>> IT'S MY GREAT PLEASURE TO INTRODUCE STEFAN ADI MULIJO, WHO WILL BE TODAY'S NIH DIRECTOR'S LECTURE. SOME OF YOU MAY WONDER WHY THIS IS CALLED THE NIH DIRECTOR'S LECTURE SERIES. AND THE NIH DIRECTOR NEVER COMES TO THE SERIES. [LAUGHTER] SINCE WE'RE COMING TO THE END OF AN ERA I'LL EXPLAIN. THIS WAS ESTABLISHED ORIGINALLY BY HAROLD VARMUS WHO USED TO INTRODUCE SPEAKERS AND SIT IN THE FRONT ROW AND ASK QUESTIONS DURING AND AFTER. SUBSEQUENT DIRECTORS HAD OTHER RESPONSIBILITIES DURING THIS TIME PERIOD WHICH IS NOON ON FRIDAY. AND DR. COLLINS WHO WOULD LOVE TO ATTEND THESE SESSIONS HAS HIS OWN LAB MEETING AT THIS TIME, APPARENTLY THE ONLY TIME IN THE WEEK THAT HE CAN ATTEND. SO HE HASN'T BEEN ABLE TO ATTEND THESE. BUT HE KEEPS AN EYE AND ACTUALLY REVIEWS AND APPROVES ALL THE NAMES OF PEOPLE WHO COME UP FOR CONSIDERATION. SO THIS IS THE NIH DIRECTOR'S LECTURE AND I'M PLEASED TO BE ABLE TO SO OFFICIATE. DR. STEFAN ADI MULIJO GOT A Ph.D. IN IMMUNOLOGY FROM HOPKINS SCHOOL OF MEDICINE AND SPENT SOME TIME AT UNIVERSITY OF CALIFORNIA BERKELEY WORKING ON HIS THESIS, POSTDOC WORK AT HARVARD MEDICAL SCHOOL, AND WAS RECRUITED FOR THE LABORATORY OF IMMUNOLOGY AND THAT IS INCREDIBLE INCUBATOR. HE ACHIEVED TENURE THIS YEAR AND IS FACULTY MEMBER OF NIH PARTNERSHIP PROGRAM WHICH INCLUDE HOPKINS PROGRAM AND UNIVERSITY OF PENNSYLVANIA. HIS WORK OVER THE YEARS HAS BEEN FOCUSED ON REGULATION OF CELLS IN THE IMMUNE SYSTEM, SPECIFIC REFERENCE TO MICRORNA, AND RNA BINDING PROTEINS, THE TALK IS ROLES OF REGULATORY RNAs AND RNA BINDING PROTEINS IN REPROGRAMMING PATTERNS OF GENE EXPRESSION. >> THANK YOU VERY MUCH. LIKE MANY LABS, WE'VE BEEN OBSESSED WITH HOW GENOTYPE GIVES RISE TO PHENOTYPES. AND THIS IS ACTUALLY A DAUNTING PROBLEM IF YOU LOOK FROM ORGANISM LEVEL. WHAT ARE THE REGULATORY MECHANISMS THAT GIVE RISE TO THAT PROGRAM. SO NOWADAYS IT'S BECOME QUITE ROUTINE TO SEQUENCE GENOMES AND TRANSCRIPTOMES SO WE HAVE HUGE CATALOGS OF PARTS, BUT IT'S NOT THAT TRIVIAL TO MAKE SENSE OF ALL THIS DATA A MAIN OBSTACLE, WE DON'T KNOW THE FUNCTION OF MOST OF THESE PARTS SO IF YOU USE CRISPR TO MUTATE CODING GENES THAT WOULD ADDRESS 5% OF THE GENOME, WOULDN'T KNOW WHAT 95% IS DOING. THAT'S MY LITTLE PLUG FOR FUNCTIONAL GENOMICS, WE NEED TO APPROACH BECAUSE THERE'S NO APPROACH THAT FITS ALL. MY PART FOR SYSTEMS BIOLOGY, KNOWING HOW THE PARTS FIT TOGETHER. HERE IS A SIMPLE SYSTEM WHERE YOU CAN PICK AN ADULT SOMATIC CELL AND WHAT WAS DONE TO WIN THE NOBEL PRIZE, FOUR TRANSCRIPTION FACTORS THAT TRANSFORMED THE CELL INTO SOMETHING THAT VERY MUCH RESEMBLES AN EMBRYONIC STEM CELL. THE SECOND PART OF MY TALK ONE OF THE FACTORS JAMIE THOMPSON USED TO REPROGRAM WAS NOT A TRANSCRIPTION FACTOR BUT AN RNA BINDING PROTEIN 1028 AND KEEP THAT IN THE BACK OF YOUR MIND. SO SINCE I WORK ON microRNAs, IT'S ALWAYS ENCOURAGING TO SEE PAPERS THAT SAY MICRORNAs ARE IMPORTANT AND MADE ME HAPPY TO SEE THESE TWO INDEPENDENT GROUPS DEMONSTRATE THAT YOU CAN ACTUALLY ACHIEVE THIS PROGRAMMING USING ONLY microRNAs, SMALL BITS OF RNAs NO PROTEIN WHATSOEVER AND THAT PLANTED THE IDEA IN MY MIND THERE'S SUCH A THING AS POST TRANSCRIPTIONAL REPROGRAMMING. AND WHAT WASN'T ADDRESSED IN THIS PAPER IS HOW CAN THEY ACHIEVE THIS INCREDIBLE FEAT. ALL RIGHT. SO I DON'T WANT TO TAKE TOO LONG, GOING INTO THIS BACKGROUND, BUT SUFFICE IT TO SAY microRNAs ARE ENCODED IN OUR GENOME, TRANSCRIBED BY RNA POLYMERASE 2, REGENERATE 21 OLIGOS, BOUND BY ARGONAUTS, SERVING AS GUIDES FOR THE COMPLEX TO FIND COGNATE TARGETS, ONE OF THE WAYS THEY CAN DO THIS IS THEY CAN RECRUIT ADENYLASE COMPLEX. OUR APPROACH TO STUDY THIS SYSTEM IS TO SEE WHAT HAPPENS TO THE CELLS. I THOUGHT IT WOULD BE APPROPRIATE TO TALK ABOUT WORK OF MY HIGHLY TALENTED COLLABORATOR CHRYSSA, WHO DID THIS WORK AS A RESEARCH FELLOW IN MICHAEL ARNOLDO'S LAB AROUND THE CORNER FROM ME. SO POSTDOC AT HARVARD, CHRYSSA GENERATED EMBRYONIC STEM CELLS DICER DEFICIENT, SURPRISING YOU COULD GET THEM, THEY LOOK LIKE REGULAR EMBRYONIC STEM