>> GOOD MORNING. WELCOME TO THE NIH RESEARCH FESTIVAL'S 30th YEAR. THOSE WHO ARE MATHEMATICIANS MAY WONDER WHY SOMETHING WHICH STARTED IN 1986 IS NOT IN ITS 31st YEAR. WE SKIPPED 1987 BECAUSE THAT WAS THE YEAR OF THE NIH CENTENNIAL. THE ORIGINAL IDEA BEHIND RESEARCH FESTIVAL CAME FROM THE CHIEF OF THE EXPERIMENTAL MEDICINE CENTER, AT THAT POINT, ABNER WAS SCIENTIFIC DIRECTOR IN THE DENTAL INSTITUTE. ABNER SUGGESTED WE MOST A FULL DAY OF SCIENCE TO GET PEOPLE OUT OF THE LABS AND GIVE THEM AN OPPORTUNITY TO FIND OUT WHAT OTHER SCIENTISTS HAT NIH WERE DOING. THE PURPOSE OF THAT, OF COURSE, WAS TO ENCOURAGE COLLABORATIONS AND INTRODUCE NEW IDEAS WHO WERE SOMEWHAT MIRED IN THEIR OWN WORK. VERY QUICKLY, THE IDEA FOR THE FESTIVAL EVOLVEED INTO MUCH LONGER EVENT. WE HAVE BEEN TRYING TO RIGHT SIZE THIS EVENT SO IT ENGAGES MOST OF THE COMMUNITY, DOESN'T TOTALLY INTRUDE ON PEOPLE'S RESEARCH TIME, BUT GIVES PEOPLE AN OPPORTUNITY TO GET OUT AND GET TO KNOW EACH OTHER. EVERY YEAR, WE TRITO TWEET THE FORMAT A BIT. AND CERTAINLY CHOOSE THE SESSIONS BASED ON WHAT IS THE HOTTEST SCIENCE. AND TODAY'S PLENARY SESSION, THE FIRST PLENARY SESSION, SUPER ENHANCERS IN CELL IDENTITY AND DISEASE IS AN EXAMPLE OF AN AREA WHICH IS EXTREMELY [INDISCERNIBLE], SHALL WE SAY. NOW, NIH ITSELF HAS BEEN A MAJOR CONTRIBUTOR TO THIS FIELD. AND THERE ARE A NUMBER OF NIH SCIENTISTS WHO HAVE PARTICIPATED. I'LL MENTION THEM IN A FEW MINUTES. IN THIS PARTICULAR RESEARCH FESTIVAL, IN ADDITION TO THE WORK ON SUPER ENHANCERS, WE HAVE CONCURRENT SYMPOSIA ON CHRONIC DISEASE, THE MICROBIOME, PRECISION MEDICINE, BENCH TO BEDSIDE HOME RUNS, LONG TERM EFFECTS OF DEVELOPMENTAL EXPOSURE, COMPUTATIONAL BIOLOGY. ALL THOSE ARE AREAS THAT NIH SCIENTISTS HAVE MADE MAJOR CONTRIBUTIONS. I HOPE YOU'LL ATTEND AT LEAST SOME OF THOSE. THE POSTER SESSIONS RUN THE GAMUT OF INTRAMURAL SCIENCE. THEY'RE MOSTLY SESSIONS THAT ARE -- POSTER S GIVEN BY POSTDOCTORAL FELLOWS. WE ALSO HAVE THE -- ACCOMPANIED BY THE FAMOUS BAKE OFF. AND TODAY, WE HAVE A POSTER SESSION WHICH IS DEDICATED, FOCUSED ON NIH FUTURE RESEARCH LEADERS CONFERENCE, WHICH IS ONGOING AT THE NIH. THIS IS A GROUP OF HIGHLY TALENTED, DIVERSE RESEARCHERS WHO ARE BROUGHT HERE TO HAVE AN OPPORTUNITY TO LEARN ABOUT THE NIH, AND TO BE RANKED, SO THAT WE CAN PERHAPS RECRUIT THEM HERE. AND I URGE YOU TO COME, LOOK AT THEIR POSTERS THIS AFTERNOON. TALK TO THEM AND ENCOURAGE THEM TO SEEK CAREERS AT THE NIH. ALL TOLD, IN ADDITION TO THE PLENARY SESSIONS AND THE CONCURRENCE SYMP POSIA. THERE WILL BE NEARLY 30 TALKS, 400 POSTERS. THIS ARE LIGHT REFRESHMENTS PROVIDED BY THE TECHNICAL SALES ASSOCIATION. YOU SEE THE BIG AT THE PRESENT TIME TO THE SOUTH OF THE BUILD -- TENT TO THE SOUTH OF THE BUILDING. WE HAVE THE NIH GREEN LABS FAIR AND EXHIBITS, THE TECHNICAL SALES ASSOCIATION TENT SHOW I JUST MENTIONED, AND THIS IS A TASTE OF BETHESDA LUNCH TOMORROW AND FRIDAY. AND THERE WILL BE A SPECIAL TRIBUTE TO THE ANIMAL RESEARCH COMMUNITY ON FRIDAY AS PART OF THE END OF THE RESEARCH FESTIVAL. I ALSO NEED TO ACKNOWLEDGE OUR CO-CHAIRS OF THIS YEAR'S RESEARCH SYMPOSIUM, AND THE SCIENTIFIC DIRECTOR FOR THE NATIONAL INSTITUTE OF NURSING RESEARCH, AND ANDY GRIFFITH, NATIONAL INSTITUTE OF DEAFNESSES AND OTHER COMMUNICATION DISORDERS. TO BE FAIR, AND THEY WOULD ADMIT THIS, RUNNING THE SHOW IS JACKIE ROBERTS, JACKIE, WHERE ARE YOU? I THINK EVERYBODY KNOWS. THERE SHE IS MAKING SURE NOBODY LEAVES. AND, OF COURSE, CHRIS, YOU ALL WILL RECOGNIZE, THE HEAD OF COMMUNICATIONS IN MY OFFICE AS WELL. SO LET ME GET TO TODAY'S PLENARY TALKS. SUPER ENHANCERS, ALSO KNOWN AS STRETCH ENHANCERS ARE EXCEPTIONALLY BUSY PARTS OF THE GENOME THAT CONTROL LINAGE DEFINING GENES. NIH RESEARCHERS HAVE BEEN AT THE FOREFRONT OF THE FIELD. IN 2013, DOCTORS COLLINS LAB IN THE GENOME IDENTIFIED STRETCH INTIONSERS AS GENE GROUPS, ANALOGOUS TO SUPER ENHANCERS. WITH O'SHEA LAB, T-CELL RELATE TO THE FUNCTION OF THE SPECIALIZED CELL. REAMLY, HENNINGHAUSEN NIDDK LAB COULD DISCOVERED SUPER ENHANCERS IN THE MAMMARY GENOME. THOSE HERE FOR THE FAIR AWARD, THERE WAS A [INDISCERNIBLE] UP, DEPENDING ON THE NUMBER OF TIMES THAT THE ABSTRACTS REFERENCED THE WORD, THE WORD CHANGES IN SIZE. IN THE MIDDLE WAS CHRISER, BIGGER THAN ANYTHING ELSE. HERE IS DR. FRANCIS COLLINS, IT'S ENTITLED TO BE HERE. FOLLOWED BY TWO OTHER SPEAKERS WHO I WILL INTRODUCE WHEN THEY COME TO THE PODIUM, RAFAEL CASELLAS, AND KEIKO OZATO, WELL-KNOWN TO YOU AS SENIOR. FRANCIS IS SHY ABOUT BEING INTRODUCED. HE IS SOMEBODY AT NIH WHO NEEDS TO NO TRODUCTION. IN ADDITION TO ALL HIS OTHER ROLES HE IS THE HEAD OF THE MOLECULAR GENETICS SESSION AND MEDICAL GENOMICS AND MET BOBBLINGS GENETICS BRANCH. HIS CURRENT WORK IS FOCUSING ON TYPE II DIABETES, USING NEXT GENERATION SEQUENCING TECHNIQUES. AND HE WILL BE THE FIRST SPEAKER. SO WITHOUT FURTHER ADO, DR. COLLINS, PLEASE COME TO THE PODIUM. [APPLAUSE] >> THANK YOU. GOOD MORNING TO EVERYONE. IT'S A A PLEASURE TO BE HERE AND TELL YOU ABOUT SOME OF THE WORK GOING ON, AND WITH OUR COLLABORATORS ON THIS TOPIC OF STRETCH ENHANCERS, SUPER ENHANCERS, LET'S CALL THEM SEs. I WANT TO PAINT A PICTURE OF HOW WE GOT INTERESTED IN THIS, BASED UPON A PROJECT IN TYPE II DIABETES, WHICH HAS INVOLVED MY LAB FOR 23 YEARS MOOCH AND WHICH HAS MOVED US FROM LOOKING AT THE GENETIC RISK FACTORS INTO TRYING TO UNDERSTAND THE FUNCTIONAL BASIS OF THOSE RISKS WHICH VERY MUCH REQUIRES US TO BEGIN TO UNDERSTAND GENE REGULATION. I'LL WALK YOU THROUGH SOME OF THE PATH WE HAVE TRAVELED, AND TELL YOU ABOUT NEW DATA THAT'S NOT YET PUBLISHED THAT RELATES TO THE EPIGENOME OF THE HUMAN PANCREATIC IL THINGS WE HAVE LEARNED THAT ARE RELEVANT TO OUR UNDERSTANDING OF THIS COMMON CITIES, TYPE TWO DIABETES. DIABETES, AS YOU KNOW, IS INCREDIBLY PREVALENT, BECOMING MORE SO ALL THE TIME. PEOPLE LIKE ME GIVING TALKS LIKE THIS OFTEN START OUT WITH MAPS THAT LOOK LIKE THAT, PRETTY SCARY WHEN YOU SEE WHAT'S HAPPENING ACROSS THE U.S. THIS IS ALSO HAPPENING IN MOST OF THE REST OF THE WORD, INCLUDING COUNTRIES THAT WE THOUGHT PREVIOUSLY WOULD NOT BE AFFLICTED BY THIS CONDITION, BUT NOW WITH INCREASING ADOPTION OF WESTERN BEHAVIORS, ARE ALSO SEEING THE INCIDENCE OF TYPE II DIABETES GROW. HERE IN THE U.S. WE HAVE 30 MILLION INDIVIDUALS WITH THE DIAGNOSIS OF TYPE TWO DIABETES, ANOTHER 80 WHO HAVE A HIGH RISK IN THE NEXT FIVE YEARS OF CONVERTING TO DIABETES IF NOTHING ELSE IS DONE. WE DO KNOW FROM THE WORK OF NIDDK THAT THOSE INDIVIDUALS CAN PULL BACK WITH INTERVENTIONS AS SIMPLE AS EXERCISE AND DIET. WE NEED TO UNDERSTAND THIS CONDITION. WE'VE KNOWN A LONG TIME THAT DIABETES TENDS TO RUN IN FAMILIES. IT'S INHERITANCE IS VERY MURKY. THIS IS NOT THE SORT OF THINK THAT MENDAL WOULD HAVE APPROVED OF. WHEN YOU LOOK AT FAMILY HISTORIES, YOU SEE WHAT APPEARS TO BE A POLY GENETIC PATTERN. IN THE PAST WE HAD VERY FEW TOOLS TO IDENTIFY WHAT THOSE CONTRIBUTING GENETIC VARIANTS MIGHT BE. THE DISEASE INVOLVES PRIMARILY THE TISSUES THAT YOU SEE HERE, PLUS THE BRAIN PLAYS A ROLE AS WELL. PANCREAS, THE PRODUCER OF INSULIN, NECESSARY FOR GLUCOSE METABOLISM TO GO WELL. THAT RESULTS IN UPTAKE IN MUSCLE, SHUT DOWN IN GLUCOSE PRODUCTION IN THE LIVER AND STOPPAGE OF LIPOLOGISTIS. IN FACT, IF YOU HAVE A PROBLEM OF OVER NUTRITION, WHICH OF COURSE IS A BIG AFFLICTION, OF MODERN TIMES, IN THIS COUNTRY AND MANY OTHER PLACES, BASICALLY, INSULIN RESISTANCE INSPIRED BY OBESITY PUTS FURTHER STRESS ON THE PANCREAS AND THOSE ILETS, AND BETA CELLS FAIL TO KEEP UP. GLUCOSE RISES AND DIABETES OCCURS, TO UNDERSTAND THIS DISEASE, ONE NEEDS TO PAY A ROT OF ATTENTION TO THESE TISSUES. AS I SAID OVER MORE THAN 20 YEARS, WE HAVE BEEN PART OF A WONDERFUL COLLABORATIVE EFFORT, TRYING TO UNDERSTAND THE GENETICS AND NOW EPIGENETICS OF WHAT WE USED TO CALL NIDDM, NOW CALLED TYPE II DIABETES WITH ACRONYM FUSION. THE PATIENT POPULATION WE HAVE BEEN BENEFITED IN BEING ABLE TO STUDY IS PRIMARILY FROM FINLAND, FINLAND, UNITED STATES INVESTIGATION OF NIDDM GENETICS OR FUSION, PARTICIPANTS IN THIS INCLUDE MIKE, UNIVERSITY OF MICHIGAN, SCHOOL OF PUBLIC HEALTH. [SEE SLIDE] IT WAS HOPELESS FOR A CONDITION OF THIS SORT THAT IS NOT INHERITED IN A VERY PREDICTABLE WAY. ON THE OTHER HAND, ONE COULD BEGIN TO ADOPT THE GENOME WIDE ASSOCIATION STUDIES, SO-CALLED GWAS, CONSIDERABLE BENEFIT. SO UTILIZING MORE THAN 30,000 DNA SAMPLES THAT WE HAVE IN THE FREEZER, AND WORKING WITH MANY COLLEAGUES AROUND THE WORLD TO BOOST UP THAT NUMBER TO NOW MORE THAN 300,000 DNA SAMPLES STUDIED BY GWAS ANALYSIS, IT HAS BEEN POSSIBLE TO FIND NO LESS THAN 86 VARIANTS THAT PLAY A ROLE IN RISKS FOR TYPE II DIABETES, ALBEIT, ALL WITH RATHER MODEST ODDS RATIOS IN TERMS OF QUANTITATIVE RISK TO THE DISEASE. THE GOOD NEWS IS WE HAVE BEEN SUCCESSFUL IN UTILIZING THIS STRATEGY TO AGAIN FEW TO NAIL DOWN WHAT THE RISK FACTORS ARE FOR THIS CONDITION. HERE IS A DEMOCRAT NOW, A BIT OUT OF DATE BUT CLOSE, OF WHERE THOSE VARIANTS FALL ACROSS THE GENOME. AND AT LEAST GUESS WHICH JOAN MIGHT BE INVOLVED. WHY WOULD WE HAVE TO GUESS SOME THE PROBLEM IS, 90%, MORE THAN 90% OF THE VARIANTS THAT ARE DISCOVERED IN THIS WAY ARE NON CODING. THERE ARE A FEW TO BE SURE THAT ALTER AN AMINO ACID IN A PROTEIN CODING REGION. MAYBE THOSE ARE THE EASIEST TO UNDERSTAND BUT IT'S A MINORITY. SO YOU HAVE THIS CIRCUMSTANCE OF FINDING VARIATIONS THAT DON'T LAND IN AN EXON, AND OFTEN TIMES