WE'LL START THE DNA REPAIR INTEREST GROUP. WELCOME TO THE SPEAKER, WELCOME TO ALL THE ATTENDEES. MY CO-CHAIR KEN KRAMER IS NOT ABLE TO BE HERE TODAY. SENDS HIS MANY REGARDS. I JUST WANTED TO MENTION IF YOU WERE NOT ON THE DNA REPAIR INTEREST GROUP LIST, YOU CAN SEND AN E-MAIL AND GET ON THE LIST WHICH HAS USEFUL INFORMATION ABOUT THINGS THAT RELATES TO DNA REPAIR AND MEETINGS THAT OCCUR AND THESE TALKS AND WE HAVE SOME INTERESTING TALKS LINED UP FOR THE NEXT FEW MONTHS IN FEBRUARY AND MARCH AND HOPEFULLY YOU CAN FIND TIME TO ATTEND THESE LECTURES. DAZE IT'S A GREAT PLEASURE TO HAVE JESSICA TYLER THE TALK IS NOVEL MECHANISMS REGULATING DNA END RESECTION AT DNA DOUBLE BREAKS AND PUBLISHED SIGNIFICANT AREAS IN THE AREA OF DNA REPLICATION AND CHROMATIN TRANSCRIPTION AND AGING, WHICH SHE SAYS SHE'S NOT GOING TO TALK ABOUT TODAY CAN WE'RE INTERESTED IN AND CURRENTLY A TENURED PROFESSOR AT THE DEPARTMENT OF PATHOLOGY AND LABORATORY MEDICINE AT WEILL CORNELL MEDICINE NEW YORK. AND BEFORE THAT SHE HAS BEEN AT A NUMBER OF OTHER IMPORTANT PLACES IN THE COUNTRY IN CALIFORNIA, IN COLORADO AS ASSISTANT PROFESSOR. SHE'S BEEN IN TEXAS BEFORE SHE ENDED UP FOR NOW AT CORNELL AND COMES FROM THE U.K. SHE'S DONE A BACHELOR OF SCIENCE IN SHEFFIELD AND YOU WON A HAND SOME PRICE AT THAT UNIVERSITY. YOU'RE ALREADY AS A YOUNG PERSON QUITE ACCOMPLISHED. AND INTERESTING SHE MENTIONED IN THE CITY OF TRENT, THERE'S A VERY ABOUT ENVIRONMENT FOR GETTING INTO SCIENCE TO CHROMATIN STRUCTURE RELATED THINGS IT. AND WE STARTED A DISCUSSION ABOUT WHAT THAT MIGHT BE. ONE OF THE THINGS IS A FAMOUS PLACE FOR MANY BREWERIES AND WE DON'T KNOW IF THAT AFFECTS US BUT THAT'S A QUESTION FOR THE FUTURE. THANK YOU FOR COMING AND THANK YOU FOR TALKING TO US AND NOW I'LL LET YOU SPEAK, JESSICA, PLEASE. >> ALL RIGHT, THANK YOU FOR JOINING US. THANKS TO KEN FOR THE OPPORTUNITY TO PRESENT AND WILL FOR THAT NICE INTRODUCTION. SO DNA DOUBLE STRAND BREAKS ARE ONE OF THE MOST DELETERIOUS TYPES OF DNA LESION. WE KNOW THAT IN EUKARYOTES THEY'RE CARRIED BY TWO PATHWAYS, NON-HOMOLOGOUS ENZYMES WHERE THE ENDS OF THE DNA ARE SUCK BACK TOGETHER BY DNA LIGASE 4 AND THE OTHER AS MORE END PROCESSING. INCLUDING RESECTION OF THE DNA ENDS FOR SHARING THEM BACK TO SINGLE STRAND OVERHANGS WHICH THEN FOUND IN THE HOMOLOGOUS RECOMBINATION. WE'RE INTERESTED IN CHROMATIN IN THE EPIGENETIC REQUIREMENT REGULATES DOUBLE STRAND BREAKS. WE AND OTHERS HAVE SHOWN CHROMATIN IS DISASSEMBLED TO ALLOW H.R. TO OCCUR AND THIS IS MEDIATED BY ATP HISTONE CHAPERONES AND THEY GET REASSEMBLED AFTER THE PATHWAY COMPLETE TO RE-ESTABLISH THE CHROMATIN ENVIRONMENT AND TO ALLOW RE-ENTRY INTO THE CELL CYCLE. RECENTLY, WE'VE BECOME INTERESTED IN DNA END PROCESSING BECAUSE IT'S A CRITICAL PATHWAY THAT DETERMINES PATHWAY CHOICE FOR DNA REPAIR. THIS IS RELATED TO THE CELL CYCLE DIFFERENCES IN DNA DOUBLE DOUBLE-STRAND BREAK REPAIR WHERE IT CAN HAPPEN IN MOST PHASES OF THE CELL CYCLE THOUGH INEFFICIENT IN N PHASE AND HR ONLY OCCURS IN S PHASE IN G2 AFTER REPLICATION. AND THE MAIN DECISION POINT BETWEEN WHETHER THEY'RE USED REPAIR DOUBLE STRAND BREAKS IS THIS END PROCESSING THAT OCCURS. TO IF IT'S EXTENSIVE END PROCESSING THE DOUBLE STRAND BREAKS IS COMMITTED TO REPAIR. WE'RE INTERESTED IN HOW END PROCESSING IS PREVENTED IN G0 AND G1 PHASED CELLS. WE KNOW FROM WORK OF MANY OTHERS IN THE FIELD THE IT PROTEINS 53BP1 AND THE SHIELDING COMPLEX PLAY AN IMPORTANT ROLE IN BLOCKING EXTENSIVE END RESECTION IN G1 PHASE. IF THERE IS EVER AN END RESECTION IN G1 OR G0 PHASED CELLS THEY BLOCK END JOINING AND WOULD NECESSITATE HOMOLOGY DEPENDENT REPAIR AND IF REGIONS OF HEMOLOGY ARE IN THE SAME CHROMOSOME IT LEADS TO CHROMOSOMAL DELETIONS OR IF IN A DIFFERENT IT CAN LEAD TO TUMOR GENESIS. AND WE'VE ABOUT READY IN G0 PAYSED CELLS FOR