CELLS, MOLECULARLY THEY EXPRESS PLURIPOTENCY MARKERS SUCH AS OCT-4, IT WAS DISHEARTENING, PERHAPS microRNAS ARE NOT SO IMPORTANT. WE CAME TO THE NIH AND DECIDED TO CHARACTERIZE CELLS FURTHER AND ONE OF THE FIRST THINGS WE DID WAS WE DID RNA SEQUENCING TO COMPARE THE TRANSCRIPTOMES OF EMBRYONIC WITH microRNA AND WITHOUT, PLURIPOTENCY MARKERS ARE EXPRESSED, ONE IS NOT IN THE ABSENCE OF DICER BUT I WON'T TALK ABOUT THAT TODAY. SHE BECAME INTERESTED IN THIS GROUP OF HOX GENES, WHICH ARE ALL AS A GROUP UPREGULATED WHEN DICER IS ABLATED. HOX GENES EXIST FOR CHROMOSOMAL CLUSTERS, THEY ARE REQUIRED FOR SETTING UP THE ANTERIOR TO POSTERIOR BODY PLAN, SO THIS IS WHAT'S FIRST CHARACTERIZED IN DROSOPHILA, IF YOU HAVE MUTATIONS IN HOX GENES YOU CAN GET LEGS GROWING OUT OF THE HEAD INSTEAD OF ANTENNA, AND SO IT'S PRETTY IMPORTANT FOR BODY PLAN PATTERNING. SO WE WERE INTERESTED IN FIGURING OUT THE MECHANISM FOR THIS, AND SO SINCE WE HAD THE RNA-SEQ DATA WE USED THE PROGRAM FROM THE LAB CALLED SYLMER LOOKING FOR DEPLETION OF MOTIFS, AND SPECIFICALLY SINCE WE'RE INTERESTED IN microRNA REGULATION WE ASKED THE PROGRAM TO LOOK FOR MOTIFS IN THE TRANSCRIPTS. AND WE DO THIS AGNOSTICALLY, NOT JUST FOR microRNA SEQUENCE BUT ALL 4096 POSSIBLE HEXIMER MOTIFS WHICH CORRESPOND TO THE SEQUENCE FOR microRNA TARGETING, ONE MOTIF SCORES POSITIVE, GCA CPT AND THAT WAS INTERESTING BECAUSE IT ACTUALLY CORRESPONDS, IT'S COMPLEMENTARY TO THE FAMILY OF microRNAs, WHICH ARE THE MOST ABUNDANTLY EXPRESSED IN EMBRYONIC STEM CELLS. SO THIS MADE GREAT SENSE SIPS THE microRNAs ARE HOW WE EXPRESS, WOULD BE PLAYING A DOMINANT ROLE IN SCULPTING TRANSCRIPTOME. WE HAVE A LONG LIST OF TRANSCRIPTS WHICH TENDS TO HAVE microRNA BINDING SITE AND BY RNA-SEQ WE CAN SEE THAT THEY ARE REPRESSED BY THIS microRNA. ALL RIGHT. SO LET'S GO BACK TO THE PHENOTYPE THAT CHRYSSA SAW, WHEN YOU DO RNA-SEQ ALIGNING TO HOX GENE CLUSTERS, WHEN DICER IS ABSENT, NOT IN ISOGENIC CONTROLS, WE WANTED TO KNOW IF microRNAs ARE INVOLVED AND IF SO WHICH ONE, SO IT'S PROBABLY THIS FAMILY. WHAT SHE DID TO TEST WAS USE SYNTHETIC INTO DICER KNOCKOUT, NOW HOX GENES ARE SILENT AGAIN. THIS IS A CONUNDRUM BECAUSE YOU MIGHT REMEMBER FROM DROSOPHILA GENETICS THAT THIS PART HAPPENS MOSTLY VIA FOLLICLE, WHICH ACTS AS TRANSCRIPTIONAL OR EPIGENETIC LEVEL, microRNAs ARE WELL KNOW TO BE REPRESSED TRANSCRIPTIONALLY SO HOW DO THE TWO PATHWAYS CONNECT? HE SO THIS IS VALIDATING THIS AGAIN YOU CAN SEE THAT PROVISION IS SUFFICIENT TO SILENCE THE HOX GENES. WE CONSIDERED POLY CHROME AND WHEN YOU MUTATE SUBUNIT OF POLY CHROME IN EMBRYONIC STEM CELLS YOU GET THE SAME PHENOTYPE AS THE DICER KNOCKOUT HOX GENES UPREGULATED SO CONSISTENT WITH WHAT'S KNOWN IN THE LITERATURE POLY CHROME IS PLAYING A MAJOR ROLE IN SILENCING HOX GENES. AND IN CASE YOU DON'T KNOW POLY CHROME IS THE WHOLE ENZYME MULTI-PROTEIN COMPLEX THAT IS RESPONSIBLE FOR TRIMETHYLATING HISTONE H-3. SO A SIMPLE EXPLANATION MIR 291 IS SUPPRESSING, BY WESTERN YOU CAN SEE THAT'S NOT HAPPENING. HOWEVER, WHEN CHRYSSA DID ChIP-SEQ WHAT WE SAW ACROSS THE GENOME IS THAT RECRUITMENT OF POLYCHROME SUBUNITS IN THE ABSENCE OF DICER AND CONSEQUENTLY HGK 27 LEVELS ARE REDUCED, TO MEAN POLYCHROME IS PRESENT IN THE CELLS BUT THERE'S A DEFECT IN RECRUITMENT TO CHROMATIN AND CAN VALIDATE BY QCR. AND SHE ASKED WHETHER AGAIN THE MYC 291 IS SUFFICIENT TO RESCUE THIS DEFECT AND WHAT WHAT'S SHOWN HERE IS YOU CAN, IN THE ABSENCE OF microRNAs, TRANFECTION RESCUES THAT DEFECT. WE HAVE A HYPOTHETICAL PATHWAY WHERE IN THE ABSENCE OF DICER YOU DON'T HAVE EXPRESSION OF THE MIR 290 CLUSTER AFFECTING POLY CHROME RECRUITMENT, DEFECTIVE HOX GENE SILENCING, STILL A MISSING PIECE IN THE PUZZLE. AND TO ADDRESS THIS WE WENT BACK TO OUR ANALYSIS WHERE WE HAVE THIS LIST OF TRANSCRIPTS REPRESSED BY MYC 291, LIKELY TO BE DIRECT TARGETS. AND WE OVERLAP THAT WITH DAVID'S TARGET PREDICTIONS WHICH LOOKS FOR EVOLUTIONARY CONSERVATION OF THIS microRNA BINDING MOTIF. AND WHEN WE DO THAT WE STILL GET A VERY