IN A GENE DENSE REGION WHERE YOU CAN'T EVEN BE SURE WHICH GENE IS ACTUALLY EFFECTED BY THIS VARIATION. IF YOU REALLY WANT TO UNDERSTAND TYPE TWO DIABETES ON A FUNCTIONAL BASIS, HOW GENETIC RISKS PLAY OUT YOU HAVE TO ROLE UP YOUR SLEEVES AND SAY WE'RE GOING TO GET INTO EPIGENOMICS. THAT'S THE ONLY WAY TO MAKE SENSE OUT OF THIS. YOU MAY WONDER, ARE WE MISSING SOMETHING? BY GWAS. PEOPLE HAVE ASKED THAT QUESTION. GWAS CAN ONLY FIND VARIANTS PRESENT IN THE POPULATION AT A FREQUENCY, OF ONE OR TWO PERCENT. IS THERE HIDING IN THIS ARCHITECT OF THE GENOMICS OF TYPE II DIABETES. >> THEIR MAN A PUBLICATION IN THE LAST MONTH. OUR CONCLUSION IS THERE DOES NOT SEEM TO BE A SIGNIFICANT LEVEL WITH TENS OF THOUSANDS OF SAMPLES OF CASES AND CONTROLS THAT ARE SUBJECTED TO SEQUENCE, THERE IS NOT THIS HIDDEN GROUP OF RARE VARIANTS OF LARGE EFFECTS. IT LOOKS AS IF MOST OF THE ARCHITECT OF TWO -- TYPE II DIABETES ARE THESE COMMON VARIANTS, AND THE MORE REASON TO GET BUSY AND UNDERSTAND HOW THEY WORK. THIS PAPER WITH MANY, MANY AUTHORS CAME OUT WITHIN THE LAST MONTH OF THE MY GROUP WAS HAPPY TO HAVE CONTRIBUTED SUBSTANTIALLY TO THIS FINDING. SO WHAT ARE WE DOING? HERE IS A DEMOCRAT OF THE KINDS OF -- DEMOCRAT OF THE KINDS OF WORK GOING ON IN THE LAB, MANY YEARS OF EFFORT TO COLLECT SAMPLES TO PUT TOGETHER PRIORITY EXPERIMENTAL APPROACHES AND DEVELOP THE ANALYTICAL CAPABILITY TO UNDERSTAND THE MEANING OF THE DATA. THIS IS BIG DATA IN A BIG WAY. WE HAVE ALREADY PUBLISHED A PAPER JUST ABOUT A MONTH AGO ABOUT ANALYSIS OF MUSCLE IN DIABETICS AND NON, BECAUSE OF THE TIME I'M NOT GOING TO TELL YOU ABOUT THAT BUT ENCOURAGE YOU TO LOOK AT NATURE COMMUNICATIONS, IF THAT'S OF INTEREST. THIS IS THE LARGEST STUDY OF GENE EXPRESSION IN HUMAN SKELETAL MUSCLE EVER DONE, AND DID REVEAL SOME INTERESTING THINGS ABOUT DIABETES SUSCEPTIBILITY. IN THAT CASE, MUSCLE WE'RE LOOKING AT IS A POTENTIAL CONTRIBUTOR TO DIABETES BECAUSE OF ITS ROLE IN INSULIN RESISTANT. WHAT I WANT TO TELL YOU ABOUT IS UNPUBLISHED DATA WHICH IS FOCUSED ON PANCREATIC EYE LETS. THIS DOES SEEM TO BE -- IF YOU WANTED TO PICK AMONGST TISSUES INVOLVED IN TYPE II DIABETES, PROBABLY THE MOST IMPORTANT ONE. WE'VE HAD ARGUMENTS OVER DECADES ABOUT INSULIN RESISTANCE IS THE PROBLEM INDICTION. I THINK MANY ARE LEADING TOWARD BETA CELLS STRONGLY BASED UPON THE DATA. SO I'M GOING TO TELL YOU ABOUT SOME OF THE DATA TYPES THAT WE'RE GENERATING, PARTICULARLY ABOUT DOING RNA SEEK, TO LOOK AT ILETS TO SEE WHAT GENES ARE BEING EXPRESSED. THOSE ARE ALSO EYE LETS THAT WE HAVE GENOTYPES ON SO WE CAN LOOK AT THE EFFECT OF GENE VARIATION ON THOSE GENE EXPRESSION LEVELS, THEN I WANT TO TELL YOU ABOUT DATA WE HAVE GENERATED ON CHROMATIN STRUCTURE, WHERE RA THE INTERESTING AREAS. HOW DOES THIS ALL FIT TOGETHER TO TRY TO EXPLAIN THE GWAS FINDINGS. LET ME START WITH THIS ISSUE ABOUT HOW WE COULD USE EQTLs TO TRY TO IDENTIFY WHICH ACTUALLY OF THE MANY GENES IN THE VICINITY OF A TYPE II DIABETES RISK VARIANT IS ACTUALLY THE ONE RESPONDING TO THAT VARIATION AND CONTRIBUTING TO DISEASE RISK. WE WANT TO FIGURE OUT HOW TO GO FROM A GWAS FINDING TO PHENOTYPE, WHAT'S THE MECHANISM. YOU START WITH A VARIANT THAT TELLS YOU THAT THIS PARTICULAR VARIANT CONVEYS A HEIGHTENS RISK OF TYPE II DIABETES ADMITTEDLY OFTEN TIMES RATHER MODEST. ODDS RATIOS WE'RE TALKING ABOUT, IN THE NEIGHBORHOOD OF 1.1 OR 1.2. CERTAINLY NOT 3 OR 4 FOLLOWED ELEVATED RISK. WE DON'T HAVE THOSE FOR TYPE II DIABETES. THAT'S STILL IMPORTANT TO FIGURE OUT IN TERMS OF PATHWAYS. YOU WANT TO KNOW WHICH OF THE GENES IN THE VICINITY IS ACTUALLY THE TARGET AND WHAT'S THE DIRECTION EFFECT, THE RISK ALLELE MAKE OVER-EXPRESSION OR UNDER-EXPRESSION. THAT WOULD BE USEFUL TO KNOW IF YOU WANT TO UNDERSTAND FUNCTION. SO IT'S BEEN TEMPTING, MUCH OF THE FIELD HAD TO RELY UPON USING PROXIMITY AS YOUR BEST INDICATOR WHICH IS THE TARGET GENE. YOU LOOK AROUND AND SAY WHAT'S CLOSEST? AND IT'S OFTEN THE CASE THAT YOU'RE RIGHT BUT ACTUALLY, ALMOST HALF THE TIME YOU'LL BE WRONG. THESE ARE, IN FACT, VARIANTS THAT OPERATE SOMEWHERE IN NON CODING DNA, OFTEN TIMES AT A DISTANCE FROM THE RESPONSIBLE GENE. THEY'RE LOCATED IN ENHANCERS, MORE ABOUT THAT SHORTLY. AND SO UNLESS YOU'VE DONE THE EXPERIMENT, YOU CAN EASILY BE MISLED. IT MAY BE THAT THERE IS ANOTHER GENE FURTHER OFF THAT'S ACTUALLY THE ONE WHICH WHEN THE RISK ALLELE IS PRESENT, VARIES AS GENE EXPRESSION AS SHOWN IN THIS CARTOON, IN THIS CASE THE RISK ALLELE SHOWING OVER EXPRESS. SO WE WOULD LIKE TO BE ABLE TO APPLY THAT STRATEGY FOR ALL OF THOSE TYPE II DIABETES G WAS HITS WHICH ARE NOT IN A CODING REGION WHICH IS MOST OF THEM. IT GETS MORE COMPLICATED. I LEFT OUT UNTIL NOW WHEN YOU GET A GWAS HIT, YOU DON'T HAVE A SINGLE SNIP THAT IS DRIVING THAT EFFORT. WE LEARNED A LONG TIME AGO THAT THERE IS STRONG LINKAGE IN LOCAL NEIGHBORHOODS. WHEN YOU FIND A SNIP ASSOCIATED WITH SOME KIND OF OUTCOME, GENE EXPRESSION, IN MOST INSTANCES THERE WILL BE MULTIPLE OTHER SNIPS TIGHTLY LINKED TO IT THAT ARE ALSO VERY STRONG LINKAGE TO EQUILIBRIUM AND ABOUT AS GOOD AS PREDICTING THE OUTCOME AT YOUR ORIGINAL SNIP. THAT MEANS THAT MAYBE MORE THAN ONE OF THOSE IS RESPONSIBLE AND CAUSATIVE, BUT YOU CAN'T NECESSARILY KNOW WITHOUT DOING MORE FUNCTIONAL DATA WHICH ONE IT IS. SO WE HAVE SORT OF THESE 2 GOALS. PHYSICIAN, TRY TO FIGURE OUT WHAT GENE IS INVOLVED. THEN WHICH SNIP, WHICH VARIANT IS FUNCTIONALLY RESPONSIBLE FOR THE OUTCOME. I'LL TRY TO WALK YOU THROUGH A COUPLE EXAMPLES WHERE WE THINK WE HAVE BEEN ABLE TO DO THAT WITH PANCREATIC EYE LET OUTRAGE ANALYSIS. THE FIRST ONE, TARGET OF THE GENE AND DIRECTION OF EFFECT. WE ANALYZED SOME 31 ILET SAMPLES, ON A DISTRIBUTION CENTER. WE RECEIVED HUMAN PANCREATIC ILETS, OVER MANY YEARS. HAD THE OPPORTUNITY TO STUDY THOSE FOR GENOTYPING CHROMATIN UNLESS, GENE EXPRESSION, SO ON. WE WERE ABLE TO THEN MERGE OUR DATA WITH 81 EYE LETS THAT HAD BEEN SUBJECTED ALSO TO GENOTYPING AND RNA SEEK THAT WERE PUBLISHED ABOUT A YEAR AGO. THEY MADE THEIR DATA FREELY ACCESSIBLE. WE PUT THAT TOGETHER TO DO THIS ANALYSIS. AROUND HERE IS AN EXAMPLE OF ONE OF THE THINGS THAT WE FOUND. I WILL TELL YOU, WE FOUND IN ABOUT 20 INSTANCES, THAT WE COULD SEE THAT THERE IS A SNIP WHICH IS ASSOCIATED WITH TYPE II DIABETES, WHICH ALSO IS CONNECTED WITH A CHANGE IN EXPRESSION OF A NEARBY GENE. AND IN A FEW INSTANCES WE WERE ABLE TO AGGREGATE SOME CONFUSION. HERE IS ONE. WE'RE LOOKING AT CHROME 6. THIS IS A VARIANT, WHICH BY GWAS, YOU SEE THE PICTURE HERE, THE ANALYSIS LOG BASED MP VALUE ON THE Y AXIS, THIS DOESN'T LOOK LIKE IT QUITS GET TO SIGNIFICANCE BY POOLING WITH OTHER LARGE DATASETS, IT DOES. ON THE Y AXIS IN THE BLUE PEEKS, RECOMBINATION RATE TELLS YOU THE BOUNDARIES. IF YOU'RE BETWEEN 2 OF THOSE PEAKS A LOT OF SNIPS WILL TRAVEL IN LOCKSTEP. YOU CAN SEE A PILEUP OF THOSE COLORED SNIPS WHICH ARE ALL GIVING YOU A SIGNAL. ONE OF THEM IS THE [INDISCERNIBLE], NOT BY MUCH. YOU WANT TO KNOW, OKAY, THAT TURNS OUT NOT TO BE A CODING VARIANT. WHICH OF THE GENES IN THE VICINITY IS ACTUALLY DRIVEN BY THIS? AND WHAT GENES ARE HERE? THIS HAS TRADITIONALLY BEEN CALLED THE KCNK16 SNIP BECAUSE IT'S CLOSEST TO THAT GENE, ALTHOUGH NOT BY MUCH. KCNK16 IS A POTASSIUM CHANNEL EXPRESSED IN PANCREATIC ILET. THERE IS A GUY RIGHT NEXT STORE, AND ANOTHER THAT COULD BE INVOLVED. WHAT TO DO. WELL, BASICALLY YOU WANT TO KNOW DOES THIS SAME SNIP OR AT LEAST DO SNIPS IN HIGH LD WITH THIS ORIGINAL SNIP HAVE AN EFFECT ON GENE EXPRESSION? LET'S ASK THAT FOR KCNK16, THE ONE THAT EVERYBODY ASSUMED WOULD BE THE ANSWER. NO. THIS IS A DIFFERENT PLOT. ON THE Y AXIS, NOW, WE'RE PLOTTING ASSOCIATION WITH EXPRESSION OF THAT PARTICULAR GENE, KCNK16. THIS IS NOTHING THERE. SO EVEN THIS VARIANT SEEMED LIKE IT PROBABLY WOULD END UP MODIFYING THIS GENE, IT CLEARLY DOES NOT BY THIS ANALYSIS HAVE MUCH IF ANY EFFECT. SAME THING HAPPENED WITH KIP 6, NOTHING. THERE KCNK17 YOU GET A VERY STRONG SIGNAL. SO ESSENTIALLY, WE HAVE BEEN MISLED UNTIL THIS ANALYSIS ABOUT WHICH IS THE APPROPRIATE TARGET FOR THIS. THIS IS ANOTHER POTASSIUM CHANNEL, NOT MUCH KNOWN ABOUT IT. I WILL TELL YOU ALSO THE ANALYSIS SHOWS THE RISK ALLELE RESULTS IN OVER-EXPRESSION WHICH FOR ANYBODY INTERESTED IN THERAPEUTICS IS PARTICULARLY INTERESTING. THAT SAYS THAT OVER-EXPRESSION OFTH POTASSIUM CHANNEL, ON THE SELF-SURFACE, COULD BE A TARGET FOR A SMALL MOLECULE IS ASSOCIATED WITH INCREASED RISK OF DIABETES. THAT MAY MAKE YOU THINK, TO SEE IF THIS TARGET COULD BE USEFUL IN PRESERVING FUNCTION OF BETA CELLS, AVOIDING THE RAVAGES OF DIABETES. THAT'S A WRONG PATH, OF COURSE. BUT INTERESTING MOMENT TO SORT OF CONTEMPLATE. THAT IS AN EXAMPLE OF HOW HAVING GENOTYPES IN THE GWAS CAN SHE HAD LIGHT ON WHAT HAD BEEN -- SHED LIGHT ON CONFUSING DATA. IT DIDN'T TELL YOU HOW THIS WORKED OR HOW THE SNIPS RESULTED IN THIS FINDING OF OVER-EXPRESSION. WE'D LIKE TO UNDERSTAND THAT. DOES ONE OF THESE CAUSATIVES -- THE CAUSATIVE SNIP INTERFERE WITH WINDING OF SOME IMPORTANT TRANSCRIPTION FACTOR? THAT WOULD BE A PLAUSIBLE HYPOTHESIS. HOW DO YOU GET THERE? WELL, NOW I NEED TO STEP INTO THE SPACE OF IDENTIFYING CAUSAL SNIPS. I NEED TO STEP INTO THE SPACE OF CHROMATIN ANALYSIS AND I'M SURE OTHER SPEAKERS WILL TALK ELEGANTLY ABOUT THIS WHOLE AREA, BECAUSE A LOT OF TECHNOLOGIES HERE THAT WE'RE ALL USING IN COMMON. ONE OF THEM IS SO-CALLED CHIP SEEK, GIVES YOU AN