SEVERAL REASONS. MOST THE CELLS IN YOUR BODY AS YOU SIT THERE ARE IN G0 AND SECONDLY, WE KNOW VERY LITTLE ABOUT DNA END PROCESSING REGULATION IN G0 CELLS. SO TO START OUR ANALYSES, WE ADOPTED USING TRANSFORMED PRE B CELLS BECAUSE THEY CAN BE INDUCED EFFICIENTLY TO ARREST IN WHAT WE NOW KNOW AS G0. AND SO YOU CAN ADD STI TO PRE B CELLS AND THE RESULT IS AN EFFICIENT ARREST WITH THE TWO END DNA CONTENT AND THE WORK WAS DONE BY A TALENTED POST-DOC AND HE IS NOW AN ASSISTANT PROFESSOR AT THE UNIVERSITY OF ALABAMA IN BIRMINGHAM MEDICAL SCHOOL AND IF YOU'RE LOOKING POR A GREAT PLACE TO HAVE GRADUATE STUDENTS I RECOMMEND BO BECAUSE HE'S A GREAT MENTOR WITH GREAT IDEAS. BO FOUND THE TWO-END CELLS ARE IN G0 NOT G1 PHASE BECAUSE THEY LACK PHOSPHORYLATION OF CDK4 AND LACK OF DIAGNOSTIC OF G0 CELLS. SO HE TAKES THESE IT G0 ARRESTED CELLS, IRRADIATES THEM AND PERFORMS AN ASSAY DEVELOPED 10 YEARS AGO IN STEVE JACKSON'S LAB WHERE YOU TREAT THE CELLS TO EXTRACT THE SOLUBLE PROTEINS INCLUDING SOLUBLE RPA. THE ONLY REMAINING RPA IS FOUND IN SINGLE STRAND IN THE CHROMATIN. THEN YOU ADD ANTIBODIES TO RPA AND SECOND FLUORESCENT CELLS AND HERE WE DEPLETED THE CELLS AND FOLLOWING IRRADIATION YOU GET LOTS OF RPA BOUND CHROMATIN OR CHROMATIN BOUND RPA COMPARED TO IRRADIATION IN RED. NOW, WHEN 53BP1 IS PRESENT ON THE LEFT, YOU SEE NO SIGNIFICANT CHROMATIN BOUND RPA AFTER IRRADIATION AND POST THE EXPERIMENTS ARE PERFORMED IN CELL LINES THAT LACK LIGASE 4 AND ALLOWS FOR ACCUMULATION OF DNA ENDS. WE SEE THE SAME RESULTS IN WILD TYPE CELLS BUT MORE EXTENSIVE AFFECTS IN CELLS LACKING DNA LIGASE 4. WE DECIDED TO DO A CRISPR SCREEN TO IDENTIFY ADDITIONAL GENE PRODUCTS THAT PREVENT END PROCESSING IN G0 CELLS. HE TOOK THE CELLS AND INTRODUCED CAS9 AND THIS INDUCES THE FLAG IN ALL THE CELLS AS SHOWN BY THIS BLUE PEAK HERE FOLLOWED BY ARREST IN G0 IRRADIATION AND THEN CELLS SORTING FOR THE CELLS THAT HAVE THE MOST CHROMATIN BOUND RPA THE RPA HIGH CELLS AND THOSE WITH THE LEAST CHROMATIN BOUND RPA THE LOW CELLS AND SEQUENCED THE GUIDE RNAs IN THE POPULATIONS AND THEN IT HE DEVELOPED AN RPA HIGH SCORE FOR EACH GUIDE RNA TOOKING THE NORMALIZED READS FOR HIGH AND LOW TO GET TE FOLD ENRICHMENT OF GUIDE RNA IN THE RPA HIGH CELLS. AND WE FOUND HIGH ENRICHMENT POR GUIDE RNAs AGAINST 53BP1 AND RIF1 AND CAN DETECT FACTORS THAT NORMALLY PROTECT ENDS IN G0 CELLS AND FOUND MANY OTHER INTERESTING HITS. TODAY I'M GOING TO TELL YOU ABOUT LIN 37 AND LIN 53 ENRICHED IN THE RPA HIGH CELLS AND THE COMPLEX. IN G1 AND G0 CELLS THIS DREAM COMPLEX HAS MANY COMPONENTS AND ACTUALLY FUNCTIONED AS A TRANSCRIPTIONAL REPRESSOR OF MANY IMPORTANT GENES AND REPRESSED BY DREAM IN ORDER TO MAINTAIN CELLS IN G0 AND G1 PHASE. SO LIN 37 AND LIN 52 CAME UP IN OUR COMPLEX SO WE WANTED TO VALIDATE THEM AND MADE CELL LINES LACKING LIN 37 AND YOU CAN SEE AFTER IRRADIATION BY THIS IT READ PEAK YOU SEE SIGNIFICANT AMOUNTS OF CHROMATIN BOUND RPA COMPARED TO EN BLACK BEFORE IRRADIATION. YOU SEE A COMPARABLE AFFECT COMPARED TO DEPLETION OF 53BP1 WHERE YOU DELETE NEITHER OF THESE YOU SEE TO SIGNIFICANT CHROMATIN BOUND RPA AFTER IRRADIATION AND THIS APPEARS TO BE GOING TO DNA REPAIR FOSI BECAUSE WE SEE THE DEPLETED CELLS SIMILAR TO WHAT YOU SEE IN THE ABSENCE OF 53BP1. AND ALSO WE SEE THE SAME AFFECT WHEN WE DEPLETE THE LIN 52 COMPONENT OF DREAM. WE SEE AFTER IRRADIATION THE CHROMATIN BOUND RPA AND THIS IS NOT AN OFF TARGET EFFECT TO THE RNA BECAUSE WE CAN COMPLEMENT THE BY ADDING A PLASMID EXPRESSING WILD TYPE LIN 37. AND FURTHERMORE WE KNOW IT'S NOT A MOONLIGHTING ROLE FOR LIN 37 AND 52 BECAUSE WE CAN ADD A VERSION OF LIN 37 THAT HAS A SPECIFIC MUTATION THAT PREVENTS INTERACTION WITH THE REST OF THE DREAM COMPLEX. IN THAT CASE IN LIN 37 DEPLETED CELLS WHILE THE WILD TYPE LIN 37 CAN LARGELY