LONG LIST OF POTENTIAL TARGETS, SO IT TOOK US QUITE A WHILE TO FIGURE THIS OUT, AND EVENTUALLY WE SETTLED ON ASH 1-L, THIS JAPANESE GROUP HAD ALREADY FOUND EVIDENCE THAT ASH 1L CAN COUNTERACT POLYCOMB SILENCING. CHRYSA CONSIDERED RISK 1 AND PERHAPS THAT TOGETHER WITH ASH 1L COULD BE CONTRACTING POLYCOMB. HERE IS IS THE VALIDATION. UPON TRANSVECTION YOU CAN REPRESS THE TARGETS, NO PROTEIN IN THE WILDTYPE CONTROLS, BUT IT GETS UPREGULATED IN THE DICER KNOCKOUT AND YOU CAN REPRESS THAT WITH MIC 291 AND TO SOME EXTENT USING THIS SRNA, AND SHE WANTED TO SEE IF THIS RESCUED THE HOX GENE SILENCING DEFECT AND INDEED YOU CAN PHENOCOPY BY FOGGING DOWN ASH 1L OR WHSC 1, THE KNOCKOUT WAS NOT AS EFFECTIVE. NOW WE HAVE THIS PATHWAY, AND IF YOU'RE INTERESTED IN DETAILS YOU CAN READ ABOUT IT IN CHRYSSA'S PAPER. SHE'S ON THE JOB MARKET, BY THE WAY, IF YOU HAVE ANY TIPS FEEL FREE TO LET ME OR HER KNOW. ALL RIGHT. IN SUMMARY, I'M NOT GOING TO GO THROUGH THIS BECAUSE I'VE ALREADY SAID IT PRETTY MUCH. EVERYTHING I WANTED TO SAY. BUT I JUST WANTED TO GIVE AGAIN THE TAKEHOME MESSAGE THAT A SINGLE microRNA CAN SIGNIFICANTLY REPROGRAM THE EPIGENETIC LANDSCAPE WITHIN A CELL, IN THIS CASE TIPS THE BALANCE BY REPRESSING HGK 37 METHYL TRANSFERASE AND THERE BY ENHANCES POLYCOMB ACTIVITY IN HG 27 TRIMETHYLATION. NEXT I WANT TO BRIEFLY TALK ABOUT THE WORK OF A TALENTED GRADUATE STUDENT FROM HOPKINS, ONLY BECAUSE IT'S VERY MUCH REMINISCENT OF THE STORY I JUST DESCRIBED BUT IN A DIFFERENT CONTEXT. SO THAT WOULD BE CD43 HELPER CELLS, SIMILAR LIKE STEM CELLS, ABILITY TO DIFFERENTIATE INTO MULTIPLE LINEAGES AND QUITE A BIT IS KNOWN ABOUT TRANSCRIPTION BUT THELMA WANTED TO KNOW ABOUT THE PROCESS. SIMPLISTIC PATHWAY IN PH 17 CELL DIFFERENTIATION YOU GET ACTIVATION OF A JAK-STAT PATHWAY, LEADING TO ACTIVATION OF TRANSCRIPTION FACTORS, NAMELY GAMMA T., EXPRESSION OF SIGNATURE CYTOKINES, A MAJOR TARGET IS microRNA GENE CALLED MIR 155 AND SO WE WANTED TO KNOW WHAT THIS microRNA MIGHT BE DOING IN THIS CONTEXT. THIS IS JUST THE EVIDENCE FOR THAT, IF YOU LOOK AT ChIP-SEQ DATA, WHAT THIS SUGGESTS IS THAT THE MIR155 LOCUS IS SIMILAR TO THE GENE, A BONA FIDE DIRECT TARGET JAK-STAT PATHWAY. THE EXPRESSION YOU CAN SEE IN THE HEAT MAP, MIR155 IS HIGHLY EXPRESSED, BUT NOT NOT SUBSETS. DOES THIS HAVE ANY IMPORTANCE FUNCTIONAL? IF YOU LOOK IN VIVO, SMALL INTESTINE IN THE WILDTYPE SITUATION YOU SEE PLENTY OF TH17 AND TH2 2 CELLS, IN THE ABSENCE OF MIR155 PRETTY MUCH ABLATED. THE CELLS ARE THERE BECAUSE YOU CAN SEE R R GAMMA P. EXPRESSION, THEY FAIL TO MAKE SIGNATURE CYTOKINES. YOU HAVE THE HYPOTHETICAL PATHWAY WHERE SOMEHOW MIR155 IS AFFECTING PH17 FUNCTION, AND THE QUESTION IS WHAT IS THE MISSING PIECE OF THE PUZZLE. WE OVERLAPPED THIS WITH BARTEL'S TARGET PREDICTION, A SHORT LIST OF CANDIDATES HERE, IMMEDIATELY DISCI ZEROING IN, IT HAS TO DO WITH POLYCOMB. WE THINK THIS RESULTS IN RECRUITMENT OF POLYCOMB AND SILENCING OF TARGETINGS AND TH17 CELLS, IDENTIFIED BY ChIP-SEQ IN TH17 CELLS. I LIST A FEW YEAR. ONE PIECE OF DATA TO SHOW YOU IS WHAT HAPPENS WHEN WE DELETE THE CELLS BECAUSE IT HADN'T BEEN DONE BEFORE SO YOU GET VERY NICE ABLATION OF JARID2 PROTEINS IN CD4 T CELLS. I DON'T SHOW THE ChIP-SEQ DATA BUT THIS LEADS TO DEFECT IN POLYCOMB AND REPRESSION OF TARGET GENE EXPRESSION AND HERE I SHOW YOU THREE CYTOKINE GENES AS EXAMPLES. SO FINALLY WE TRIED TO MAKE MORE ACCURATE REGULATORY NOTE WORK TO EXPLAIN, PUBLISHED A NETWORK IN 2012 BUT IT HAD NO microRNA OR EPIGENETIC REGULATORS, NOW WE CAN ADD MIR155 NETWORK AND INTEGRATE THE JARID2 INTO MORE ACCURATE AND PREDICT PREDICTIVE MODEL OF WHAT'S GOING ON. WE SEE A SINGLE microRNA CAN REPROGRAM THE EPIGENOME IN TH17, THERE ARE PROBABLY HUMAN SUBJECTS IN THE WORLD WITH MUTATIONS IN THIS microRNA GENE AND THAT WOULD RESULT IN PRIMARY AMINO DEFICIENCY, AND SINCE TH17 TH17 CELLS ARE IMPLICATED THIS MIGHT BE A GOOD TARGET FOR THOSE HUMAN DISEASES. I DON'T HAVE TIME TO TALKING ABOUT IT TODAY BUT WE'VE STARTED A