>> SO I AM VERY PLEASED TO INTRODUCE TODAY'S SPEAKER. ALISA WAS AN UNDERGRADUATE AT THE UNIVERSITY OF TEXAS AT OFF THEIN. AND THEN DID HER GRADUATE WORK AT HARVARD WITH M SILVER AND THEN POST DOC AT HOPKINS WITH CONVIVICTOR CORSI.^ AND YOU CAN TELL FROM THAT INCREDIBLE PEDIGREE THAT SHE'S INTERESTED IN USING THE MODEL SYSTEM. AND HAS BEEN STUDYING WHAT /I WOULD SAY IS PROBABLY THE MOST IMPORTANT, INTERESTING PROBLEM IN BIOLOGY, HOW A SINGLE CELL NEURONS A DIFFERENT /SHAEIATED ORGANISM. AND HER INTEREST IN PARTICULAR IS FOCUSED ON CHROME /* CHROMAT CHROMATIN INSULATEORS AND I THINK PROBABLY THE BEST WAY TO INTRODUCE THE SUBJECT IS TO HAVE ALISA COME UP AND TALK ABOUT IT. SO IT'S REGULATION OF CHROMATIN REGULATED FUNCTION. >> SO I WANT TO THANK YOU FOR /EUINVITING ME TO GIVE THIS LECTURE TODAY. IT'S REALLY AN HONOR AND SO NICE TO SEE SO MANY FRIENDS AND COLLEAGUES HERE AND ALSO THOSE STREAMING ONLINE. SO NORMALLY WHEN I GIVE A TALK, I LIKE TO TALK ABOUT THE LATEST AND GREATEST RESEARCH FROM MY LAB, BUT I THOUGHT FOR THE PURPOSE OF THIS TALK, IT'S SUCH A BROAD AUDIENCE, IT WOULD BE NICE TO EP/* TAKE A STEP BACK AND TALK ABOUT THE PAST FEW YEARS OF WORK THAT WE'VE DONE REALLY TO UNDERSTAND HOW RNA AND RNA BINDING PROTEINS REGULATE CHROMATIN IN/SSULATION FUNCTIONS AND TODAY I AM GOING TO TRY TO CONVINCE YOU THAT CERTAIN MESSENGER RNAS ACTUALLY HAVE NON-COATING FUNCTIONS IN PROMINENTING CHROMATIN ACTIVITY. AND I AM ALSO GOING TO TELL YOU ABOUT OUR LAB FINDINGS THAT PROMOTEIN -- CHROMATIN IN/SSUL ATE INSULATORS ARE REGULATE BID CERTAIN RNA BINDING PROTEINS. OKAY. SO THE OVERARCHING QUESTION IN MY LAB IS TO /TRAOTRY TO UNDERSTAND, AS THE DOCTOR SAID, HOW A SINGLE GENOME CAN GIVE RISE TO MANY DIFFERENT CELL TYPES WITH DIFFERENT FUNCTIONS AND PROPERTIES. WE BELIEVE THAT, THIS INCLUDES CHROMATIN ARK ARCHITECTURE AND KEY DRIVERS OF THIS PROCESS. RNA IS WRAPPED AROUND /THE HIST HISTONE TO FORM NUCLEOSOMES. BUT WHAT HAPPENED BEYOND THIS PAGE IS NOT REALLY UNDERSTOOD AND WE'RE INTERESTED IN UNDER UNDERSTANDING HOW CHROMATIN IN IN/SSUL ATATORS ARE INVOLVED IN THESE HIGHER ORGANIZATIONS AS /AA GENOME GENOME. SO HE, WITH ADEN AND RECENTLY SPOKEN TO US ABOUT -- HAVE SPOKEN TO US ABOUT HOW RECENT /SKWHRAOEUPBLD STUDIES USING CHROMATIN CONFORMATION CAPTURE TECHNIQUES HAVE REALLY HELPED US IN OUR UNDERSTANDING IN HIGHER RESOLUTION. THE FACT THAT THE CHROMOSOME IS ACTUALLY ORGANIZED INTO THESE LOOSE STRUCTURES. AND THESE CHROMATIN LOOP DOMAINS GENERALLY CORRESPOND TO REGIONS OF THE GENOME THAT ARE EITHER ACTIVELY TRANSCRIBED OR CONVERSE CONVERSELY REGIONS THAT ARE TRANSCRIPTION /HRA*EUL REEXPRESS REEXPRESSED -- REPRESSED. AND AT THE BASE OF THESE LOOPS WE OFTEN FIND IN/SSUL ATATORS PROTEINS HERE. SO IF THIS CASE THE VERT BREA IN IN/SSUL ATATOR IS SITUATED AT THE BASE OF THESE LOOPS. AND C /T-FPTCS ACTIVITY IS NEEDED TO HELP THEM. SO A NUMBER OF GENOME-WIDE PROFILES HAVE NOW BEEN DETERMINED IN DIFFERENT MODELS, DIFFERENT ORGANISMS, AND IT'S CLEAR THAT THIS LOOP STRUCTURE IS QUITE CONSERVED, AT LEAST BETWEEN CLOSELY RELATED SPECIES. AND SO THERE ARE THESE UNDER UNDERLYING PRINCIPLES THAT THESE INSUL AATORS ARE VERY IMPORTANT FOR OVERALL CHROMATIN ORGANIZATION. SO WHAT I AM SHOWING YOU HERE IS A CHROMOSOME FROM A HIGHLY REPRESELICATED CHROMOSOME FROM THE GLAND. JUST TO SHOW THAT YOU IF YOU LOOK AT THE ENTIRE GENOME, WITH ANTIBODIES THROUGH A COUPLE OF THE ANTIBODY PROTEINS THAT I AM GOING TO TALK TO YOU ABOUT TODAY TODAY. AND WHAT YOU CAN SEE IS THESE TWO PROTEINS ROOK ALIKE IN THE GENOME. AND IF YOU COULD LOOK -- IF YOU COULD SEE ON A HIGHER MAG MAGNIFICATION LEVEL -- IT'S NOT VERY EASY TO SEE HERE -- BUT THE LOCALIZATION OF THESE COMPLEXES TENDS TO BE AT THE BOARDERS BETWEEN CONDENSED REGIONS OF THE GENOMES. OKAY. AND THAT'S -- FOR THIS PARTICULAR COMPLEX, PARTIALLY INDICATIVE OF THEIR PROPOSED FUNCTION AS ORGANIZERS OF THE GENOME. THE CHROMATIN INFLATEERS ARE THESE DNA COMPLEXES SITUATED ABOUT THE GENOME AND THEY ARE BELIEVED TO BE DEFINED DESCRIPTIAS TRANSCRIPTIONAL DOMAIN. AND THEY ARE REALLY KNOWN TO BE IMPORTANT IN A NUMBER OF LOCI THAT ARE QUITE COMPLEX. FOR EXAMPLE, THESE ARE SOME VERY WELL-STUDIED LOCI. FOR EXAMPLE, THE VERTEBRATE, WHICH CHANGES AN EXPRESSION THROUGHOUT DEVELOPMENT. AND THE FOCUS OF ANOTHER RECENT LECTURE, THE H 19 IMPRINTED REGION, AS WELL INFLATEERERS KNOWN TO BE VERY IMPORTANT FOR THE REGULATION OF CLUSTERS. AND EACH OF THESE LOCI IS HIGHLY COMPLEX. THERE IS EITHER A NUMBER OF DIFFERENT PROMOTERS THAT NEED TO BE ACT VATED AT THE RIGHT TIME OF DEVELOPMENT OR DIFFERENT ENHANCERS AND I'LL GIVE YOU AN EXAMPLE OF THAT IN THE NEXT SLIDE. BUT WE KNOW THAT STUDIES IN HUMANS, ALTERATION OR MUTATION SITES, CAN BE ASSOCIATED WITH CERTAIN DISEASES. FOR EXAMPLE, IN THE 2 H 19 PRINT PRINTED REGION AGAIN, THESE MUTE MUTATIONS ARE ASSOCIATED WITH THE SYNDROME AND WILLS TUMOR. AND /AA RECENT STUDY BY BRAD'S LAB HAS SUGGESTED THAT THESE INFLATE INFLATEER ACTIVITIES ARE KEY TO THESE ID H 1 -- THE PROGRESSION OF THESE MUTANTS. AND WE KNOW THAT LOSS OF IN/S*UL ATOR PROTEINS WITCAN CAUSE DIFFER DEFECTS. THE SOME OF THE PROTEINS ARE ESSENTIAL FOR VIABILITY AND YOU CAN LS -- MUTATIONS CAN ALSO AFFECT BODY PASSENGER OR GENESIS GENESIS. AND -- CAN MESSAGING. BECAUSE IT'S SUCH A POWERFUL ORGANISM, IT'S VERY PRACTICAL FOR BOTH GENETICS AND KECHEMISTRY IN BUILDING A CELL LINE. AND IT ALSO CONTAINS THE LARGE NUMBER OF KNOWN IN/SSUL ATATOR COMPLEXES. ALL RIGHT. SO TO GIVE YOU A -- AN EXAMPLE OF WHERE IN/SSUL ATATOR PROTEINS ARE EXTREMELY IMPORTANT, I AM SHOWING YOU THE