COMPLEMENT THE PHENOTYPE IS NOT THE CASE IN THE DREAM COMPLEX. THIS IS DUE TO LIN 37'S FUNCTION WITHIN THE DREAM COMPLEX. AND THE POTENTIAL END RESECTION IN GO CELLS COULD BE ENTRY THE CELL CYCLE WE KNOW IT'S MOT THE CASE LOOKING AT THE DNA CONTENT. THERE'S STILL IN G0 PHASE. NOW WE WANTED KNOW. WHAT'S THE END RESECTION OF G0 CELLS THAT OCCURS IN THE ABSENCE OF LIN 37 AND ACT IF THE CANONICAL FACTORS ARE INVOLVED AND THIS IS DATA FOR C TIP. THE END OF RESECTION YOU GET IS SHOWN UP GREEN HERE IN CELLS THAT ARE EITHER DEPLETED FOR 53BP1 OR LIN 37 BUTTON RED THE END RESECTION YOU SEE WHEN YOU ADDITIONAL ADDITIONALLY DEPLETE CTIP AND SAME IN THE CELLS LACKING LIN 37 AND CTIP AND THIS IS REQUIRES IN THE ABSENCE OF LIN 37 IN G0 CELLS. TO KNOW WHETHER THIS IS END RESECTION, THE ULTIMATELY PROOF IS PROVIDED BY END SEQ. THIS WAS A METHOD DEVELOPED SO WE COLLABORATED WITH A GROUP WHERE THEY PERFORMED END SEQ FOR US AND IN THE ABSENCE OF LIN 37 YOU SEE THE LONG STRETCHES OF RECESSION -- RESECTION AND YOU SEE THIS ONE BREAK BUT ON THE RIGHT I'M SHOW FOR THE 200 MOST EFFICIENT CUT SITES AND YOU CAN CLEARLY SEE UPON DEPLETION OF LIN 37 THE END RESECTION IS GOING UP FURTHER DISTANCES. THIS IS IN END ZERO CELLS IN THE ABSENCE OF THE IT END COMPLEX. THIS IS ALSO AND THIS IN THE ABSENCE OF LIN 37 AND 53BP1 GOING TO REPAIR AND THIS IS QUANTITATED ON THE RIGHT. FURTHERMORE, 53BP1 AND LIN 37 APPEAR TO BE ACTING IN DISTINCT PATHWAYS IN G0 CELLS BECAUSE THE RED PEAKS SHOW THE ABSENCE OF LIN 37 AND 53BP1 TOGETHER. ON THE LEFT SIDE ARE CELL LINES STABLY DEPLETED FOR 53BP1 IN GREEN IS THE EXTEND OF END RESECTION AND IN RED WHEN YOU ADDITIONALLY GET RID OF LIN 37 AFTER IRRADIATION YOU GET EVEN MORE END RESECTION. AND HERE ON THE RIGHT WE'RE SHOWING THE CELLS DEPLETED FOR LIN 37 STABLY AND TRANSIENTLY DEPLETE FOR 53BP1 AND THIS MOVES TO THE POSITION OF THE RED LINE WHEN YOU DEPLETE IT INDICATING 53BP1 ARE FUNCTIONING BY A DISTINCT PATHWAY FROM LIN 37 DREAM TO PROTECT DNA ENDS IN G0 CELLS. GIVEN DREAM IS TRANSCRIPTIONAL REPRESSOR WE WANTED TO FIND THE TRANSCRIPTS THAT GET REDUCED IN THE ABSENCE OF LIN 37 THAT MIGHT EXPLAIN THE PHENOTYPE. SO WE DID RNA SEQ AND IN RED IS THE IT TRANSCRIPTS THAT ARE SIGNIFICANTLY INCREASED OR DECREASED UPON DEPLETION OF DREAM AND THE ONES LABELLED HERE ARE FACTORS THAT PLAY ROLES IN HOMOLOGOUS RECOMBINATION AS IN BRCA 1 OR BLOOM OR HOMOLOGOUS RECOMBINATION SUCH AS BAR 1. WE WANTED TO KNOW ARE THEY ALSO INCREASED IN THE PROTEIN LEVEL IN LIN 37 DEFICIENT CELLS AND THESE ARE PRESENT AT LOW LEVELS IN G0 CELLS COMPARED TO THEY'RE ABUNDANCE AND THEN BECOME ABUNDANT AS IN CYCLING CELLS. WE WONDERED IF THE ABSENCE OF BRCA 1 CAN EXPLAIN THE LACK OF PROCESSING IN G0 CELLS AND WHETHER THEIR ABUNDANCE CAN EXPLAIN THE END PROCESSING CAN NOW OCCURS. WE DEPLETED BRCA 1 TRANSIENTLY WITH GUIDE RNAs IN CELL LINES LACKING 53BP1 WITH NO AFFECT AFTER RADIATION OR THOSE LACKING LIN 37 WHERE THE AMOUNT OF RPA CHROMATIN SHOWN AFTER LIN 37 DEPLETION AND RADIATION NOW MOVES TO THE POSITION IN RED WHEN WE ADDITIONALLY DEPLETE BRCA 1. THE IT CELLS LACKING LIN 37 IS DEPENDENT ON BRCA 1 IS DEPENDENT ON BAR 1 IN CELLS LACKING LIN 37 AS SHOWN BY THE RED LINE AND LARGELY DEPENDENT ON BLUE. SO WHAT I'VE SHOWN YOU SO FAR IS IN LIN 37 DEFICIENT G0 CELLS THE ABSENCE OF LIN 37 LEADS TO IT HIGH LEVELS OF ALL THESE PROTEINS SHOWN IN RED WHICH EXPLAINS THE END PROCESSING THAT WE SEE IN G0 CELLS IN THE ABSENCE OF LIN 37 AND ALSO LEADS TO RPA LOADING. SO GIVEN THE BRCA 2 AND BARD 1 ARE ABUNDANT IN G0 CELLS DO YOU GET SEQUENCE EVENTS SUCH AS RAD 51 LOADING. IN COLLABORATION WITH NCI WE SEE RAD 51 GOING -- [NO AUDIO] AS QUANTITATED HERE. SO WE WANTED TO KNOW IF THE ROLE OF LIN 37 UNIQUE TO G0 CELLS. CYCLING CELLS AND YOU CAN SEE BY WESTERN BLOTS THE CELLS