COLLABORATION WITH NCATS USING THE REPORTER MOUSE THAT WE'VE DEVELOPED TO TRY TO LOOK FOR SUCH SMALL MOLECULES. ALL RIGHT. NOW I'M GOING TO SWITCH TOPICS AND TALK ABOUT WORK THAT THIS TALENTED POSTDOC DID WHEN SHE WAS IN MY LAB. SHE'S NOW LEFT AND HAS HER OWN LAB AT STEM CELL CENTER IN SWEDEN, BUT BASICALLY WE WERE INTERESTED IN HEMATOPOIESIS EARLY IN LIFE, SPECIFICALLY IN THE FETUS. QUITE A BIT IS KNOWN ABOUT LYMPHOCYTE DEVELOPMENT IN ADULT BUT THIS PROCESS IN THE FETUS IS RELATIVELY UNDERSTUDIED, SO WE THOUGHT THIS WAS A GOOD NICHE FOR US TO FILL, AND WE WANTED TO LOOK FOR SPECIFIC FACTORS THAT ARE INVOLVED, AND PERHAPS IDENTIFY A SWITCH, MOLECULAR SWITCH THAT CAN ACCOUNT FOR THIS FETAL TO ADULT TRANSITION. IT'S ACTUALLY QUITE CHALLENGING TO WORK WITH HEMATOPOIETIC STEM CELLS BECAUSE THEY ARE SO RARE. WE DECIDED TO START USING A MORE TRACTABLE SYSTEM, ALREADY NOPE THAT IF YOU TAKE PROGENITOR B CELTS FROM FETAL LIVER YOU GET DEVELOPMENT PRIMARILY OF THE INNATE CELLS, IN CONTRAST FROM ADULT BONE MARROW TRANSPLANTED TO IMMUNODEFICIENT HOST YOU GET DEVELOPMENT PRIMARILY OF CONVENTIONAL FOLLICULAR B2 CELLS. THIS WAS REPORTED DECADES AGO BUT NO ONE SEEMED TO BE FOLLOWING THIS UP SO WE DECIDED TO SEE WHAT MIGHT BE HAPPENING. I HAD SOME EVIDENCE microRNAs MIGHT BE INVOLVED, WHEN I DELETED DICER SPECIFICALLY IN PROGENITOR B-CELLS, YOU GOT NO -- WE DIDN'T SEE ANY DEVELOP -- COMPLETELY BLOCKED DEVELOPMENT OF CONVENTIONAL B-2 CELLS. HOWEVER, THE B-1 CELLS DEVELOPED JUST FINE, SO THAT SUGGESTED TO ME THAT THERE MIGHT BE microRNAs THAT ARE EXPRESSED AND REQUIRED FOR THIS ADULT DEVELOPMENTAL PATHWAY BUT MIGHT BE NOT EXPRESSED AND DISPENSABLE FOR THE FETAL PATHWAY, AND SO JOAN LOOKED FOR SUCH microRNAs, SHE SORTED PROGENITOR B-CELLS AND FOUND microRNAs DIFFERENTIALALLY EXPRESSED, BUT WE FOCUSED ON microRNAs LABELED IN RED BECAUSE THEY ALL BELONG TO THE SAME FAMILY, THE LET-7 FAMILY OF microRNAs, AND ALSO FIT OUR HYPOTHESIS IN THAT THESE MICRORNAs ARE HIGHLY EXPRESSED IN ADULT PRO B-CELLS BUT NOT FETAL LIVER. SEVERAL microRNAs WERE QUITE INTERESTING, EVOLUTION SEEMS TO THINK THEY ARE QUITE IMPORTANT SO WE'VE DUPLICATED 12 COPIES OF THE THIS microRNA GENE, SCATTERED THROUGH DIFFERENT CHROMOSOMES AND FOR THAT REASON THOUGHT PROBABLY THE DIFFERENTIAL EXPRESSION WE'RE SEEING IS PROBABLY NOT HAPPENING THROUGH A TRANSCRIPTIONAL MECHANISM BECAUSE IT WOULD BE HARD TO COHERENTLY REGULATE ALL THESE DIVERSE GENES IN ONE SHOT. HOWEVER, IT WAS LIKELY THAT POST TRANSCRIPTIONAL REGULATOR COULD DO SUCH A THING, AND INDEED IT WAS ALREADY KNOWN AT THE TIME THAT THERE'S AN RNA BINDING PROTEIN CALLED LIN28 THAT CAN BIND TO PRECURSORS OF LET-7 RNAs AND LIMIT CROSS MATURATION. LIN HAS A COLD SHOCK DOMAIN, TWO ZINC FINGERS, A GROUP AT HARVARD SOLVED THE CRYSTAL STRUCTURE WITH LET-7 PRECURSOR MICRORNAs AND BELIEVE THE ZINC FINGERS RECOGNIZES THE SPECIFIC MANNER WHEREAS THE COLD SHOCK DOMAIN RECOGNIZES SOME SORT OF STRUCTURE. TOGETHER, WE CAN MEDIATE VERY SPECIFIC BINDING TO COGNATE TARGETS WITH HIGH AFFINITY. AND SO YOU CAN IMAGINE WE WERE ECSTATIC TO FIND LIN28B WAS EXPRESSED IN FETAL HEMATOPOIETIC PROGENITORS AND STEM CELLS BUT IT WAS NOT DETECTABLE IN THE ADULT BONE MARROW COUNTER PARTS, SO THIS IS CONSISTENT WITH OUR MODEL THAT LIN28 IS INHIBITING LET-7 IN FETAL LIVER AND IT'S ABSENT IN BONE MARROW AND YOU CAN GET HIGH EXPRESSION OF LET-7 RNA. THIS DICHOTOMY IS CONSISTENT IN HUMANS AS WELL. WE WANTED TO SEE WHETHER THIS IT FUNCTIONAL RAMIFICATIONS, AND THIS IS AGAIN A CRITICAL JUNCTURE WHEN CHRYSSA SUGGESTED THAT WE DO THIS EXPERIMENT FOR SOME STUPID REASON SO I SAID IT WASN'T A GOOD IDEA, AND EVENTUALLY JOAN DID THIS EXPERIMENT AND IT WORKED BEAUTIFULLY. SO WE CAN TAKE ADULT BONE MARROW PROGENITORS AND TRANSDUCE THEM USING RETROVIRUS WITH LIN28 AND SO IF YOU REMEMBER ADULT BONE MARROW IS ESSENTIALLY LIKE A LIN28B KNOCKOUT BECAUSE WE CAN'T DETECT EXPRESSION AND NOW WE ECTOPICALLY EXPRESS LIN28 TO SEE WHAT MIGHT BE HAPPENING. THIS WORKED INCREDIBLY