OPPORTUNITY TO LOOK ACROSS THE GENOME AND SEE WHICH PARTICULAR PROTEINS ARE BINDING WHERE. IF YOU HAVE A GOOD ANTIBODY AGAINST THAT PROTEIN. AND A FAVORITE WAY TO LOOK AT THIS, LOOK AT MODIFIED HISTONES. THOUGH ARE VERY GOOD MARKERS, ALBEIT COMPLEX, WHICH PARTS OF THE GENOME ARE INVOLVED IN PROMOTERS, TRANSPORTATION, AND IN -- TRANSCRIPTION AND ENHANCERS, AS WELL AS INSULATORS. THIS IS A CARTOON TO SHOW YOU OUR CURRENT UNDERSTANDING OF TYPICAL KINDS OF MARKS THAT YOU WOULD SEE DEPENDING ON WHICH PART OF THE GENOME YOU'RE LOOKING AT. SO FOR INSTANCE, THE INSULATOR THAT YOU SEE ON THE LEFT THERE IS ONE WHERE YOU WOULD EXPECT TO SEE PARTICULARLY CTCF, SORT OF THE LANDMARK. I WANT TO POINT TO ENHANCERS, PARTICULARLY ACTIVE, ARE MARKED BY H3K27AC, AND [INDISCERNIBLE] A3K4, AS WELL AS HYPER SENSITIVE TO DNAS AND ATTACK, WHICH I'LL COME TO IN A MINUTE. THERE ARE OTHER MARKERS THAT ALLOW YOU TO SEE WHAT'S ACTIVELY TRANSCRIBED GENE. IT'S HANDY TO LOOK AT THOSE AND THEY'RE GOOD ANTIBODIES TO ALL OF THESE. SO A SKILLED POST-DOC, IN MY CASE, SEVERAL WHO HAVE ADOPTED THIS APPROACH ARE ABLE TO GENERATE THIS KIND OF DATA FOR A CELL LINE OR TISSUE AS LONG AS WE HAVE ACCESS TO SEQUENCING CAPABILITY AND BIG SHUTOUT TO THE SEQUENCING CENTER WHO HAS DONE ALL THE SEQUENCING OF THE LIBRARIES WE PRODUCED. ONES YOU HAVE THIS DATA ACROSS THE GENOME, THERE NEEDS TO BE A WAY TO MAKE SENSE OUT OF IT. IN THAT REGARD WE HAVE DEPENDED VERY MUCH ON A PROGRAM CALLED CHROME HMM, A HIDDEN MARK OFF MODEL THAT TAKES THIS KIND OF DATA AND THEN BREAKS DOWN THE GENOME INTO VARIOUS CATEGORIES OF CHROMATIN STRUCTURE. WE FOUND THOSE TO BE VERY USEFUL. YOU SEE AT THE TOP OF THIS SLIDE, THE 9 CATEGORIES THAT COME OUT OF HMM, WHEN YOU DO THIS ACTIVE PROMOTERS, AND SO ON. BUT AGAIN I'M PARTICULAR LAG FOCUSING ON ENHANCERS, STROKE AND WEAK ONES. IN THIS DIAGRAM I'M SHOWING YOU A PART OF THE GENOME AROUND A JOAN CALLED GCK, VERY IMPORTANT GENE IN EYE LETS, FOR THE WAY IN WHICH IT HANDLES GLUCOSE. NOTICE, HERE, I'VE GOT MULTIPLE DIFFERENT CELL TYPES. THE ILET DATA IS OURS. THE REST OF THE DATA COMES FROM THE PUBLIC DOMAIN, FROM THE ROAD MAP PROJECT AND THE COMMON FUND PROJECTS ON EPIGENOMICS. IT'S GREAT TO HAVE ALL THIS DATE AT OUR FINGER TIPS. YOU CAN SEE HERE WHAT THE ANATOMY IS OF THIS REGION. GCK HAS TWO DIFFERENT PROMOTERS, EYE LET SPECIFIC 1P1 AND LIVER 1P2. YOU'LL NOTICE IF YOU LOOK AT THE GREEN AREAS, WHAT IS ACTUALLY TRANSCRIBED, THERE ARE TWO GENES ON EITHER SIDE OF THIS THAT ARE HOUSEKEEPING GENES, SO THOSE ARE ON IN ALL THESE CELL TYPES. ILETS STAND OUT RATHER DRAMATICALLY AS DIFFERENT. YOU CAN SEE THE RNA SEEK DATA, AGAIN, GCK DRIVEN BY THE P1 PROMOTER IS ACTUALLY ON BUT PRETTY MUCH OFF IN THE OTHER CELL TYPES LOOKED AT HERE. PAY ATTENTION TO ONE THING ABOUT THIS, WHICH IS THAT BIG WIDE STRETCH 0 YELLOW ENTERED BY OTHER COLORS, THIS IS AN ENHANCER. NOTICE THE SIZE MARKER. THIS EXTENDS ACROSS ALMOST 50 KILABBASIS, A VERY WIDE OPEN CHROMATIN. WE SAW THAT INITIALLY, AND IT KEPT POPPING UP IN INTERESTING PLACES. WE REALIZED PLACES YOU SEE THIS ARE ALMOST ALWAYS IN AREAS OF TISSUE SPECIFIC HIGH LEVEL TRANSCRIPTION OF A GENE. I THINK THAT IS A THEME THAT MANY PEOPLE HAVE NOW BUMPED INTO IF YOU'RE LOOKING AT GENE EXPRESSION OF A PARTICULAR TRANSCRIPT THAT IS FAIRLY EXQUISITELY TISSUE SPECIFIC, WHERE THE RNAS PRODUCED AT A PRETTY DECENT RATE. VERY LIKELY YOU'LL FIND ONE OF THESE ENHANCERS THAT STRETCHES OVER A LONG DISTANCE. WE NAMED THESE STRETCH ENHANCERS, ABOUT THE SAME TIME THAT RICK YOUNG'S LAB WAS DISCOVERING A SIMILAR PHENOMENON, NAMING THEM SUPER ENHANCERS, SUPER ENHANCERS WERE INITIALLY DEFINED IN A DIFFERENT WAY THAN WHAT WE'RE DOING HERE. THEY HAVE EVOLVED A BIT. INITIALLY, THEY WERE PARTICULARLY TAGGED HAVING HIGH LEVELS OF BINDING OF MEDIATORER. WE HAVE BEEN NOT BEEN ISSUING THAT BY WE HAVE BEEN MESHING OTHER MARKS AND OUR DEFINITION OF A STRETCH ENHANCERS IS BASED UPON THIS CHROME HMM ANALYSIS, SEEING A LONG DISTANCE THAT APPEARS TO MATCH THE FEATURES OF A STRONG ENHANCER. WHAT THAT MEANS IS NOW THAT THIS HAS BEEN LOOKED AT BY DIFFERENT METHODS IN THE SAME TISSUE, MOST ENHANCER ARE INCLUSIVE OF SUPER ENHANCERS, BUT THERE ARE MORE STRETCH ENHANCERS THAN SUPER ENHANCERS. EVERY SUPER ENHANCERS TO A CERTAIN LEVEL OF PRECISION, TURNS OUT TO BE A STRETCH ENHANCERS BUT THERE ARE MORE STRETCH ENHANCERS IN THE GENOME BY THIS ANALYSIS THAN SUPER ENHANCERS WHICH ARE IDENTIFIED INITIAL LAY BE MEDIATOR BINDING AND RECENT BY H3K27AC. IN TERMS OF THE DISTRIBUTION OF THIS, STEVE PARKER AND MICHAEL DID THIS EXPERIMENT TO SEE ACROSS THE GENOME, THERE AN OVERABUNDANCE OR IS THIS JUST A LONG TAIL OF A DISTRIBUTION. DON'T WORRY TOO MUCH ABOUT THE SQUIGGLES HERE, ABOUT THE WAY THE ANALYSIS WAS DONE. PAYING ATTENTION TO THE FACT THAT THERE IS AN UNACCEPTABLY LONG TAIL OF THESE. THIS IS NOT JUST WHAT YOU MIGHT EXPECT FROM A NORMAL DISTRIBUTION. THOSE ARE THE ONES THAT WE DECIDED TO CALL STRETCH BECAUSE THEY GO ACROSS A LONGER DISTANCE. AND BETWEEN, WE SHOULD PROBABLY BE CAREFUL ABOUT OUR TERMINOLOGY. WHEN WE STRETCH ENHANCERS WE'RE NOT MEANING THE SAME THING WHEN SOMEBODY SAYS A SUPER ENHANCERS BUT THERE IS A STRONG OVERLAP. THE THEMES HERE ARE SIMILAR. THESE ARE DOMAINS THAT ARE RESPONSIBLE FOR OPEN CHROMATIN WITH A HIGH LEVEL OF EFFECTIVENESS ON NEARBY GENES EXPRESSION. ANOTHER THING WE DISCOVERED, MADE IT EVEN MORE INTERESTING TO LOOK AT THESE, IS IF YOU TAKE THE CASE OF TYPE II DIABETES, AND YOU ASK THE QUESTION WITH THOSE GWAS HITS, DO THEY MAP IN THE STRETCH ENHANCERS DISPROPORTIONATELY, THE ANSWER IS QUESTION. LOOK AT THIS DIAGRAM. YOU SEE SAME DIFFERENT TYPES OF CELLS, OUR DATA FOR ILETS ON THE LEFT. AND THEN IN THE Y PILEUP HERE ARE DIFFERENT PHENOTYPES, THE FIRST 3 BEING DIABETES OR CLOSELY RELATED TRAITS. THE OTHERS, OTHER TRAITS THAT HE WOO DIDN'T EXPECT NECESSARILY WOULD HAVE THEIR TISSUE OF RESPONSIBLE CONTRIBUTION PRIMARILY EYE LETS. WHAT YOU CAN SEE, TYPE II DIABETES AT THE TIME THIS WAS DONE, THERE WERE 86GWAS SIGNALS THERE. 61 OF THOSE FELL WITHIN A STRETCH ENHANCERS THAT WAS IN THE EYE LET, AND ALSO, SOME OF THEM IN LIVER WHICH WAS INTERESTING. IF YOU LOOK AT THE OTHER CONTROL HERE, GM12878 WELL-KNOWN TO MANY AS A LIMBO PLASTID LINE. WHEN YOU LOOK AT AUTOIMMUNE DISEASES, IT'S THOSE PARTICULAR DISEASES THAT HAVE THEIR GWAS SIGNALS IN STRETCH ENHANCERS THAT YOU FIND IN THIS LYMPHOBLAST. ENCOURAGES YOU TO THINK WE'RE ON TO SOMETHING. IF YOU'RE TRYING TO IDENTIFY FUNCTIONALITY, GOING TO THOSE STRETCH ENHANCERS WOULD BOW A GOOD THING TO DO. SO THAT'S WHAT WE HAVE BEEN TRYING TO DO. OF COURSE THOSE STRETCH ENHANCERS ARE BIG SPRAWLING AREAS. THEY'RE NOT GOING TO TELL YOU FUNCTIONALITY AT THE LEVEL OF A SPECIFIC FACTOR. YOU WANT SOMETHING WITH MORE PROCEEDCISSION TO BE ABLE TO IDENTIFY THE ACTION. IN THAT REGARD, WE HAVE BEEN VERY BENEFITED AS MANY OTHERS WORKING IN THIS FIELD HAVE BY THE DEVELOPMENT AND WIDE AVAILABILITY OF TECHNOLOGY CALLED ATTACK SEQ. IT GIVES YOU A MORE PRECISE LOCALIZATION OF WHERE OPEN CHROMATIN LIES. THIS IS BASED ON THE IDEA YOU HAVE A [INDISCERNIBLE] THAT TARGETS OPEN CHROMATIN AND CLEAVES IT, AND YOU MAKE A LIBRARY OF THAT, SEND IT TO THE SEQUENCING CENTER AND LOOK TO SEE WHAT ENDS HAVE APPEARED IN YOUR SEQUENCE, THAT MUST MEAN WHERE THE TN5 CLEAVED. THIS HAS ADVANTAGES OVER DNA HYPER SENSITIVITY APPROACH WHICH ACTUALLY MY LAB PLAYED A SIGNIFICANT ROLE IN DEVELOPING 15 YEARS AGO, WHICH WE DON'T USE MUCH BECAUSE ATTACK SEQ IS EASIER, REQUIRES FEWER CELLS AND GIVES YOU THE PRECISE LOCALIZATION OF THE ACTION. WE'VE APPLIED THAT. NOT EASY TO DO THIS ON LOTS OF SAMPLES. I'LL TELL YOU ABOUT TWO WHERE WE DID ATTACK SEQ, LOOKING TO SEE THINGS IN COMMON. AND WE WANT TO SEE CAN THIS DO A BETS JOB OF SHINING A LIGHT ON THE ACTUAL FUNCTIONAL ACTIVITIES GOING ON IN STRETCH ENHANCERS THAT PLAY A ROLE IN THESE GWAS RISK FACTS. BECAUSE ATAK SEQ PRECISE, ONE CAN LOOK AT THE DATA. WHEN YOU HAVE A WHOLE GENOME WORTH AND BEGIN RECOGNIZABLE MOTIFS. AS YOU CAN SEE HERE, CTCF, THAT INSULATOR AND RFX, A TRANSFACT IDENTIFY PRECISELY WHERE THE OPEN CHROMATIN IS, AND YOU GET THIS SIGNATURE AS YOU SEE THERE WITH RFX. YOU HAVE DO YOU THINK UP, DOWN, AND YOU CAN IMAGINE WHAT THAT MEANS HOW IT'S POSITIONED. YOU CAN SHOW HOW IT FITS NICELY FOR THAT PARTICULAR TRANSCRIPTION FACTOR MOTIF. SO THIS IS THE KIND OF THING YOU'D LIKE TO BE ABLE TO UTILIZE TO GET MORE CLOSE TO THE ACTION. IN A PARTICULAR INTERESTING DEVELOPMENT, WORK DONE BY FORMER POST-DOC STEVE PARKER, NOW ASSISTANT PROFESSOR AT MICHIGAN AS WELL BROOK, A FORMER POST-DOC HERE IN MY LAB, NOW A GRADUATE STUDENT. WHEN YOU HAVE A WHOLE GENOME'S WORTH OF DATA, AND YOU HAVE ALL THESE ATAK READS, YOU CAN LOOK TO SEE IS THERE A BIAS IN THE READS FOR A PARTICULAR TRANSCRIPTION SITE. IF YOU HAVE AN AREA THAT EFFECTS THE BINDING AND HETEROGUYING THE FOR THAT VARIANT, YOU'LL SEE AN ABUNDANCE OF THE VARIANT THAT FITS, AND REDUCTION IN THE AMOUNT OF THE ONE THAT DOESN'T FIT. WITHOUT GOING INTO GREAT DETAILS THIS IS GATE ELOQUENT. YOU CAN RECONSTRUCT THE MOTIFS THAT THE TRANSCRIPTION FACTOR LIKES TO BIND TO USING THAT BIAS, WHEN YOU HAVE A WHOLE GENOME'S WORTH OF DATA TO DO THAT HERE SHOWN FOR CTCF AND RFX. NOW, OF COURSE, WE WANTED TO KNOW DO THESE FOOT PRINTS HELP YOU IN TERMS OF UNDERSTANDING WHICH OF THOSE SNIPS THAT YOU'RE IDENTIFYING