WE ISOLATED HAVE BRCA 1, BARD 1 EXPRESSED AT DETECTIBLE LEVELS AND WE DON'T THINK IT LIN 37 IS EXPRESSING TRANSCRIPTION OF THESE PROTEINS IN G1 PHASED CELLS. ACCORDINGLY WHEN WE LOOK AT THE RPA BOUND CHROMATIN AS AN INDICATION OF DNA RESECTION IN G1 PHASED CELLS WE SEE RESECTION IN CELLS THAT LACK 53BP1 AND SEE MINOR EFFECT IN CELLS LACKING LIN 37 AND WE DON'T THING IT'S PLAYING A ROLE IN END RESECTION IN G1 PHASED CELLS AND SEE THE SAME RESULT IN G2 CELLS. SO WE THINK DREAM IS SPECIFICALLY PROTECTING DNA ENDS IN G0 CELLS. WHAT I'VE SHOWN YOU SO FAR IN G0 CELLS THERE ARE TWO PATHWAYS OF END PROTECTION. THE CANONICAL SHIELD PATHWAY AND IF YOU LOSE HIS PATHWAY YOU GET END RESECTION BY CTIP AND RPA LOADING BUT YOU DON'T GET SUBSEQUENT STEPS SUCH AS RAD 51 LOADING. BUT WE FOUND THERE'S ALSO AN YOU GET EXPRESSION OF RAD 51 IN THE ABSENCE OF DREAM WHICH THEN LEADS TO RAD 51 LOAD. A REMAINING QUESTION IS DO YOU GET COMPLETION OF HR LEADING TO EVENT SUCH AS CHROMOSOMAL TRANS LOCATION IF THE ABSENCE OF LIN 37 DREAM IN G0 CELLS. AND WE FIND IT TO BE THE CASE IN 10A CELLS. IT'S NOT A WEIRD PHENOTYPE SHEEN IN THESE CELLS. SO WE'LL TAKE A PAUSE NOW BECAUSE THIS IS THE END OF THE FIRST PART OF MY TALK. AND WHAT I HAVEN'T TOLD YOU IS EVERYTHING I'VE SHOWN YOU SO FAR IS LOOKING 18 HOURS AFTER IRRADIATION. YOU SEE IT IF YOU DEPLETE 53BP1 OR LIN 37 OR DREAM. WE FOUND A DIFFERENT PHENOTYPE WHEN WE LOOK AT EARLIER TIMES AFTER IRRADIATION IN G0 CELLS AND EVERYTHING IN THE SECOND PART OF THE TALK ARE PERFORMED AT THE EARLIER TIME SUCH AS THREE HOURS AFTER IRRADIATION. WHERE IN CELLS THEY HAVE 53BP1 AND HAVE DREAM. WHAT WE SEE IN RED IS WE SEE SIGNIFICANT ACCUMULATION OF CHROMATIN BOUND RAP INDICATING RESECTION IN G0 CELLS AT EARLY TIMES AFTER IRRADIATION. SO A TALENTED POST-DOC WANTED TO DETERMINE IS THIS IT REALLY END RESECTION HAPPENING IN G0 CELLS BECAUSE THIS HAS NEVER BEEN REPORTED BEFORE AND IF SO, WHAT IS THE MECHANISM FOR THIS END RESECTION. FIRSTLY, WE WANTED TO KNOW IT IT THIS ONLY SEEN IN CELLS WHERE WE ARE SENSITIZING THE ASSAY CHROMATIN BOUND RPA SUGGESTING THE RESECTION IN FORMAL G0 CELLS SOON AFTER IRRADIATION. THIS ISN'T JUST A UNIQUE PHENOTYPE IN THE CELLS BECAUSE IN WILD TYPE MCF10A CELLS WE SEE INCREASED CHROMATIN BOUND RPA AND THINK IT'S A PHENOMENA IN G0 CELLS. THE RPA ASSOCIATING WITH THE CELLS ARRESTED SOON AFTER IRRADIATION IS GOING TO REPAIR FOCI AND THE DOTS QUANTITATE AFTER IRRADIATION. TO ASK IF IT'S GOING TO THE DNA BREAKS A LAB KINDLY PERFORMED RPA TIP SEQ AROUND THE ENDONUCLEASE SITES. YOU SEE TIS NICE STRANDED ACCUMULATION OF RPA AROUND THE ASIS1 SITES AND YOU GET THREE OVERHANGS ON EITHER SIDE DURING END RESECTION. THIS IS SHOWING TWO ASIS1 SITES AND THE 200 MOST EFFICIENTLY CUT SITES WHERE YOU SEE RPA ACCUMULATING AROUND THE DOUBLE-STRAND BREAKS IN THE G0 CELLS SOON AFTER DNA DAMAGE. BUT THE ULTIMATE PROOF OF END RESECTION IS END SEQ. SO ANDREI'S GROUP PERFORMED END SEQ IN THE G0 CELLS SOON AFTER INDUCING ASIS1 AND YOU SEE THE TRAILS OF END RESECTION BORN ON EITHER SIDE OF THE BREAKS. THIS IS FOR THE 200 MOST EFFECTIVE CUT SITES AND WE'RE SEE END RESECTION IN G0 CELLS. THE G0 CELLS HAVE 53BP1 AND DREAM YET WE'RE STILL SEEING ENDS RESECTION. WE WANTED TO FIGURE OUT THE END RESECTION AND DEPLETE HEAD CANONIC CANONICAL MRE11 AND USED BULK INFECTION SO IT'S HARD TO GET A COMPLETE DEPLETION OF THE FACTORS AND YET YOU'LL SEE IT RED LINES ARE MOVING CLOSER TO THE BLACK LINE WITHOUT IRRADIATION AS XIRD TO THE CONTROLS FOLLOWING DEPLETION OF MRE11 OR DEPLETION OF C IT IS TIP. IT APPEARS THE CANNONICAL FACTORS AFFECT THE G0 CELLS. WE THOUGHT THIS WAS A GREAT SITUATION TO DO ANOTHER CRISPR CAS 9 SCREEN TO FIND ADDITIONAL FACTORS THAT ARE REGULATING THIS IT END RESECTION TA OCCUR IN G0 CELLS AND A SCREEN WAS