WELL, SO WE LOOKED AT microRNA EXPRESSION, MOST microRNAs ARE UNTOUCHED BY LIN28B BUT ALL THE LET-7 FAMILY MEMBERS ARE SPECIFICALLY KNOCKED DOWN AND THIS BASICALLY AMOUNTED TO A 90 PERCENT KNOCKDOWN AT THE ABSOLUTE EXPRESSION LEVEL. ALL RIGHT. WE WENT TO OUR ORIGINAL HYPOTHESIS THAT THIS MIGHT HAVE SOMETHING TO DO WITH B-1 VERSUS B-2 SULFATE CHOICE AND INDEED IF WE TRACKED LIN28 TRANSDUCE B-CELLS THEY DEVELOP INTO B-1 CELLS WHICH NORMALLY WOULD DEVELOP DURING FETAL LIFE. HOWEVER, THIS HAPPENS IN AN ADULT HOST, SO WHATEVER LIN28 IS DOING IT'S DOING IT IN A CELL INTRINSIC FASHION. ALL RIGHT. NEXT WE WANTED TO KNOW WHETHER THIS MIGHT HAVE BROADER RAMIFICATIONS AND UP MIGHT BE AWARE OF A MORE FAMOUS SWITCH IN HEMATOPOIESIS, FETAL-TO-ADULT HEMOGLOBIN SWITCH. AND IS IT POSSIBLE THAT LIN28B MIGHT BE REGULATING THIS PROCESS, COLLABORATED WITH JEFF MILLER'S LAB AT NIDDK WHERE YOU IS TAKE CD34 POSITIVE PROGENITORS AND DIFFERENTIATE IN VITRO INTO ERYTHREGULATORY BLASTS, WHAT WOULD HAPPEN IF WE USED THE RETROVIRAL VECTOR, AND AGAIN TO OUR GREAT JOY WE SAW THAT IN ADULT ERYTHROBLISTS LIN28B CAN TURN ON FETAL HEMOGLOBIN EXPRESSION, AND THIS HAS CLINICAL IMPLICATIONS BECAUSE IT HAS LONG BEEN THOUGHT IF YOU CAN SOMEHOW TURN FETAL HEMOGLOBIN ON IN SICKLE CELL DISEASE YOU COULD AMELIORATE TO A LARGE PART THE SYMPTOMS IN THOSE PATIENTS. AND SO WITH LIN28 WE HAVE A WAY TO DO JUST THAT. BUT WE'RE INTERESTED IN MOLECULAR MECHANISMS AND SO WE'RE PROBABLY GOING TO -- I'LL GET INTO THAT IN A FEW MINUTES. WHAT SEEMS TO BE GOING ON IS THAT EARLY IN LIFE IN THE HEMATOPOIETIC SYSTEM YOU HAVE HIGH EXPRESSION OF LIN28B PROBABLY RESPONSIBLE FOR REPRESSING LET7 LEVELS AND AFTER BIRTH EXPRESSION OF LIN28B GOES DOWN ALLOWING EXPRESSION OF L-7 microRNA, CONSISTENT WITH THE IDEA OF TWO FACTORS MIGHT BE FORMING A BI-STABLE SWITCH. AGAIN, MY TAKEHOME MESSAGE IS THAT GENE EXPRESSION PROGRAMS CAN BE REGULATED NOT ONLY BY TRANSCRIPTION FACTORS BUT microRNAs AND RNA BINDING PROTEINS AS WELL, HOPEFULLY I'VE CONVINCED YOU OF THAT. AND AS I ALLUDED TO, THIS MOLECULAR TRICK MIGHT BE USEFUL TO TREAT SICKLE CELL AND SALICEMI, AND REJUVENATE THE HEMATOPOIETIC SYSTEM IN CERTAIN SITUATIONS. FOR EXAMPLE, WITH ADVANCES IN MEDICINE, YOU MIGHT WANT TO THINK ABOUT IN UTERO HSC TRANSPLANTATION, WITH ADVANCED DIAGNOSTICS NOT UNUSUAL TO DIAGNOSE BABIES BEFORE THEY ARE EVEN BORN AND BY TRANSPLANTING FETAL HSCs IN UTERO CAN AVOID A LOT OF THE PROBLEMS THAT HAPPENS WHEN YOU ATTEMPT HSC TRANSPLANTATION LATER IN LIFE, AND THE MAJOR UPSIDE IS THAT THE BABY IS BORN ESSENTIALLY CURED, NOT HAVING TO SUFFER ANY OF THE SYMPTOMS THAT CAN ARISE DUE TO INBORN LIVER HEMATOPOIESIS. THERE ARE ALREADY SOURCE OF FETAL LIVER, FOR HUMANS PART OF THE ANSWER IS PROBABLY NOT. HOWEVER, AS I MENTIONED, WE HAVE THIS MOLECULAR TRICK NOW WHERE WE CAN ACTUALLY TAKE ADULT HSCs AND IF SOMEHOW ELEVATE LEVELS OF LIN28B CAN REPROGRAM TO BECOME FETAL LIKE AGAIN, USEFUL FOR SUCH A CLINICAL APPLICATION. ALL RIGHT. SO NOW ON TO MECHANISMS, AND I'M GOING TO TALK ABOUT SOME UNPUBLISHED WORK NOW THAT'S LED BY THESE TALENTED POST DOCS, COMPUTATIONAL ANALYSIS SINGLE-HANDEDLY BY BRIAN CHIM, ASSISTANCE FROM YI JUN SU WHO HAS BEEN VOLUNTEERING BECAUSE I HAVE NO WAY OF HIRING HIM, BUT HE'S DONE SOME REALLY NICE WORK FOR US. SO AS I SAID IN THE TOP OF THE SLIDE, OUR STRATEGY IS TO FIND FRIENDS OF LIN28 AND ONE WAY WE CAN DO THAT IS BY LOOKING FOR TRANSCRIPTS WHICH ARE CO-EXPRESSED AND IMMEDIATELY WE FOUND THAT IGF 2-BGBP 1 ARE SO EXPRESSED LEADING TO THE HYPOTHESIS THAT THIS FAMILY OF PROTEINS MIGHT BE COLLABORATING WITH LIN28B. THEY ARE CO-EXPRESSED WITH LIN28 IN FETAL LIVER HEMATOPOIETIC PROGENITORS. AND TO GET MORE SPECIFIC WE WERE INTERESTED IN FINDING RNAs BOUND BY THESE PROTEINS AND WE'RE INTERESTED IN ADDITIONAL PROTEINS THAT MIGHT BE INTERACTING WITH LIN28B AND I GF2BP3. TO DO THAT YOU NEED INDUSTRIAL AMOUNTS OF CELLS, SO WE'RE NOT GOING TO DO THAT WITH PRIMARY STEM CELLS. WE HAVE TO RESORT TO A CELL