COMPAEXAMPLE OF THE AD BD GENE. SO THIS GENE, WHICH HERE IS TRANSCRIBED FROM RIGHT TO LEFT -- AND DOWNSTREAM OF THE GENE IS ACTUALLY ALL OF THIS MOST OF THE REGULATORY ELEMENTS. SO IT CAN BE EXPRESSED IN THE RIGHT PLACE AT THE RIGHT TIME OF A DEVELOPING EMBRYO IN ORDER FOR DEVELOPMENT TO OCCUR. SO THESE IBELEMENTS HERE ACT AS ENHANCERS, REGIONS WHICH ACT ACTIVATES THE ABBD GENE IN THE PROPER TERM SEG /*PMENT OF THE EMBRY EMBRYO. AND THE WAY THIS FUNCTIONS IS THAT THE ENHANCER ACTUALLY HAS TO LOOP TO THE PROMOTE TORE ACT ACTIVATE IT. SO THAT'S REALLY IMPORTANT ARE THESE POLYRESPONSE ELEMENTS, AND THESE RECRUIT THE POLY COMPLEX, AND THIS CAN SILENCE THE REGULAR REGULATORY ELEMENT IN PART OF THE -- PARTS OF THE EMBRYO WHERE ABD SHOULD NOT BE EXPRESSED. SO ARE FOUND IN BETWEEN THE ELEMENTS AND THEY ARE BOUND BY INSUL AATOR PROTEINS X THESE IN INFLATEER PROTEINS HELP DECIDE ESSENTIALLY WHICH OF THE ENHANCE ENHANCERS ARE LOOPING THROUGH THE PROMOTERS IN ALLOWING ACT ACTIVATION OF THE GENES. AND FURTHERMORE, THEY ARE IMPORTANT FOR CONSTRAINING THE SPREAD OF THE REEXPRESSIVE POLY REEXPRESSION. SO, HISTORICALLY, IN/SSUL ATATORS WERE DEFINED BY TWO FUNCTIONAL -- WHICH I'VE ALREADY ALLUDED TO HERE PREVIOUSLY. SO IF AN /EUINFLATEOR IS PRESENT BETWEEN AN ENHANCE AIR AND PROMOTER, IT ACTUALLY BLOCKS THEIR INTER/AOBACTION. BUT WHAT'S IMPORTANT TO NOTE IS THAT INSUL AATORS DON'T INACTIVATE INACTIVATE. THEY CAN STILL FUNCTION. IF THIS ENHANCER HAD A PROMOTER ON THIS SIDE OR THE OTHER SIDE. IT COULD STILL FUNCTION. BUT THE IN/SSUL ATATOR ACTS IN /TTHIS CASE SORT OF LIKE A WALL BROG THEIR ACTIONS. BUT WHAT WE THINK IS ACTUALLY HAPPENING DUE TO THIS LOOPING PROPERTY OF TWO INSUL AATOR COMPLEXES INTERACT WITH ONE ANOTHER, THEY CAN LOOP OUT THE DNA AND PREVENT THE ABILITY OF THE ENHANCER TO ACTIVATE THE PROMOTER. SO IT'S A PATH /HROPBOLOGICAL EFFECT ON THE DNA THAT WE SEE IS IMPORTANT FOR FUNCTION. FURTHERMORE INSUL AATORS CAN ACT AS BARRIERS TO /THE SPREAD OF RE REPRESSIVE CHROMATINS. YOU TRY TO INSERT A TRANSGENE INTO YOUR FAVORITE GENOME, CAN OFTEN LAND INTO AN UNFAVORABLE LOCATION, PERHAPS THE CHROMATIN OR THE REEXPRESS /* -- REPRESS REPRESSIVE COMPLEX AREA AND THEN IT WOULD NOT BE EXPRESSED. BUT IF YOU HAVE INSUL AATOR SITES, WHAT HAPPENS IS /THE TRANSGENE CAN BE EXPRESSED AT ESSENTIALLY THE SAME OR SIMILAR LEVEL NO MATTER WHERE IT'S INSERTED IN THE GENOME BECAUSE IT CAN BLOCK THE SPREAD OF THIS REPRESSION. WETENT TO /* DON'T KNOW EXACTLY HOW THIS WORKS. THERE ARE SEVERAL MODELS. BUT ONE THAT YOU CAN ENVISION IS PERHAPS OUT /OF THIS REGION, SO THEREBY, MOVE THE TRANSGENE AWAY FROM THEIS REPRESSIVE CHROMATIN. ALL RIGHT, SO A NUMBER OF -- OVER /THE YEARS A NUMBER OF GROUPS HAVE PROFILED IN HIGH-RES HIGH-RESOLUTION WHERE INSUL AATOR COMPLEXES BIND IN THE GENOME IN DIFFERENT CELL TYPES AND SO ONE CONCLUSION THAT HAS BEEN FOUND IS THAT WHILE THERE ARE DIFFERENCES IN BINDING SITES ACROSS CELL TYPES, OVERALL THE MAJORITY OF THE IN/SSUL ATATOR SITES ARE FAIRLY CONSTANT. BUT ONE THING WE DO KNOW IS THAT THE LOOPING OR ONE POSSIBILITY THAT WE WOULD /HRAOEULIKE TO SUGGEST IS THAT THERE ARE SOME EVIDENCE OF THIS THAT THE LOOPING OF THE DIFFERENT /EUINSUL ATATOR COMPLEXES WITH ONE ANOTHER COULD BE DIFFERENT IN DIFFERENT SITES. SO IN /THIS CASE, FOR EXAMPLE, THIS ENHANCER CAN PROMOVE TO THE PROMOTER AND ACTIVATE IT. BUT IN CELL TYPE B PERHAPS THE CONFIGURATION PREVENTS THE ENHANCER FROM ACTING ON THIS EN ENCODER. SO MY LAB IS REALLY FOCUSED HAY /* A LOT /OF EFFORT IN TRYING TO UNDERSTAND HOW CHROMATIN IN/SSUL ATEINSULATORS ARE REGULATED BY THE INTERINTER/AOBACTION WITH OTHER ASSOCIATED FACTORS. ALTHOUGH IT IS, /STKPWROERBGS VERY IMPORTANT ALSO TO UNDERSTAND HOW INFLATEER COMPLEX COMPLEXES ARE FORMED AND HOW THEY FUNCTION. OKAY. SO WHAT I AM GOING TO TELL YOU ABOUT TODAY IS OUR WORK ON THE SO-CALLED JUST THE INSUL AATOR AND IT GETS ITS NAME BECAUSE IT WAS ORIGINALLY IDENTIFIED, THE SEQUENCE IDENTIFIED AS /AA GYPSY TRANS/POPOSON AND REPEATED SEQUENCE SEQUENCE. BUT WHAT IT BASICALLY IS IS A 26 26-BASE PAIR THAT IS BOUND BY THIS SUPPRESSIVE DNA BINDING PROTEIN. AND TWO OTHER CORE COMPONENTS OF THIS INSUL AATOR COMPLEX ARE MODIFIERS MB G 4 PROTEIN, WHICH I'LL JUST REFER TO AS /AA MODIFIER FOR SIMPLE FLIELICITY AND CT 1 ID, WHICH IS THESE EACH OF THESE PROTEINS INTERACT DIRECTLY AND BOTH MODIFIERS CONTAIN THE CTP CALLED PROTEIN-PROTEIN INTER INTER/AOBACTION. WHICH ALLOWS -- WHICH MAY BE IMPORTANT FOR -- I SHOULD ALSO POINT OUT THAT -- WELL, AS I SHOWED YOU EARLIER ON THE CHROME CHROMOSOME, THESE INSUL AATOR PROTEINS DO BIND TO IN/TKOPBENDOGENOUS SITES WITHIN THE GENOME. NOT JUST IN THE CONTEXT OF INAUDIB[INAUDIBLE]. SO ONE FEATURE OF THE INSUL AATOR COMPLEX THAT IS REALLY REMARK REMARKABLE IS /THE FACT THAT IF YOU LOOK AT THE LOCALIZATION PATTERN IN /AA DIPLOID NUKCLEUS, YOU SEE IT'S NOT COMPLETELY DIFFUSED THROUGHOUT THE NUCLEUS, OKAY? SO IN /THIS FLUORESCENCE ANTI- ANTIBODIES, YOU CAN SEE CONS CONCENTRATIONS OF THE INSUL AATOR PROTEINS INTO THESE LARGE NUCLEAR LOCI, WHICH ARE TERMED YOU IN/SSUL ATATOR BODIES AND THESE BODIES ARE APPROXIMATELY 200 NAN NANOMETERS IN DIAMETER AND THEY'RE VERY LARGE AND HIGHLY -- INAUDIB[INAUDIBLE]. SO IT'S HARD TO KNOW WITH ANY KIND OF NUCLEAR VISIBLE STRUCTURE WHAT IT ACTUALLY CORRESPONDS TO AND THERE ARE SEVERAL MODELS ABOUT WHAT THE BODIES ACTUALLY ARE. BUT ONE POSSIBILITY IS THAT THE BODIES CORRESPOND TO INSUL AATOR COMPLEX THAT'S HELP ORGANIZE THE GENOME IN A PARTICULAR WAY. WHAT WE DO KNOW IS THAT THESE BODIES NEED TO TETH TORE THE NUCLEAR