DONE TWO HOURS AFTER IRRADIATION INSTEAD OF 18 HOURS AFTER IRRADIATION AND SORTS FOR THE CELLS THAT IS LOW AMOUNTS OF RPA BOUND CHROMATIN WHICH HAVE DECREASED RESECTION OR DECREASED RPA BINDING TO THE CHROMATIN AND HIGH LEVELS WITH INCREASED RESECTION AND SAW INTERESTING FACTORS IN THE SCREEN AND LOOK AT THOSE THAT WERE ENRICHED IN RPA LOW CELLS AND AS PROOF OF PRINCIPLE WE GOT ENRICHMENT FOR FIVE DIFFERENT GUIDES AND SAW GUIDES WHICH EXPRESSES A COMPONENT OF THE MRM COMPLEX WHICH MEDIATES RESECTION. SO WHAT ABOUT THE THIS UNEXPECTED FACTORS? WE FOUND ALL THREE SUB UNITS OF THE DNA COMPLEX. WE FOUND GUIDES AGAINST THESE IT THREE GENES BEING HIGHLY ENRICHED IN THE RPA LOW CELLS AND END PROTECTION IN G1 PHASED CELLS. SO IT NORMALLY PROTECTS DNA ENDS YET WE'RE SEEING IMPLICATED IN DNA END PROCESSING IN OUR SCREEN. SO IT'S IMPORTANT TO VALIDATE THESE HITS AND FIRSTLY WE LOOKED AT DNA PKCS THE CATALYTIC SUB UNITS AND MADE AN ENCODING GENE PRKKC AND IN RED IS THE AMOUNT OF END RESECTION YOU SEE AFTER IRRADIATION AND IN GREEN IS THE AMOUNT OF DNA END RESECTION YOU SEE AFTER IRRADIATION WHEN YOU DEPLETE DNA PKCS AND INDICATING IT'S CONTRIBUTING TO THE RESECTION OF THE G0 CELLS AND SEE AN INCREASED AMOUNT WITH THE DNA KCS AND IT HAS REDUCED END RESECTION WHEN WE INHIBIT DNA PK. ALSO THE RPA GOING TO CHROMATIN AFTER IRRADIATION IS OBLITERATED WHEN WE INHIBIT DNA PK AS QUANTITATED HERE. SO WE WANTED TO LOOK AT THE AFFECT OF END RESECTION WHEN WE INHIBIT DNA PK. THE GROUP PERFORMED END SEQ AND THE END RESECTION TRAILS THAT WE SEE FLANKING THESE WOULD ASIS1 SITES ARE OBLITERATED AGAIN WHEN WE INHIBIT DNA PK. YOU DON'T GET THIS PROXIMAL END PROCESSING. AND WHAT YOU SEE WHEN WE LOOK AT 200 TOP CUTTING ASIS1 SITES AGAIN YOU CAN SEE THE INHIBITION OF DNA PK GREATLY REDUCES THE PROCESSING THAT OCCURS AROUND THE ASIS1 SITES AT EARLY TIMES AFTER DNA CATH. AND WANTED TO SEE IT WAS A MOONLIGHTING ROLE OR DOES IT ATTACK THE ENTIRE DNA COMPLEX. THIS TIME WE GOT RID OF KU 70 AND INTRODUCED BULK INFECTION IN THE CELLS AND THIS IS SHOWN BY THE ORANGE LINE AFTER IRRADIATION WHICH BASICALLY MOVES ON TOP OF THE NO IRRADIATION LINE WHICH INDICATES THAT KU 70 FOR THE DNA PROCESSING IN G0 CELLS AS COMPARED TO THE RED LINE WHICH IS THE PROCESSING YOU SEE WHEN KU 70 IS PRESENT. THIS IS A ROLE FOR THE INTACT DNA COMPLEX IN END PROCESSING. SO NOW WE'RE GOING TO LOOK AT FACTORS THAT CAME UP IN THE RPA HIGH CELLS. THESE ARE PRO-DNA END PROTECTION FACTORS AND FOUND EXPECTED FACTORS INCLUDING 53BP1 AND SHIELDING AND RIF1 KNOWN COMPONENTS THAT PROTECT DNA ENDS AND CLEARLY DOING THIS IN G0 CELLS. THE STRONGEST HIT BY FAR WHERE WE SAW REALLY HIGH ENRICHMENT OF ALL FIVE RAPs WAS FOR THE THE RECOGNITION COMPLEX FOR THE COMPLEX AND IT'S BEEN SHOWN TO UBIQUINATE THE ENDS AND IT SUGGESTS IT HAS A ROLE IN PRO DNA AND PROTECTION AND MADE THEM STABLE AND COMPARED TO THE AMOUNT RESECTION IN SEE IN RED AFTER IRRADIATION, WHEN WE ADDITIONALLY DEPLETE THE CELL IN GREEN WE SEE A HUGE INCREASE IN DNA END RESECTION AFTER IRRADIATION SUGGESTING IT NORMALLY HELPS TO REGULATE THE END PROCESSING IN G0 CELLS AND WANT ASK IF IT'S THROUGH END REGULATION AND DEPLETE THE THIS DNA PKCS IN CELL LINES DEPLETED OF FBXL12 INDICATING THE RESECTION IN THE ABSENCE OF FBXL12 IT AFTER IRRADIATION MOVES TO THE BLUE AND SUGGESTING IT'S THROUGH THE SUB UNIT AND ADDITIONALLY APPEARS TO BE REQUIRING HE KU SUB UNITS BECAUSE WHEN WE MADE A CELL LINE LACKING KU 70 IN ADDITION TO DEPLETION OF FBXL12 WE SEE AFTER IRRADIATION, THIS GREEN LINE MOVES TO THE BLUE LINE UPON DEPLETION OF KU 70. SO WHAT I'VE SHOWN YOU SO FAR IS THAT THERE IS DNA END RESECTION OCCURRING QUITE EXTENSIVELY IN G0 CELLS AND IT'S REGULATED BY FBXL12. IN THE ABSENCE OF DNA PK WE SEE NO RESECTION AFTER DAMAGE IN G0 CELLS AND IN