LINE SYSTEM. AGAIN, WE TOOK BONE MARROW PRO-B-CELLS, LIN28B DEFICIENT, TRANSFORMED THEM USING ONCOGENIC ABLESON VIRUS AND CONFIRMED THEY INDEED DON'T EXPRESS THE LIN28 AND HAD TO TRANSFUSE THEM WITH 28A OR 28B, THIS WORKED. THESE NUMBERS MIGHT SEEM HIGH TO YOU BUT ACTUALLY THIS IS QUITE TYPICAL FOR RNA BINDING PROTEINS, THERE ARE SO MANY COPIES OF RNAs IN OUR CELLS THAT WE NEED CORRESPONDINGLY HIGH NUMBERS OF PROTEINS TO REGULATE THEM. AND MOST IMPORTANTLY FOR WHAT WE WANTED TO DO NEXT, USING THE FLAG ANTIBODY, SO A PULLDOWN OF LIN28 COMPLEX, AND JUST AS PRECAUTION WE TREATED THE LYSIS WITH RNAs TO GET RID OF POTENTIAL RNA BRIDGING INTRACTION AND EVEN WITH RNA TREATMENT ABLE TO FIND SPECIFIC PROTEINS THAT INTERACT WITH LIN28A, I WANT TO SHOW ONE EXAMPLE OF THE HITS WE GOT AFTER MASS SPEC AND THAT IS OUR FRIEND IGF2BP3 VALIDATED BY IP WESTERN. AND AGAIN THIS BRINGS UP THE IDEA THAT LIN28 AND IGF2BP3 SEEM TO BE COLLABORATING, WHAT RNAs ARE THE TWO PROTEINS BINDING IN THE CELLS. ALL RIGHT. BEFORE I GET TO THAT I JUST WANT TO SHOW YOU SOME OF THE MICROSCOPY HERE FROM YI JUN, YOU CAN SEE BOTH ARE PRIMARILY IN THE CYTOPLASM AND CO-LOCALIZE BUT WE WANTED TO BE SURE THAT THEY ARE ACTUALLY INTERACTING AND SO HE WORKED OUT THIS PROXIMITY LIGATION ASSAY, DEPICTED HERE, AND INDEED LIN 28 AND IGF2BP3 ARE INTERACTIVE, IN WHEN YOU DELETE IGF2BP3 THE TRANSIENT GOES AWAY. ANOTHER SPECIFICITY CONTROL LIN28 DOES NOT INTERACT WITH ANOTHER RNA BINDING PROTEIN PUM2 ALSO IN THE CYTOPLASM AND GW182. ALL RIGHT. SO NOW WE WERE CONVINCED LIN28 AND IGF2BP3 ARE INTERACTING, NEXT WE WANT TO FIND OUT WHAT RNAs MIGHT BE BOUND BY THESE TWO PROTEINS. AND LUCKILY FOR ME THERE WAS THE FORESIGHT TO HIRE THIS GUY MARCUS HAFNER WHO DEVELOPED THIS ASSAY PAR-CLIP ALLOWS TO IDENTIFY RNA BINDING SITES TRANSCRIPTOME WIDE AT SINGLE NUCLEOTIDE RESOLUTION, ONE PROBLEM IS YOU NEED A LOT OF CELLS, LUCKILY WITH THE CELL LINE SYSTEM WE HAVE SUFFICIENT NUMBER OF CELLS, METABOLICALLY LABEL THEM, INCORPORATED INTO ACTIVELY TRANSCRIBE RNAs, FACILITATES CROSS-LINKG TO PROTEIN WHEN YOU SHINE UV LIGHT THROUGH THEM. AND AT THE END OF THE DAY YOU DO RNA SEQUENCING TO SEE, TO IDENTIFY ALL THE RNAs THAT ARE BEING BOUND. ALL RIGHT. THIS IS ACTUALLY A VERY CHALLENGING TWO-WEEK PROCEDURE, AND IF YOU MAKE A MISTAKE ON ANY DAY YOU HAVE TO GO BACK TO THE BEGINNING AND REPEAT IT FROM SCRATCH. BUT HE GOT PRETTY GOOD AT THIS, WITH MARCUS' HELP. SO WE CAN SEE LIN28B AND LIN28A CROSSLINK TO RNAs, WHICH HAVE BEEN FRAGMENTED WITH RNAs, AND LABELED WITH P32, AND HE CUTS OUT THESE BENDS AND MAKES cDNA LIBRARIES OUT OF THEM. BRIAN ANALYZES DATA AND FINDS OUT -- TRIES TO MAKE SENSE OF THEM AND WHAT ONE OF THE FIRST THINGS WE SAW WAS THAT CONSISTENT WITH LITERATURE LIN28 IS BINDING TO CYTOPLASMIC MESSENGER RNA, CODING REGIONS. AND WE SAW THAT LIN28B TARGETS OVERLAP WITH LIN28A, PRETTY MUCH DO THE SAME THING IN TERMS OF TARGET RECOGNITION. AND THESE ARE AT THE LEVEL OF INDIVIDUAL TARGET SITES BUT THIS BOILS DOWN TO ABOUT 4000 MESSENGER RNAs THAT THEY HAVE IN COMMON. AS YOU REMEMBER WE'RE ALSO INTERESTED IN IGF2BP3, GOT SIMILAR RESULTS, AND CONSISTENT WITH THE IDEA THAT THESE TWO PROTEINS ARE INTERACTING THEY ALSO RECOGNIZE HIGHLY OVERLAPPING SET OF TARGETS. SO WE WANTED TO KNOW WHAT THE RAMIFICATIONS OF LIN28B ARE AND NOT SURPRISINGLY WE CONDUCTED RNA-SEQ TO DO THIS, PREPARING UNTRANSFUSED VERSUS LIN28 TRANSFUSED CELLS AND WHEN YOU CONSIDER TRANSCRIPTS, A CONTROL SET OF TRANSCRIPTS NOT BOUND BY LIN28 YOU GET THIS NICE DISTRIBUTION ON GENE EXPRESSION, BUT COMPARED TO A SET OF TRANSCRIPTS WHICH HAVE A SINGLE BINDING SITE IN THE PRIMARY, YOU SEE A SHIFT TO THE RIGHT, WHERE LIN28 BINDS YOU GET ACTIVATION OF TRANSCRIPT LEVELS. AND INTERESTINGLY, THERE SEEMS TO BE A DOSE DEPENDENT EFFECT SO THE MORE BINDING SITES YOU HAVE THE GREATER ACTIVATION. AND WE SEE THIS NOT ONLY FOR LIN28 BUT INDEPENDENTLY FOR LIN28B TARGETS. NOW I WANT TO SHOW YOU A FEW EXAMPLES OF SUCH TARGETS, SO YOU RECALL THE