AS BEING INVOLVED IN EQTLs, WHICH ARE ACTUALLY POTENTIALLY FUNCTIONALLY IMPORTANT. BECAUSE THEY'RE BINDING TO A -- THEY'RE ACTUALLY LOCATED WITHIN A MOTIF OR TRANSCRIPTION FACTOR. SO ONE CAN DO THIS KIND OF ANALYSIS ON THE Y AXIS, YOU'RE BASICALLY LOOKING FOR ENRICHMENT OF THOSE KINDS OF MOTIFS IN AREAS THAT DO, IN FACT, FIT FOOT PRINTS. ON THE X AXIS YOU'RE LOOKING TO SEE ARE THERE INSTANCES WHERE I SEE ENRICHMENT. YOU DO, 127, SOME ARE HOUSEKEEPING, SOME ARE FAIRLY TISSUE SPECIFIC. MAYBE OF MORE INTEREST, IS TO ASK THE QUESTION, IF YOU PUT ALL THIS TOGETHER, CAN YOU FIGURE OUT DO YOU HAVE THESE FOOT PRINTS FROM YOUR ATAC DATA THAT INDICATE A TRANSCRIPTION FACTOR BINDS THERE. AND IS THIS THE PLACE WHERE YOU HAVE A GWAS SNIP FROM YOUR ANALYSIS, AND IN THAT CASE, CAN YOU PUT THIS ALL INTO ONE STORY THAT YOU CAN MAKE SENSE OUT OF AND FEEL LIKE YOU HAVE FIGURED OUT FUNCTION. SO WHAT YOU WANT TO DO IN THAT CASE IS TO ASK, ARE THESE SNIPS ENRICHED IN FOOT PRINTS. IF YOU START OUT JUST WITH THE ENTIRE GENOME AND PAY NO ATTENTION TO THE FOOT PRINTS, YOU DON'T GET ENRICHMENT. YOU WOULDN'T EXPECT TO. IF YOU LOOK IN AN IRRELEVANT CELL TYPE, NOW YOU'RE ASKING ARE THERE TYPE II DIABETES GWAS SNIPS ENRICHED IN FOOT PRINTS FOR A LYMPHOBLAST I HAD LINE. THE ANSWER IS NO. IF YOU LOOK IN ANISELET, YOU SEE -- ISLET, THAT'S IS LET NUMBER ONE, I THINK I TOLD YOU WE HAD DONE TWO. HERE IS NUMBER TWO. IF I PUT THEM TOGETHER. THIS IS STUNNING. WHAT YOU SEE IS ALL THE FINDINGS THAT FALL ABOVE THE LINE SEEM TO HAVE SOMETHING IN COMMON. SOMETHING CALLED RFX. I WILL CONFESS UNTIL THIS RESULT, ONLY A FEW MONTHS OLD, I HAD NOT PAID A BIT OF ATTENTION. WE STUDIED A LOT OF OTHER TRANSCRIPTION FACTORS IN THE PANCREAS. RFX HAD NOT ATTRACTED A LOT OF ATTENTION. IT HAS NOW. SO WHAT THIS IS SAYING IS THAT THERE IS A COMMON THEME HERE. THAT A SIGNIFICANT NUMBER OF THOSE TYPE II DIABETES GWA SNIPS SEEM TO HAVE THEIR FUNCTION BY ALTERING THE BIND OF THIS FAMILY OF TRANSCRIPTION FACTS CALLED RFX, ALTAR GENE EXPRESSION OF SOME NEARBY GENE. WHO ARE THESE FACTORS -- TRANSCRIPTION FACTORS? THAT'S QUITE A LARGE FAMILY. THAT WAS INITIALLY CONFUSING. BUT IF YOU LOOK TO SEE WHAT IS THE PATTERN OF EXPRESSION, WHAT YOU WILL SEE IS THAT THERE IS ONE OF THESE RFX 6, WHICH IS ONLY EXPRESSED IN THE PAN CREATEDIC IS LET. THAT MAKES US PARTICULARLY INTERESTED IN IT. NOW, INTERESTINGLY, IF YOU ASK, THEN, OS, DOES THE RISKS VARIANCE DISRUPT OR ENHANCE RFX MOTS? YOU WOULD THINK THAT IF YOU'RE ON TO SOMETHING HERE, THE ANSWER OUGHT TO BE THE SAME, UP OR DOWN FOR MOST OF THESE FINDINGS. AND IT'S REMARKABLY TRUE. REMEMBER OUR FRIEND KCNK16, WE FIGURED OUT THERE WAS A SNIP THAT WAS MODIFYING GENE EXPRESSION BUT DIDN'T KNOW WHY. GUESS WHAT? IT'S ONE OF THESE. THAT HAS AN RFX MOTIF. IT HAPPENS TO FIT RFX 2-4. THESE ARE EXTREMELY SIMILAR IN THEIR MOTIFS. I WOULD BE SURPRISED IF THIS DOESN'T ALSO BIND RFX 6. WE'RE LOOKING AT THAT RIGHT NOW. YOU CAN SEE HERE WHAT THE MOTIF INTERRUPTING VARIANT IS, THE NON RIVING ALEGAL HAS AN A IN THE POSITION OF THAT BOX. THE RISK ALLELE HAS A G. WOULD BE EXPECTED TO INTERFERE WITH THIS AND THEREFORE, TO REDUCE THE ABILITY OF RFX TO BIND TO THAT SITE MOVE SO THAT'S A SINGLE EXAMPLE. HERE IS ANOTHER GWAS LOCUST, PH9, WHICH HAS 3 DIFFERENT SNIPS ALL IN LD WITH THE LEAD SNIP. THEY ALL 3 HAVE RFX BINDING SITES. IN EVERY INSTANCE, THE THE RISK ALLELE INTERFERES WITH BINDING, AND THE NON RISK MAKES THE BINDING PRETTY GOOD. AND THERE ARE OTHERS LIKE THIS. THEY ALL GO THE SAME WAY. IN EVER INSTANCE, THE RISK ALLELE INTERFERING WITH WINDING OF RFX. SO WE THINK WE'RE ON TO SOMETHING HERE THAT WE HAVE DISCOVERED A GENERAL PHENOMENON OF PANCREATIC BIOLOGY OF THE ISLET. THAT PLAYS A ROLE IN TYPE II DIABETES. THIS IS MORE OF A MASTER TRANSCRIPTION FACTOR THAT WE PREVIOUSLY REALIZED. MAYBE WE SHOULD HAVE. ONCE YOU KNOW WHERE TO LOOK THERE ARE PAPERS DESCRIBING, THIS PARTICULARLY, A RARE CONDITION CALLED MITCHELL REILLY SYNDROME, IN THE BINDING DOMAIN OF RFX 6, CAUSING THE DIABETES. IT ALL MAKES SENSE. SO PUTTING THAT ALL TOGETHER, AND RECOGNIZING THAT OTHER SPEAKERS HAVE LOTS HAVE THINGS TO SHARE WITH YOU AND I SHOULD NOT GO ON ANY LONGER, BUT IT'S BEEN FUN TO TELL YOU THIS STORY. I THINK IT'S FAIR TO SAY THAT DETERMINING THE FUNCTIONAL BASIS OF GENETIC RISK FACTORS FOR TYPE II DIABETES IS GOING TO REQUIRE AN UNDERSTANDING OF THE EPIGENOME OF RELEVANT TISSUES. I COULD GENERALIZE THAT TO ANY POLY GENIC CONDITION, IF YOU SORT THIS OUT IN MOST OF THOSE IT WILL BE THE NON CODING VARIANTS, THE MAJORITY OF THE GENETIC RISK FACTS. WE HAVE TO GET INTO THE EPIGENOME AND THAT MEANS YOU HAVE TO GET TO THE RIGHT TISSUE. SOMETIMES I WORRY PEOPLE SAY WE'LL DO EPIGENOMICS, FIGURE THIS OUT. YOU SAY WHAT TISSUE? BLOCK OF COURSE. YOU KNOW, BLOOD IS ONLY A WINDOW INTO A SUBSET OF WHAT'S GOING ON IN THE BODY. IF YOU'RE INTERESTED IN THE DISORDER THAT'S NOT PRIMARILY INVOLVING THE HEMATIC SYSTEM, OR THE IMMUNE SYSTEM YOU MAY NEED TO LOOK IN OUR TISSUES AS WE HAVE BEEN FORCED TO DO. I THINK IT'S FAIR TO SAY THAT ONCE YOU'RE TRYING TO SORT OUT THOSE GWAS SIGNALS, HAVINGTINO TYPING AND IDENTIFYING EQTLs GIVES YOU A GOOD WINDOW TO HOW THE RISK VARIANTS OPERATE AND WHAT GENE IS DRIVEN BY THE VARIANT. THE EXAMPLE I SHOWED YOU WITH KCNK17 IS JUST ONE. IN THIS INSTANCE, LEADS TO INTERESTING IDEAS ABOUT A NEW DRUG TARGET THAT WOULD NOT OTHERWISE HAVE BEEN SO CLEAR. I THINK IT'S FAIR TO SAY THAT CHROMATIN ANALYSIS IDENTIFYING STRETCH ENHANCERS OR SUPER ENHANCERS IS A POWERFUL WAY TO SEE SOME PRETTY INTERESTING FUNCTIONAL DATA, AT LEAST FOR TYPE II DIABETES THAT'S A LOT OF THE ACTION IS IN TERMS OF GENE REGULATION. ATAC-SEQ PROVIDES THE WINDOW WHAT'S BINDING AREA, GIVING US IDEAS HOW TO TAKE A STRETCH ENHANCERS AND BREAK IT DOWN INTO FUNCTIONAL ELEMENTS. AND WHEN YOU COULD INTERSECT THAT WITH GWAS SNIPS, YOU CAN BEGIN TO DISCERN SOMETHING THAT WAS OTHERWISE COMPLETELY OUT OF SO IN WHICH WE THINK CAN LEAD TO FURTHER UNDERSTANDING OF THE NATURE OF PANCREATIC IS NULL LET BIOLOGY IN THE NORMAL STATE AND CASE OF DIABETES. TYPE II DIABETES WAS CALLED BY JIM KNEEL IN THE 70s, THE GENETICIST NIGHTMARE. MANY PEOPLE IN MY LAB HAVE AGREED WITH THAT OVER THE COURSE OF THE LAST 23 YEARS. BUT MAYBE WE'RE FINALLY BEGINNING TO WAKE UP BY HAVING THESE TECHNOLOGIES AND THESE INSIGHTS, BEGINNINGS TO SHED LIGHT ON THE INHERITANCE AND WHAT WE CAN DO TO PRACTICE BETTER PREVENTION AND TREATMENT. FINALLY, I WANT TO THANK THE MANY PEOPLE WHO HAVE BEEN INVOLVED IN THIS WORK, MORE THAN I COULD PUT ON THE SLIDE. PARTICULARLY WANTED TO MENTION THE FOLKS IN MY LAB, YOU SEE PICTURED HERE, PARTICULARLY FOR THIS WORK, MIKE IS HERE, VERY MUCH PART OF ALL OF THIS EFFORT, GOING WAY BACK TO THE ORIGINAL EFFORT TO COLLECT SAMPLES. WE'VE HAD WONDERFUL COLLABORATION WITH STEVE PARKER SINCE HE LEFT NIH A YEAR AGO TO AND HIS GRADUATED GRADUATE STUDENT. MIKE CONTINUES TO BE A CITICAL COLLABORATOR FOR ALL THIS WORK, LAURA SCOTT AND HIS GROUP. BROOK, PICTURED RIGHT THERE, WHEN SHE WAS A POST-DOC, NOW A GRADUATE STUDENT AT MICHIGAN. MICHAEL, ANOTHER FORMER POST-DOC IN THE LAB, JACKSON LAB NOW, ANOTHER CRITICAL COLLABORATOR IN ALL THIS WORK INCLUDING THE WORK ON IS NULL LETS. WE WOULDN'T BE ABLE TO DO ANY OF THIS IF IT WASN'T FOR THE WONDERFUL COLLABORATIVE HELP AND ASSISTANCE WE HAVE FROM OUR GREAT COLLEAGUES AT THE NIH INTRAMURAL SEQUENCING CENTER. THANK YOU ALL VERY MUCH. [APPLAUSE] >> WE HAVE TIME FOR A COUPLE OF QUESTIONS. ARE THERE ANY QUESTIONS? THANK YOU FOR THE IMPRESSIVE TALK. I'M [INDISCERNIBLE]. WE -- WE PUBLISHED ABOUT THE MEMORY SUPER ENHANCERS AND TESTED THE FUNCTION OF THE SUPER ENHANCERS. SO ACTUALLY, I HAVE TWO QUESTIONS. THE FIRST ONE IS, SO HAVE YOU EVER PERFORMED OR -- DO YOU HAVE ANY PLANS TO CORRECT THE RIVE VARIANTS, USING CRISPR CAS, IN THE DIABETES MODELS, SEE IF IT GETS BACK TO NORMAL. AND MY SECOND QUESTION IS MORE GENERAL THAT -- LIKE DO YOU -- COULD YOU KIND OF PROVIDE -- GIVE US ANY ADVICE OR LIKE PERSPECTIVES TO YOUNG SCIENTISTS LIKE ME FOR THE FUTURE OF THE SCIENCE FIVE TO TEN YEARS SO WE CAN FEEL HOW [INDISCERNIBLE] WE SHOULD GO FOR IT. [LAUGHTER] >> WOW. OKAY. TWO QUESTIONS. FIRST OF ALL, AGAIN, CONGRATULATIONS TO YOU AND TO LOTHAR FOR THE WONDERFUL WORK YOU DID ON THE MAMMARY GRAND. I READ YOUR PAPER VERY CAREFULLY, AND THE ELEGANT WORK YOU DID WITH CRISPR CAS, AND SEE THERE WAS AN EFFECT BETWEEN THE VARIOUS MOTEACHS, BY KNOCKING THEM OUT ONE AT THAT TIME YOU COULD SEE HOW THAT WORKS. THAT WAS A BEAUTIFUL DEMONSTRATION OF SOME OF THE DETAILS THAT WE HAVE NOT GOTTEN INTO. WE HAVE CERTAINLY GOT CRISPR CAS IN THE LAB. WE HAVE A WITH IT OF A PROBLEM DEALING WITH DIABETES. THERE IS NOT A ROBUST CELL LINE THAT ADEQUATELY REPRESENTS THE HUMAN BETA CELL. WE HAVE ONE LINE PEOPLE ARE USING CALLED NOC. IT GROWS VERY POORLY AND HARD TO WORK WITH. IT SLOWS US DOWN. SO WE'RE FORCED TO LOOK AT RAT OR MOUSE LINES THAT ARE MANY FURTHER AWAY FROM THE HUMAN CONDITION. THAT'S IN OUR WAY. AND I WISH WE HAD OPPORTUNITIES MORE TO DO THE KINDS OF THINGS THAT YOU HAVE TON. IN TERMS OF THE BIG PICTURE. WE'RE TALKING ABOUT THE EPJOKE, I WILL LIMTHE COMMENT TO THAT. THIS FIELD IS BUSTING OPEN WITH OPPORTUNITY AND POTENTIAL. IT'S VERY ACCESSIBLE TO PEOPLE IN LABS THAT DON'T HAVE TO INVOLVE DOZENS OF COLLABORATORS ALL LABORING AWAY, BECAUSE THE