THE ABSENCE OF FBXL12 WE SEE MORE EXTENSIVE DNA RESECTION SUGGESTING IT NORMALLY PLAYS AN IMPORTANT ROLE IN HELPING REMOVE KU FROM DNA ENDS TO REGULATE HIS END RESECTION IN G0 CELLS. NOW WE WANTED TO KNOW IS THIS PATHWAY OF END RESECTION AT EARLY TIMES AFTER DAMAGE SPECIFIC G0 CELLS. THIS IS THE DATA I SHOWED YOU EARLIER IN G0 ARRESTED CELLS AND THE END RESECTION IS GREATLY REDUCED AND WE ALSO IN ARRESTED CELLS IN G2 PHASE AND FOUND THE END RESECTION BY END SEQ IS NOT AFFECTED BY DNA PK. AND THIS RESECTION DOESN'T APPEAR TO HAPPEN IN G2 CELLS AND WHEN WE LOOK AT THE TRACK LENGTHS IN THE G0 CELLS, YOU SEE THE TRACK LENGTHS IN MOST CELLS IS SEVERAL THOUSAND KILO BASES AROUND THE ZERO STRAND BREAK AND WHEN YOU GO EIGHT HOURS AFTER THE INDUCED BREAK YOU SEE GREATER LENGTH OF RESECTION DEPENDENT ON DNA PK AND IN THIS WE SEE THE END RESECTION OCCURRING THAT'S IN MINIMAL DEPLETION. WE ISOLATED G1 CELLS FROM CYCLING POPULATIONS AND ADDITIONALLY DEPLETED THE CATALYTIC SUB UNIT OF DNA PK AFTER IRRADIATION AND THE G LINE IS THE AMOUNT OF DNA RESECTION YOU SEE SOON AFTER DAMAGE AND THE GREEN LINE IS THE REDUCTION YOU SEE WHEN YOU DEPLETE THE G CELLS AND HERE YOU SEE AN INCREASE IN RESECTION CONSISTENT WITH THE ROLE THAT'S BEEN REPORTED IN PROTECTING DNA ENDS IN G2 ARRESTED CELLS WHEREAS IN G1 CELLS THERE'S NO AFFECT OF DEPLETING THIS AND WE SEE THE SAME FOR KU 70. THE EXTEND OF END RESECTION GREATLY GOES DOWN IF THE G0 ARRESTED CELLS AND INCREASES IF THE ARRESTED CELLS AFTER DAMAGE AGAIN BECAUSE OF HE ROLE IN PROTECTI PROTECTION IN G2 CELLS AND TO AFFECT IN THE G1 CELLS. THERE'S END RESECTION OCCURRING SOON AFTER DOUBLE STRAND BREAK INDUCTION SPECIFICALLY IN G0 CELLS BUT NOT IN THE G1 OR G2 PHASED CELLS THIS IT EARLY END RESECTION OCCURS THE COMPONENTS OF DNA PK AND REGULATED BY FBXL12 AND WE HAVE SHOWN AFTER DNA BREAKS AND THE FIRST PART OF THE TALK THERE IS NO DETECTABLE END RESECTION. PRESUMABLY THIS END RESECTION RESOLVES POTENTIALLY BY GAP FILLING ALLOWING COMPLETION AND DON'T THINK THE IS LEADING TO HOMOLOGOUS RECOMBINATION. MANY QUESTIONS REMAIN WHICH I CAN'T SEE BECAUSE MY NAME IS OVER THIS BECAUSE THE QUESTIONS ARE HOW IS THIS END RESECTION OCCURRING BY DNA PK AND WHY DO YOU HAVE THIS END RESECTION OCCURRING SPECIFICALLY IN G0 CELLS TO LEAD TO NHEJ? WE CAN SPECULATE THE ANSWERS TO THOSE QUESTIONS BUT I CAN ACKNOWLEDGE MY FANTASTIC TEAM. FAITH FOWLER CAN MOST THE WORK IN THE SECOND PART OF THE TALK AND WE RUN A JOINT LAB THIS IS NOW VIRTUAL WHICH IS QUITE EASY IN THESE TIMES OF COVID AND BO BO RUI CHEN DID MUCH WORK AND I'D LIKE TO ACKNOWLEDGE MANY AS PLAYING CRITICAL ROLES IN THE DATA I'VE SHOWN YOU TODAY AND WOULD LIKE TO ACKNOWLEDGE GENEROUS FUNDING BY NIH AND FINALLY END BY TELLING YOU ABOUT A CONFERENCE NOT YET ON THE DNA REPAIR E-MAIL FROM NIH THAT WE'RE ORGANIZING. FOR THOSE INTERESTED IN DNA REPAIR AND THE CHROMATIN ENVIRONMENT, THIS IS AN ASBMB ON THE INTERPLAY BETWEEN EPIGENETIC REGULATION AND GENOME STABILITY IN SEATTLE. IT WILL HOPEFULLY BE AN IN-PERSON MEETING IN THE FALL AND HAVE FANTASTIC SPEAKERS BUT THE IMPORTANT THING YOU NEED TO KNOW IS THAT MOST THE TALKS WILL BE CHOSEN FROM THE ABSTRACT. THIS IS A GREAT OPPORTUNITY FOR STUDENTS, TRAINEES AND P.I.s TO GET YOUR LATEST WORK PRESENTED ON PLATFORM SESSIONS. AND WITH THAT I WILL END MY SHARING AND GLADLY TAKE ANY QUESTIONS. >> OKAY, WELL, THANK YOU FOR A BEAUTIFUL TALK, JESSICA. THAT WAS EXCELLENT. >> THANK YOU. >> I'LL READ SOME QUESTIONS FOR YOU. I HAVE SOME MYSELF TOO. THE FIRST ONE HERE IS FROM CHRISTINA. WHY SHOULD SECTION OCCUR MAINLY IN G0 CELLS. WHAT ARE YOUR THOUGHTS ABOUT THAT? IS DSB REPAIRED DIFFERENTLY IN G0 AND CYCLING CELLS. IF YES, IS IT KNOWN THE MECHANISM BEHIND AND THE ADVANTAGE OF THIS DIFFERENCE. >> GREAT QUESTION. IT'S A QUESTION