT. AND SO WE WERE TRYING TO UNDERSTAND WHY IT WAS THAT THESE GENES WOULD GO THE WRONG WAY, WHY WOULD PAUSING ACTUALLY BE HELPFUL FOR MAINTAINING EXPRESSION OF THESE GENES? AND SO I WON'T GO THROUGH THIS YEARS OF WORK THAT DAN ABOUT TRYING TO GET TO THE BOTTOM OF THIS. I'LL JUST TELL THAT YOU WE HAD SOME REALLY USEFUL INSIGHTS INTO THIS WHEN WE STARTED TO ASK VERY SPECIFIC QUESTIONS ABOUT WHAT HAPPENED TO DISTRIBUTION IS GENOME WIDE WHEN WE KNOCKED IT OUT. AND SO GIVE US A SENSE OF WHAT HAPPENS HERE, I AM GOING TO SHOW YOU A HEAT MAP OF THE DISTRIBUTION AROUND PROMOTEERS AND I AM GOING TO SHOW YOU A NUMBER OF THESE HEAT MAPS AND I'LL TAKE YOU THROUGH THIS ONE SLOWLY. SO THIS IS A HEAT MAP OF ABOUT 16,000 NON-OVERLAPPING FLY GENES, ALIGNED BY THEIR PROMOTER, WHICH IS HERE WITH THE ARROWS. AND UP STREAM TO ABOUT 1.5 KB DOWNSTREAM WHERE THE MORE RED THERE IS IS THE MORE GREATER SIGNAL WE FOUND AT THESE GENES. THIS IS WHAT THE SIGNAL LOOKS LIKE IN WILD TYPE CELLS AND HOW DOES THIS CHANGE WHEN WE KNOCK DOWN MOUSE? SO NOW THIS HEAT MAP IS SHOWN WHERE RED GIVES YOU,, TELLS YOU WHERE THERE IS AN INCREASE IN SIGNAL AND GREEN IS A DECEASE IN THE ALL?{}. AND WHAT I THINK YOU WILL NOTICE IS ALL ACROSS THESE PROMOTEERS, PRETTY MUCH GLOBALLY IN THESE FLY CELLS, YOU SEE THE SIGNAL PROMOTEERS, AND THIS IS NOT ACCOMPANIED BY AN INCREASE IN PAUL 2 WITHIN THE JEEVENLT YOU ARE NOT SUDDENLY SEEING ALL THIS POLYMERASE MOVING INTO THE GENE. THERE IS NO RED DOWNSTREAM WITHIN THIS REGION. BUT THIS IS SHOWN AGAIN HERE BY GIVING A COM PIZ -- COMPOSITE PROFILE OF ALL THAT YOU SEE ACROSS THE GENES WITHIN THESE CONTROL CELLS. AGAIN YOU SEE SOMEHOW, WITHOUT HOLDING THE POLYMERASE PROMOTEERS, THOSE PROMOTEERS ARE LESS GOOD AT BINDING ADDITIONAL POLL 2. AND SO WE TRIED TO UNDERSTAND WHY IT WAS THAT HOLDING POLYMERASE PROMOTING KIND OF MADE THAT PROMOTER FRIENDLY FOR POTENTIALLY SUBSEQUENT ROUNDS OF PAUL 2 RECRUITMENT. AND THE ANSWER CAME WHEN DAN STARTED LOOKING AT CHROMATIN. AND SO HERE I AM GOING TO SHOW YOU A STRAIGHTFORWARD ASSAY HE DID AT ONE OF OUR TARGET GENES WHERE HE DID DIGESTION. SO THIS IS AN ENZYME THAT CHOOSE UP ALL THE DNA THAT'S NOT PROTECTED. AND THEN HE LOOKED AT THE PROTECTED FRAGMENTS IN THIS EXPERIMENT AND SO WHAT WELCOME BACK SEE IS THAT THE POLYMERASE IS DEPICTED HERE SITS WITHIN THIS REGION OF OPEN CHROMATIN AND THAT DOWNSTREAM OF THIS THERE IS A NICE PLUS 1 AND PLUS 2 NUKESOME WITHIN THE GENE POISONED. THIS ISN'T WHAT YOU'D EXPECT FOR AN ACTIVE GENE. AND WHAT YOU SEE IS WHEN YOU LOSE IT IN THESE CELLS YOU LOSE POLYMERASE AND YOU GAIN NUCLEARSOMES RIGHT OVER WHERE THE POLYMERASE WAS SITTING AS WELL AS OVER THE UPSTREAM ACTIVATED REGION. AND WHAT THIS TOLD US WAS THERE WAS SOME KIND OF DYNAMIC COMPETITION GOING ON BETWEEN THE PAUSED POLYMERASE AND WHEN THAT WAS LOST, NUCLEARSOMES DMAM AND SO THAT SET UP THIS MODEL WHERE PAUSING COULD HAVE A POSITIVE EFFECT ON GENE STROIPGS BY PREVENTING NUCLEARSOME ASSEMBLY OVER THE PROMOTER. AND WHEN IT WAS LOST, THAT OCCUPANCIY WOULD COULD -- HISTOOCCUPANCIY COULD INCREASE FOR THESE GENES AND CAUSING THE DECEASE IN TRANSCRIPTION. NOW, OUR INITIAL WORK WAS DONE JUST A FEW SELECT GENES, BUT OF COURSE WE WANTED TO LOOK AT HOW GLOBALLY THIS WAS TAKING PLACE. AND THE NEXT THING THAT DAN DID WAS HE TOOK HIS DIGESTION OF NUCLEARSOMES AND HE SUBJECTD THAT DNA TO HIGH THROUGHPUT SEQUENCING TO MATCH THE PRIMARY STRUCTURE OF CHROMATIN IN THESE CELLS. AND OF COURSE WE WANTED TO LOOK AT CELLS WHERE THEY WERE TREATED WITH RNAI. AND HERE I AM SHOWING YOU ANOTHER EXAMPLE OF A GENE WHERE THE DELETION CAUSES AROUND THE PROMOTER AND YOU CAN SEE THAT IN THE MOCK TREATED CELLS THERE IS VERY LITTLE OCCUPANCIY AND WE SEE THE OCCUPANCIY INCREASING. SO NOW IF WE LOOK AT ALL OF THE GENES THAT ARE DOWNREGULATED AND SO WE LOOK AT A COMPOSITE PROFILE OF THESE OPEN CHROMATIN REGION.^ AND UP WANT TO ASK WHAT HAPPENS WHEN IT IS DEPLETED IN THESE SAMPLES. AND THIS IS A STRIKING CHANGE WHERE NUCLEARSOMES ARE COMING IN TO OCCUPY THE PROMOTER AND IN ADDITION THESE NUCLEARSOMES ARE ACTUALLY SLIDING BACK OVER THE PROMOTER. SO THIS REALLY UNDERSCORES HOW DYNAMIC NUCLEARSOME YOIPS IS AT WITH PROMOTEERS AND WHEN YOU MAP THE OCCUPANCIY, YOU NEED TO TAKE INTO ACCOUNT THE TRANSCRIPTION MACHINERY'S ROLE IN THE OCCUPANCIY AND POSITION. OKAY, SO THIS IS THE CASE FOR THESE GENES THAT ARE DIRECTLY AND VERY RAPIDLY DOWNREGULATED BY GETTING RID OF IT. WHAT WE WANTED TO ASK NEXT WAS WHETHER THIS WAS A COMMON FEATURE AND IS THIS PERHAPS A GENERAL ROLE FOR POLYMERASE PAUSING IS TO KEEP NUKESOMES AT BAY? WE TOOK ALL OF THE GENES THAT HAD SIGNIFICANT OCCUPANCIY NEAR THE PROMOTEERS AND RANK-ORDERED THEM BY THIS PAUSING INDEX SO THAT THE MOST PAUSED GENES ARE AT THE TOP AND THE LEAST PAUSED GENES ARE AT THE BOTTOM. AND WE ASKED WHAT HAPPENS TO THE