TECHNOLOGY IS WORKABLE IN THE HANDS OF A FAIRLY CAPABILITY EXPERIMENTIST. THE ONLY ADVICE I WOULD SAY IS THAT ANYBODY WHO WANTS TO WORK IN THIS FIELD, VIRTUALLY ANY OTHER AREA OF THE WAY BIOLOGY IS GOING, NEEDS TO GET SOPHISTICATED ABOUT THE COMPUTATIONAL PART. THAT IS WHERE THE INSIGHTS WILL COME FROM, BIG DATA SETS ARE OUT THERE BEING INCREASINGLY MADE AVAILABLE. NIH IS INSISTING ON THAT KIND OF OPEN DATA ACES. BUT THE EXCITING DIFFICULT PART IS MAKING THE MOST OUT OF IT WITH DESIGNING THE RIGHT ALGORITHMS, BUILDING THE RIGHT PROGRAMS, KNOWING HOW TO CODE, AND THEN COMING UP WITH THOSE BIOLOGICAL INSIGHTS THAT OTHERWISE MIGHT HAVE BEEN MISSED. THANK YOU. >> THANK YOU. >> CONGRATULATIONS FOR FINDING THE VARIANTS. WHAT IS THE ROLE OF THE CHAN THE [INDISCERNIBLE] >> THERE IS NOT A LOT NOPE. ROB FORD 6, NOW THAT WE'VE SEEN THE POTENTIAL TARGETS, HAVE MANY DIFFERENT GENES THAT IT MODIFIES THE EXPRESSION OF. IT IS A GENERAL TRANSCRIPTION FACTOR. IT'S CITY TELLING YOU IT'S DOING SOMETHING INTERESTING. IF YOU LOOK AT A MOUSE KNOCK OUT THERE IS A SIGNIFICANT EFFECT ON DEVELOPMENT AND FUNCTION OF PANCREATIC BETA CELLS. IT IS ONE OF THOSE MASTER TRANSCRIPTION FACTS THAT WE PROBABLY SHOULD HAVE PAID MORE ATTENTION TO. >> HOW ABOUT THE MUSCLE [INDISCERNIBLE] [INAUDIBLE] >> WE CAN'T UNFORTUNATELY LOOK AT MUSCLE IN THE SAME PATIENTS. THE ISLETS WE GET ARE THE RESULTS OF NETWORKS THAT ARE HARVESTING TISSUE AT THE TIME OF TRANSPLANTATION. THESE ARE KADAVERS, WE HAVE AN ELOQUENT COLLECTION OF SKIN THAT CAN BE TURNED INTO IPS CELLS FROM MORE THAN 300 VOLUNTEERS IN FINLAND. WE HAVE DONE A LOT OF WORK ON THE MUSCLE TO SAY THAT AT LEAST AT THE MOMENT, ROB FORD 6 OR ANY OF THE -- RFX 6 OR NUMBER OF THE RFXs HAVE POPPED HAVE AS MAJOR PLAYERS. >> OUR TIME IS A LITTLE LIMITED. WE CAN TAKE SHORT QUESTIONS AND SHORT ANSWERS. OTHER HERE. >> DO YOU SEE ANY [INAUDIBLE] >> RFX 6 HAS FINDING SITES FOR ITSELF. SO THERE IS SOME AUTO REGULATION GOING ON HERE. AND WE HAVEN'T SORTED OUT WHAT THAT MEANS. I THINK IT'S PROBABLY PRETTY SIGNIFICANT. >> A LOT OF INTERESTING WORK [INAUDIBLE] >> WE HAVE DATA JUST COMING IN FROM A COLLABORATION WITH WASH U, DURING WHOLE GENOME OH IS PRIME MINISTER LETS OF MUSCLE -- [TECHNICAL DIFFICULTIES]. I'M DIEING TO SEE WHAT THE RESULT LOOKS LIKE. >> THANK YOU. >> HAVE YOU SEEN TISSUES SPECIFIC ENHANCERS IN REGIONS IN MICE WHERE YOU MIGHT BE ABLE TO HAVE A LITTLE BIT MORE EXPERIMENTAL CONTROL? >> YES. I DON'T KNOW IF INNED SAY SYSTEMATICALLY. I COULD CERTAINLY CALL UP IN MY BRAIN HERE SEVERAL EXAMPLES WHERE THE [INDISCERNIBLE] IS VERY IMPRESSIVE. THIS ARE LIMITS THERE. WE HAVE TO ADMIT THAT FOR DIABETES MICE HAVE NOT BEEN THE GREATEST MODEL. WE HAVE TO BE CAREFUL. THERE IS NO KCNK17 GENE GENE IN MICE AT ALL. WE WOULD NOT HAVE LEARNED ABOUT THAT IF WE STUCK TO THE MOUSE. I AGREE IT WOULD BE GREAT, IN PARTICULAR, IF WE WANT TO DO SOMETHING LIKE CRISPR CAS MODIFICATIONS, WE SHOULD PAY ATTENTION TO THAT. GOOD QUESTION. >> THANK YOU ALL. THANK YOU. [APPLAUSE] >> THANK YOU DR. COLLINS FOR STARTING OFF THIS SESSION SO WELL. OUR NEXT SPEAKER IS DR. RAFAEL CASELLAS, CHIEF OF THE LABORATORY MOLECULAR IMMUNOGENETICS. BORN IN BINIS ARES, LIVED IN GENEVA, PARIS, AND HELL SINKY BEFORE COMING TO THE UNITED STATES. HIS Ph.D. IN MOLECULAR IMMUNOLOGY FROM THE ROCKEFELLER UNIVERSITY IN 2012, WHERE HE WORKED WITH MICHELLE. FROM 2002 TO 2003, HE DID POSTDOCTORAL WORK WITH DAVID BALTIMORE AT CAL TECH. WITH DAVID, HE WORKED ON MOLECULAR STUDIES OF B CELL ACTIVATION. FINALLY, HIS WORLD TOUR LANDED HIM IN BETHESDA IN 2003, HE'S -- AJUNK VER IN ADDITION TO SENIOR STATUS, AN ADJUNCT INVESTIGATOR FOR CANCER RESEARCH. THE MAIN GOAL OF THE LAB IS TO UNRAVEL THE MOLECULAR MECHANISMS DRIVING EARLY DEVELOPMENT AND PERIPHERAL ACTIVATION OF B LYMPHOCYTE AND TO UNDERSTAND HOW DEREGULATION OF THESE REACTIONS LEADS TO B CELL TUMOR GENESIS. TO ACHIEVE THESE GOALS, THIS PLANT COMBINES A VARIETY OF MODERN TECHNIQUES, AND HIS TALK TODAY IS ENTITLED THE ROLE OF NUCLEAR ARCHITECT IN GENE EXPRESSION. PLEASE JOIN ME IN WELCOMING DR. CASELLAS. [APPLAUSE] >> SO THE STORY THAT I WANT TO SHARE WITH YOU TODAY RELATES TO THE FUNCTION HOW SUPER ENHANCERS WORK. AND WHAT WE HAVE BEEN DOING IS STUDYING B CELL ACTIVATION, BUT THE QUESTIONS THAT WE ANSWER RELATE TO ALL CELL TYPES, PARTICULARLY TRANSCRIPTION. SO B CELL ACTIVATION TAKES PRAISE WHEN THIS NAIVE BONE MARROW CELLS THAT MIGRATE TO THE PERIPHERY IN [INDISCERNIBLE] STATE, THEY ENCOUNTER ANTIGENS AND BECOME ACTIVATED. YOU CAN SEE BY THE CELL SIZE, THERE IS BIG CHANGES HAPPENING DURING THIS PROCESS. AND IT ACTUALLY HAPPENS WITHIN 24 HOURS. VERY QUICK, VERY DYNAMIC PROCESS. OF COURSE THE END GOAL OF THIS DIFFERENT ASIAN IS TO RELEASE ANTIBODIES TO BLOCK THE [INDISCERNIBLE]. NOW, THIS PROCESS IS MEDIATED AT LEAST IN PART BY THE AMPPLICATION OF A [INDISCERNIBLE] OF NAIVE B CELLS. WHEN WE TALK ABOUT AMP FLYIFICATION, PROPORTIONAL AMPLIFICATION, MEANING THAT THE VAST MAJORITY ARE TRANSCRIBED ABOUT TEN FOLD HIGHER UPON ACTIVATION. HOW THIS PROCESS TAKES PLACE, WE PUBLISHED THIS A FEW YEARS AGO, BUT WHAT WE HAVE BEEN STUDYING SINCE IS HOW IN CHROMATIN DECON SENIDATION IS PHOSPHORYLATED IN THE AMPPLICATION OF THE TRANSCRIPTOMY, AND HOW WE STUDY THIS IS BY SUPER RESOLUTION MICROSCOPY. THIS IS A MICROGROW GAFFE TAKEN FROM ANALYSIS OF NAIVE AND ACTIVATED B CELLS. WE'RE IMAGING H2B. WHAT WE FOUND IS THE CHROMATIN TAKES PLACE IN 2 DIFFERENT STEPS. INDEPENDENTLY REGULATED STEPS. THE FIRST ONE IS SPREADING OF -- [INDISCERNIBLE]. YOU CAN SEE THE SPREADING GOING FROM THE NUCLEAR ENVELOPE ESSENTIALLY TO THE REST OF THE NUCLEOPLASM. THIS IS A VERY DYNAMIC PROCESS, ABOUT TEN MICROMETER PER HOUR. WITHIN 24 HOURS, [INDISCERNIBLE] AND THE WHOLE CHROMATIN SPREADS ACROSS THE ENTIRE NUCLEUS. BUT THERE IS A SECOND STEP. AND THAT IS THE DECOMPACTION OF NUCLEAR CLUSTERS, WE CAN VIRAL LIZ THIS FOR THE FIRST TIME BECAUSE OF THIS NEW TECHNIQUE. WHAT THAT MEANS IS THAT THE CLUSTERS, WHICH ARE ESSENTIALLY GROUPS OF NUCLEOSOMES, THEY COMPACT, INCLUDE SINGLE FIBERS. AND WE CAN SEE HERE THIS PROCESS, WHEN WE LOOK AT THIS, H 3K9 TRIMETHYLATION. WE CAN SEE THE CLUSTERS ARE SPREADING AND DECOMPACTING, AND THIS IS THE CASE WHEN YOU LOOK AT [INDISCERNIBLE], WHERE THERE IS ACTIVE GENE EXPRESSION AND THE SAME PROFILE. DECOMPACTION, SPREADING OF THE NUCLEOSOME CLUSTERS. NOW, THE QUESTION, OF COURSE, IS THAT THIS ACTIVE PROCESS SHOULD IMPACT HOW PROMOTER AND ENHANCER INTERACTS. AND THIS TEST TEST. NOW YOU'VE CREATED [INDISCERNIBLE]. SOMETIMES THEY CAPTURE BITS OF DNA THAT WERE FAR APART, IN 1D, CLOSE IN 3D. THEN YOU CAN ANALYSIS THESE THINGS, FIGURE OUT IF THE EXPERIMENT WORKED WELL. BASICALLY, I'M TRYING TO HIGHLIGHT THE MAJOR FEATURES THAT YOU SEE THAT EXPLAIN 99% OF WHAT YOU SEE WHEN YOU LOOK ON AUTOSOMES IN -- DURING INTERPHASE AT THESE CONTACT MAPS. I'LL SHOW THEM AS CAREY TUNES ON THE LEFT, ON THE RIGHT I'LL SHOW EXAMPLES OF THESE EXPERIMENTS WE HAVE BEEN DOING. SO FIRST THING YOU SEE WHEN YOU LOOK AT WHOLE CHROMOSOMES USING HIGH C MAPS, WHAT YOU SEE IS THESE LONG RANGE PATTERNS, INDIVIDUAL LOCI HAVE LONG RANGE PATTERNS, THE NEIGHBORS THEY INTERACT WITH, MOSTLY WITH PEOPLE ON THE NIH CAMPUS. I SPEND A LOT OF TIME INTERACTING WITH BAYLOR COLLEGE. YOU FIGURE OUT WHERE YOU LIVE, HOW MANY NEIGHBORHOODS THERE ARE. THERE IS TWO MANY DOMINANT SPATIAL COMPARTMENTS, HALF A DOZEN SUBCOMPARTMENTS, VERY, VERY OBVIOUS. HERE, THIS IS SHOWING YOU CHROME 19. YOU CAN SEE THE -- CHROMOSOME 19. YOU CAN SEE THE AT THIS PATTERNS, THEY'RE STRONGLY ASSOCIATED WITH UCHROMATIN AND HETEROCHROMATIN. THE CONSEQUENCE OF THE FACT THAT INTERVALS OF THE GENOME THAT LIE IN THE SAME COMPARTMENT, THEY'RE GOING TO THE SAME PLACE, INTERACTING WITH THE SAME FOLKS, AND INTERACT WITH ONE ANOTHER. CONTIGUOUS INTERVAL, SO INTERACTING WITH LEADS TO FORMATIONS OF SQUARES ON THE DIAGONAL OF THESE MAPS. ONE THING WE DR. COLLINS ALLUDED TO HIS TALK, WE STARTED WORKING ON HIGH C, WHEN HBO STARTED WORK ON THE GAME OF THRONES. SOME OF THE CHARACTERS IN THE EARLY EPISODES ARE STRIKING, VERY INTERESTING. THEY ACQUIRE HUGE NUMBERS OF NAMES. THESE SQUARES ON THE DIAGONAL THAT APPEAR HAVE ACCRUED A TRULY DIZZYING ARRAY OF NAMES, PROBABLY MOTHER NAMES THAN . -- MORE NAMES. I DON'T UNDERSTAND SOME OF THE NAMES. THE MAJOR POINT IS THEY'VE GOTTEN PEOPLE TO PAY ATTENTION TO THIS PHENOMENON. SIMILAR ALSO TO [INDISCERNIBLE], THEY HAVE BECOME ASSOCIATED WITH MANY MAGIC POWERS. I'M NOT COMPLETELY SURE THAT'S ALL OF THE MAGIC POWERS THEY'RE SUPPOSED TO POSSESS BUT THEY'RE INTERESTING, THESE INTERVALS OF UNBROKEN -- UNBROKEN INTERVALS, ON THE SAME COMPARTMENT. YOU KNOW, WHICH GO BY MANY NAMES, CONTACT DOMAINS, ASSOCIATED DOMAINS, CHROMATIN INTERACTION DOMAINS, ET CETERA, ET CETERA, ET CETERA. IF YOU ASSUME IN ON THESE DOMAINS, YOU'LL SEE THEY MANIFEST HAS SMALL CARES. ONE TYPE LOOKS LIKE THE SQUARE, THE OTHER IS A BRIGHT PEEK IN THE UPPER RIGHT-HAND CORNER, THAT PEEK IS A LOOP. TWO BITS OF GENOME THAT TENDS TO BE IN VERY, VERY CLOSE CONTACT IN SPACE. MANIFESTS VERY CLEARLY. HERE IS AN EXAMPLE FROM B CELL DATA. YOU CAN SIGH THERE ARE LOOP DOMAINS, BIG DOMAINS WITH LOOPS IN THE CORNER. SO IT'S SAYING THE FORMATION OF LOOP SEEMS TO BE ASSOCIATED WITH THE PREPARESANCE OF AN INTERVAL IN WHICH ALL THE LOCI, HAVING CONTACT FREQUENCIES WITH ONE ANOTHER. THIS