RNA TO FIND CROSS-LINK THE RNAs CROSSLINK TO LIN28, ALIGN TO GENOME, HITS SUCH AS THIS. THIS HAPPENS TO BE WITHIN LIN28A ITSELF, AND THIS IS VERY INTERESTING TO US BECAUSE IT'S A HALLMARK OF A MASTER REGULATOR, FREQUENTLY MASTER REGULATORS AMPLIFY THEIR OWN EXPRESSION, THAT'S EXACTLY WHAT LIN28 IS DOING. WE SEE THIS FOR LIN28B, AXON 3, AND ALSO FOR IGF2BP3. IN FACT, AS YOU WOULD PREDICT SINCE LIN28 AND IGF2BP3 INTERACT PHYSICALLY, THEY BIND AT THE VERY SAME LOCATION. AND HERE IS A LOOK THE AT THE EVOLUTIONARY CONCENTRATION TRACK, HIGHLY CONSERVED, IF YOU ZOOM IN YOU CAN IDENTIFY A POTENTIAL MOTIF THAT IS BEING BOUND BY THE ZINC FINGER OF LIN28, AND IT'S HIGHLY CONSERVED IN A NUMBER MUCH SPECIES AND YOU CAN IDENTIFY A MOTIF FOUND BY IGF2BP3, ALSO CONSERVED. JUST AN EXAMPLE OF HOW IT'S ACTIVATING EXPRESSION YOU CAN SEE FOR IGF2BP3 UPON LIN28 TRANSDUCTION EXPRESSION GOES UP, RNA LEVEL ALSO AT THE PROTEIN LEVEL, AND SO THAT LEADS TO THE MODEL WHERE LIN28 AND IGF2BP3 COLLABORATE TO RECOGNIZE COMMON TARGETS AND IT SEEMS TO BE PROMOTING mRNA STABILITY PERHAPS BY PREVENTING LET7 BINDING OR HOW PREVENTING MESSENGER RNA DECAY AND WE'RE EXPLORING THE POSSIBILITY IT MIGHT BE ALSO ACTIVATING TRANSLATION BY DOING SOME PROFILING. ALL RIGHT. SO HOPEFULLY YOU GOT A FLAVOR OF WHAT WE'RE TRYING TO DO IN THE LAB AND NOW WE HAVE THIS GIANT HAIR BALL AND WE HAVE TO MAKE SENSE OUT OF IT. THERE ARE MANY OPEN QUESTIONS THAT NEED TO BE ADDRESSED, IN THE INTEREST OF TIME I'M NOT GOING THROUGH EVERY ONE OF THESE POINTS BUT I WANT TO REITERATE THAT IF YOU'RE INTERESTED IN MODELING REGULATORY NETWORKS THAT ORCHESTRATE GENE EXPRESSION PROGRAMS, YOU NEED TO CONSIDER WHAT RNA BINDING PROTEINS AND NON-CODING RNAs MIGHT BE DOING AND HOPEFULLY YOU'LL GET A MORE ACCURATE AND PREDICTIVE MODEL THAT WAY. ALL RIGHT. SO I ACKNOWLEDGED MAIN PLAYERS AS I WENT ALONG. NEEDLESS TO SAY I CAN'T DO THIS WITHOUT THE HARD WORK OF MIGHT HAVE LAB MEMBERS, AND FUNDING FROM THE INTRAMURAL PROGRAM, AND I'D BE REMISS IF I DIDN'T ACKNOWLEDGE MY DAD WHO PASSED AWAY EARLIER THIS YEAR AND ALSO MY LAB CHIEF. AND -- BILL PAUL, AND LOTS OF HELP FROM MY NIH COLLEAGUES. [APPLAUSE] >> THERE'S TIME FOR QUESTIONS. GO TO THE MICROPHONE BECAUSE WE'RE BROADCASTING SO PEOPLE CAN HEAR THE QUESTIONS. >> THAT'S GREAT. THE IGF2BP3 PROTEIN, DOES THAT FOLLOW THE EXPRESSION PATTERN OF LIN28, ONLY EXPRESSED IN FETAL LIVER OR -- >> YEAH, I ACTUALLY SHOWED THAT. >> YOU DON'T THINK THERE'S AN INDEPENDENT FUNCTION OF IGF2BP3 EXCEPT FOR IT BINDS TO LIN28 IS THE QUESTION I WANTED TO ASK. >> WELL, FROM THE ANALYSIS BRIAN DID FIND SOME EXCLUSIVE TARGETS, SO THERE ARE TARGETS WHICH ARE EXCLUSIVELY BOUND BY IGF2BP3 BUT NOT LIN28B, IT MIGHT HAVE INDEPENDENT FUNCTIONS BUT FOR THE MOMENT WE'RE INTERESTED IN FOCUSING ON THE SHARED TARGETS BECAUSE I LIKE THIS IDEA THAT IF THESE TWO FACTORS ARE COLLABORATING PERHAPS WE CAN INCREASE THE EFFICIENCY OF OUR REPROGRAMMING. SO FAR WE'RE USING ONE FACTOR, YAMANA USED FOUR, WE MIGHT GET A CLUE HOW TO MAKE THE REPROGRAMMING OCCUR MORE EFFICIENTLY. >> SO MY QUESTION, VERY NICE WORK, BY THE WAY. LIN 28 FACILITATES, SO THIS IS THE microRNA, A REGULATOR WOULD HAVE TO BE A CHANGE WITH PHYSIOLOGIC STIMULI TO MAKE SOMETHING GO BACK AND FORTH. WHAT IS GOING ON? YOU HAVE OTHER SUBUNITS, RECRUITED SPECIFICALLY IN RESPONSE TO PHYSIOLOGIC SIGNALS OR I DON'T SEE THE DYNAMISM. >> WE HAVE NO IDEA WHAT'S UPSTREAM OF LIN28 AND WHAT REGULATES ITS ACTIVATION. >> ANY POST-TRANSLATIONAL? >> WELL, WE DON'T KNOW. ONE IDEA WE HAVE IS THERE'S SOMETHING SPECIAL ABOUT THE FETAL LIVER ENVIRONMENT, PERHAPS SECRETED FACTORS, CYTOKINES OR GROWTH FACTORS WHICH ACTIVATE A SIGNAL PATHWAY AND TRANSCRIPTIONAL FACTORS THAT ACTIVATE LIN28B. IT CAN ACTIVATE ITS OWN EXPRESSION BUT -- THAT'S TOO MUCH. SOMETHING HAS TO INITIATE A LITTLE BIT OF EXPRESSION AND IT CAN AMPLIFY ITSELF AND WE DON'T KNOW YET AT THIS TIME WHAT THOSE PLAYERS MIGHT BE. GREAT QUESTION. WE'RE VERY INTERESTED IN FINDING OUT. >> GREAT