NUKESOME OCCUPANCIY SDMKS IT TURNS OUT THAT THESE PAUSED GENES, YOU SEE A REALLY SIGNIFICANT INCREASE IN NUKESOME OCCUPANCIY CENTERED RIGHT OVER THE TRANSCRIPTION START SITE. AND SO WHAT THAT TELLS US AGAIN IS THAT THERE IS A DYNAMIC COMPETITION, SO WE KNOW THAT IT TURNS OUT THAT COULD BE EXTENDED TO THE PROMOTER REGION. IF THE POLYMERASE IS THERE, NUKESOMES AREN'T GOING TO COME IN. AND YET WHEN A NUKESOME SITS TOWN, YOU NEED A MODELER TO GET RID OF IT IN ORDER TO BE ABLE TO RESTART THE TRANSCRIPTION PROCESS. OKAY, SO WHAT THAT TELLS US THEN IS THAT PAUSING HAS A MUCH MORE COMPLICATED OR SOPHISTICATED EFFECT ON TRANSCRIPTION THAN WE'D ORIGINALLY ASSUMED. SO WE KNOW, WE KNEW THAT PAUSING COULD BE CONSIDERED REEXPRESSIVE FOR TRANSCRIPTION BECAUSE IT LIMITS THE RELEASE OF PAUL 2 DOWNSTREAM INTO THE GENE. BUT WHAT THAT TELLS US, THOUGH, IS THAT THIS COULD BE A POSITIVE EFFECT OF THIS MINOR DELAY IN THAT IT CAN MAINTAIN ITS OPEN CHROMATIC ARCHITECTURE. AND IF YOU CAN DELAY THIS RELEASE UNTIL A NEW POLYMERASE COMES IN AND IS WAITING IN THE WINGS SO THAT THIS PROMOTER REGION IS ALWAYS OCCUPIED, YOU CAN MAINTAIN THAT GENE IN A CONSTANTLY ACCURATE CAN STATE. SO I WANT TO BELABOR THIS POINT JUST A LITTLE BIT BECAUSE WHAT I THINK THIS TELLS US THEN IS THAT WHETHER OR NOT PAWINGS IS GOING TO HAVE A POSITIVE OR A NEGATIVE EFFECT ON GENE EXPRESSION IS REALLY THEN GOING TO BE DEPENDENT ON THE CHARACTERISTICS OF THE PROMOTER CHROMATIN. AND SO THAT REALLY IS AFFECTING THE RATE OF YOU CAN INSOME REASSEMBLY SO THAT IF A POLYMERASE IS RELEASED INTO THE GENE, HOW LONG IS IT WITH THAT PROMOTER EMPTY BEFORE A YOU CAN INSOME FINDS THAT TO BE A DELIGHTFUL PLACE TO CONSTITUENT SIT ITSELF DOWN? AND THAT IS GOING TO BE A REFLECTION OF A COUPLE OF DIFFERENT THINGS. ONE IS THE SEQUENCE OF THAT PROMOTER. SO THERE ARE SOME CONSEQUENCES THAT ARE KNOWN TO BE HAPPILY WRAPPING AROUND NUKESOMES AND OTHERS THAT ARE RIGID THAT DON'T ASSEMBLE VERY WELL INTO NUKESOMES AND WERE THOUGHT TO BE INTRINSICALLY NUKESOME-DEPRIVED AND THIS IS GOING TO BE DEPENDENT ON THE AVAILABILITY OF REMODELERS. SO GENES LIKE THE HITCHOCK GENES WILLR ALMOST BOUND BY A CHROMATIN REMODELER. MAY NOT NEED PAUSE POLYMERASE TO KEEP CHROMOSOMES AWAY AND LET ME EXPLAIN WHY THOSE GENES ARE EXPRESSED BY PAUSING. AND SO THERE CAN BE THESE DIFFERENTIAL EFFECTS AND I'LL GET BACK TO THIS LATER, THAT ARE GOING TO BE LINKED TO THE INHERENT ASPECTS OF PROMOTER CHROMATIN. OKAY, SO WHY WOULD YOU HAVE HAVE A PAUSE 2 AND WHAT PURPOSE WOULD THIS SERVE IN A CELLULAR CONTEXT AND ESPECIALLY WITHIN THESE SIGNAL RESPONSIVE GENES THAT WORK? I TOLD YOU IT'S ENRICHED IN THESE NETWORKS BUT I DIDN'T TELL YOU THE FUNCTION THAT IT'S SERVING. BUT OF COURSE BY ANALOGY THE IDEA THAT IT'S POISONING THE DOWNSTREAM TARGETS OF THESE PATHWAYS FOR RAPID ACTIVATION. BUT THIS HAS NEVER REALLY BEEN TESTED IN ANYTHING OTHER THAN THE MODEL. SO WE WANTED TO TEST THIS AND THE MODEL SYSTEM THAT WE DECIDED TO DO THIS IN WAS IN THE NOT AATE IMMUNE RESPONSE IN THE FLY, BECAUSE THIS WAS ENRICHED IN BOTH NFL TARGETS AND IN GENES AND I'LL START JUST BY SAYING THAT THE FLY IMMUNE SYSTEM IS HOMMOLGUS TO THE MAMMALIAN SYSTEM, AND SO THESE TWO PATHWAYS, BOTH OF THEM CONVERGE ON THE TRANSCRIPTION FACTORS, OF WHICHREL IS THE FOUNDING MEMBER. AND SO WE HAVE THESE IMMUNE PATHWAYS THAT ARE HIGHLY ENRICHED IN PAUSING. AND IN ANALOGY IT SHOULD BE THE DOWNSTREAM TARGETS, THESE ANTI-MICROBE BALAND THAT SHOULD HAVE POLL 2. OF COURSE, WE'D CHIP AND SHIP AND A BUNCH OF DIFFERENT CELL TYPES, AND WE LOOKED AT THEM AND UNFORTUNATELY, THE ANTI-MICROBIAL PEPTIDE GENES LOOKED LIKE THIS, WHICH DIDN'T HAVE ANY PRELOADING SO WE LOOKED IN FLIES AND LOOKED EVERYWHERE AND WE COULD NEVER SEE EVIDENCE THAT THE ANTI-MICROBIAL PEP TIDE GENES EXHIBITING PAUSING. ALL RIGHT, SO WE THOUGHT THAT MAYBE THERE ARE OTHER GENES THAT ARE PAUSED AND SO WE LOOKED AT A MIO ARRAY OF ALL THE GENES INDUCED IF A COUPLE OF HOURS AND HERE I'M SHOWING YOU ALL THESE GENES RANK ORDERED BY THE MOST INDUTIESED TO THE LEAST INDEUCED. AND THESE ARE PRIOR TO INDUCTION. AND WHAT YOU WILL NOTICE IN PARTICULAR THOSE GENES THAT ARE HIGHLY INDUCED AREN'T PRELOADED WITH POLL 2 SO THERE IS NOT PAUSING OF THESE GENES BEFORE INDUCTION. IN FACT, THESE ARE LESS LIKELY TO BE PAUSED THAN YOUR AVERAGE GENE. SO THEN START SCRATCHING YOUR HEAD AND YOU SAID WE'RE STILL GETTING THESE GO CATEGORIES TELLING US THAT THERE IS SOMETHING GOING ON IN THIS PATHWAY. WE HAD TO START THINKING ABOUT THE SIGNAL TRANSCRIPTION CASCADE AND THE OTHER PARTS OF THIS PATHWAY BEYOND JUST THE DOWNSTREAM EFFECTERS AND ASK WHETHER OR NOT THESE GENES WERE POTENTIALLY BEING TARGETED BY THE COMBROIL MERRASE. SO WE STARTED LOOKING AT THE PROMOTEERS OF THESE GENES AND FOUND SOMETHING STRIKING, WHICH IS THAT MANY, MANY OF THEM, THE INHIBITER