THAT WE'RE ABOUT TO WRITE AN NIH GRANT ON AND HOPEFULLY IT WILL BE FUNDED TO ADDRESS THAT. IT WAS UNEXPECTED TO FIND THIS. THERE MUST BE A DIFFERENCE IN REPAIR. WE HOPE TO LEARN WHAT THE DIFFERENCE IS. EVEN FOR NORMAL NEHJ YOU DO NEED END PROCESSING BECAUSE IT'S FACTORS LIKE SHIELD 2 THAT BINDS TO SINGLE STRAND DNA TO RECRUIT 53BP1 AND FACTORS LIKE HELB WHICH LIMITS BLOOM MEDIATED END RESECTION. YOU DO NEED SOME END RESECTION TO PROMOTE END PROTECTION FACTORS TO GET NHEJ AND WHY THERE'S MORE IN ZERO GOING OUT THOUSANDS OF BASE PAIRS IS A BIG QUESTION. WHETHER 53BP1 IS LESS EFFICIENT IN FILLING IN THE ENDS IN G0 IS ONE POSSIBILITY AND WE INHIBITED THE POLYMERASE THAT IT STIMULATE TO DO END FILLING. WE FIND GREATER EXTENSIVE RESECTION AND THERE'S A BALANCE BETWEEN END RESECTION AND FILLING IN THAT IN G0 CELLS IS TIPPED AT LEAST MORE TOWARDS THE END RESECTION BEING MORE EFFICIENT. AT LATER TIMES AFTER DAMAGE THIS THE END FILLING THE GAP FILLING SEEMS TO BE TIPPED TO BE MORE EFFICIENT. IT'S A KEY QUESTION AND IF YOU WANT TO JOIN MY LAB OR BARRY'S TO TRY AND ADDRESS THOSE QUESTIONS I CAN TELL YOU AN ANSWER IN THE. . >> I'LL READ THESE CHATS AND QUESTIONS AND YOU'LL GET THEM FROM US IN AN E-MAIL SO YOU CAN CONTACT SOME PEOPLE IF YOU'D LIKE OR THEY MAY CONTACT YOU FURTHER TO CONTINUE DISCUSSIONS. FROM GORDON, WHAT PRIMES RESYNTHESIS OF END SECTION ENDS TO ALLOW REJOINING. >> IT'S DEPENDENT ON PAL ALPHA AND WE SEE MORE DNA END RESECTION. SO THAT'S THE FIRST FACTOR WE'VE LOOKED AT. ALSO, IF YOU GET RID OF 53BP1 THAT STIMULATES THE GAP FILLING WE SEE ADDITIONAL END RESECTION. WE THINK IT MAY BE FOR THE USUAL PATHWAY THAT IS USED IN LIKE G1 AND G2. BUT POTENTIALLY IT'S LESS ACTIVE IN G0 CELLS. THAT'S SOMETHING WE NEED TO LOCK AT IF IT'S LESS ABUNDANCE OF THOSE FACTORS OF G0 CELLS. >> AND ASK THE END RESECTION IN NORMAL G0 CONDITIONS LEAD TO CHROMOSOME ABERRATIONS? >> BASICALLY IN G0 CELLS THERE'S NOT HIGHER LEVELS OF CHROMOSOMAL ABERRATIONS OR HOMOLOGOUS TYPE OF REPAIR. WE THINK THIS THAT INDICATES THAT IS NOT THE CASE. THAT'S WHERE 53BP1 WAS PRESENT. WHERE WE DEPLETE DREAM, AT LEAST LIN 37, WHEN YOU GET THE END RESECTION OCCURRING, WE'VE DONE HR ASSAYS USING A CONSTRUCT AND LOOKED AT THE EFFICIENCY OF GENE TARGETING TO GUIDE RNAs AND SEE A TWO TO THREEFOLD INCREASE IN GENE TARGETING WHEN WE DEPLETE DREAM. WE WERE HOPING FOR MUCH MORE AND IT SEEMS IN THE LIGASE 4 MUTANTS THERE'S NON-CANONICAL END JOINING BUT WITHOUT DEPLETING LIN 37 OR 53BP1 WE DON'T SEE MORE HOMOLOGOUS EVENT AND THINK THEY'RE STILL GOING THROUGH NHEJ. >> HERE'S A QUESTION. GREAT TALK. WHY DO THE CELLS STILL RESET THE ENDS AFTER EIGHT HOURS WHERE THEY SHOULD TRY TO REPAIR THEM? >> IT'S A GREAT QUESTION. THEY THAT'S THE END SEQ EIGHT HOURS. THE IT END SEQ WE DID THAT WAS 18 HOURS, WE DON'T SEE THE RESECTION. SO THERE'S A TIME POINT WHERE THERE'S EFFICIENT GAP FILLING MORE EFFICIENT THAN THE END RESECTION. EITHER THAT OR THE DNA PK STOPS RESECTING ENDS. SO I THINK WE NEED TO DO -- WE'LL FIGURE OUT THE MECHANISM FIRST OF HOW DNA PK IS DOING IT WITH MORE CAREFUL TIME COURSES. >> LET ME FOLLOW-UP BECAUSE I WAS THINKING ABOUT ASKING WHY DID YOU CHOOSE THE TIME POINTS. 18 HOURS IS NOT REPAIR COMPLETED BUT ALMOST COMPLETED. THREE HOURS IS VERY EARLY ON. HOW WAS THAT DECIDED SOME >> IT WAS EMPIRICAL. THE CELLS THAT WE'RE MAINLY USING ARE LIGASE 4 DEFICIENT. THEY'RE REALLY NOT GOING TO DO CANONICAL NHEJ. >> THIS IS A TIME POINT WHEN YOU THINK REPAIR IS COMPLETE. >> WE THINK IT'S COMPLETE IN WILD TYPE CELLS BUT NOT THE LIGASE 4 DEFICIENT CELLS. WE LOOKED AT A BUNCH OF TIME POINTS IN BETWEEN ONE, TWO, THREE, EIGHT, 12, 16. JUST CHOSE THE TIME POINTS WHERE WE SEE NO RESECTION AND THE MOST WHICH WAS TWO OR THREE. >> I WOULD THINK IT'S MOST COMPLETED. >> WITH LIGASE 