ISN'T THE ONLY WAY. YOU SEE MEANDER DOMAINS THAT DO THE NOVEMBER -- MANY OTHER DOMAINS THAT DO NOT HAVE LOOPS IN THEIR CORNER. IF YOU ZOOM IN YOU CAN FIGURE OUT HOW THESE THINGS ARE ESTABLISHED. IN PARTICULAR WE'RE LOOKING AT A PARTICULAR LOOP. YOU CAN MAP WHERE BINDS BINDING IS, IN THESE CELLS. TCPF IS A DNA BINDING PROTEIN, THOUGHT TO BE ASSOCIATED WITH LOOP FORMATION AND INSULATION, AND LOTS OF DIFFERENT THINGS. WITH VERY, VERY STRIKING -- IF YOU LOOK LOOP ANCHORS YOU SEE THE BINDING SITES PERFECTLY LINED UP. IF YOU LOOK CAVALLY AT THE MOTIFS ASSOCIATED WITH THE BINDING SITES, THEY DO A STRIKING THING. YOU HAVE MOTIFS, ABOUT TEN BASIS LONG, LIE MILLIONS OF BASE PAIRS, BUT TO BE FORMED THEY HAVE TO BE POINTING THE. THIS IS THE CONVERGENT RULE. WE THINK WHEN WE DON'T SEE IT BECAUSE WE MADE AN ERROR. ALSO SHOWED BY ENGINEERING, WHEN YOU WHITTLE THESE, IF YOU INVERT THEM, THE LOOPS WILL DISAPPEAR AND THE DOMAINS WILLTIST APPEAR. THAT IS A VERY, VERY STRANGE THING. HOW ARE TWO MOTIFS SO FAR APART SUPPOSED TO KNOW WHETHER THEY'RE POINTING AT ONE ANOTHER AND WHETHER THEY SHOULD LOOP. THIS IS HARD TO EXPLAIN GIVEN THE OLD MODEL OF LOOP FORMATION, THAT LOOPS FORM BY DIFFUSION. THIS LED US TO PROPOSE A DISTINCT MODEL OF HOW LOOPS MIGHT FORM IN THE GENOME. WE CALL IT THE EXDRUGS MODEL. THIS IS WHAT IT LOOKS LIKE. SO WE THINK THAT THERE IS THIS PAIR OF DOUGHNUTS THAT LIVE IN THE GENOME. THEY CAN LAND AT ANY PARTICULAR POSITION. THE TWO SUBUNITS SLIDE IN OBSTHE DIRECTIONS. YOU WANT TO STOP SLIDING. WHERE DO YOU STOP? I STOP WHEN I'M DRIVING AT A STOP SIGN. SO THESE WILL STOP AT STOP SIGNS, TOO. THEY ONLY STOP AT STOP SIGNS POINTING TOWARD ME. THERE IS THE MOTIF POINTING TOWARD THE SUBUNIT. THIS IS POINTING THE WRONG WAY, THE BOTTOM SUBUNIT WON'T STOP SLIDING. NOW IT'S GOING TO STOP. IF SUCH A THING EXISTS, AND I'M NOT SAYING THERE IS ANY DIRECT POSITIVE EVIDENCE IT DOES, IT WOULD EXPLAIN WHY YOU SEE THESE LOOPS WITH THE ANCHOR IN THE CONVERGENT ORIENTATION. SO WE THINK THAT THIS IS AROUND, BUT WE ACTUALLY DON'T HAVE DIRECT EVIDENCE FOR IT. ANYWAY, THAT, I THINK, HOPEFULLY ENABLES YOU TO UNDERSTAND WHAT YOU SEE, THE VAST MARINETTE OF THE TIME WHEN YOU'RE LOOKING AT A MAP. NOW WE'RE GOING TO TALK ABOUT SOME OF THE THINGS WE HAVE BEEN SUING THAT DON'T FIT IN SOME OF THOSE NEAT CATEGORIES. >> THANK YOU. [APPLAUSE] >> SO GOING BACK TO THIS RESTING ACTIVATED B CELLS, THESE ARE THE MAPS FROM THE TWO CELL TYPES. WHAT YOU SEE RIGHT AWAY IS THAT THERE ARE BIG DIFFERENCES IN THIS INTERDOMAIN INTERSECTION. THE LONG RANGE INTERACTIONS. AND YOU CAN SEE IT HERE IN A COMPOSITE DIAGRAM, ON THE LEFT, SINGLE ON THE RIGHT. YOU SEE THE CELLS, THEY FORM LONG RANGE CONTACTS THAN THE ACTIVATED COUNTERPART. AND THE OPPOSITE APPEARS TO BE TRUE FOR SHORT-RANGE CONTACT. WE ZOOM INTO THE TWO DOMAINS. WE CAN EXPLAIN HOW THAT HAPPENS. SO IN TERMS OF SHORT-RANGE CONTACTS THAT WAS EXPLAINED, THEY TAKE PLACE IN TWO WAYS, FIRST OF ALL, YOU CAN SEE THE APPEARANCE OF THESE LOOPS IN ACTIVATED B CELLS PARTICULARLY. ALSO, YOU CAN SEE THIS IN A GLOBAL VIEW. THERE IS ABOUT 600 OR SO LOOPS THAT APPEAR TO BE SPECIFIC FOR [INDISCERNIBLE] CELLS BUT UPON ACT VASE, YOU GAIN MORE THAN 4,000 LOOPS. SECONDLY, PARTICULARLY BETWEEN -- OR WITHIN THIS DOMAIN FORMED BY LOOPS, THE INTERACTIONS ARE INCREASED UPON THE ACTIVATION. SO THIS ACTIVATION FAVOR THE SHORT-RANGE CONTACT. THIS LEADS TO A STRONGER LOOP AND DOMAIN. WHICH IS IN CONTRAST TO WHAT WE SEE IN CELLS WHICH FAVOR LONG RANGE CONTACTS. NOW, THE QUESTION IS WHETHER THIS TWO CHANGES IN ARCHITECT, THEY ARE IMPACTING GENE GENE EXPRESSION. SO WHAT WE DID IS WE CLASSIFIED DOMAINS RELATIVE TO THE EXTENT OF INTERDOMAIN INTERACTION. TO THE RIGHT YOU'RE SEEING THE DOMAINS THAT CHANGE THE MOST. TO THE LEFT, CHANGE THE LEAST. WHEN WE PLOT IN GENE EXPRESSION, SURPRISINGLY WE FIND THAT GENE EXPRESSION WITHIN THOSE DOMAINS IS HIGHER THAN THOSE THAT INTERACT THE LEAST. WE'RE SEEING HERE, WHY THIS WOULD BE THE CASE, ONE WAY TO EXPLAIN THIS IS BECAUSE INTERACTIONS ARE INCREASED WITHIN THE DOMAINS [INDISCERNIBLE] ARE GOING UP. NOW, THE PROBLEM OR IMPLICATION IS THAT ACTIVATED B CELLINGS PROGRESSIVELY WITHIN 24 HOURS FAVOR THIS SHORT-RANGE INTERACTION, AND THERE BY INTENSIFY DYSFUNCTIONAL CONTACT BETWEEN REGULATORY ELEMENTS. THE CAVEATS OF THIS IS THIS IS A CORRELATION, AND THERE ARE TOO MANY CHANGES GOING ON DURING THIS PROCESS OF ACTIVATION, THEREFORE, WE CANNOT REALLY BE SURE THAT ARCHITECT IS INDUCED IN THIS, INCREASE GENE EXPRESSION. TO ADDRESS THIS, ONE CAN PERHAPS DELETE SOME OF THE ARCHITECTURAL [INDISCERNIBLE] AND MEASURE TRANSCRIPTION IN THESE CELLS. THE PROBLEM WITH THAT EXPERIMENT THAT DELETION OF CTCF LEADS TO CELL MORPHOLOGY, SO WE CAN NOT DO THAT. BUT WE RELY ON ONE FEATURE ABOUT CTCF BINDING. IT HAS TO DO WITH THE FACT THAT THERE ARE 11 FINGERS IT USES TO RECOGNIZE MOTIF DIVERSITY ACROSS THE GENOME. SO IT USES SIMILAR OR DIFFERENT FINGERS TO RECOGNIZE DIFFERENT FINDING MOTIFS. THEREFORE, ONE CAN DISRUPT SOME OF THE SAME FINGERS. IN THESE KINDS OF EXPERIMENTS THEY PRECLUDE CTCF BINDING TO SOME BUT NOT ALL BINDING MOTIFS. WHEN WE DO THIS AND TEST IT FOR SEVERAL DIFFERENT COMBINATIONS, THIS MUTANT LEADS TO AN ABROGATION OF BINDING TO SOME DOMAINS WITHOUT AFFECTING CRITICAL VIABILITY. SO WE CAN WORK WITH THIS SYSTEM. WHEN WE DO THAT, WHAT WE CAN SEE RIGHT AWAY, WHEN WE DO THIS MAP FOR THE -- WHENEVER SEE SEE A LOT OF LOOP, OR LOTS OF BINDING, THE LOOPHOLES STILL GOES AWAY, SO DOES THE DOMAIN. IN A GLOBAL VIEW, HERE LOOPS THAT ARE UNEFFECTED. HERE ARE LOOPS THAT ARE EFFECTED. AND WHAT WE SEE GOING BACK TO OUR QUESTION ABOUT GENE EXPRESSION IS THAT THOSE UNEFFECTED LOOPS, THEY DON'T CHANGE GENE EXPRESSION. THOSE WHERE THE LOOPS ARE LOST, GENE EXPRESSION GOES DOWN. IT'S A SMALL EFFECT BUT APPEARS TO BE REAL. AND NOW THE BEAUTY OF THIS EXPERIMENT IS THAT ALL THINGS ARE BEING EQUAL. WE HAVE NOT MODIFIED CHROME ATEEN OR ANY OTHER THINGS THAT HAPPEN DURING THIS CELL ACTIVATION. THIS IS [INDISCERNIBLE] SO THE IDEA HERE IS THAT CHANGES IN INTRADOMAIN CONTACTS APPEAR TO IMPACT GENE EXPRESSION, LIKELY BY PROMOTING [INDISCERNIBLE] BETWEEN PROMOTERS AND ENHANCER. THE QUESTION IS HOW. HOW DOES ARCHITECT DO THIS? HOW DOES THIS PHYSICAL TETHERING OF PROMOTING ENHANCERS TAKE PLACE? WELL, FOR THAT, WE GOT A CLUE FROM ANOTHER FEATURE OF ARCHITECT AND WE CALL THIS ARCHITECTURAL STRIPES. YOU CAN SEE HERE 3 STRIPES. GOING BACK TO OUR BINDING SITES. SO THERE ARE 4 BINDING SITES HERE THAT I'M HIGHLIGHTING. THESE TWO, THEY FORMED THIS LOOP. THESE TWO, THIS LOOP, AND THIS, THE OTHER ONES, THEY FORMED THE OTHER LOOP HERE. BUT ALSO WHAT YOU SEE IS A STRIPE -- WITHIN THIS DOMAIN, THIS ANCHOR, ANOTHER ONE ASSOCIATED WITH THE LEFT ANCHOR, AND THIRD ONE HERE. COMING THE OTHER WAY FROM THIS ANCHOR. SO HOW DO WE EXPLAIN THIS? GOING BACK TO THIS MODEL, THIS CTCF CLOSEST TO THE PROMOTER OF HS3, A COMPLEX COMES ASSOCIATED WITH DNA EXTRUEDING ALL THE WAY TO THE [INDISCERNIBLE] AS WAS EXPLAINED, THAT CTCF BINDING SITE. IT WILL KEEP [INDISCERNIBLE] IN THE DNA UNTIL IT REACHES THE SECOND BINDING SITE, AND NOW THE LOOP IS FORMED. SO THE STRIPE CAN BE EXPLAINED, PERHAPS, BY REDUCED PROCESSIVETY OF THIS EXTRUSION ACTIVITY. SOMETHING WITHIN THAT DOMAIN IS SLOWING DOWN THE ACTIVITY ALLOWING THE INTERACTION FREQUENCY GOING UP. THIS IS HOW WE DETECT THE STRIPE. WHAT COULD THAT SOMETHING BE? IN THIS PARTICULAR EXAMPLE, THAT'S -- DOES SOMETHING APPEAR, OR MAYBE ASSOCIATED WITH [INDISCERNIBLE]. AND WHAT I'VE DONE HERE, IF YOU LOOK AT THE Y AXIS OF THOSE TWO TRACKS, WE'RE ZOOMING IN. THE UPPER ONE, THE UPPER IS 10, LOWER IS 2. YOU CAN SEE THOSE VERY TINY CTCF PEEKS, BARELY IN THE UPPER TRACK. NOW YOU CAN SEE IN THE LOWER ONE. SO IN ADDITION TO THAT, THESE DOMAINS ARE ASSOCIATED WITH ENHANCER CHROMATIN MARKS. AS A GLOBAL VIEW, THIS IS EXACTLY WHAT WE'RE SEEING. IT'S 80% OF ALL STRIPES DETECTED IN THE GENOME, ASSOCIATED WITH THESE PEEKS. SOME OF THEM ASSOCIATED WITH ACTIVATION MARK ONLY, ALSO MINORITY WITH NONE OF THEM. ALSO IMPORTANT TO POINT OUT, THE SIZE OF THOSE PEEKS -- SO WE COMPARE THE CTCF PEEKS, LOOPS, MUCH HIGH YOU ARE THAN STRIPES. SO THIS IS A [INDISCERNIBLE] OF CTCF BINDING. PERHAPS EXPLAINS HOW OR WHY OR GIVEN A RATION TO PERHAPS ACTIVITY OF THOSE TINY PEEKS THAT WE HAVE SEEN MANY TIMES BEFORE WE COULD NOT EXPLAIN WHAT THEY WERE THERE FOR. ANOTHER IMPORTANT POINT ABOUT THE DOMAINS THAT ARE DECORATED BY THIS LOW CTCF BINDING, MOST FREAKILY THEY ARE ASSOCIATED WITH ACTIVATION MARK. THOSE ACT EVALUATION MARKS ARE NOT MARKS THAT DEFINE PROMOTERS, BUT MARKS THAT DEFINE ENHANCERS. SO THE CTCF ANCHORS IN THE MIDDLE. AND TO YOUR RIGHT, IS THIS STRIPE. YOU CAN SEE TRIMETHYLATION, WHICH DEMARCATES PROMOTERS, NOT ENRICHED WITHIN THIS DOMAIN. BUT DOES -- BUT IS H3K4 METHYLATION ASSOCIATED WITH ENHANCER. NOW HERE IS ONE EXAMPLE. THIS IS WHERE SUPER ENHANCERS COME AROUND. I LOVE SUPER ENHANCERS BECAUSE THEY'RE THE MOST STRIKING EXAMPLES OF THE STRIPES WE FIND IN THE GENOME. BEAUTIFUL CONSOLATIONS AS YOU SEE THIS MAP. AND HERE IS THE EXAMPLE OF AN ONCOGENE, HIGHLY EXPRESSED IN ACTIVATED B CELLS. P TV 1 IS A RNA APPEARING TO REGULATE EXPRESSION BETWEEN DIFFERENT SPECIES. AND HERE IS THE SUPER ENHANCERS WHICH IS WITHIN THE RNA GENE. FRANCIS MENTIONED WE CAN DEFINE THESE SUPER ENHANCERS BE [INDISCERNIBLE] ASSETLATION. AND HERE IS CTCF. THE LOW BINDING THERE, WE CAN ZOOM IN AND SEE IT. IN CORRELATES WITH THAT STRIPE YOU SEE ON THE TOP. THE STRIPE OR THE EXTRUSION ACTIVITY HAPPENS FROM THE PROMOTEER OF MYC. AND THE SUPER ENHANCERS REELED IN TOWARD THE PROMOTER. THAT'S THE MODEL WE'RE WORKING WITH. HERE IS ANOTHER EXAMPLE, INTERFERON 2 BINDING PROTEIN 2. THIS IS THE RNA, THE EXPRESSION OF THIS GENE. HERE IS A SI, PRETTY FAR AWAY. SUPER ENHANCERS. PRETTY FAR AWAY. 500 KILLA BASES AWAY. AGAIN, AND YOU CAN SEE TYPY [INDISCERNIBLE] ASSOCIATED ASSOCIATED WITH THE SUPER ENHANCERS. AS THAT DOMAIN IN OUR MODEL IS REELED IN, IT WOULD SLOW DOWN THE PROMOTING DRIVE WITH THE SUPER ENHANCERS. HERE IS ANOTHER EXAMPLE. THIS IS JOHN OSHA'S FAVORITE ONE, AND THIS IS A SUPER MASSIVE [INDISCERNIBLE], CALLED SUPER ENHANCERS. IT HAS WORKED IN FOUR STRIPES ASSOCIATED WITH IT. HIGHLY EXPRESSED INACTIVATED CELLS, FUNCTIONS BEAUTIFUL IN B CELLS, T CELLS AS JOHN SHOWED. THE SUPER ENHANCERS DOWN STREAM OF THE PROMOTER. THIS IS THE COUNTER EXAMPLE OF MYC. THE FIRST EXAMPLE I SHOWED YOU IN THAT THE EXDRUGS ACTIVITY APPEARS TO BE FROM THE SUPER ENHANCERS INSTEAD OF BEING THE PROMOTER, WE'RE LOOKING AT INTERPRET THE SUPER ENHANCERS PERSPECTIVE. THIS IS THE CTCF, ZOOMED IN. THIS IS THE VIEW IN B CELLS WHERE THIS GENE IS HIGHLY ACTIVE. IF YOU LOOK AT [INDISCERNIBLE] WHERE THE GENE IS TRANSCRIPTIONALLY SILENT. THERE IS NO SUPER ENHANCERS ACTIVITY, NO TRANSCRIPTION. CTCF IS NOT REALLY PRESENT. THAT CORRELATES WITH DNA DEMETHYLATION WHICH WE'RE PLOTTING HERE. SO IN THESE CELLS THE SUPER ENHANCERS AREA IS HEAVILY DEMETHYLATED, WHICH WE BELIEVE [INDISCERNIBLE] THE RECRUITMENT OF TRANSCRIPTION FACTOR INCLUDING CTCF AT THE LOW BINDING SITES. THAT'S ACTUALLY NOT THE CASE WHEN THE CELLS [INDISCERNIBLE] METHYLATED. HERE IS ANOTHER EXAMPLE. TICK TECH. THE LONGEST STRIPE WE HAVE FOUND IN ACTIVATED B CELLS, 2.3 MEGABASIS. ASSOCIATED WITH THE IGH LOGO H IS A DOMAIN WHERE IMMUNOGLOBULIN IS TRANSCRIBED FROM. AND HERE IS CTCF. AND IN FACT THE CTCF BINDING MOTIF, THEY'RE ALL FORWARD. THERE ARE 109 OF THEM ALL LOOKING FORWARD. THE ENHANCER, THE -- ACTUALLY, THE OCR, I WOULD CHANGE THAT, THE OCR WHICH IS THE RIGHT SIDE OF THE IGH LOGO. IN FACT, DOWN STREAM OF THAT LCR., THERE IS WHAT IS CALLED A SUPER ANCHOR. EVERYTHING IS SUPER HERE. THERE IS ACTUALLY TEN CTCF BINDINGS SITES. SO PRESUMABLY, THAT IS -- THOSE TWO STRIPES HERE YOU'RE SEEING THERE IS THE ACTIVITY OF THOSE COMPLEXES OF THEIR DNA TOWARD THE ENHANCER INDUCED. SO TO TEST THE FUNCTION OF THESE STRIPES INDIRECTLY, OF COURSE, WHAT WE DID IS DELETED THE ENTIRE REVERSE MOTIF, SO BINDING SITES, TO ABOUT 15 KILO BASIS. SO WE DELETE THAT HAD WITH CRISPR CELLS AND LOOK AT THE FUNCTION. FIRST OF ALL, WE CHECKED [INDISCERNIBLE]. INTERACTION OF THAT DOMAIN. THE ENHANCER DOMAIN, JUST UPSTREAM OF THE UNDELETED SUPER ANCHOR. AND WE SEE THAT INTERACTION GOES DOWN, CONSISTENT WITH THE FACT THAT CTCF ANCHORS ARE RESPONSIBLE FOR THOSE STRIPES WE'RE SEEING. AND THEN WHAT WE DID IS WE ACTIVATED THESE CELLS IN THE PRESENCE OF LPS [INDISCERNIBLE] BETA. WHAT THAT DOES AMONG OTHER THINGS IS TRANSCRIPTION, THE ACTIVATION OF IGM AND IgA. AND SUBSEQUENTLY, THE RECOMBINATION BETWEEN THESE TWO DIFFERENT IMMUNOGLOBIN ISISO TYPES. SO THAT'S THE VIEW WE GET IN WILDTYPE B CELLS. THIS IS WHAT HAPPENS WHEN WE REMOVE THE SUPER ANCHOR, THAT RED BAR THERE, GOES DOWN. SO THAT RECOMBINATION BETWEEN THESE TWO ICO TYPES. SO WHAT THIS DOES, DELETION OF THE STRIPED ANCHOR REDUCES GENE EXPRESSION AND RECOMBINATION OF HEMOGLOBIN GENES. THIS IS OVER A VERY LARGE AREA IN THE GENOME. SO THIS IS OUR SMALL MODEL THAT IS AN EXTENSION OF THE [INDISCERNIBLE] MODEL. WHAT WE BELIEVE IS GOING ON, AS THE COMPLEX ASSOCIATED WITH CTCF BINDING SITES, THESE ARE ASSOCIATED WITH PROMOTERS. THE DNA IS EXTRUDED. THIS WOULD [INDISCERNIBLE], IN THIS CASE, THE ENHANCER TOWARD THE RIGHT WITH THAT PROTEINER ON THE -- PROMOTER ON THE LEFT. THIS IS A ACTIVITY, CTCF TINY BINDING SITE FUNCTION OF THE SPEED BUMP FOR THAT ACTIVITY, WHICH HAPPENS WHEN DOSED TO ELEMENTS ARE INTERACTED. PERHAPS THIS IS THE MODEL TO EXPLAIN HOW THESE THINGS ARE TETHERED WHEN B CELLS ACTIVATING GENE EXPRESSION ACROSS THE GENOME. WHAT WE'RE SEEING -- WHETHER WE'RE SEEING THIS PARTICULARLY FOR STRIPES IS ONE THING. WE BELIEVE EXDRUGS IS HANG ON THE GENOME. AND THESE ARE THE PEOPLE THAT DID THE WORK FROM MY LAB. [LIST OF NAMES] THANK YOU FOR YOUR ATTENTION. [APPLAUSE] >> AGAIN, WE HAVE A COUPLE MINUTES FOR SOME QUESTIONS. >> WHEN YOU LOOK AT THE WEAK CTCF BINDING SITES, IS THERE A SEQUENCE MOTIF YOU CAN RECOGNIZE THERE? >> WE LET MICHAEL ANSWER THIS QUESTION. >> ACTUALLY, SO WE HAVE BEEN LOOKING AT WHETHER THERE IS PREFERENTIAL DIVIRGINS. IT'S A BIT 5050. SO IT'S UNCLEAR AT THIS TIME WHETHER IT'S DUE TO CONVERGENCE. BUT YES, MAY NOT PLAY A ROLE THERE. >> NICE TALK. [INDISCERNIBLE] QUESTION, ARE YOU SURE [INDISCERNIBLE] I JUST WONDER, DO YOU THINK IT'S JUST ASSOCIATION OR SUPER ENHANCERS DEMETHYLATION. >> IT'S PRETTY WELL ESTABLISHED IN THE FIELD THAT METHYLATION FOS LATED BINDING OF TRANSCRIPTION FACTOR. NOT ALL TRANSCRIPTION FACTORS. THERE IS SOME OF THEM THAT -- WE COOL THEM PIONEER THAT CAN BIND, REGARDLESS OF DEMETHYLATION STUDIES. THAT'S WHAT FACILITATES THE BINDING, WHAT OPENS THE CHROME TEEN TO BINDING OF FACTORS. THAT ALSO HAS BEEN SHOWN FOR CTCF. SO WE BELIEVE IT'S DOING TWO THINGS THERE. IN TERMS OF THE ACTIVATION OF THE ENHANCER ITSELF, ALSO THE ARCHITECTURAL ROLE OF THE CTCF. >> THANK YOU. >> I'M CURIOUS, THE DEPLETION OF CTCF, YOU SEE DISRUPTION OF THE [INDISCERNIBLE]. CAN YOU RESTORE THOSE ORIGINAL LOOPS AFTER WILDTYPES IN THE CTCF? >> YES. SO THIS IS KIND OF IN DEPTH. AT THIS POINT, I THINK THAT ACTUALLY IT'S EXPERIMENTS THAT WERE DESCRIBED IN THE SECOND HALF OF THE TALK WERE DECISIVE RESOLUTION OF THIS. SO WHAT WE HAD DONE IN MY LAB IS TO DISRUPTIONS OF INDIVIDUAL CTCF MOTIFS. YOU CAN SEE IN A PREDICTABLE FASHION, AN THE CONVERGENT ROLE WILL ELIMINATE LOOPS. SIMILARLY, WILL ELIMINATE DOMAINS. VERY PREDICTABLE. SO WE DID 13 SUCH EXPERIMENTS. THEY ALL WORKED AS YOU EXPECTED. DELETE MOTIFS, YOU ADD, YOU INVERT, AND YOU ALWAYS HAVE THE EXPECTED RESULTS. THE RESULTS TOGETHER HAVE SHOWN BEHIND ANY REASONABLE DOUBT, VERY, VERY CLEARLY IN A GENOME-WIDE, THE WALL OF CTCF BINDING. PARTICULAR SITES WILL LEAD TO THE DESTRUCTION OF LOOPS AND DOMAINS AT PARTICULAR SITES. I SHOULD MENTION THERE ARE OTHER GROUPS, 2015 PAPER ON THIS CAME OUT THAT DID A COUPLE OF INDIVIDUAL SOCIETIES AND CONCLUDING -- SITES, I THINK CELL. SO THERE HAS BEEN A WORK OF WORK ON -- BUNCH OF WORK ON TARGETED SITES. NOW YOU CAN SEE HUNDREDS OF RECEIPTS, VERY MUCH -- SITES, VERY MUCH CLOCK WORK TYPE RESULTS IN TERMS OF LOSS OF LOOPS AND DOMAINS UPON DISCORRUPTION OF CFCF BINDING. >> MY UNDERSTANDING THAT [INAUDIBLE] >> THAT'S A TRAFFIC QUESTION. ALL METHODS FOR CONTACT MAPPING PRIOR TO HIGH SPEED, CHROMOSOME, 2002, CIRCULAR CHROMOSOME CAPTURE, AND CHROMOSOME CARBON COPY, ALL THOSE WERE TARGETED METHODS. HIGH C, INTRODUCED IN 2009 IS AN UNBIASED METHOD, NOT TARGETED. WE USE IT FOR DISCOVERY. WE'RE USING IT TO MAP LOOPS GENOME-WIDE, MAP DOMAINS, OTHER FEATURES GENOME-WIDE IN A HYPOTHESIS FREE FASHION. SO I THINK THAT WE HAVE ACTUALLY TURNED THE CORNER WHERE WE CAN RELIABLY MAP ALL SORTS OF FEATURES WITHOUT KNOWING WHAT IT IS THAT WE'RE LOOKING FOR. >> ONE SHORT LAST QUESTION. >> I THINK THE LOOPS ARE VERY NICE. WHAT IS THE FUNCTION? TWO TAKE A MARM [INDISCERNIBLE] SITE IN VIVO, DOES IT REALLY EFFECT THE ENHANCER, A BOUNDARY? >> YEAH, SO THERE IS AN INCREDIBLY STRONG ASSOCIATION BETWEEN THE FORMATION OF LOOPS AND THE ACTIVATION OF GENES THAT LIE AT THE ANCHORS OF THOSE LOOPS. IN PARTICULAR, THE BEST WORK ON THIS IS OUT OF [INDISCERNIBLE] GROUP. THEIR PAPER IN MOLECULAR CELL AT THE END OF LAST YEAR, THEY DID AN ADDITION TO ENGINEERING, CAREFULLY TRACED THE TRANSCRIPTIONAL CONSEQUENCE, PART OF THEIR ENGINEERING EXPERIMENTS THAT WE DID NOT IN OUR EXPERIMENTS. THEY SHOT THE TRANSCRIPTIONAL -- SHOW THE TRANSCRIPTIONAL EFFECT. NOW WE CAN LOOK AT THE BROADER FEATURES TOGETHER, STRIPES POTENTIALLY DO REFLECT -- MAYBE THEY'RE A SUPER ENHANCERS FLAVOR OF LOOPING WHERE YOU HAVE AN ARRAY OF SMALL LOOP ANCHORS, LITTLE LOOP HERE, THEN A LITTLE LOOP THERE, THEN, THERE ALLOWS THE PROMOTER INSTEAD OF THE SINGLE TYPE TO SORT OF SAMPLE AN ARRAY OF TYPES. SO THAT'S THE IDEA. THAT OBVIOUSLY IS GOING TO CREATE ITS OWN FLAVOR OF TRANSCRIPTIONAL CONSEQUENCES. >> [INAUDIBLE] >> LET'S DISCUSS THIS LATER. WE NEED TO MOVE ON. THANK YOU VERY MUCH. THANK YOU FOR THIS WONDERFUL TALK. [APPLAUSE] SO OUR FINAL SPEAKER TODAY IS DR. KEIKO OZATO, LEADS THE SECTION ON MOLECULAR GENETIC OF IMMUNEITY. RECEIVED HER Ph.D. IN DEVELOPMENT BIOLOGY, TRAINED AT THE CARNEGIE INSTITUTION OF WASHINGTON, HOPKINS AND LATER AT THE NCI, POSTDOCAL FELLOW. IN SERVING YEARS AGO SHE JOINED NICHD, AND BECAME A SENIOR INVESTIGATOR THERE. THROUGH HER CAREER SHE'S BEEN INTERESTED IN HOW TRANSCRIPTION OF REGULATION OF GENE EXPRESSION EFFECTS INNATE IMMUNITY. HER LABORATORY DISCOVERED IRF8, A TRANSCRIPTION FACTOR CRITICAL FOR HOST RESISTANCE IN PATHOGENS IN MARC PAGES AND DENY DREADIC CELLS. ALSO IDENTIFIED THE ROLE OF BRD4, A PROTEIN THAT BINDS TO SETLETED HISTONES, AND RECENTLY, HAS BEEN LOOKING AT THE COUPLING