MA/TREUBTRIX THROUGH INTER INTER/AOBACTION WITH LAMIN. WHAT'S IMPORTANT IT KNOW IS /THE INSUL AATOR BODIES, AT LEAST THE PROPER LOCALIZATION IS HIGHLY CORRELATED WITH GYPSY INSUL AATOR ACTIVITY. SO MUTATIONS THAT AFFECT INSUL ATOR BODY LOCALIZATION ALSO AFFECT IN/SSUL ATATOR ACTIVITY, ALTHOUGH THEY'RE NOT EFFICIENT FOR ACTIVITY. IF NOTHING /SKWRERBLGS THEY SERVE AS /AA USEFUL FENE /T*EPHENOTYPIC READ OUT FOR S/EUPINSUL ATATOR ACTIVITY. C OKAY. SO MY LAB HAS REALLY BEEN INTERESTED IN HOW RNAS REGULATE ACTIVITY AND WORK FROM WHEN I WAS A POST DOC IDENTIFIED AN RNA INDEPENDENT INTER/AOBACTION WITH THE GYPSY INSUL AATOR COMPLEX. AND MUTATIONS AFFECT INSUL ATATOR ACTIVITY AND IN/SSUL ATATOR BODY LOCALIZATION. SO OTHER WORK FROM MY LAB, THAT I AM NOT GOING TO REALLY GO INTO TODAY, IDENTIFIED THE MAJOR RNA I COMPONENT IS PROMOTING CT 190 IN/SSUL ATATOR AND IT SEEMS TO HAVE A COMPLETELY RNA-I INDEPENDENT LIFE IN THE NUCLEUS PROMOTEING THIS INT/SUSUL ATATOR ACTIVITY. THE ONE QUESTION THAT REMAINS IS WHETHER THE RNA BINDING ACTIVITY IS REALLY IMPORTANT FOR CHROMAT CHROMATIN IN/SSUL ATATOR ACTIVITY /SWREFPLT ALSO IDENTIFIED THE GENOME-WIDE PHYSICAL ASSOCIATION BETWEEN THE EXOSOME INTERACTING WITH THIS PARTICULAR INSUL AATOR COMPLEX. SO INTRIGUELY IT'S RECRUITD TO A NUMBER OF TRANSCRIBED GENES AS PROMOTERS. ALTHOUGH WE DON'T KNOW WHAT THE RELEVANT RNA SUBSTRATE IS. IT'S AN /EUINTRIGUING CONNECTION WITH THE COMPLEX. MEANWHILE, IN MAMMALS FOUND THAT THE HUMAN HOMOLOG OF THE R 52 CASE ALONG WITH ITS NON-COATING RNA SRA PROMOTE INSUL AATOR ACTIVITY BY HELPING. FURTHER MOMORE, THE LAB SHOWED SEVERAL YEARS AGO THAT THE NON-COATING RNA GPS, WHICH IS INVOLVED IN AX /O*ON ACTIVATION CAN FIBIND IT DIRECTLY AND /SKWAOEFRT IT OFF OF CHROMATIN, THERE BY NEGATIVE REGULATING ACTIVITY. SO YOU CAN SEE AT LEAST AT SOME LEVEL THERE IS SOME CONSERVATION OF MECHANISMS, ALTHOUGH DIFFERENCES IN THE DETAILS IN BOTH POSITIVE AND NEGATIVE LEVELS OF REGULATION BY /RAFPLS /* RNA. OKAY. SO TODAY I AM JUST GOING TO TELL YOU THREE PARTS TO THE STORY, WHICH IS THE FIRST IS THIS POTENTIAL NON-COATING FUNCTION FOR MESSENGER RNA INSUL AATOR ACTIVITY AND THEN OUR FINDING OF A NOVEL MODE OF REGULATION OF IN IN/SSUL ATATORS IN TERMS OF SPECIFIC REGULATION BY THESE RNA BINDING PROTEINS. THE FIRST CALLED CHEF, WHICH IS THE CENTRAL NERVOUS SYSTEM SPECIFIC AN /TTAGONIST AND THE OPPOSITE. SO WE STARTED WITH A FAIRLY UNBIAS ADD /PREFP TO /TRAOTRY TO IDENTIFY WHAT THESE RNAS ACTUALLY ARE AND WE TOOK EM EMBRYONIC NUCLEAR HE CAN TRACTS AND PASSED THEM OVER SEQUENTIAL IMMUNOAFFINITY FIRST IN ANTI- ANTI-SUPPRESSORS, ANTIBODY COLUMNS, THROUGH BINDING AND THEN RELEASEING AND GOING OVER 1 190 COLUMN. SO THE ISOLATED RNA WAS THEN N LABELED AND RUN ON A HIGH- HIGH-PERCENTAGE GEL. SO THE REASON FOR THIS GEL, BECAUSE WE WERE ACTUALLY LOOKING FOR HISTORICAL REASONS AT THE TIME LOOKING FOR SMALL RNAS, WHICH WE THOUGHT /PHAOUBT IN. BUT WHAT WE FOUND, WHEN WE LABELED TOTAL NUCLEAR EXTRACT VERSUS IP, THAT WE FOUND A PECULIAR CLASSES OF RNAS COMING DOWN FROM 35 TO 55 NUCLEAR TYPES RNAS WHICH ARE OBVIOUSLY /STHRAEUPBGS LARGER THAN SMALL R RNAS. BUT IN ORDER TO IDENTIFY THEM, WE CUT THEM OUT /-OF THE YELLS, CLONEED THEM DIRECTIONALLY AND SUBJECTED THEM TO HIGH THROUGH PUT SEQUENCE. SO THE FIRST SURPRISE WE GOT IS WHEN WE MAPPED THESE RNAS BACK TO THE GENOME, OKAY, SO WHAT I AM SHOWING YOU IS /THE PILOT IN THE TOTAL FRACTION VERSUS THE IP IP, FRACTION AND YOU CAN SEE THERE IS EN/REURICHMENT OVER THIS PROTEIN-COATING GENE. SO THAT WAS THE FIRST SURPRISE. AND SINCE THIS IS DIRECTIONAL, WE CAN SEE THAT IT'S ON THE GRAM AND WE ALSO SEE AX /O*ON-AX /O*ON JUNCTION IN THE READ. INDICATING THAT THESE RNAS ARE ACTUALLY PROCESSED. SO WHAT WE REALIZED, THROUGH DOING AND CONFIRMED BY BT PURIFICATION, THAT THESE ARE ACTUALLY FULL-LENGTH MESSENGER R RNAS THAT WE WERE PULLING DOWN AND THE REASON WE WERE GETTING THESE FRAGMENT IS BECAUSE WE WERE LOOKING AT THE SUBCLASS. THAT'S WHAT WE WERE FOCUS ON. BUT REALLY THESE ARE FULL-LENGTH TRANSCRIPTS. AND THE SECOND SURPRISE WE GOT IS THIS M /R-FPRNA CORRESPONDS WITH THE GENE ITSELF. OKAY, THAT SEEMED TO BE NOT A COINCIDENCE. SO WHEN HE LOOKED AT /THIS GENOME GENOME-WIDE, WE SEE A FURTHER SURPRISE IS THAT WHEN WE LOOK AT THE IT FRACTION COMPARED TO THE TOTAL IN/PPUT, EACH INDIVIDUAL KNOT IS AN ANNOTATED GENE IN THE GENOME, WHETHER IT'S COATING -- CODING OR NOT /* NON-CODING /SKPWHRAFPLT WE SEE ENRICHED ACROSS TWO BIOLOGICAL REC SITS, SIGNIFICANT THRESHOLD, ARE ONLY TWO MESSENGER RNAS THAT ARE TWO RNA THAT'S HAPPEN TO BE THE MESSENGER RNA IS /THE SUPPRESSIVE AND TYPICAL 190 CELL. SO THESE ARE NOT THE MOST ABUNDANT M /R-FPRNAS IN THE CELL. FOR EXAMPLE, HIGHLY ABUNDANT PROTEIN TRYPT 32 ARE NOT EN ENRICHED. SO OF COURSE THE FIRST THING YOU ARE WONDERING IS THERE MUST BE SOME TYPE OF ARTIFACT AND OF COURSE WE ASKED OURSELVES THAT QUESTION. AND SO WE DID A LARGE NUMBER OF CONTROLS TO ADDRESS THIS. AND I WON'T GO THROUGH ALL OF THEM, BUT JUST KEEP IN MIND THAT WE WERE DOING NUCLEAR PREP IN PASSING OVER TWO SEQUENTIAL IMMUNOAFFINITY COLUMNS SO IT WAS VERY UNLIKELY WE WERE PULLING DOWN, TRANSLATING RIBOSOMES, FOR EXAMPLE, WHICH IS ONLY THE ART ARTIFACT THAT SEEMS REASONABLE TO US. BUT I THINK THE CONTROL THAT REALLY CONVINCES PEOPLE THAT THIS IS NOT AN ARTIFACT IS /THE FACT THAT WE ALSO DID A PARALLEL PURIFICATION OF /AA TRANSCRIPTION FACTOR DORSAL, PULLED IT DOWN USING THE SAME TECHNIQUE AND DID NOT PULL DOWN ITS MESSENGER RNA. SO OKAY. SO NOW THAT WE'VE ESTABLISHED THAT THIS IS NOT LIKELY TO BE