4. >> REALLY BEAUTIFUL TALK. DID UP STREAM FACTORS CONTROLLING C TIP END RESECTION ACTIVITY IT I'M THINKING OF KINASE COME UP ON YOUR SCREENS? >> THANK YOU FOR THE COMPLIMENT. NO, THEY DID NOT. WE'RE INTERESTED IN THE PHOSPHORYLATION EVENTS REGULATING THESE PROCESSES. WITH THE LAB WE'RE DOING PHOSPHORYL PROTEOMICS AT THE G0 CELLS DEPLETED OF ATM INHIBITED FOR ATM OR FOR INHIBITION OF DNA PK. WE HAVE PHOSPHATE TARGETS TO LOOK AT THERE BUT WE DIDN'T GET KINASES IN OUR SKREN. >> DOES XO1 PLAY A ROLE? >> THEY PLAY A ROLE IN THE END RESECTION THAT OCCURS IN THE ABSENCE OF DREAM, AS I SHOWED YOU. WE LOOKED AT XO1 JUST THIS WEEK IN THE EARLY END RESECTION THIS OCCURS AFTER DNA DAMAGE AND IT IS DEPENDENT ON XO1. WE'VE DONE THE RPA ASSAY. THE AMOUNT OF RESECTION IS GREATLY REDUCED WHEN WE INHIBIT XO1 AT EARLY TIMES. >> IS THE ALT END JOINING PATHWAY UNDER CONDITIONS OF DEPLETION OF THE DREAM COMPLEX? >> GREAT QUESTION. WE HAVEN'T LOOKED AT ALTA. >> CAN I FOLLOW UP WITH THE DREAM COMPLEX YOU USED LIN 37 AS THE PART OF THE DREAM COMPLEX AND YOU MAKE A CONCLUSION BASED ON LIN 37. WILL OTHER COMPONENTS OF THE TE DREAM COMPLEX HAVE THE SAME AFFECT? HOW DO YOU KNOW IT'S ALL DREAM COMPLEX? >> GREAT QUESTION. SO ONLY GOT THOSE TWO COMPONENTS IN OUR SCREEN. THE KEY REASON WHY WE THINK IT'S THE WHOLE OF DREAM COMPLEX IS WE USED A MUTANT THAT CAN NO LONGER ASSOCIATE WITH THE DREAM COMPLEX AND THAT WAS NOT ABLE TO RESCUE THE PHENOTYPE. WE ALSO LOOKED AT LIN 52 AND A LOT OF THE OTHER COMPONENTS ARE INVOLVED IN A LOT OF DIFFERENT PATHWAYS WHEREAS LIN 52 AND 37 ARE SPECIFIC TO THE DREAM COMPLEX. WE DIDN'T LOOK AT ANYTHING LIKE RB OR E2. >> SO YOU DON'T THINK IT'S A SEPARATE FUNCTION? >> WE DON'T THINK IT'S A SEPARATE FUNCTION. >> CREAM COMPLEX IS ALSO INTERESTING IN AGING. JESSICA, NICE TALK. WE FOUND DNA PK PROMOTES C TIP DEPENDENT INITIATION OF RESECTION BUT IN OUR WORK WE SEE THAT HE HE IT DNA PK INHIBIT HAD HAD -- INHIBITS RATHER THAN PROMOTES. >> I'M FAMILIAR WITH THE WORK WHICH IS IMPORTANT TO REMOVE DNA PK TO GET THE IT END RESECTION. BEAUTIFUL WORK. ALL I CAN SPECULATE IS THE G0 CELLS ARE DOING SOMETHING DIFFERENT AND WE HAVE THE PROTEOMIC RESULTS FROM A LAB AND WE ARE NOW MUTATING SOME OF THOSE HITS TO SEE IF THEY PLAY A ROLE. WE DIDN'T SPECIFICALLY SEE NOVEL DNA PK PHOSPHORYLATION SITES OR SITES ON ARTEMIS OR THE N OF H HAD NHEJ FACTORS. >> IT COULD BE THEY'RE CLIPPED OFF AND DEGRADED. THIS IT HAPPENS IN YEAST. >> OKAY. YEAH. THE END SEQ WOULD NOT DIFFERENTIATE THAT. >> IS THERE A ROLE FOR PRO Q? I MIGHT HAVE MISSED THAT. >> WE FOUND A ROLE FOR IT. WE FOUND A DIFFERENT POLYP OUR SCREEN. I DON'T REMEMBER IF IT WAS POL Q. >> IN THE REPAIR OF THE RECEPTOR BREAKS? >> WE CAN'T GET REPAIR ALL THE WAY BECAUSE THERE'S NO LIGASE 4 BUT THAT'S KIND OF WHAT THE SCREEN'S MEASURING. I'LL E-MAIL YOU BECAUSE I'D LOVE TO HEAR WHY YOU THINK POL Q WOULD BE INTERESTING BECAUSE I THINK IT WAS A HIT ON OUR SCREEN. I THINK IT VALIDATED AS WELL. >> AND ASK YOU BRIEFLY ABOUT THE F6X PROTEIN PLAYING A ROLE IN THE UBIQUINATION BUT CAN IT PLAY A ROLE YOU FIND AS A TARGET. DOES IT DO OTHER THINGS? DOES IT AFFECT OTHER PATHWAYS. >> IT'S TOTALLY A POSSIBILITY. Q IS A MAJOR SUBSTRATE THAT'S BEEN IDENTIFIED. THE INCREASED RESECTION WE SEE WHEN YOU GET RID OF THE FBXL12 SHOWS THAT IT REMOVES KU FROM DNA ENDS. >> FINALLY HERE, NICOLE IS ADDING BECAUSE OF THE MMEJ IN THE QUESTION BEFORE. THIS IS AN ADDITION TO THE QUESTI QUESTION. >> HOW IT PLAYS A ROLE. >> ANYWAY, I THINK WE GOT THROUGH THE QUESTIONS. THANKS TO THE ATTENDEES FOR THE QUESTIONS. THANKS TO YOU FOR AN EXCELLENT TALK AND AGAIN YOU WILL RECEIVE THE QUESTION SO YOU CAN FOLLOW-UP IF YOU'D LIKE OR THEY CAN CONTACT YOU LATER. THANKS TO ALL. THANKS FOR ATTENDING THIS IT SESSION HERE AND SEE YOU NEXT MONTH.