BETWEEN TRANSCRIPTION AND HISTONE EXCHANGE WITH PARTICULAR REFERENCE TO THE REPLACEMENT HISTONE H3.3. I ALSO WANT TO MENTION THAT DR. OZATO HAS BEEN ONE OF OUR PRIME MECHANISMS OF COMMUNICATION BETWEEN OUR JAPANESE COLLEAGUES AND THE UNITED STATES. SEVERAL YEARS AGO SHE WON THE ORDER OF SACRED TREASURE THIRD CLASS FROM THE GOVERNMENT OF JAPAN FOR HER WORK IN PROMOTING COMMUNICATION BETWEEN U.S. AND JAPANESE SCIENTISTS, AND ALSO BECAUSE OF HER IMPORTANT WORK DURING THE EARTHQUAKE IN WHICH SHE HELPED TO ORGANIZE EFFORTS TO HELP OUR SCIENTIFIC COLLEAGUES IN JAPAN. I'M PARTICULARLY GRATEFUL TO HER FOR HER SERVICE FOR MANY YEARS AS CHAIR OF THE JSPC, JAPAN SOCIETY FOR PROMOTION OF SCIENCE PROGRAM, TO BRING JAPANESE FELLOWS TO THE NIH AND SHE'S DONE A WONDERFUL JOB. SO WE ARE HERE TO HEAR HER TALK ABOUT HER SCIENCE, ENTITLED BRD4 COORDINATES RAPID REORGANIZATION OF SUPER ENHANCERS TO CONTROL INFLAMMATORY GENE EXPRESSION. DR. OZATO. [APPLAUSE] I THANK [INAUDIBLE] FOR THE INVITATION. [INAUDIBLE] VERY KIND INVITATION OKIFYED BY TRANSCRIPTION FACTOR SHOWN IN THIS CARTOON. AND [INDISCERNIBLE] CURRENTLY USED FOR IDENTIFICATION OF ENHANCER. THAT SUPER ENHANCERS FORMS [INDISCERNIBLE] MOST IMPORTANTLY, DRIVES HIGH SECRETION OF [INDISCERNIBLE] STRONGLY REGULATED BY BRD4, A COMPONENT OF [INDISCERNIBLE]. AND THIS FACTOR HAS BEEN EXISTENTIAL BY [INDISCERNIBLE]. SO I WILL BE TALKING ABOUT BRD4 AND THE MAIN POINT BEING [INDISCERNIBLE]. ADJUSTING TO CHANGEABLE STANDARD ENVIRONMENT. HOW DOES BRD4 COME TO CENTER OF [INDISCERNIBLE] THIS EXPRESSION AND EFFECTIVELY REDUCING CANCER GROWTH INCREASING THE [INDISCERNIBLE] FOR CANCER. SO, BRD4 IS THOUGHT TO BE ESSENTIAL FOR FORMATION AND [INDISCERNIBLE]. NOW, SMALL MOLECULE INHIBITORS THAT ARE [INDISCERNIBLE] THESE ARE SHOWN TO INHIBIT INFLAMMATION, AND NEURO INFLAMMATION. [INDISCERNIBLE] POSSIBILITIES FOR DISEASES. SO THE NEW GENERATION BET INHIBITERS ARE ARE IN CLINICAL CLINICAL TRIALS. SO THESE INHIBITORS [INDISCERNIBLE] UNDERSTANDING BRD4. HOWEVER, DO HAVE SOME LIMITATIONS INCLUDING THE DEFENDY IN DISTINGUISHING -- DIFFICULTY IN DISTINGUISHING [INDISCERNIBLE]. ALSO, THE REAL EFFECTS, IMPACTS ON NORMAL CELLS, SPECIFICALLY, HEMATOPOIETIC STEM CELLS. NOW, WE HAVE WORKING ON BRD4 FOR MANY YEARS. BEFORE THE [INDISCERNIBLE]. VERY MUCH OBSCURE. BY OVER THE YEARS, WE HAVE SHOWED THAT BRD4 BINDS TO ACETYLATED HISTONES, AND STAYS ON CHROMOSOMES DURING MITOSIS, IMPLYING [INDISCERNIBLE]. IN ADDITION WE SHOW THAT BRD4 INTERACTS WITH P50 AND THE MEDIATOR TO PROMOTE [INDISCERNIBLE]. HOW ROLE HAS BEEN TO [INDISCERNIBLE] KNOCKOUT MICE. AND YOU WILL SEE TODAY [INDISCERNIBLE] MACRO PAGE, BUT BEFORE PROCEEDING, I DO HAVE TO SAY THAT THIS STUDY WAS DONE PRIMARILY BY DEY IN THE LABORATORY. NICHD, IN COLLABORATION WITH JUN ZHU, NHLBI. WE NOW KNOW THAT BRD4 IS IMPORTANT FOR NORMAL CELL GROWTH, PARTICULARLY CELL GENERATION USING THESE JOCK NOT NOT -- KNOCKOUT MODELS. IT IS IMPORTANT WITH [INDISCERNIBLE] USE OF INHIBITOR S FOR THERAPY. NOW WE KNOCKED OUT BRD4 IN [INDISCERNIBLE] MACROPHAGES BY [INDISCERNIBLE], AND TESTED UNSIMULATED LPS STIMULATED MACRO PAGES. WE FOUND THAT 200, 300 GENES DOWN REGULATED AND THOSE CONDITIONS IN BRD4 KNOCKOUT MACRO PAGES. [INDISCERNIBLE] [INDISCERNIBLE]. BECAUSE OF THIS, INFORMATION, FACTORS ASSOCIATED WITH NOT ONLY A FEW [INDISCERNIBLE], BUT VASCULAR DISEASES, CANCER, AUTOIMMUNE DISEASE, AND EVEN SOME NEURO [INDISCERNIBLE] DISEASES SUCH AS ALZHEIMER'S, AND PARKINSON. NOW, WE FOUND BY [INDISCERNIBLE] BRD4 LOCALIZES TO ENHANCER. YOU SEE BRD4 ALONG [INDISCERNIBLE] OF DNA, AND BOTH [INDISCERNIBLE] THESE ERD4 FACTORS MEET THE [INDISCERNIBLE] DESIGNATION. AND THAT'S [INDISCERNIBLE] DISTRIBUTION OF [INDISCERNIBLE] AND H3 [INDISCERNIBLE] OVER [INDISCERNIBLE]. THERE ARE OTHER MARKS YOU CAN SEE, INCLUDING [INDISCERNIBLE] H3K9, H 4, ALSO DID NOT CORRELATE THAT WELL WITH [INDISCERNIBLE]. AS NOTED BY MANY OTHER INVESTIGATORS IN THE FIELD. SO [INDISCERNIBLE] WE FOUND THAT THE -- THERE ARE MANY MORE SMALL [INDISCERNIBLE] ASSOCIATING WITH BRD4 THAT ARE CALLED [INDISCERNIBLE], HOWEVER, THE TITLE OF BRD4 ASSOCIATED WITH [INDISCERNIBLE] ARE MUCH GREATER. SO EVEN THOUGH THE SUPER ENHANCERS RECOVER ONLY 10% OF THE ENTIRE ENHANCER LOCI, [INDISCERNIBLE] AND BRD4 [INDISCERNIBLE] DISPROPORTIONATELY HIGH. NOW, WE FOUND THAT [INDISCERNIBLE] STIMULATING SUPER ENHANCERS IN MACROPHAGES. HERE, WE [INDISCERNIBLE] BINDING ACCORDING TO INCREASING DLD, POSING [INDISCERNIBLE] AXIS AND YOU SEE THE DLD4 ACCUMULATE BOTH IN UNTREATED AND LPS STIMULATED [INDISCERNIBLE]. ALTHOUGH THESE LOOKED VERY SIMILAR, IN FACT, THEY ARE ON [INDISCERNIBLE] NONE OF OUR [INDISCERNIBLE] THIS WOULD INDICATE THAT THE MAJORITY OF ENHANCERS ARE NEWLY CREATING WITHIN 4 HOURS OF LPS STIMULATION DERIVING TRANSCRIPTION OF A NEW SET OF GENES. THIS IS AN EXAMPLE HERE. DLD4 BINDING IS ABSENT IN UNTREATED MACROPHAGES OVER DNS [INDISCERNIBLE], AS YOU CAN SEE, DLD [INDISCERNIBLE], AFTER LPS STIMULATION FORMING OR [INDISCERNIBLE] ENHANCER. THIS [INDISCERNIBLE] VERY QUELL KNOWN PRO INFLAMMATORY TYPE SIGN ASSOCIATED WITH CANCER [INDISCERNIBLE]. IL1 IS ANOTHER INFLAMMATORY SITO KIND, ALTHOUGH IMPORTANT FOR [INDISCERNIBLE] ALSO ASSOCIATED WITH DAMAGE IN MUSCULAR CELLS. THAT'S [INDISCERNIBLE] CONDITIONS. AGAIN, YOU SEE INCREASE OF BINDING IN [INDISCERNIBLE] AND H 3 AASSETLATION. THIS CASES THAT FORMATION IS [INDISCERNIBLE] [INDISCERNIBLE]. NOW, DLD4 ENHANCERS ARE ALL [INDISCERNIBLE] IMMUNE iAND INFLAMMATORY RESPONSES. AND [INDISCERNIBLE] LPS STIMULATED AND [INDISCERNIBLE] CELLS. IMMUNE RESPONSE, RESPONSE TO INJURY, AND INFLAMMATORY RESPONSES. THESE CATEGORIES HAD OVER-ACTING SET OF CYTOKINES, [INDISCERNIBLE] AND. NOW, WHAT WAS INTERESTING IS THAT P VALUES IN THE ENHANCERS ARE INCREASED, EVEN IN UNTREATED CONDITIONS OVER CELL ENHANCER. THIS WOULD [INDISCERNIBLE] INNATE IMMUNE AND INFLAMMATORY RESPONSES. SO THIS INVOLVED [INDISCERNIBLE] ARE PU1, [INDISCERNIBLE] AND IMPORTANT FOR RESPONDING TO SIGNALS INCLUDE AD1 AND IRFs, AND STAT1, UNTREATED [INDISCERNIBLE] STIMULATED MACROPHAGES. THE ONLY MOTIF THAT SHOWED UP AFTER [INDISCERNIBLE] WAS NFKB1. NOW, ONE IMPORTANT QUESTION IS WHETHER BRD4 IS REQUIRED FOR GENERATEING ALL SUPER ENHANCERS, AS DESCRIBED BY THE INHIBITOR STUDIES. OUR ANSWER WAS NO [INDISCERNIBLE]. THIS IS ANOTHER WAY OF LOOKING AT SUPER ENHANCERS. YOU SEE THAT BRD4 SUPER ENHANCERS ARE GONE [INDISCERNIBLE] AS YOU WOULD EXPECT, ON THE LEFT SIDE OF THE GRAPH. HOWEVER, WE FOUND THAT IF YOU LOOK FOR SUPER ENHANCERS [INDISCERNIBLE] H3K27 ACETYLATION, YOU SEE THAT KNOCKOUT MACROPHAGES DO HAVE SUPER ENHANCERS. ALTHOUGH THE NUMBER [INDISCERNIBLE] AND H3K27 SUPER ENHANCERS SHOWED CLASSICAL FEATURES [INDISCERNIBLE] YOU DON'T SEE THE [INDISCERNIBLE], IMPORTANT IN THE PROTEIN. AND [INDISCERNIBLE] TO MAKE THIS POINT, THIS IS -- THESE PLOT BASED ON H3K27 SIGNALS. AND YOU CAN SEE ACCUMULATE OVER THE [INDISCERNIBLE] SHOWING AUTHENTICATE ENHANCER FEATURE, AND ALSO, AGAIN, THESE [INDISCERNIBLE] LOOK SIMILAR. [INDISCERNIBLE] INDEPENDENT SET OF GENES. FOUND THAT GENES UNIQUE TO KNOCKOUT MACROPHAGES SHOWN BY [INDISCERNIBLE] UNRELATED TO INNATE OR INFLAMMATORY RESPONSES. THEREFORE, GENERAL METABOLISM OR SIGNALING [INDISCERNIBLE] DICK SETTING. SO MY TEAM [INDISCERNIBLE] SITUATION TAKES PLACE IN SOME DISEASE CONDITIONS. SO IN SUMMARY, SUPER ENHANCERS ARE FLUID, LPS STIMULATION, NEW SUPER ENHANCERS IN MACROPHAGES. BY RAPIDLY REDISTRIBUTING BRD4 OVER THE GENOME. AND ESTABLISH A NEW GENE EXPRESSION PARADIGM IMPORTANT FOR INMATE IMMUNE AND INFROMRY RESPONSES. AND SUPER ENHANCERS ARE DIVERSE. IN THE ABSENCE OF BRD4, ALTERNATIVE SUPER ENHANCERS ARE FORMED, SETTING ABERANT EP GENETIC STATES AND GENE EXPRESSION PROGRAMS. SO MY [INDISCERNIBLE] FOR THESE RESULTS ARE SUPER ENHANCERS REVEAL A DOMINANT AND CONSINGLE GENE EXPRESSION PARADIGM IN A GIVEN CELL SYSTEM. AND [INDISCERNIBLE] THAT STUDIES OF SUPER ENHANCERS PROVIDE DEEPER, MORE COHESIVE INSIGHT INTO VARIOUS BIOLOGICAL PROCESSES, INCLUDING DISEASES, AND MAY POTENTIALLY LEAD TO NEW THERAPEUTIC AVENUES. AND THESE THE [INDISCERNIBLE]. THANK YOU FOR YOUR ATTENTION. [APPLAUSE] >> WE HAVE A FEW MINUTES FOR SOME QUESTIONS, QUESTIONS FOR DR. OZATO. ANY QUESTIONS? OKAY. IF NOT, LET ME THANK YOU FOR A WONDERFUL PRESENTATION. AND LET'S THANK ALL THE SPEAKS IN THE FIRST PLENARY SESSION FOR REALLY ENLIGHTENING TALKS. OBVIOUSLY AN EXCITING AREA OF RESEARCH THAT NIH IS CONTRIBUTING TO IN A MAJOR WAY. I NEEDED TO MAKE A COUPLE OF QUICK ANNOUNCEMENTS. THE EFFICIENCY IS THAT AS WE SPEAK, RIGHT BEHIND ME IN THE SOUTH LOBBY, NIH GREEN LABS FAIR AND EXHIBITS IS GOING ON UNTIL 2:00. AND THE NIH LIBRARY THERE IS A HAND'S ON VIRTUAL REALITY AND 3D PRINTING EXHIBIT. AND THEN FROM 12:30 TO 2:30, WE WILL OF CURRENT SYMPOSIA HERE IN LIPSETT AND FAS CLASSROOMS. BENCH TO BEDSIDE, EXPERIMENTAL EXPOSURE. AND AFTER THAT, FROM 3-4:30, A POSTER SESSION. AT ALL THESE EVENTS, THERE WILL REREFRESHMENTS. IF YOU'RE GETTING HUNGRY, THEY'LL BE OPPORTUNITIES TO GET SOMETHING TO EAT. TOMORROW AT SOCK WE HAVE OUR SECOND -- 10:00, WE HAVE OUR SECOND PLENARY SESSION, NEW INSIGHTED THROUGH CLINICAL MANAGING. AS YOU LEAVE MAKE SURE YOU GET A SCHEDULE TO THE FESTIVAL EVENT. THANK YOU ALL FOR ATTENDING AND PARTICIPATING. AND WE LOOK FORWARD TO TWO MORE DO IS OF WONDERFUL ACT -- DAYS OF WONDERFUL ACTIVITIES.