AN ARTIFACT, THE QUESTION THEN IS WELL, WHY ARE WE PULLING DOWN THESE MESSENGER RNAS IN THE NUCLEUS WITHIN THE COMPLEX? SO ONE POSSIBILITY IS THAT THESE R /R-FPR -- M /R-FPLRNAS ARE ASSOCIATED WI TH SOME KIND OF SURVEY LENGTH MECH MECHANISM. BUT ANOTHER POSSIBILITY IS PERHAPS THESE MESSENGER RNAS ARE THERE FIRE FUNCTION /AAL REASON. MAYBE THEY ARE PARTICIPATING IN INSUL AATOR, SIMILAR TO THE WAY NON-CODING RNAS HAVE BEEN SITUATED. SO BUT YOU CAN'T TAKE A STANDARD APPROACH OF MUTATING THE GENE BECAUSE YOU WILL AFFECT THE PROTEINS. SO WE HAD TO COME UP WAY DIFFERENT STRATEGY. SO WE DECIDED TO TAKE A GENOME GENOME-FUNCTION APPROACH, WHICH IS TO EXPRESS THESE RNAS IN AN UNTRANSLATABLE MANNER AND WE TOOK ADVANTAGE OF THE T 7 SYSTEM SYSTEM. SO IT'S BEEN KNOWN IN A NUMBER OF DIFFERENT ORGANISMS THAT IF YOU SUPPLY CELLS WITH A T 7 SUM SUMMARY AND YOU SHOWED IT PROMOTER IT WILL TRANSCRIBE HIGH LEVELS OF RNAS. BUT SINCE THEY'RE UN/KPHOUPLD FROM THE TRANSCRIPTION, THEY'RE NOT PROCESSED PROPERLY SO THEY'RE NOT VENTILATEED AND THEY ARE NOT EFFICIENTLY ASPIR AATED. SO WE WERE ABLE TO TAKE ADVANTAGE OF THIS SYSTEM THAT WE SET UP BY A LOCAL LAB. WHAT WE DID IS WE CLONED THE CD CDNAS ALONG WITH THEIR UTRS UNDER /THE T 7 PROMOTER CONTROL AND ENGINEERED A TERMINATOR. WE PUT IN /AA MIXED SEQUENCE TAG JUST TO BE ABLE TO DISTINGUISH IT FROM THE IN/TKOPBENDOGENOUS TRANSCRIPT. THE SECOND THING WE PUT IN IS THISOR AND THE THIRD TRANSGENE IS A TISSUE-SPECIFIC PROMOTER DRIVING 4. SO YOU ARE ABLE TO EXPRESS THESE TRANSCRIPTS IN SLIDES BY A APPLYING A PARTICULAR RIGHT 4 DRIVER, WHICH BINDS TO THE UAF, WHICH BINDS TO THE T 7 PROMOTER AND MAKES THE TRANSCRIPT. SO I WON'T SHOW YOU ALL THE CONTROLS, BUT SUFFICE TO TO SAY THAT WE DID NOT -- WHEN WE EXPRESSED, WE DID NOT SEE ANY EVIDENCE OF /AA PROTEIN BEING MADE AND NO CHANGES IN INSUL AATOR PROTEIN LEVELS, SUGGESTING THAT THERE IS PROBABLY NOT THIS /TPAO*EG FEEDBACK OF THE RNA IN INSUL AATOR PROTEIN TRANSCRIPTION. SO WHAT HAPPENS WHEN WE EXPRESS THESE TRANSCRIPTS? SO, WE HE GROW THESE TRANSCRIPTS IN THE DEVELOPING LARVAE IN /PRUFRPTIVE-LIKE TISSUE. SO WE LOOKED AT THE LOCALIZATION OF INSUL AATOR BODIES USING CT 190 ANTIBODIES. AND WHAT HAPPENED WHEN WE LOOKED AT THE CONTROL SITUATION WHERE THE EXPRESSING THE POLY/PHMERASE BUT NOT GIVING ANY TO TRANSCRIBE, WE SEE ON AVERAGE ONE IN/SSUL ATATOR BODY PER NUCLEAR AND THIS IS NORMAL. WHEN WE DRIVE THE UNTRANSLATABLE TRANSCRIPT, WE GET MULTIPLE /TPOFOCI THAT ARE MUCH MORE INTENSE AND THESE ARE AB/ERRANT-LOOKING. WITH THE T 7 UNTRANSLATABLE TRANSCRIPTS, WE GET IT. AND IT IS /* AS /AA NEGATIVE CONTROL, WE ALSO DRONE THE MODIFIER TRANSCRIPT, WHICH IS NOT BE IN THE PURIFICATION. AND WHEN WE EXPRESS THAT, WE SEE SIMILAR TO THE CONTROL, ONLY ONE INSUL AATOR BODY. SO WE ARE LITERALLY SEEING CHANGES IN THE BODY LOCAL /WHAOEUFGS WE DRIVE THESE TRANSCRIPTS. WE CAN QUANTIFY THESE RESULTS BY COUNTING THE NUMBER OF INSUL AATOR BODIES PER NUCLEAR IN EITHER THE LATE TISSUE WHEN WE'RE DRIVING THE TRANSCRIPT VERSUS THE /SKPWRAOEUFPLT WHAT WE CAN SEE IS THERE IS A CLEAR INCREASE IN THE NUMBER OF THE IN/SSUL ATATOR BODIES WHEN YOU DRIVE EITHER EXPRESS OR THE CT 190 TRANSCRIPT TRANSCRIPT. BUT WHAT ABOUT INSUL AATOR ACTIVITY? SO WE TOOK ADVANTAGE OF /AA NATURALLY OCCURRING ALLELE OF CUT, WHICH IS CALLED CUT 6 IN ORDER TO STUDY THE ENHANCER-BROG ABILITY OF IN/SSUL EATOR 190. SO THE IN/SSUL ATATOR IS PRESENT BETWEEN THE CUT AND DOESN'T HAVE THEIR INTER/AOBACTION AND THEN DE DECREASES THE EXPRESSION OF THE CUT. ALL YOU NEED TO KNOW ABOUT CUT IS THAT IT'S IMPORTANT FOR THE PROPER FORMATION OF THE MARGIN. SO THIS IS A NORMAL WING. AND AS WE GET MORE NOTCHED, THAT MEANS CUT EXPRESSION IS DECREASE DECREASED, WHICH ALSO MEANS THAT INSUL AATOR ACTIVITIES IS IN INCREASED. SO WHAT YOU CAN SEE IS THAT WHEN WE COMPARED TO NON-DRIVING THE TRANSCRIPT, WE DRIVE THE T 7 TRANSCRIPT, HE SEE A STRONG IN INCREASE IN ENHANCER-BLOCKING ACTIVITY. AND WITH THE T 7 CT 190 TRANSCRIPT, WE GET AN INTERMED INTERMEDIATE FEPHENOTYPE. SO THE SIMPLEST WAY TO INTERPRET THESE RESULTS IS THAT THESE JUST THE PRESENT /* PRESENCE OF THESE RNAS IS ACTUALLY PROMOTING ENHANCER-BLOCKING ACTIVITY. SO HOW DOES THIS WORK? WE HAVE -- WE DON'T KNOW. BUT WE CAN PROPOSE -- AT LEAST THIS PROVIDES A PROOF OF PRINCIPLE THAT PERHAPS SOME MESSENGER RNAS ARE ACTUALLY HAVING THESE NON-CODING FUNCTION FUNCTIONS AND SO THE MODEL THAT WE SUGGEST IS A SIMPLE ONE, SIMILAR TO AS I SAID, HOW NON-CODING RNAS HAVE FUNCTIONS. BUT ACT AS /AA SCAFFOLD, PERHAPS TO PROMOTE THE FORMATION OF IN IN/SSUL ATATOR COMPLEXS AND TO PROMOTE THEIR ACTIVITIES, PERHAPS DIV /KWRAO*EIDE INFORMATION IN PARTICULAR. SO ONE THING THAT BE /* -- THAT -- SO WE KNOW ACTUALLY AND I WILL TALK ABOUT THIS DATA TOWARDS THE END /-OF THE TALK -- THAT WE DON'T THINK THAT THE R RNAS ARE INTERACTING DWELL /* DIRECTLY WITH THE IN/SSUL ATATOR PROTEINS AND THAT'S BECAUSE OF SOME OF OUR CROSS LINKING STUDIES THAT WE'VE DONE. SO WE THINK THERE IS AN RNA BINDING PROTEIN THAT'S LEADING THIS ACTION AND SO WE /* FAR WE DON'T KNOW WHAT IT IS. BUT WE HAVE MANAGED TO DEFINE A MINUIMAL SEQUENCE ABOUT 150 NUKE NUCLEOTIDES NOW THAT IT SEEMS TO BE RESPONSIBLE, AT LEAST IN THE T 7 ASSAY. IS SUFFICIENT FOR THE FEPHENOTYPE. SO WE'RE STARTING TO NOW TRY TO FISH OUT RNA BINDING PROTEINS THAT /PWAOEUPD TO THIS SEQUENCE, TO PERHAPS IDENTIFY THIS SET /OF FACTORS. SO ANOTHER QUESTION THAT REMAINS ALSO IS -- AND THIS IS VERY PEEK PECULIAR -- HOW DO THESE RNAS GET HERE? ARE THEY TRANSCRIBED, AS /AEA SEPARATE POOL OF RNAS? AND THERE