ITSELF, LIGANS FOR THE TOLL PATHWAY, WERE BEING EN CODED BY GENES THAT HAD HIGH LEVELS OF PAUSE POLL 2. AND NOW I AM SHOWING YOU A HEAT MAP OF ALL OF THE GENES IN THIS PATHWAY THAT I SHOWED YOU EARLIER WHERE YOU CAN SEE THAT THERE IS A GREAT EN RICHMENT OF POLL 2 AND IN FACT THESE GENES ARE ABOUT FOUR TIMES MORE LIKELY THAN THE AVERAGE GENE TO HAVE A POLYMERASE. SO THIS I FIND KIND OF COOL IN THAT HERE I AM SHOWING YOU THIS PATHWAY WHERE I AM COLOR-CODING THE PROTEIN BY THE DWIKS DISTRIBUTION AT ITS PROMOTER. AND WHAT I THINK YOU CAN SEE IS THAT THE PROTEINS THAT FLOWED FLOAT AROUND IN EXTRA CELLULAR SPACE AREN'T SO MUCH TARGET BY PAUSING BUT THINGS LIKE THE RECEPTOR AND THE CAP SPACES AND ALL OF THE TRANSCRIPTION FACTORS ARE EN CORRODE -- EN CODED BY GENES THAT HAVE PAUSE PROMOTER RIGHTS. SO WE WANTED TO ASK WHETHER OR NOT PAUSING MIGHT AFFECT BASAL EXPRESSION OF THESE GENES IN A WAY THAT IT COULD AFFECT THE PATHWAY ACTIVITY. AND WE'D DONE A MIKO ARRAY AND I'M SHOWING YOU VALIDATION OF THAT ARRAY WHERE YOU CAN SEE THAT LOSS OF PAUSING IN THESE CELLS CAUSED A DOWNREGULATED EXPRESSION OF A NUMBER OF THESE DIFFERENT PATHWAY COMPONENTS. AND SO THAT SUGGESTS THAT PAUSING MAY NOT BE ABOUT PRIMING EVERY DOWNSTREAM TARGET FOR ACTIVATION BUT IT MAY BE ABOUT PRIMING THE SIGNAL TRANSDUCTION NETWORK AND MAINTAINING ITS BASAL ACTIVITY. AND SO ONE OF THE THINGS THAT REALLY CAUGHT OUR EYE WAS THE DECREASED EXPRESSION OFREL IN THESE CELLS, BECAUSE IN CONTRESS TRAS TO A LOT OF THESE THINGS, WE ACTUALLY HAD ANTIBODIES AND WAYS TO MANIPULATE THIS TRANSCRIPTION FACTOR. SO WE WANTED TO LOOK AT WHETHER OR NOT MOUSE DELEGS LODE IT A MEANINGFUL LOSS OF PROTEIN IN THESE CELLS AND IN INDEED IT DOES. AND WHAT THAT SUGGESTS IS PAUSING SOMETHING SOMEHOW MAINTAINS THE LEVEL OF THE TRANSCRIPTION FACTOR IN THESE CELLS IN A MANNER NALOSS OF PAUSING WOULD DAMPEN EVERYTHING ABOUT THIS PATHWAY. SO IF THIS TRANSCRIPTION FACTOR IS NECESSARY FOR TURNING ON ALL GENES, INCLUDING THOSE ANTI-MICROBIAL PEP TIDE GENES, NONETHELESS, DELETION BY AFFECTING THIS HUB OF THIS NETWORK SHOULD DAMPEN EXPRESSION OF ALL GENES OF THE IMMUNE CHALLENGE. SO WE TESTED THIS. HERE, I AM SHOWING THIS IN CELLS BUT WERNL WELSH ABLE TO DO THE SAME EXPERIMENT IN FLIES. AND WHAT WE TESTED WERE A NUMBER OF THESE PEP TIDE GENES THAT AGAIN DIDN'T HAVE THE MEDIATED PAUSING BUT WERE NONETHELESS NOT EXPRESSED AS WELL IN THE ABSENCE. AND THIS WAS AS TRUE AS THE GENES IN TARGETS THAT HAD PAUSE POLL 2. AND THIS SUGGESTS THAT IT'S THE PATHWAY THAT'S BROKEN, NOT THE DOWNSTREAM TARGET. AND TO REALLY NAIL THAT DOWN, WE HAD AN ACTIVE PROTEIN SO WE COULD TRANSECT THE CELLS WITH THESE AND SHOW THAT NOW THESE GENES COULD BE ACTIVATED JUST FINE IN A MANNER THAT WAS -- THAT DID NOT DEPEND AT ALL ON MELF. AND THE DELEGS WAS CAUSING BASAL FOR DYSFUNCTION IN THE PATHWAY AND NOT AT THE SPECIFIC DOWNSTREAM TARGET GENE. ALL RIGHT, SO TO WRAP THIS PART UP, I WANT TO GIVE YOU AN OVERVIEW OF WHAT WE'RE THINKING ABOUT DOING IN THESE SIGNAL RESPONSIVE NETWORK, WHERE WE MOVED FROM THINKING THAT PAUSING IS REALLY JUST FACILITATING RAPID ACTIVATION OF DOWNSTREAM TARGET GENES AND THINKING OF THIS AS HAVING MUCH PRODDER ROLES WHERE IT'S PREVALENT ACROSS THE GENES WHERE IT'S MAINTAINING THE BASAL EXPRESSION AND COORDINATING THE EXPRESSION OF GENES WITHIN THIS CASCADE TO ENABLE EITHER ENHANCED OR ATTENUATED TRANSCRIPTION ACROSS THIS NETWORK. ALL RIGHT, SO -- BROADER. THAT'S FINE. THAT'S NICE FOR THE DRE CEPHALUS SYSTEM BUT YOU MIGHT HAVE ASKED WHAT HAPPENS IN MAMMALS AND WE WANTED TO ADDRESS THAT QUESTION OURSELVES AND ONE OF THE MOST OBVIOUS PLACES TO LOOK WAS IN MAMMALIAN DEVELOPMENT, BECAUSE THE MOUSE AND EMBRYONIC STEM CELLS HAD BEEN EXTENSIVELY INVESTIGATED AND POLL 2 PAUSING HAD BEEN SHOWN TO BE HIGHLY ENRICHED IN CELL TYPES. NOW, THE MODEL FOR WHAT PAUSING WAS DOING IN EMBRYONIC STEM CELLS IS REALLY BASED ON SOME BEAUTIFUL DATA BY MIKE LEVINE AND DRE SOLVA, WHERE THE IDEA THAT PAUSING WOULD OCCUR AT DEVELOPMENTAL REGULATORS, THAT GENES WOULD BE TURNED ON DURING DEVELOPMENT AND THAT PAUSING WOULD HELP POISE THEM FOR THIS ACTIVATION BUT THAT IT WOULD REPRESS THEM IN THE ABSENCE OF A DIFFERENTIATION SIGNAL. SO IT WOULD REPRESS THE PROCOCIOUS TRANSCRIPTION. SO THE PREDICTION WOULD BE IF YOU GOT RID OF THE EMBRYONIC STEM CELLS OR ANOTHER CELL TYPE, THAT THIS WOULD RESULT IN SPONTANEOUS DIFFERENTIATION. AND THAT THE STATE WOULD BE LOST BECAUSE THE GENES WOULD COME ON. AND IN FACT, THERE IS A PAPER PAPER SUGGESTING THAT THIS MIGHT BE THE CASE WHERE LEE'S LAB KNOCKED DOWN THIS MOUSE B SUBUNIT IN THE COMPLEX OF THE EMBRYONIC STEM CELLS AND THEY SHOWED THAT THIS CAUSED A LOSS IN PROLIFERATION IN EMBRYONIC STEM CELLS AND ALSO IN STAINING, WHICH IS GENERALLY USED AS A MARKER OF THIS STATE. HOWEVER, WE WANTED TO GET AT THE MOLECULAR