TALK. I HAVE A QUESTION REGARDING THE FIRST PART OF YOUR TALK. YOU SHOWED NICELY THE MIR 155 CONTROLS CHROMATIN STRUCTURE AT TARGET LOCI. DOES THAT MEAN THAT MOST OF THE EFFECTS YOU'RE SEEING ARE TRANSCRIPTIONAL CHANGES OR MIR 55 HAS IT IN POST-TRANSLATIONAL -- >> WE COULD COMPARE THE GENE LIST, I DON'T REMEMBER HOW CAREFULLY WE DID THAT AND CAN'T ANSWER YOUR QUESTION. CERTAINLY MIR 155 WILL HAVE POST TRANSCRIPTIONAL EFFECTS, MODESTER CHANGES ON THE ORDER OF TWO-FOLD, BUT ONCE WE UNLEASH THIS TRANSCRIPTIONAL EFFECT, THEN YOU GET MUCH GREATER CHANGES IN GENE EXPRESSION. >> THANKS. >> GREAT TALK, STEPHAN. YOU SAID ONE THING EARLIER IN YOUR TALK THAT REALLY EXCITES ME WHICH IS THAT THESE SMALL RNAs MAY UNDERLIE GENETIC DISEASES OF THE IMMUNE SYSTEM. SO HAVE YOU THOUGHT ABOUT WHAT WOULD BE THE MOST EFFECTIVE WAY TO TRY TO GET AT THAT, BECAUSE OBVIOUSLY THEY TOLERATE A LOT OF SINGLE NUCLEOTIDE VARIANTS, THERE'S FAMILY OF THESE RNAs INSTEAD OF A SINGLE ONE, SO -- >> WHAT WE KNOW FROM C. ELEGANS YOU CAN GET SINGLE NUCLEOTIDE VARIANTS IN THE C REGION, THE BUSINESS CHANNEL FOR TARGET RECOGNITION FOR microRNAs, IF YOU GET A SINGLE POINT MUTATION THERE YOU WILL AFFECT ITS ACTIVITY, AND SO -- >> A PATIENT WITH TH17 YOU WOULD GO THROUGH THE REGIONS IN YOUR CIRCUIT FOR TH17 PHENOTYPE? >> YES, DEFINITELY I WOULD FOCUS ON THE C REGION OF MIR155. >> OKAY. >> JUST NEED A LOT OF PATIENCE TO SEQUENCE. >> DID YOU TRY TO LOOK WHAT THE INTERACTION OF LIN28B AND IGF2BP3 ACTUALLY DOES? ARE THEY REQUIRED TO STABILIZE THE COMPLEX, TO EFFICIENTLY REGULATE mRNA? >> WE DON'T HAVE GREAT DATA BUT, YEAH, WE'RE OBVIOUSLY THINKING ALONG THOSE LINES. WHAT I DIDN'T SHOW THE DATA, BUT WHEN BRIAN DOES THESE CDF PLOTS LOOKING AT THE SHAPE TARGETS, THEIR SHIFT IS GREATER COMPARED TO THE EXCLUSIVE TARGETS SUGGESTING THAT WHEN THEY CO-BIND, YOU'RE GETTING GREATER EFFECT. WHETHER THAT'S THROUGH STABILIZATION AT THE BICHEMICAL LEVEL WE DON'T KNOW. AS YOU KNOW, WE'RE GOING TO COLLABORATE WITH LEEMORE AT COLD SPRING HARBOR TO TRY TO ADDRESS THIS. >> THANK YOU. >> I KNOW -- CAN YOU HEAR ME? >> YEAH. >> I NOTICED IN THE MICROSCOPY IMAGES THAT (INDISCERNIBLE) MAYBE IT'S IN SOME KIND OF GRANULES, THE PROTEIN, NOT JUST SPREAD IN THE ENTIRE (INDISCERNIBLE) IF YOU TRY TO CHARACTERIZE WHAT HAPPENS WHAT'S HAPPENING? MAYBE YOU CAN GET INSIGHT ON THE MECHANISM. >> YEAH, OUR FIRST ATTEMPT WAS TO LOOK AT GW182 A MARKER FOR CYTOPLASMIC T BODIES BUT THE ASSAY DOESN'T SEEM TO BE AN INTERACTION THERE. ALTHOUGH IF YOU LOOK CONFOCAL YOU COULD CONVINCE YOURSELF THERE MIGHT BE SOME LOCALIZATION BUT IT'S NOT A HUNDRED PERCENT CLEAR. IF YOU HAVE ANY IDEAS WHAT ELSE WE SHOULD LOOK AT PLEASE LET ME KNOW. >> OKAY. THANK YOU. >> IN YOUR COMMENTS ABOUT MIR155 DEFECTS SHOULD RESULT IN IMMUNODEFICIENCY, ARE THERE PATIENTS WITH microRNA DEFECTS THAT ARE DEFICIENT AND IF NOT ARE THERE TARGETS OUTSIDE THE IMMUNE SYSTEM THAT MIGHT BE ESSENTIAL FOR OTHER FUNCTIONS? >> YEAH, INTERESTINGLY SO FAR NONE HAVE BEEN REPORTED, BASED ON THE MOUSE WORK WE THINK MIR155 IS VERY SPECIFIC FOR THE IMMUNE SYSTEM SO PROBABLY, BUT SURPRISINGLY NO HUMAN YET. I'M TRYING TO CONVINCE MY COLLEAGUES MIKE AND STEVE. >> THEY ARE HAPPY TO LOOK. >> I DID NOTICE IN THE SLIDE YOU HAD OF YOUR COLLABORATORS THAT WAS THE MOST INCREDIBLE LIST OF PEOPLE BOTH AT NIH AND EIGHT DIFFERENT INSTITUTES AND OUTSIDE OF THE NIH. CAN YOU GO BACK TO THAT SLIDE? THE LAST SLIDE. I WANT TO MAKE THE POINT IT REALLY DOES TAKE A VILLAGE TO DO THIS KIND OF VERY TECHNICAL AND VERY SOPHISTICATED WORK. LOOK AT THIS LIST OF PEOPLE. UNBELIEVABLE. ANY SYMPOSIUM WOULD BE DELIGHTED TO HAVE THIS LIST OF PEOPLE PRESENTING. MORE AND MORE WE'RE GOING TO SEE THESE INTERACTIONS AND NETWORKS IN THE WORK THAT YOU'RE DOING, THE SCIENCE, BUT WITHIN THE PEOPLE WHO YOU'RE INTERACTING WITH BOTH AT THE NIH AND ELSEWHERE. SO I WANT TO THANK YOU FOR A TERRIFIC SEMINAR AND THANK ALL THESE PEOPLE WHO HAVE MADE THIS POSSIBLE AS WELL. >> THANK YOU. [APPLAUSE]