IS A DIFFERENT POOL THAT GOES OUT FOR TRANSLATION? OR DO THEY STAY IN THE CONFLICT FOR SOME TIME AND GET EXPORTED? COULD THEY EVEN POSSIBLY GO OUT AND COME BACK IN? SO THERE IS A LOT OF QUESTIONS ABOUT BIOGENESIS AND WHAT'S THE BEHAVIOR OF THESE RNAS. B ONE THING I WANT TO POINT OUT IS THIS IS NOT LIKELY -- EXAMPWELL, IT'S POSSIBLE THAT THIS COULD BE A MORE GENERAL FEN /OPHENOMENON AND NOT JUST A WEIRD THING WITH THE IN INSUL AATOR COMPLEX. BECAUSE WHEN WE REPEATED OUR PURIFICATIONS AND INSTEAD OF RUNNING A GEL, WHICH IS VERY IN INEFFICIENT, WE USED AN /* A PT SELECTION AND WE WERE ABLE TO PULL IT DOWN A NUMBER OF ADDITIONAL TRANSCRIPTS. OF THESE M /R-FPLRNAS THAT WE PULLED DOWN, IT'S A SMALL SAMPLE SIZE, OF COURSE, BUT SEEMED TO BE ALSO NOT A COINCIDENCE THAT MAYBE HALF OF THEM ACTUALLY CODE FOR PROTEINS THAT ASSOCIATE WITH CHROMATIN, HAVE CHROMATIN FUNCTIONS. SO IT'S POSSIBLE THAT YOU KNOW, THIS IS NOT JUST /AA WEIRD IN/SUSUL ATOR FEN /OPPHENOMENON AND COULD BE MORE GENERAL. IT'S THE SORT OF THING THAT IF YOU -- IT'S NOT EASY TO STUDY. SO IT COULD BE SOMETHING THAT HAS BEEN SO /TPFAR GONE UNNOTICED. SO ANOTHER POINT I WANT TO MAKE IS THAT WHERE ARE THESE PRESCRIPTIV TRANSCRIPTS COMING FROM? SO WE DID LOOK TO SEE WHETHER THE GENES THAT CODE THESE TRANSCRIPTS HAVE INSUL AATOR SITES BINDING NEARBY. AND ONLY A COUPLE /OF THEM DO. AND IT SEEMS /TTHAT PROBABLY FOR THE MOST PART THAT THESE TRANSCRIPTS ARE BEING SIN /TYNTHESIZE SYNTHESIZED IN THE COMPLEX AND THEN COMING TO ASSOCIATE SO WE DON'T KNOW HOW THAT HAPPENS BUT IT'S KIND OF INTRIGUING THAT THIS COULD OCCUR. AND ANOTHER THING I JUST WANT TO POINT OUT THAT WILL BE RELEVANT LATER IN THE TALK IS THAT A COUPLE /OF THESE RNAS CODE FOR DEVELOPMENT AND A NUMBER OF THESE TRANSCRIPTS ARE HIGHLY EXPRESSS IN THE CENTRAL NERVOUS SYSTEM. OKAY. SO FOOD FOR THOUGHT. OKAY. SO AND THE NEXT PART OF THE TALK I AM GOING TO TELL YOU ABOUT THIS RNA-BINDING PROTEIN CALLED CHEF. SO THIS PROTEIN WAS ACTUALLY PULLED OUT /-OF THE TWO HYBRID SCREENS THAT WAS PERFORMED IN CONVIVICTOR'S LAB LOOKING FOR INTER INTERACTORS OF THE MODIFIER PROTEIN. THIS INTERACTOR REALLY CAUGHT MY ATTENTION BECAUSE AT THE TIME IT WAS A COMPLETELY UN UN/KAEURCHARACTERIZED GENE, BUT IT WAS CLEAR THAT IT CONTAINED TWO HIGHLY CONSERVED RNA RECOGNITION MOTIFS. SO WE STUDIED TO FOLLOW THIS UP FURTHER. AND DECIDED. AND THIS CHEF ACTUALLY HAS A NUMBER OF DIFFERENT IS/OFORMS AT THE TIME AND /* THAT WE PUBLISHED THIS. THERE WERE ONLY A FEW IS/OFORMS BUT THERE WERE PROBABLY -- EVERY TIME YOU /HRAOLOOK AT THE DATABASE THERE IS MORE IS/OFORMS. IT'S A PRETTY COMPLEX PROTEIN. WE DID VERIFY THAT CHEF INTER INTERACTS DIRECTLY WITH BOTH MODIFIER, EITHER MODIFIER OR SUPPRESSIVE IN VITRO. OKAY. SO IT DOES ASSOCIATE WITH THOSE FACTOR. BUT WHAT WAS REALLY INTERESTING TO US IS WHEN WE LOCALIZED THE CHEF PROTEIN, WE GENERATED ANTI- ANTIBODIES TO IT AND THEN STAINED LATE-STAGE EMBRYOS WITH THE ANTIBODIES. COULD SEE A STRONG EN/REURICHMENT OF CHEF IN THE CENTRAL NERVOUS SYSTEM. SO THIS IS THE CENTRAL NERVOUS SYSTEM AND WE CO-STAIN WITH A MARKER AND YOU CAN SEE NICE OVER OVERLAP. SO THIS IS INTRIGUING BECAUSE SO FAR ALL INTERLAYER PROTEINS THAT WE HAVE STUDIED ARE KNOWN TO BE /AOUUBIQUITOUSLY EXPRESSED. SO THIS IS THE FIRST TISSUE- TISSUE-SPECIFIC ENRICHED FACTOR. SO THIS LED US TO THE QUESTION, WELL, PERHAPS IT COULD ACTUALLY BE REGULATING AT A TISSUE- TISSUE-SPECIFIC MANNER. BUT IN ORDER TO STEST THAT, WE HAD TO DEVELOP AN ASSAY TO DRESS /* ADDRESS THAT. SO WE DEVELOPED AN ASSAY THAT WAS BASED ON SOME TRANSGENES THAT WERE DEVELOPED IN THE LAB. SO THIS IS A BARRIER ASSAY AND /* THAT I'LL EXPLAIN TO YOU. SO THERE IS A UA /TP-FPF DRIVING RECORD RECORDER. IT'S EITHER FLANKED WITH SUPPRESSIVE HARROWING BINDING SITES OR NOT. AND THESE TWO DIFFERENT TRANS TRANSGENES ARE INSERTED INTO THE SAME GENOME. AND IT'S INSERTED INTO A POLY POLYCHROME REPRESSED REGION SO THAT IF IT'S NOT INSULATED, IT BASICALLY WON'T GET EXPRESSED. THE SECOND IS A HAIRPIN SO WE COULD KNOCK DOWN ANY PARTICULAR FACTOR OF INTEREST AND THE THIRD IS A TISSUE-SPECIFIC PROMOTING DRIVER FORE. SO WE CAN KNOCK DOWN FACTORS OF INTEREST AND THEN SEE IF WE CAN DRIVE THIS REPORTER. SO THE EXPRESSION IS QUITE VARIABLE ACROSS INDIVIDUALS. SO WE DID SPECIFIC ANALYSIS OF AN INDIVIDUAL TO EXPRESS THE DATA. SO JUST TO SHOW YOU THAT WE ARE ABLE TO KNOCK DOWN THE FACTORS OF INTEREST -- TO /* SO THESE ARE THE NON-/EUIN/SUSUL ATATED LINES AND THEN THIS IS A /AOUUBIQUITOUS DRIVER DRIVER. SO WHEN WE KNOCKED DOWN SUPPRESS SUPPRESSER, WE GET GOOD LOSS OF PROTEIN. AND THEN WHEN WE KNOCK DOWN CHEP CHEP, YOU CAN SEE THE DELETION, ALTHOUGH NOT ALL ARE DEPLETED. WE CAN ALSO OVEREXPRESS THIS PARTICULAR IS/OFORM OF CHEF. SO WHAT HAPPENS WHEN WE LOOK AT THE LOSE /EUIVORUS ACTIVITIES IN /TH THIS LAR VIRGINIA? SO WE USED THE DRIVER TO ASSESS THE AFFECT AND IN -- YOU WOULD EXPECT TO GET MORE ACTIVITY AND WE GET A BIT HIGHER EXPRESSION. THIS IS A SMALL FRACTION OF THE LARVAE AND THE LEVELS ARE FAIRLY LOW. AS A PROOF OF PRINCIPLE, YOU CAN SEE THAT THE BARRIER ACTIVITY IS LOSS /* LOST. SO THE EXPRESSION IS REALLY DOWN DOWN. AND WHEN WE KNOCK DOWN CHEF, WE ACTUALLY SAW AN /EUINCREASE IN THE BARRIER ACTIVITY. SO THIS ACTUALLY INDICATES THAT THIS CHEF IS A NEGATIVE -- IS AN AN TAGONIST. WHEN WE DO THIS EXPERIMENT USING A MUSCLE GAL 4 DRIVER -- SO THIS IS A TISSUE WHERE CHEF IS NOT EXPRESSED HIGHLY. L AGAIN MOST EXPRESSION IN THE LINE, IS DEPENDENT ON SUPPRESSOR BUT WHEN WE KNOCK DOWN CHEF THERE IS NO EFFECT. BUT IF WE OVEREXPRESS CHEF, WE CAN