DETAILS UNDERLYING THIS AND REALLY UNDERSTAND WHAT THE GENE TARGETS OF THIS MIGHT BE. AND SO TO TRY TO UNDERSTAND WHAT GENES MIGHT BE PAUSED IN EMBRYONIC STEM CELLS AND WHETHER OR NOT THIS INCLUDED A LOT OF DEVELOPMENTALLY RESPONSIVE GENES, WE USED EXPRESSION DATA THAT WAS PREVIOUSLY PUBLISHED AND ASKED ARE GENES TURNED ON QUICKLY AFTER A DIFFERENTIATION SGLASHLGS WERE THEY PAUSED PRELOADED WITH POLL 2 PRIOR TO DIFFERENTIATION? RETINOIC ACID IS ONE OF THE COMMONLY USED WAYS AND WE USED A MICRO ARRAY TELLING US ALL OF THE GENES THAT WERE UPREGULATED WITHIN A COUPLE OF HOURS OF TREATMENT AND WE LOOKED AT THE PALTIC DISTRIBUTION AROUND THESE PROMMERS. AND IT WAS NOT LESS IMPRESSIVE AS FAR AS THE ROLE OF PAUSING IN THESE INDUCED GENES. AND SO VERY LITTLE POLL 2 RECRUITED TO THESE GENES PRIOR TO THE DIFFERENTIATION SIGNAL. IS IN FACT, THESE GENES ARE ABOUT AS HALF AS LIKELY AS THE AVERAGE GENE TO HARBOR POLL 2. ALL RIGHT, SO IF IT'S NOT -- IF PAUSE POLYMERASE ISN'T SITTING THERE WAITING, THESE GENES THAT GET TRIGGERED TO GO ON UPON SOMETHING LIKE RETIN CANOIC ACID SIGNALING AND WHERE THE GENES THAT ARE PAUSED IN THESE CELLS? AND SO WE TOOK AGAIN RICK YOUNG'S DATA AND WE TOOK THE TOP 10 PERCENT, THE MOST HIGHLY PAUSED GENES AND RAN THEM THROUGH AN INGENUITY PATHWAY AND WHAT WE CAME UP WITH WAS SIGNALING PATHWAYS LIKE THAT WERE HIGHLY PAUSED AND AND SO NOW I'M SHOWING YOU THE SIGNALING PATHWAYS WITH THIS MAP IRK COMPONENT WITHIN IT AND THIS IS THE POLL 2 DISTRIBUTION AROUND THE PROMOTERS OF THESE GENES AND NOW WE'RE FINDING WITHIN THIS SIGNAL TRANSDUCTION CASCADE SIMILAR TO THE SITUATION IN OUR FLY CELLS, THAT THE SIGNAL TRANSDUCTION CASCADE IS MUCH MORE LIKELY THAN AVERAGE TO ACTUALLY BE -- HAVE THIS EN RICHMENT OF THE PROMMER THAN THE AVERAGE GENE. AND THIS IS ALSO IN THE ENGAGED ELONGATION CONFIDENCE POLYMERASE AROUND THE PROMOTERS OF KEY REPRESSERS OF THE SIGNALING PATHWAY. OKAY, MAP IRK WAS AN EXCITING THING TO HIT UPON, BECAUSE THIS IRK PATHWAY PLAYS A REALLY IMPORTANT ROLE IN EMBRYONIC STEM CELLS. AND SO IN THESE CELLS THERE IS THIS TRANSITION BETWEEN THIS GROUND STATE OR THIS MOST NAIVE STATE AND INTERMEDIATES THAT ARE ON THE PATHWAY TO DIFFERENTIATION. AND SIGNALING THROUGH MAP IRK, LARGELY THROUGH THESE FGF PROTEINS, IS WHAT TRIGGERS THE BEGINNINGS OF THE DIFFERENTIATION PROCESS. AND SO THIS INTERMEDIATEIAT CAN TURN INTO PRIMITIVE ENDODERM AND A MARKER FOR THIS, I'LL TALK ABOUT IN A FEW MINUTES. AND WHAT WE KNOW IS THAT IN IRK KNOCKOUT CELLS, YOU CAN DERIVE EMBRYONIC STEM CELLS AND THEY STAY IN THIS STATE. THEY REALLY DON'T LIKE TO GO THROUGH DIFFERENTIATION BECAUSE IRK SIGNALING IS A BIG PART OF GETTING YOU TOWARDS THE RIGHT-OF-WAY THIS DIAGRAM. AND SO IN FACT PEOPLE MAKE USE OF THIS AND THE MEDIA THAT IS USED TO GROW EMBRYONIC STEM CELLS AND SO THE MEDIA THAT IS REALLY NICE FOR ENHANCING THIS NAIVE OR GROUND STATE IS CALLED 2 I'MS AND ONE OF THEM IS AN INHIBITOR OF THE MAP IRK PATHWAY. AND INHIBITION OF IRK PREVENTS DIFFERENTIATION, AND SO WE HAVE THIS ROLE FOR PAUSING IN THE MAP IRK PATHWAY. SO A ROLE FOR PAUSING YES, IN DIFFERENTIATION BUT NOT IN THE WAY THAT WE'D INITIALLY ANTICIPATED BUT IN A DIFFERENT WAY WHERE IT MAY BE FUNCTIONING THROUGH THE SIGNAL TRANSDUCTION CASCADE. OKAY, SO TO TRY TO GET A HANDLE ON THIS OURSELVES, WE DID SOMETHING THAT AS A FLY BIOCHEMIST I NEVER THOUGHT WE WOULD DO BUT WE MADE A KNOCKOUT MOUSE AND A LOT THE WORK IS THE WORK OF GEORGE FRUM, WHO HAS HELPED IMMENSELY IN THE BREEDING OF THE INITIAL MOUSE GENERATION BY GINGER AND WITH THE EMBRYOLOGY, I'LL SHOW AT THE END WITH LUCY WILLIAMS. WE AND OTHERS, HAD GENERATED A SLAURP MOUSE, DISCOVERED THAT THE HETEROZYGOUS MICE WERE ABSOLUTELY FINE AND COMPLETELY SUFFICIENT FOR LIFE. BUT WHEN YOU MATE THESE MICE TOGETHER, YOU ABSOLUTELY CANNOT GET NULL MICE. SO THE LAUGHALITY CAN OCCURS POST IMPLANTATION. AND WHEN YOU DO OUTGROWTH EXPERIMENTS, WE OFIND THAT THE DEFECT IS NOT IN THINGS LIKE THE CELLS BUT IN A MASS-SPECIFIC DEFECT AND THIS IS THIS CELLS FROM WHICH EMBRYONIC STEM CELLS ARE DERIVED. AND SO THIS WAS EXCITING, BUT -- AND IT'S CONSISTENT AGAIN WITH WORK FROM LEE'S LAB WHERE THEY GENERATE A SIMILAR MOUSE. WE WANTED TO UNDERSTAND WHAT THE MOLECULAR DEFECTS WERE THAT WERE LEADING TO THIS LETHALITY. AND THAT IS A TOUGH PHENOTYPE TO STUDY. SO WHAT WE WANTED, TOO, IS GET A HANDLE ON THE MOLECULAR ASPECTS OF THIS IN A CELL-PACED SYSTEM SO WE CROSSED OUR MOUTH MOUSE WITH A RECOMBINATION AND WHEN TAM OX FINN IS ADMINISTERED IF SUBCULTURE IN THE MOUSE, IT CAUSES RECOMBINATION AND WE CAN VERY RAPIDLY THEN ASK WHAT HAPPENS TO OUR CELLS IN THE ABSENCE OF MAVL. ONE OF THE FIRST THINGS THAT GEORGE DID WAS HE DERIVED EMBRYONIC CYBER BLASTS FROM THESE MICE AND TRIED TO REPROGRAM THEM INTO CELLS USING THIS COCKTAIL OF FACTORS THAT CAUSES PLURIPROTON STEM