ACTUALLY DRIVE DOWN IN/SSUL ATOR /SRAOBGACTIVITY IN /TTHIS TISSUE AND WE CAN ACHIEVE A SUFFICIENT LEVEL OF CHEF TO AN /TTAGONIZE IN INSUL AATOR ACTIVITY IN THE TISSUE TISSUE. ALL RIGHT, WE ALSO LOOKED AT THE LOCALIZATION OF IN/SSUL ATATOR BODIES IN DIFFERENT TISSUES IN THE DEVELOPING LARVAE. SO WE LOOKED FIRST IN THE LARVAE AND STAINING AGAIN IN WILD TYPE PSYCHE A SINGLE FOCUS OF THE IN IN/SSUL ATATOR BODY. WE LOOKED AT A SYNTHESIZED GENT GENETIC BACKGROUND. THIS IS A KNOWN MUTANT FOR THE MODIFIER GENE. AND WHAT HAPPENS IS IT'S KNOWN THAT IN /THIS GENOTYPE INSUL ATATOR BODIES BECOME SMALLER AND MORE NUMEROUS AS IF PERHAPS THEY ARE FALLING APART WITH JUST AN INTERPRETATION. BUT IN THE CHEF HE, A STRONG LOSS OF FUNCTION, WHICH MEANS VERY LITTLE PROTEIN, IN COMBINATION THAT MODIFIER MUTE MUTATION, THIS DOUBLE MUTANT ACTUALLY RESTORES BAINSUL AATOR BODY BACK TO /TTHAT ONE FOCUS. SO THAT'S CONSISTENT WITH CHEF ACTING AS AN /AAN /TATAGONIST OF INSUL ATOR ACTIVITY AND THIS IS ONLY SEEN IN THE LARVAEAL BRAIN BECAUSE IF WE LOOK AT TISSUES THAT ARE ARE SEEING THIS PAIR PARALLEL, WE DON'T SEE THIS EFFECT. SO WILD TYPE 1 FOCUS 1 AND MUTE MUTANT AND IN THE CHEF MODIFIER, DOUBLE MUTANTS, HE STILL SEE MULTIPLE /TPOFOCI SO THE REST IS ONLY SEEN IN THE BRAIN. OKAY /SWRORBGS WE WANTED TO KNOW WHERE DOES CHEF LOCALIZE ON CHROMATIN? SO WE PERFORMED A CHIP C PROFILING CHEF USING OUR ANTI- ANTIBODIES IN /AA CNS DRIVE CELL CALLED BG 3. SO WHAT I AM SEEIHOWING YOU ARE JUST A COUPLE /OF SCREEN SHOTS OF CHEF LOCALIZATION COMPARED TO SUPPRESSIVE ORDERING MODIFIER IN THE GENOME. SO THE FIRST THING THAT WE NOTED IS THAT CHEF-BINDING PROFILES ARE A BIT BROADER IN GENERAL. PERHAPS BECAUSE IT'S IN AN RNA- RNA-BINDING PROTEIN. VERSUS MODIFIER. BUT ANOTHER THING THAT WE NOTICE NOTICED IMMEDIATELY IS THAT IN MANY PLACES IN THE GENOME, WE SAW OVERLAP OF CHEF WITH EITHER SUPPRESSIVE OR WITH MODIFIER BUT NOT ALL THREE FACTORS TOGETHER. AND THE INSUL AATOR PROTEIN TOGETHER BY THEMSELVES WITHOUT CHEF. SO WE LOOKED THAT THE ON A GENOME-WIDE LEVEL AND WHAT I AM SHOWING YOU IS A BINARY HEAT MAP, WHICH INDICATES INDIVIDUAL -- EACH COLUMN IS AN INDIVIDUAL GENOMIC SITE AND IF YOU SEE A BLACK MARK IN THAT ROW, THAT MEANS THIS FACTOR OF INTEREST IS PRESENT AT THAT SITE. SO THERE ARE A NUMBER OF CHEF- CHEF-BINDING SITES IN THE GENOME THAT ARE INDEPENDENT OF IN/SSUL ATOR PROTEINS. SO I WANT TO CALL YOUR ATTENTION TO THIS SET, WHICH IS WHERE CHEF OVERLAPS STENEXTENSIVELY WITH EITHER MODIFIER OR SUPPRESSIVE HARROW HARROWING, BUT NOT ALL THREE TOGETHER. SO YOU CAN SEE THE TWO IN/SSUL ATATOR GENES ALSO TOGETHER /STKPWRAOEFPBLT THERE IS SOME 3 WAY OVERLAP, WHICH IS PARTIALLY DUE TO THE MAPPING. BUT THIS 3 WWAY OVERLAP IS HIGHLY ENRICHED SPECIFICALLYING SPECIFICALLYING-SPEAKING. SO I JUST WANT /TO STOP HERE /TPIN THIS SECTION AND TALK ABOUT WHY DO YOU HAVE AN /AANTAGONIST OF IN INSUL AATOR ACTIVITY? SO WE SUGGEST THAT PERHAPS A NON-CNS TISSUE YOU HAVE SOME GENES THAT NEED TO BE KEPT OFF AND THESE COULD BE PREVENTIVE EXPRESSION BY A PARTICULAR CON CONFIGURATION OF INSUL AATOR ACTIVITY. IN /THIS CASE I AM DRAWING ENHANCING ACTIVITY. THEN YOU WANT /TO TURN THESE GENES ON. YOU HAVE HIGH LEVELS OF CHEF PRESENT AND THIS CAN AN /TTAGONIZE FURTHER THERE BY RELEASING AND A ALLOWING THIS ENHANCER TO ACT AS A MOTOR. SO WE DON'T KNOW -- SEVERAL QUESTIONS HERE. WE DON'T KNOW WHAT SPEC /TPEIFICITY HAS THE CHEF RECOGNIZED THESE PARTICULAR IN/SSUL ATATORS? COULD IT BE THE RNAS? COULD THERE BE OTHER FACTORS AND I WILL TRY TO GET BACK TO THIS AT THE END, IF THERE IS TIME. SO I AM GOING TO MOVE /OON TO THE THIRD SECTION, WHICH IS THE SECOND RNA-BINDING NINE WE IDENTIFIED, WHICH IS CALLED SLUM SLUM. SO LUMP IS ACTUALLY THE HNR PN HOMO/SKPHROG IT AS WELL HAS ACTUALLY HAS THREE CONSERVED RNA RECOGNITION MOTIFS. SO THE REASON THAT WE STARTED LOOKING AT RUMP IN THE FIRST PLACE IS WE LOTOOK A CANDIDATE A APPROACH BECAUSE IT HAD ALREADY BEEN SHOWN BY THE ADVIVISA LAB THAT RUMP BINDS THIS TRANSCRIPT AS CR 18054, WHICH WE WILL PULLED DOWN WITH THE IN/SSUL ATATOR COMPLEX. NOW WITH WE DON'T HAVE ANY EVIDENCE THAT THIS PARTICULAR R RNA IS ASSOCIATED EITHER WITH THE PROTEIN OR IN THE CONTEXT OF THE INSUL AATOR COMPLEX. AND RUMP NICELY HAD ALREADY BEEN CHARACTERIZED BY THE LAB AND SEEN AS AN IMPORTANT FACTOR IN THE LOCALIZATION. FIRST THEY SHE'D IT'S AN IMPORTANT FACTOR FOR THE LOCAL LOCALIZATION IN THE EARLY EMBRYO EMBRYO. BUT ALSO, INVOLVED IN THE LOCAL LOCALIZATION IN THE DENDRITE SENSORY NEWEURON. SO IT'S REALLY NICE TO HAVE A SET /OF VALID /A*EATION AVAILABLE THAT WE COULD USE. OKAY, THE FIRST QUESTION IS HOW DOES THIS RUMP AFFECT IN/SSUL ATATOR ACTIVITY? SO WE DECIDED TO ASSESS THE BLOCKING ACTIVITY USING THE A ASSAY. SO AGAIN, WE'RE LOOKING AT IN INCREASED -- INCREASE OF INSUL ATOR ACTIVITY ON THIS SCALE. SO, THIS FONT IS A LITTLE BIT -- HERE BUT -- OKAY. SO WE FIRST TESTED -- AGAIN THIS IS BACKGROUND AS /AA MODIFIER E1. SO IN /THIS PARTICULAR BACKGROUND BACKGROUND, THE IN/SSUL ATATOR ACTIVITY IS QUITE LOW, SO MOST OF THE SLIDES HAVE 0 SCALE. BUT WITH A RUMP MUTATION IN /TTHIS GENETIC BACKGROUND, ACTUALLY HAS STRONGLY INCREASED INSUL AATOR ACTIVITIES. AGAIN, INDICATING THAT RUMP IS AN /AAN /TATAGONIST OF ENHANCER- ENHANCER-BLOCKING ACTIVITY. AND THIS CAN ACTUALLY BE RESCUED WITH EXPRESSION OF THE GENOMIC COPY OF RUMP, WHICH AGAIN, DECREASES THE INSUL AATOR ACTIVITY ACTIVITY. AND WHAT ABOUT THE BARRIER A ASSAY? SO WE TESTED BARRIER ASSAY USING A /AOUUBIQUITOUS GAL 4 DRIVER, AGAIN NOT INSUL AATED LINES AT VERY LOW EXPRESSION AND DEPENDENT ON BOTH THE -- AND WHEN WE KNOCKED DOWN RUMP, WE SEE A STRONG INCREASE IN THE ACTIVITIES. AGAIN, CONSISTENT WITH THIS BEING AN /TTAGONIST OF THE ACTIVITY ACTIVITY. WHEN WE LOOKED IN THE CNS, WE ACTUALLY WERE SURPRISED TO FIND THAT WHEN YOU KNOCK DOWN RUMP, WE SEE NO CHANGE IN THE BARRIER ACTIVITY. THIS IS DIFFERENT FROM WHAT WE SEE WITH CHEF. FURTHER MOMORE, CONTRASTING WITH CHEF IS /THE FACT THAT WHEN YOU DO THIS ASSAY IN THE MUSCLE, WHEN YOU KNOCK DOWN RUMP, YOU GET AN /EUINCREASE IN THE BARRIER SO IT HAS THE OPPOSITE SPEC FIS FISITY. IT'S ANTAGONIZING IN THE MUSCLE BUT NOT IN THE RNF. CNS. WHEN WE LOOK AT INSUL AATOR BODY CONSISTENT WITH OUR RESULTS. SO WHEN WE LOOK IN EYE TISSUE, AN /EUIN/S*UL AATOR BODY WE GET THE WILD FOCUS AND WE HAVE MULTIPLE LOCI. IN RUMP 1 THIS IS A KNOWN MUTANT MUTANT, MODIFIER AND DOUBLE MUTE MUTEANT AND GET THE RESPIRATION BACK TO /TTHAT BODY. BUT THIS IS NOT SEEN IN THE BRAIN SO THE INSUL AATOR IN THE DOUBLE MUTANTS, THE BODIES REMAIN MORE NUMEROUS. OKAY. BUT STRANGELY, RUMP IS ACTUALLY /AOUBT LOIS EXPRESSED. UNLIKE CHEF, WHICH IS HIGHLY EN ENRICHED IN THE CENTRAL NERVOUS SYSTEM. WE DID FIND THAT RUMP DOES INTER INTERACT PHYSICALLY WITH THE PROTEINS WHEN WE TAKE EMBRYONIC NUCLEAR EXTRACT AND IT RUMP, WE CAN FOLD DOWN THE IN/SSUL ATATOR PROTEIN, WHICH IS NOT -- INAUDIB[INAUDIBLE]. SO THEY DO FORM SOME TYPE OF COMPLEX. AND WHEN WE DO THE PROFILE OF RUMP, WE CAN SEE THAT IT BINDS ONLY TO 300 OR SO SITES IN THE GENOME. BUT WHAT WE SEE IS REMARKABLE IS THE VAST MAJORITY OF THESE SITES OVERLAP WITH THE OTHER INSUL AATOR INSULATORS, AND WE CAN SEE THAT RUMP ACTUALLY LOCALIZES NICELY TO A VERY WELL-CHARACTERIZED BINDING SITE INSUL AATOR IN THE GENOME. OKAY. SO I JUST WANT /TO STOP HERE AND TALK ABOUT HOW WE THINK ALL THIS IS WORKING. OKAY. SO I TOLD YOU ABOUT THIS CHEF AN TAG NIST, WHICH SEEMS TO AN /TAG ANTAGONIZE, HIGHLY EXPRESSED THERE. I JUST TOLD YOU ABOUT RUMP, WHICH ANTAGONIZE IN NON-TISSUE. THIS STRANGELY IS EXPRESSED AT HIGH LEVELS FHERE. SO FOR SOME REASON IT'S NOT FUNCTION HERE. PERHAPS IT'S MISSING AN RNA OR SOME OTHER PROTEIN FACTOR OR MAYBE IT STILL TRANSLATES TO BE A MODIFIER. THESE ARE QUESTIONS THAT WE'RE TRYING TO ADDRESS. THEN I TOLD YOU IN THE FIRST PART ABOUT THE ASSOCIATION OF CERTAIN MESSENGER RNAS, IN /THIS CASE 190 CELLS TO PROMOTE INSUL ATOR ACTIVITY AND WE ACTUALLY FOUND THAT THIS ONLY WORKS IN NON-CNS TISSUE BECAUSE WHEN WE DID THE EXPERIMENT IN THE CNS, WE DON'T SEE ANY EFFECTS. SO WE CAN SEE THAT THERE SEEMED TO BE MULTIPLE LEVELS OF REGULATION OF THE ACTIVITY BY R RNAS AND RNA-BINDING PROTEINS IN DIFFERENT TISSUES AND YOU CAN IMAGINE THERE ARE COMMON INTER INTER/AOBACTIONS THERE. BUT BOTH POSITIVE AND NEGATIVE INTER/AOBACTIONS. OR RELATIONSHIPS. IN THE LAST FEW MINUTES I JUST WANT /TO TELL YOU ABOUT SOME OF OUR DATA AND KIND OF GIVE YOU A FLAVOR WHERE WE'RE GOING WITH THE CHEF STORY IN PARTICULAR. SO I TOLD YOU EARLIER THAT WHEN WE DID THE UV CONFLICTING STUDIES -- SO WE DID THE CROSS LINKING STUDIES OF NUCLEAR EX EXTRACT TO LOOK TO SEE -- WHETHER DIRECT INTER/AOBACTIONS EXIST WIN SUL AATOR FACTORS OF THE RNA BINDING PROTEINS. SO THE THEY WERE TREATED WITH U C AND THEN BOUND TO /THE -- WASHED WITH VERY HIGH STRINGEN E ENCY AND WHAT YOU CAN SEE IS THAT IN THE CROSS LINKS MATERIALS, WE NEVER SEE THE IN IN/SSUL ATATOR PROTEINS BUT WE CAN SEE CONTROL PROTEINS, WE CAN ALSO SEE THE PRESENCE OF MULTI MULTIPLE CHEF. SO AGAIN, WE THINK THERE HAS TO BE SOME KIND OF ADAPTER BINDING TO THE RNA, FOR THE IN/SUSUL ATATOR COMPLEXES TO ASSOCIATE WITH RNA. ONE THING THAT WE WOULD REALLY LIKE TO ADDRESS IS SO /TPFAR WITH OUR LOSS OF FUNCTION MUTANTS IS A TOTAL LOSS OF PROTEIN. BUT WE WANTED TO KNOW WHETHER R RNA BINDING ITSELF IS IMPORTANT FOR THE FUNCTION OF IN/SSUL ATATOR ACTIVITY. SO FAR WE'VE BEEN ABLE TO MAKE /THE POINT WITH MODELS ON OTHER /PHAO*UPBTS THAT HAVE BEEN PART OF THE LITERATURE LITERATURE. BUT NOT PROTEIN HOLDING. AND THEN ASSESS -- SO /TPFAR WE'VE BEEN ABLE TO ASSESS CERTAIN GENT GENETIC INTER/AOBACTIONS. SO NORMALLY WHEN YOU EXPRESS WILD TYPE CHEF IN /AA COMPLETELY NORMAL GENETIC BACKGROUND, THERE IS NO PROBLEM WITH THE EXPRESSIONS. BUT IF YOU OVEREXPRESS A WILD TYPE CHEF IN MODIFIER KNOWN MUTE MUTANT, YOU GET A COMPLETE LOSS OF VIABILITY. AND WHEN WE EXPRESS TO RNA MUTE MUTANT WHICH EXPRESSES AT THE SAME LEVEL AS THE WILD TYPE, AGAIN, WE GET 100% VIABILITY IN EITHER GENETIC BACKGROUND. SO IT'S ONLY TOXIC IN /THIS SITUATION WHEN THE RNA BINDING IS IN TACT. AND IN OTHER INTER/* GENETIC INTER/AOBACTIONS, THEY MENTION IS THE FACT THAT DOUBLE MUTATION IS MUTUALLY AS WELL. SO THERE IS A CLEAR GENETIC RELATIONSHIP, OR THERE IS A CLEAR IMPORTANT BALANCE OF THE LEVELS OF THE PROTEINS THAT SEEMS TO BE DEPENDENT ON RNA BINDING. SO WE'VE ALSO PERFORMED A NUMBER OF -- TESTS BUT ONE THING THAT I WANTED TO SHOW YOU IS THAT OUR R RNA IT SEQUENCING OF CHEF IN THE CNS DRIVES THE CELL LINE AND WE PULLED DOWN A HANDFUL TRANSCRIPTS BUT A COUPLE /OF THEM ACTUALLY TURN OUT TO BE TWO OF THE SAME TRANSCRIPTS THAT WE FOUND WITH THE IN/SSUL ATATOR COMPLEX SO THAT 555. WE COLLABORATED WITH T-- WHO ARE DOING A VERY LARGE STUDY LOOKING AT THE RNA BINDING SPEC /TPEIFICITY WITH A NUMBER OF PROTEINS ACROSS DIFFERENT SPECIES. THEY WERE ABLE TO IDENTIFY CHEF BINDING. SO THIS IS AN /EUINV VITRO ASSAY, WHERE YOU TAKE A RECOMBINANT PROTEIN AND MIX IT WITH A HUGE LIBRARY /OF RNAS AND THEN INVOLVE THE SPECIFIC BINDING ON MICROA MICROARRAY. WE WERE ABLE TO DO THAT FOR /AA COUPLE OF THE DIFFERENT ISOFORMS WITH