CELLS. AND WHAT HE FOUND IF YOU KNOCK MOUSE OUT IN THE FIBER BLASTS, YOU CANNOT MAKE INDUCE YOU HAD STEM CELLS FROM THEM. SO THAT'S NOT HAD BUT IT DOESN'T TELL US WHETHER IT IS IMPORTANT FOR GETTING TO POTENCY OR MAKING IT. WHAT HE DID WAS HE REPROGRAMMED THEM INTO THE PLUROPOTENT STEM CELLS AND THEN VALIDATE D THEM AND THEN KNOCKED OUT MOUSE TO ASK IN THE MAINTENANCE PHASE NOW WHAT HAPPENS TO THESE CELLS, TO THE SELF-RENEWAL CHARACTERISTICS AND TO THEIR POTENCY AND REALLY KEEPING A FOCUS ON WHETHER OR NOT THERE IS THIS SPONTANEOUS DIFFERENTIATION. SO HERE ARE SOME PICTURES OF OUR MOUSE KNOCKOUT CELLS AND YOU CAN SEE FOR ABOUT A WEEK AFTER THE RECOMBINATION EVENT, CELLS LOOK FINE. BUT AFTER THAT, THEY START TO LOSE THE STEM CELL MORPHOLOGY AND REALLY DRAMATICALLY LOSE THEIR PROLIFERATIVE CAPACITY. AND I SHOULD NOTE THAT THIS WAS OBSERVED EARLIER, BUT IT WAS THOUGHT TO BE SORT OF AN ADDON EFFECT OF THE FACT THAT THESE CELLS WERE DIFFERENTIATED AND THEY WERE VERY PROLIFERATION RATE AND AS THEY DIFFERENTIATE, THE PROLIFERATION RATE DECREASES QUITE CONSIDERABLY. SO WE SAW THE SAME LOSS OF STEM CELL MORPHOLOGY, THES LOSS OF PROLIFERATION, BUT WHAT WE WERE LEFT CONVINCED ABOUT WAS THIS WAS REALLY A SPONTANEOUS DIFFERENTIATION FENE OR-TYPE THAT WAS DRIVING THIS. AND SO WHAT WE WANTED TO DO THEN WAS TO INVESTIGATE GENE EXPRESSION IN THESE CELLS WHEN WE KNEW THAT THE MOUSE PROTEIN WAS GONE BUT BEFORE THE ONSET OF THIS KIND OF FUNNY PHENOTYPE. AND SO GEORGE DID AND AN KERMIT AND FIRST THING THAT HE NOTICED WAS THAT THE PLUROPOTENCY GENES WERE NOT CHANGED AT ALL IN THEIR EXPRESSION, NOR WERE ANY OF THE TRADOISHL MARKERS OF DIFFERENT LINEAGE OF UPREGULATED CELLS. SO THIS DIDN'T ARGUE FOR SPONTANEOUS DIFFERENTIATION. NOW, THESE GENES WEREN'T CHANGED IN THEIR EXPRESSION BUT NEARLY 1,000 GENES WERE SO THE KNOCKOUT WAS CLEARLY AG AN EFFECT IN THESE CELLS BUT WE WEREN'T FINDING THIS TO BE ASSOCIATED WITH THE DIFFERENTIATION PHENOTYPE. INSTEAD, AND THIS IS WHERE THINGS GOT EXCITED, WHEN WE DID AN ANALYSIS OF THE KINDS OF GENES THAT WOULD BE AFFECTED, WE GOT THE PATHWAY AND A PATHWAY WHERE WE HAD PREVIOUSLY SEEN AN EN RICHMENT IN OFF ANDING NOW WHEN WE KNOCKED MOUSE OUT, WE ARE SEEING THAT THIS IS A PATHWAY THAT IS EFFECTIVE, AND AGAIN, THESE ARE THE MOST DOWN-AND-UPREGULATED GENES ON OUR MICRO RNA. I HAD TOLD YOU THAT A LOT OF GENES ARE PAUSED IN EMBRYONIC STEM CELLS AND WE WENTED TO MAKE SURE TO CHECK THAT THAT WAS TRUE AND HERE I AM JUST SHOWING YOU AN EXAMPLE OF TWO GENES WHERE GEORGE DID A CHIP AND HE IS USING TO DETECT THE AMOUNT OF PROMOTER, WHICH IS QUITE HIGH AND LESS AS IT'S MAKING ITS WAY INTO ELONGATION. AND YOU CAN DETECT MAVM D. MORE IMPORTANTLY, WHEN WE KNOCK MOUSE OUT, WE CAN SEE THE EXPECTING CHANGES IN POLYMERASE DISTRIBUTION IS. AND WHAT I MEAN BY THAT IS IF WE LOOK AT A GENE THAT'S DOWNREGULATED, WE SEE LESS POLYMERASE NEAR THE PROMOTER, LIKE THE EXAMPLE I SHOWED YOU IN THE SLIDE, WHERE THE POLYMERASE IS LESS AND YOU REALLY DON'T SEE ANY INCREASE OF POLYMERASE GOING INTO THE GENE N CONSDWRASHGS THE GENES THAT ARE UPREGULATED, WE DO SEE AN INCREASE IN POLYMERASE BEING RELEASED INTO THE GENE, TELLING US THAT THE EFFECTS OF MAVL ON THESE GENES ARE DIRECT THROUGH DISTRIBUTION. AND THE LOSS OF MAVL IS LEADING TO INCREASES AND DECREASES IN EXPRESSION. OKAY, SO NOW IF I SHOW YOU THE PATHWAY AND I COLOR-CODE THE PROTEEPZ HERE BY WHETHER THE GENES ARE UP OR DOWNREGULATED, WE FOUND SOMETHING KIND OF INTERESTING HERE. SO AGAIN, LYINGANS AND RECEPTORS FROM THIS PATHWAY AND THINGS LIKE THE IMPORTANT TRANSCRIPTION FACTORS TENDED TO BE DOWNREGULATED. AND THINGS SHOWN AS THIS SORT OF STOP SIGN KIND OF SHAPE, WHICH INDICATES REPRESSORS, TENDED TO BE UPREGULATED. AND SO THIS WAS INTERESTING TO US BECAUSE IT RAISINGS A QUESTION ABOUT WHY SOME GENES ARE UP OR DOWNREGULATED. AND THINGS THAT EXPRESSED PATHWAY ACTIVITY WERE UPREGULATED, SUGGESTING THAT WE MAY HAVE A COHERENT EFFECT IN THESE CELLS. I'LL GET TO THIS FIRST QUESTION FIRST AND I WANT TO TAKE YOU BACK TO WHAT I WAS SAYING EARLIER ABOUT THE ROLE OF CHROMATIN AND DICTATING WHETHER OR NOT PAUSING HAS A NEGATIVE OR POSITIVE EFFECT ON GENE EXPRESSION AND THAT THIS CAN BE CONSTRUED AS AN EFFECT OF THE CONSEQUENCE OR THE CHROMATIN REMODELING CAPACITY OF THAT PROMOTER. AND THIS IS REALLY KIND OF A FUN THING TO LOOK AT IN MAMMALS BECAUSE A LOT OF MAMMALIAN PROMOTERS LIES WITHIN THIS CPG ISLAND, WHICH ARE FOUND TO BE NUKESOME-DEPRIVED. AND THERE ARE ARGUMENTS OUT THERE ABOUT WHO WL IT'S THE SEQUENCE THAT'S CAUSING THEM TO BE NUKESOME DEPRIVED OR FACTORS BUT EMPIRICALLY IT'S TRUE THAT THESE THINGS ARE RATHER NUKESOME DEPRIVED AND WE THOUGHT THAT MAYBE GENES IN CPG ISLAND DON'T NEED PAUSING TO KEEP THEM OPEN. SO MAYBE THESE GENES WOULD BE MORE LIKELY TO BE REPRESSED BY MEFL AND UPREGULATED UPON THE LOSS. AND WE LOOK ALL THE GENES THAT WERE IN ENGIN SXOOUT WE ASKED FOR THE UPREGULATED, UNCHANGED OR DOWNREGULATED GENES, HOW MANY OF THESE FALL WITHIN CP G ISLAND. AND SO THERE IS A STRIKING AMOUNT OF UPREGULATED GENES WITHIN THESE CPG ISLANDS. AND SO THIS DOESN'T LEND ITSELF VERY WELL TO STATISTICS AND SO WHAT WE DID WAS COUNTED THE NUMBER OF CP G NUCLEOTIDES AND THERE IS A HUGELY STATISTICAL SIGNIFICANT INCREASE IN PC -- CPG CONTENT SUGGESTING THAT THE SEQUENCE ITSELF HAS IT COVERED AND YOU DON'T NEED THE PAUSED POLYMERASE TO KEEP IT AT BAY. IN CONTRAST, GENES WHERE THERE IS A VERY CP G CONTENT AND NUKESOMES MAYBE LIKE THAT REGION, THEN PAUSING IS GOING TO BE MORE IMPORTANT FOR ACTIVATING THE GENE OR KEEPING THE GENE ON. OKAY, SO THAT SUGGESTED THAT THERE MAY BE SOME METHOD TO THE MADNESS OF WHAT'S GOING ON HERE. AGAIN AND THAT THESE GENES ALL LIE WITHIN CPG ISLAND PROMOTERS. AND SO AGAIN, GETTING BACK TO THIS QUESTION OF WHETHER OR NOT THERE IS A COHERENT EFFECT ON MAP IRK SIGNALING IN THESE CELLS. WE STARTED BY DOING -- AND WHAT YOU CAN SEE IS WHEN WE LOOKED AT CELLS THAT HAVE BEEN INDUCED AND HAVE A PULSIVE IRK SNAULG SEE LOT OF NICE IRK ONE AND TWO AND IRK TWO IS THE GUY THAT GETS POSFOR LATED IN STEM CELLS. AND WE SEE SIGNIFICANTLY LESS OF THIS IN OUR MEFL KNOCKOUT CELLS SUGGESTING THAT THERE IS A DECEASE IN THE NICHING THESE CELLS. SO LET'S GO BACK TO IRK THE ROLE OF IRK IN THE DIFFERENTIATION PROCESS. WHAT I SAID IS THAT INHIBITING IRK SIGNALING, EITHER BY THE USE OF THIS TWO-LINE MEDIA KIND OF PUSHES ITSELF TOWARDS THIS NAIVE STATE. AND SO WE WONDERED WHETHER OR NOT THE MEFL NEW YORKOUT IN A FUNCTIONAL PHYSIOLOGICAL ASPECT WOULD MEMMIC -- MIMIC THIS IN PREVENTING DIFFERENTIATION. AND SO WE ASKED' VERY SPECIFIC QUESTION IN THE EMBRYO ABOUT WHETHER OR NOT LOSS OF MEFL WOULD BLOCK FORMATION AND IT'S SHOWN. SO THIS IS AN EXPERIMENT FROM AUSTIN'S LAB AND YOU SEE HOW EXPERT HE DOES THESE EXPERIMENTS. AND HERE HE'S TAKEN EMBRYOS THAT HE'S GROWN EITHER IN CONTROLLED MEDIA OR THE IRK INHIBITOR. AND YOU CAN SEE A PSYCHOLOGIST OF THE POTENCY MARKERS WITHIN THIS INNER CELL MASS AND YOU SEE AN ABSENCE OF STAINING, WHICH IS AROUND OUTSIDE OF THIS MASS. AND SO AGAIN, THE IDEA IS IF MEFL KNOCKOUT REDUCES SIGNALING, THEN IN CONTROL MEDIA WE MAY SEE THIS EFFECT. AND SO THIS IS SOME EMBRYOLOGY DONE BY LUCY WILLIAMS WHERE I AM SHOWING YOU THESE EMBRYOS FROM E 3.75 AND YOU CAN SEE IN THE MEFL KNOCKOUT THERE IS A NUMBER OF POSITIVE CELLS AND THE MASS IS SLIGHTLY EXPANDED, AND THERE IS AN ABSENCE OF STAINING. SO IN THE CONTROLS YOU SEE THIS NICE STAINING AROUND THE IC M, AND IN THE MEFL KNOCKOUT CELLS. SUGGESTING THAT WITH THE INHIBITOR, EXPRESSION OF THE PATHWAY REALLY ACTUALLY PREVENTS THE DIFFERENTIATION IN THE EARLY EMBRYO, SUGGESTING A POTENTIAL CONTRIBUTOR TO THE EMBRY YONK KNOCKOUT. THE TAKE-HOME IS THAT MEFL MEDIATED PAUSING, THE LOSS OF PAUSING ACTUALLY, IT DOESN'T CAUSE MUCH DIFFERENTIATION. IN FACT, WE SEE THE OPPOSITE OF THAT. IN FACT, MEFL IS REALLY ESSENTIAL FOR MAINTAINING THE IRK PATHWAY IN A STATE THAT IS -- MAMAKES IT CAPABLE OF TRIGGERING THIS DIFFERENTIATION SIGNAL AND THAT MEFL KNOCKOUT PREVENTS THIS BY ATTENUATING IRK SIGNAL. SO WHAT I ALSO THINK THAT THIS MEANS IS THAT THE PROLIFERATION DEFECT THAT WE SEE BOTH IN THESE EMBRYOS AND IN STEM CELLS CAN'T BE ATTRIBUTED SIMPLY TO A DIFFERENTIATION PHENOTYPE. BUT THAT MEFL KNOCKOUT IS REALLY FUNDAMENTALLY EFFECTING THE SELF-RENEWAL PROPERTIES OF THE EMBRYONIC STEM CELLS SO THAT PAUSING ISN'T JUST IMPORTANT FOR THE STEM CELLS BUT SEEMS TO BE IMPORTANT FOR THE KEY FUNCTION UGZ OR THE KEY CHARACTERISTICS OF STEM CELLS, WHICH IS SELF-RENEWAL, THEY'RE RAPID -- THEIR RAPID PROLIFERATION RATE AS WELL AS THEIR ABILITY TO DIFFERENTIATE. I HOPE I'VE CONVINCED YOU THAT PAUSING HAS BROAD RULES IN DIFFERENT SPECIES, AND FUNCTIONING THROUGH THESE STIMULUS RESPONSIVE PATHWAYS IN A BROADER CONTEXT THAT THAN WE INITIALLY SUSPECTED AND THAT IT'S AFFECTING THE BASAL EXPRESSION OF A NUMBER OF VERY IMPORTANT GENES, LIKELY BY AFFECTING THEIR PROMOTER CHROMATIN STRUCTURE. AND I SHOULD JUST NOTE THAT THE GENES THAT ARE BEING TARGETED BY MEFL MEDIATED PAUSING ARE THINGS LIKE RECEPTORS AND TRANSCRIPTION FACTORS WHERE IN FACT WE KNOW THAT A TWO-FOLD OR A THREEFOLD CHANGE IN BOTH OF THESE TRANSCRIPTION FACTORS OR RECEPTORS ACTUALLY MAKES A DIFFERENCE TO THE PHYSIOLOGY OF THE CELL. THIS IS NOW SIGNAL TRANSDUCTION NETWORKS GET TUNED. SO YOU DON'T NEED A TENFOLD UPREGULATION TO MAKE THAT CELL MORE RESPONSIVE. I THINK THAT THIS IS REALLY AN ELEGANT WAY TO FINE TUNE THE PATHWAYS IN A COORDINATED CONTEXT. MY MODULATING THE EXPRESSION OF THESE HUBS OF NETWORKS, THE HUBS OF THE SIGNALING CASCADES AND YOU CAN REALLY TUNE THE BEHAVIOR OF A CELL TYPE IN A WAY THAT I THINK ALLOWS FOR SOME OF THE