CHEF. THEY ALSO VERIFIED THAT THIS MO MOTIF IS PRESENT IN OUR RNA IT, THE TRANSCRIPTS THAT WE PULLED DOWN BY RNA IT. AND THEN FURTHER MOMORE, WE FOUND THAT THERE IS -- WE FOUND A FRAG FRAGMENT OF CT 10555 WITH ONE PREDICTED BINDING SITE OF THESE BINDING SITES PRESENT AND WE WERE ABLE TO DO JUSTICE AFTER IT SHOWED THAT THE WILD TYPE CHEF PROTEIN BINDS TO THIS RNA WITH THE MUTE -- BUT THE MUTANT DOES NOT. OKAY. SO THIS IS JUST TO GIVE YOU AN IDEA OF WHERE WE'RE HEADING AND A BIT MORE DETAILED ANALYSIS OF TRYING TO UNDERSTAND HOW THE RNA BINDING CAPABILITIES OF A PROTEIN AFFECT THIN SUL AATOR ACTIVITY AND ONE THING THAT WE ARE REALLY EXCITED ABOUT -- THIS IS JUST /AA MODEL OF HOW CHEF MAY BE FUNCTIONING. ONE THING THAT I DIDN'T MENTION SO FAR IS THAT THE CHEF GENE ACTUALLY STANDS FOR ALLAN SHEP SHEPHERD, THE SAME AS THE RESTAURANT A/STKPRAUPBT IT WAS PULLED OUT OF SCREEN. SO CLEARLY, SHEP HAS IMPORTANT ROLES AS BEHAVIOR. SO WE'RE ACTUALLY NOW STUDYING SHEP COMPLETELY IN THE CONTEXT OF THE NERVOUS SYSTEM. AND I HOPE TO BE ABLE TO TELL YOU MORE ABOUT WHAT THE SPECIFIC FUNCTION IS IN THE NERVOUS SYSTEM BUT HOPEFULLY WE CAN TEST SOME OF THESE MODELS THAT WE USED TO HAVE -- THAT WE SUGGESTED. OKAY, I'LL STOP HERE. AND ACKNOWLEDGE THE PEOPLE WHO CONTRIBUTED TO THE WORK. SO THIS IS AN OLDER LAB. SO WE HAD A FAIRLY BIG TURNOVER, A COUPLE /OF YEARS AGO. SO A FORMER POST DOC IN THE LAB WHO CONTRIBUTED TO ALL THE PROJECTS. THE MAIN DRIVER OF THE SHEP PROJECT. MATT CANE, A FORMER POST DOC. HE ALSO GOT HELP FROM SU JUNE, WHO IS A FORMER POST DOC. ALL OF OUR COMPUTATIONAL ANALYSIS WAS DONE BY DALE. AND I JUST WANTED TO PLUG ONE OF HIS PACKAGES -- THERE ARE MANY PACKAGES THAT ARE AVAILABLE, WHICH IS CALLED MEDASEEK, WHICH WAS REALLY INSTRUMENTAL IN ANALYZING A NUMBER OF OUR GENOME GENOME-WIDE DATA /SK*ET AND EXPLORATION IF YOU HAVE SOME OF YOUR OWN. DATASET. SO I'D LIKE TO ACKNOWLEDGE OUR COLLABORATORS AND INVESTIGATORS FOR PROVIDING IMPORTANT -- I THANK YOU FOR YOUR ATTENTION AND I'LL TAKE ANY QUESTIONS. PLA [APPLAUSE] >> QUESTIONS. YES. >> CAN YOU GO TO THE MICROPHONE MICROPHONE? >> I'M ACTUALLY LOUDER WITH OUT WITH IT /* WITHOUT IT. >> SO VERY NICELY TALKED ABOUT. OKAY. SO FOR SHEP, FOR EXAMPLE, MOST OF ITS PROPERTIES SUGGESTS IT PLAYS A ROLE IN ANTI-INSULATEOR ACTIVITY. IS IT POSSIBLE OR IS THERE ANY EVIDENCE TO SUGGEST THAT IT MAY HAVE A DIRECT ROLE IN TRANSCRIPT RE/SRAOEUT /HRAOEVITALIZATION? >> THAT'S A GOOD QUESTION. WE DON'T HAVE ANY EVIDENCE TO THAT END. IN TERMS OF -- SO WE DID STUDY CAREFULLY ITS RECRUITMENT TO DNA -- SO IT DOES SEEM TO HAVE A TRANSCRIPTIONAL DEPENDENCE RE RECRUITMENT IN GENES. THAT'S PROBABLY SO FAR THE ONLY CLUE THAT WE HAVE. OTHER GROUPS HAVE STUDIED ITS ROLE WITH RESPECT TO PLACING, BUT THAT COULD BE POST TRANSCRIPTIONAL. BUT YEAH. SO FAR THERE IS NO CLEAR EVIDENCE TO /TTHAT END BUT THAT'S AN INTERESTING POSSIBILITY. >> I WAS JUST CURIOUS WITH THE T 7 TRANSCRIPTS APPROACH, YOU MENTIONED THAT YOU'D BEEN ABLE TO NARROW DOWN THE SEQUENCE. I WAS WONDERING IS IT IN THE CODING REGION? >> IT IS IN THE CODING REGION. YEAH. SO ARE NOT REQUIRED. >> IS THAT CODING REGION IMPORTANT FOR THE FUNCTION OF THE PROTEIN? I'M WONDERING WHETHER YOU COULD MUTATE IT. >> YEAH. SO RIGHT. THAT'S THE EVENTUAL GOAL IS TO GET REALLY HONED DOWN TO WHICH NUKE TYPES ARE IMPORTANT. THE ASSAY IS TAKING LOT OF YEARS AND POST DOC MANPOWER. BUT YEAH, EVENTUALLY -- SO I THINK -- SO EVEN -- IT IS IN THE CODING REGION. IT'S IN /AA CONSERVED REGION. WE LOOKED AT THE CONSERVATION THAT'S A PROTEIN AND SO THAT REGION IS CONSERVED ACROSS SPECIES. SO IT'S PROBABLY IMPORTANT FOR PROTEIN FUNCTION, BUT WE COULD MUTE ATE AND POTENTIALLY MAKE SILENT CHANGES IN RNA. THAT WOULD BE REALLY HE WILL /TKPWAPT BUT WE JUST HAVEN'T GOTTEN TO /TTHAT POINT YET. >> DID YOU LOOK TO SEE WHEN YOU OVEREXPRESSED, DID YOU ACTUALLY FIND IT ASSOCIATED WITH THE IN INSUL AATOR PROTEINS OR AT THE IN INSUL AATOR SIZE /* SITES IN THE CHROMOSOME? WHERE /* >> YEAH, WE TRIED REALLY HARD TO DO THAT. YOU CAN SEE THE TRANSCRIPT BY -- IN THE TISSUES BECAUSE WE'RE DRIVING SO MUCH OF IT AND WE HAVE THE TAG THAT WE CAN SEE IT AND WE SEE ONE BIG FOCUS WHERE THEY KIND OF GET STUCK. BUT UNFORTUNATELY, AS MANY TIMES AS I TRIED, I WASN'T ABLE TO CODE THE INSUL AATOR BODIES WITH THE TRANSCRIPT BECAUSE THE PROT PROTOCOLS ARE JUST NOT COMPATIBLE. AND BY OTHER ASSAY THAT'S WE HAVE TRIED, HAVEN'T SO /TPFAR HAD LUCK SEEING ASSOCIATION. SO WE DON'T TECHNICALLY KNOW FOR THESE TRANSCRIPTS THAT WE ARE ASSOCIATING WITH THE BODIES OR THE COMPLEX. IT IS POSSIBLE. MORE COMPLICATED POSSIBILITIES THAT THE TRANSCRIPTS ARE ACTUALLY ACTING AS SOME KIND OF DOMINANT NEGATIVE. SO THEY ARE INTERFERING WITH AN RNA BINDING PROTEIN THAT'S A NEGATIVE REGULATOR. IT'S JUST TOO COMPLICATED TO EXPLAIN. BUT THAT IS DEFINITELY POSSIBLE. >> SO ONE OF THE FACTORS THAT SEEMS TO REGULATE RNA, M /R-FPRNA LOCALIZATION OF CELLS IS POST TRANSLATIONAL MODIFICATION OF R RNAS. IS THERE ANY EVIDENCE THAT THE R RNAS THAT ARE ASSOCIATED WITH THESE REGULATORS ARE MODIFIED IN ANY WAY? >> YEAH. SO WE HAVEN'T ADDRESSED THAT AT ALL. IT IS POSSIBLE CERTAINLY. AND I THINK THAT'S REALLY A FAS FASCINATING QUESTION. SO /TPFAR WE DON'T HAVE THE TOOLS TO REALLY ADDRESS IT. I THINK THAT'S REALLY EXCITING. BUT JUST WOULD BE VERY TECHNICAL TECHNICALLY CHALLENGING TO ADDRESS. >> FIRST OF ALL /SKWR-RB, LET ME THANK YOU FOR /AA TERRIFIC SEMINAR. PLA[APPLAUSE] AND I KNOW SOME OF YOU ARE SHY SO IF YOU COULD STAY FOR /AA FEW MINUTES, YOU CAN COME UP AND SPEAK TO HER AFTER LUNCH. THANK YOU.