FINE TUNE REGULATION YOU NEED DURING DEVELOPMENT AND IMMUNE CHALLENGES, ET CETERA. OTHER RESPONSES TO ENVIRONMENT. ALL RIGHT, SO THE FINAL THING I WANT TO DO IS THANK THE PEOPLE THAT ACTUALLY DID ALL THE WORK. GEORGE, WHO I WHOSE WORKER TALKED ABOUT AT THE END. LUCY WILLIAM, WHO DID THE EMBRYOLOGY THAT I SHOWED. JIFRMER WHO DID THE MOUSE PRECEDING AND THE CHIP CHIP. THELMA, WHO JUST STARTED, AND VAN, WHOSE WORK I TALKED ABOUT RELATIVELY SIGNIFICANTLY THROUGHOUT. I WANT TO THANK PEOPLE WHO HELPED WITH THE BIOINFORMATICS AND ALL THE COLLABORATORS AND THANK YOU FOR YOUR ATTENTION. [APPLAUSE] >> PEOPLE WITH QUESTIONS, PLEASE GO TO THE MICROPHONES. >> THIS IS GREAT. TWO SORT OF RELATED QUESTIONS ON THE MAP KINASE RESULTS. DO YOU HAVE ANY EVIDENCE WHETHER THIS IS JUST AN IPS CELL-SPECIFIC MODE OF REGULATING MAP KINASE SIGNALING OR DO YOU SEE IT LET'S SAY IN CANCER CELLS OR ANYTHING LIKE THAT? >> I DON'T KNOW. THE CELL TYPES THAT WE'VE LOOKED AT FROM THESE MICE HAVE ALL BEEN PRIMARY CELLS FROM MICE AND SO WE DON'T SEE THE SAME EN RICHLT IN SIGNALING IN MACROPHAGES, WHICH ARE DIFFERENT TO CELL TYPE. BUT THEY HAVE NOT DONE SYSTEMATIC STUDY OF A BUNCH OF DIFFERENT CELL TYPES BASED ON PROLIFERATION RATE AND/OR DIFFERENTIATION. SO THAT'S AN INTERESTING THING TO DO. WE HAVEN'T GOTTEN THERE. I SUSPECT I KNOW THE ANSWER TO THE NEXT ONE BECAUSE I WAS GOING TO ASK IF YOU HAVE ANY EVIDENCE IN EITHER NORMAL DEVELOPMENT OR EVEN A DISEASE, WHERE THE CELL IS TUNING A WAY OF A KIND OF REA STAT NATURALLY. NOT YOUR KNOCKOUT BUT NATURALLY THAT IT'S TUNING THE PATHWAY THAT WAY? >> YES, SO WE HAVE PROVOCATIVE BUT UNCLUSIVE DATA ABOUT THIS IN THAT IF YOU DO -- IF YOU LOOK FOR MEFL STAINING BY IMMUNOHISTIC CHEMISTRY AND DIFFERENT TISSUES IN THE MEFL, RAPIDLY DIVIDING CELLS TEND TO EXPRESS A LOT MORE MEFL THAN CELLS THAT ARE NOT DIVIDING. BUT EVEN WITHIN A DIVIDING SPECULATION POPULATION, THE LEVEL IS QUITE HETEROGENEOUS AND WHAT THAT SUGGESTS TO ME IS THAT MEFL IS NOT MONOLITHIC IN EITHER THE GENES THAT IT'S TARGETING OR THE NETWORKS THAT IT'S TARGETING OAR IN ITS EXPRESSION LEVEL. AND SO BOTH MEFL AND P TEST B ARE HIGHLY DIFFERENTIALLY EXPRESSED IN DIFFERENTTEL CELL TYPES IN A WAY THAT AS FAR AS WE CAN TELL, IT CLEARLY IS LINKED TO PROLIFERATION, BUT EVEN WITHIN PROLIFERATIVE TISSUES, IT'S SORT OF VARIABLE AND THAT'S NOT SOMETHING WE HAVE A HANDLE ON. >> HI. THAT WAS A REALLY GOOD TALK. TWO QUESTIONS. SO MEFL SEEMS TO REGULATE ESSENTIALLY ALL OF THE PAUSING, RIGHT? SO WHAT TARGETS MEFL AND HOW DOES MEFL KNOW WHICH GENES TO PAUSE A POLYMERASE AT? AND DO YOU EN VISION THAT BEING ONE POLYMERASE PAUSE OR MULTIPLE POLYMERASE STACKED UP? >> OKAY, SO THE FIRST QUESTION, WE IN FLY CELLS AND RICK YOUNG AND HAVE MAPPED MEFL ACROSS THE GENOME, AND THE LEVELS OF MEFL CORRESPOND SO WELL TO THE LEVELS OF POLL 2 THAT I KIND OF THINK MEFL IS STUPID^-- OR MAYBE VERY SMART, DEPENDING ON HOW YOU WANT TO THINK ABOUT IT. IT GOES EVERYWHERE THAT POLL 2 GOES. SO I DON'T THINK THAT THERE IS A LOT OF SPECIFIC TARGETING OF MEFL. NOW, THERE MAY BE FACTORS LIKE DPAGA FACTOR IN THE FLIES THAT START TO INTERACT WITH MEFL, AND SO I THINK THAT IT MAY MAIN MEAN IT'S THERE BEFORE THE POLL MY POLYMERASE GETS THERE. I DON'T WANT TO ARGUE THAT THERE IS A DIFFERENCE NUT KINNETICS. SO I THINK, IN FACT, THE CLEVER PART OF THIS, THE TARGETING PART OF THIS IS REALLY P TEST X AND I THINK THAT IT'S RECRUITMENT IS KINNETICS AND WHERE IT GOES AND HOW FAST. THAT'S DETERMINING THE HALF LAUGH OF PAUSING. I THINK MEFL IS KIND OF A DEFAULT PROCESS IN THAT RECRUITMENT IS REALLY WHAT'S REGULATORY. AS FAR AS WE CAN TELL, THERE IS ONE POLYMERASE THAT IS ENGAGED. I DON'T WANT TO SAY THAT THERE IS NOT ANOTHER ENGAGED BUT THINGS THAT CAN HAVE RESOLUTION LIKE OUR -- WE DON'T SEE TWO PEAKS OF POLYMERASE OR TWO PEAKS. WE JUST SEE ONE CENTERED AROUND 25 TO 40 AND THAT WOULD SUGGEST IF THERE WAS ANOTHER POLYMERASE, WOULD BE EITHER NON-ENGAGED OR MAYBE AND POTENTIALLY LESS CHIPABLE. IN THAT THE LEVELS OF POLL 2 EVEN BY CHIP NEAR THE PROMOTER SEEM TO BE MUCH LOWER. IT DOESN'T MEAN THAT THERE IS SOMETHING WAITING IN THE WINGS. I DON'T WANT TO ARGUE AGAINST THAT, WHETHER IT'S PHYSICALLY THERE OR COMING IN THROUGH A LOOP WITH AN HEARNSER, WHICH I THINK IS AN INTERESTING THING TO TEST AS KIND OF A FAVORED MODEL, WE WE DON'T THINK THAT THERE ARE TWO ENGAGED POLYMERASES BASED ON THE DATA THAT WE HAVE. >> ALL RIGHT, GOING BACK TO THE MODEL, TWO QUESTIONS. ONE, I'M WONDERING IF ONE OF THE OTHER LABS HAVE MUTANTS AND IF SO, WHAT ARE THEY TELLING YOU ABOUT THE FUNCTIONAL ROLE? AND ALSO, GETTING TO THE ISSUE OF WHETHER THE PROTEINS JUST SORT OF IN ITSELF IS KIND OF NOT SMART AND JUST EVERYWHERE IT OR THE REGULATION OF IT IS ALSO IMPORTANT IF YOU HAVE ANY INFORMATION ABOUT ITS EXPRESSION THROUGHOUT DEVELOPMENT. >> SO IN THE FLY MEFL KNOCKOUT -- MUTANTS DIE. THEY CLOSE AND SO IF YOU ARE DOING SOMETHING LIKE CROSSING HATS OR SOMETHING, THE PROBLEM IS THAT THE MATERNAL MODE, THE MATERNAL DEPOSITION OF THE MEFL RNA IS HUGE AND SO THEY DIE WHEN THE MEFL RUNS OUT BUT THEY DIE WITH THIS KIND OF UNINTERESTING PHENOTYPE. SO WHAT GILL DESANTOS DID WAS SUVED A TRICK TO GET RID OF THE MATERNAL DEPOSITION, THEN THE EGGS CAN'T EVEN BE FERTILIZED. SO WE'RE KIND OF STUCK IN A SENSE THAT WE CAN'T FERTILIZE THE EGGS OR WE DIE WITH THIS MUSH PHENOTYPE. AND SO THAT HAS NOT BEEN PARTICULARLY INFORMATIVE. NOW, WE CAN KNOCK MEFL DOWN USING DIFFERENT TISSUES, AND OFTENTIMES THIS CAUSES -- SO IN THE FAT BODIES BECAUSE THAT'S RELEVANT TO THE IMMUNE STUFF THAT WE'VE DONE AND THEY SEEM MUCH SMALLER AND LESS FATTY. SO I DON'T KNOW. I WAS TRYING TO CONVINCE GILL THAT MEFL KNOCK DOWN COULD BE USED FOR FAT REDUCTION BUT HE SUGGESTED MAYBE YOU COULDN'T MIX IT FROM FAT BODIES -- WE DON'T HAVE A TON OF INFORMATION ON DIFFERENT TISSUES THAT WE'VE USED. THE SECOND QUESTION IS AS FAR AS MEFL EXPRESSION OVER DEVELOPMENT, IT'S A HUGE MATERNAL LOAD THAN THEN THE LEVELS DROP AND THEN THEY INCREASE AND STAY RELATIVELY HIGH AND CONSTANT THROUGH THE THIRD END STAR AND THAT'S AS FAR AS IT WENT. >> SO SPEAKING ABOUT SPECULATING, I HAVE SOME QUESTIONS ABOUT CANCER. JUST A COUPLE. IF I UNDERSTAND CORRECTLY OR HYPOTHESIS, RNA POLYMERASE PAUSING MAY BE RELATED TO LESS COMPLEX PATHWAYS AND IMPORTANT REGULATORY POINTS IN THE PATHWAY. SO ONE OF THE BIG PROBLEMS FOR TREATING CANCER NOW IS TRYING TO UNDERSTAND COMPLICATED GROWTH-PROMOTING NETWORKS. THIS IS, AGAIN, A THEORETICAL QUESTION. WOULD LOOKING FOR PAUSING BE A WAY OF FIGURING OUT HOW TO TARGET SPECIFIC SMALL MOLECULE CHEMOTHERAPY, FOR EXAMPLE, TO DIFFERENT PARTS OF THE PATHWAY THAT ARE MORE LIKELY TO BE REGULATORY THAN OTHERS? IS THAT POSSIBLE? >> SO I MEAN, IF THE HYPOTHESIS IS RIGHT, THEN THAT WOULD WORK, AND WE ARE STILL KIND OF A WAYS FROM KNOWING IF THAT TRUE. BUT WE ARE ACTUALLY ENGAGED IN A COLLABORATION WITH THE EXPERT IN THE IRK PATHWAY AND THE NETWORK DIE GRAMS AND SHE'S INTERESTED IN TRYING TO IDENTIFY WHAT THE MEFL TARGETS ARE IN IRK PATHWAY TO SEE WHETHER OR NOT CABIN TORORRIAL TARGETING OF TWO OF THEM AT THE SAME TIME WOULD BE INFORMATIVE AND UNDERSTANDING OR WOULD BE HELPFUL IN TREATING CANCER. WE'RE TRYING, BUT I DON'T KNOW WHETHER OR NOT WE'RE GOING TO HAVE -- WHETHER OR NOT THAT'S GOING TO BE INDICATIVE OF SUCCESS. >> IS THERE ANY ASSOCIATION BETWEEN DNA'S HYPERSENSITIVITY SITES AND THESE PAUSING SITES? >> MOST OF THE PLACES WHERE WE SEE PAUSING ARE DNA HYPERSENSITIVE SITES. >> 100 PERCENT? >> SO IT DEPENDS A LITTLE BIT. IF YOU ARE LOOKING AT THE BROAD SCALE, YES. USUALLY THE REGIONS AROUND THE PAUSE POLL 2 ARE HYPERSENSITIVE. IF YOU ARE LOOKING FOR SOMETHING LIKE EMINNASE, IT TURNS OUT THAT THE POLYMERASE ITSELF PROTECTS A LITTLE REGION AND YOU CAN SEE ACTUALLY SHORT EMINNASE FRAGMENTS IN A HYPERSENSITIVITY ASSAY, YOU SEE A SMALL AMOUNT OF PROTECTION FROM THIS SORT OF STATIC POLL 2. BUT THEN THE REGION AROUND IT IS HYPERSENSEIVEN. >> AND FINALLY, THERE IS A LOT OF EXCITEMENT NOW ABOUT SOME YOU?{}DISCOVERIES AMERICA MICK KEFRNZ ITS ACTIVITY TO STIMULATE TRANSCRIPTION. HAVE YOU LOOKED AT ALL OF MICK INTERACTIONS WITH ALL OF THE COMPLEX THAT YOU ARE DEALING WITH? >> SO THE IDEA FROM -- THIS IS RICK YOUNG'S STUFF AND DAVID LEVINE'S, EXACTLY, AND SO THE IDEA IS THAT MICK MAY BROADLY STIMULATE TRANSCRIPTION BY INCREASING P TEST B RECRUITMENT TO PROMOTERS AND THAT SO AGAIN AND THE IDEA THAT MEFL IS SORT OF EVERYWHERE IN POLY MER MERRASE, IF YOU COULD INCREASE IT TO GENES IN GENERAL -- >> MEFL-DEPENDENT PROCESS AND IT HAS TO BE THERE? >> YES. BUT AT LEAST THE STUFF FROM RICK'S LAB HAS BEEN DONE WHERE HE'S SHOWN THAT EVERYWHERE POLYMERASE IS, MEFL IS AND I'D BE STRIVED SURPRISED IF THAT'S DIFFERENT IN THE LEVINE'S STUDY. AND IF PAUSING REALLY IS BROAD, SOME GENES MAY NOT BE THAT AFFECTED BY MICK OR BY INCREASED DID YOU BUT THERE ARE THINGS THAT ARE REGULATING THROUGH PROLIFERATION, MAY VERY WELL-TARGETED BECAUSE IT'S AN EASY WAY TO TURN THEM OFF. >> HI. I WAS JUST WONDERING IF YOUR MEFL KNOCKOUT IN IPS OR ES CELLS DEFECT IN PROLIFERATION DIFFERENTIATION. CAN YOU RESCUE THAT BY JUST OVEREXPRESSING MAP IRK COMPONENTS TO STIMULATE THE PATHWAY, OR DO YOU THINK MEFL IS ALSO REGULATE AGO THINGS? >> THIS IS A GREAT QUESTION. THIS IS A FABULOUS QUESTION. SO IT IS PROBABLY NOT GOING TO BE THAT JINX BUT THAT WE COULD RESCUE PROLIFERATION, BECAUSE IN FACT, DECREASING IRK'S SIGNALING ENHANCES PROLIFERATION. SO WE'RE DECREASING SO IT'S GOING TO MAKE DIFFERENTIATION HARDER BUT PROLIFERATION SHOULD BE BETTER. AND SO WE'RE NOT GOING TO BE ABLE TO RESCUE THE PROLIFERATION DEFECT. I THINK THAT'S ENTIRELY SEPARATE. BUT THE QUESTION ABOUT WHETHER