>>WELCOME TO THE DNA REPAIR INTEREST GROUP VIDEO CONFERENCE SERIES. THOSE OF YOU WHO GOT OUR MESSAGE CAN JOIN THE LISTSERV BY SENDING E-MAIL TO KRAEMERK@NIH.GOV, AND THE OTHERS WILL KNOW THE UPCOMING MEETINGS. DR. BOHR, MY CO-HOST WILL BE SPEAKING ABOUT MID O CHONT RIA AND NEURODEGENERATION AND IN MAY, DR. DEIN, ARDO FROM ITALY WILL TALK ABOUT MULTIFACETED ROLES OF N. E. R. PROTEINS AND THEIR IMPLICATIONS IN HUMAN PATHOLOGIES. AND IN JUNE TOM KONKLE FROM NIEHS WILL SPEAK ABOUT EXTRINSIC PROOF READING OF THE DNA REPLICATION PROCESS. TODAY WE'RE HAPPY TO HAVE DR. RACHEL FLYNN HERE FROM BOSTON UNIVERSITY. SHE GREW UP IN NEW HAMPSHIRE AND GOT A LIBERAL ARTS DEGREE IN ST. MICHAELS COLLEGE IN VERMONT WHICH SHE TELLS US IS NEAR BURLINGTON AND SHE WENT TO THE UNIVERSITY OF--WHERE SHE'S STILL LIVING AND SHE WENT AND DID A POST DOCTORAL FELLOWSHIP AT THE MASS GENERAL AND AT HARVARD AND THEN, JOINED THE BOSTON UNIVERSITY IN THE DEPARTMENT OF PHARMACOLOGY AND MEDICINE AND SHE'S BEEN THERE EVER SINCE. SHE'S IN ADDITION A CO-DIRECTOR OF THE GENOME SCIENCE DEPARTMENT AT BOSTON UNIVERSITY AND NOW IS AN ASSOCIATE PROFESSOR IN PHARMACOLOGY AND MEDICINE IN THAT DEPARTMENT. SHE'S DONE SOME VERY NICE WORK ON TELOMERE BIOLOGY AND TODAY, SHE WILL SPEAK ABOUT DEFINING THE DNA DAMAGE RESPONSE IN ALT TELOMERES. >> TAKE IT AWAY, RACHEL. THANK YOU VERY MUCH INTERNATIONAL CLASSIFICATION ON ROUGH ATOM DUKS AND THE INVITATION TO COME AND TALK AND TELL YOU ABOUT THE WORK I'VE BEEN DOING IN MY LAB. SO I REALLY HAD A LONG STANDING INTEREST IN UNDERSTANDING HOW CELLS ACQUIRE DAMAGE, HOW THEY RESPOND TO DNA DAMAGE AND ULTIMATELY HOW THEY REPAIR THAT DNA DAMAGE. EARLY ON IN MY CAREER, I WAS REALLY FOCUSED ON THIS, THE WHOLE GENOME LEVEL AND I THINK OVER THE YEARS I'VE SORT OF EVOLVED THIS INTO UNDERSTANDING THE DYNAMICS OF THE DNA DAMAGE RESPONSE SPECIFICALLY AT TELEO MERRIC DNA AND SO TODAY I DON'T HAVE AN OUTLINE HERE BUT I WANTED TO GIVE YOU A FEW VIGNETTES FROM THE LAB IN OUR EFFORT TO COMBINE DEFINING THE GENETIC ALTERATIONS IN CERTAIN TYPES OF CANCERS AND HOW THAT AFFECTS THE DNA DAMAGE REPAIR PATHWAYS. AND SO DNA DAMAGE AT THE TELOMERE IS NOT UNLIKE ANY OTHER DNA DAMAGE, REPLICATION BREAKS, SINGLE STRANDED DNA, BREAKS AND ALL THESE INVOKE A DNA DAMAGE RESPONSE IN TELOMERES SO REALLY UNDERSTANDING THIS MECHANISM OF THE RESPONSE AND THE REPAIR IS SOMETHING THAT I BUILT MY LAB UPON. SO THE MOST OBVIOUS SOURCES OF DNA DAMAGE AT TELOMERES REALLY ARISE IN THE TELOMERE REPEAT THEMSELVES SO BEING THESE HEXAMORE TTA, GGO, G REPEATS, THEE ARE PRONE TO REPLICATION SLIPPAGE, THEY FORM SECONDARY STRUCTURES LIKE G-DUPLEXS AND THEY HAVE THIS SINGLE STRANDED DNA TAIL AT THE END AND THAT COMBINED WITHLET LINEAR NATURE OF THE CHROMOSOME AND THE TERMINAL POSITION OF THE TELOMERE ITSELF, REALLY RENDERS THIS REGION SUSCEPTIBLE AS RECOGNITION TO A DNA DOUBLE STRAND BREAK. SO AT TELOMERES THEY FORM A SECONDARY STRUCTURE REALLY FOLDING THAT SINGLE STRANDED DNA OVERHANG BACK INTO ITSELF, CREATING A LOCALIZED D-LOOP OR THESE ARE T-LOOPS AND THIS IS TO PROTECT THE END OF THAT CHROMOSOME FROM BEING RECOGNIZED AS A DNA DOUBLE STRABD BREAK. THIS DOESN'T HAPPEN ON ITS OWN. THE FORMATION OF THAT T-LOOP IS FACILITATED BY THE PROTEIN COMPLEX REFERRED TO AS SHELTERIN, IT'S A MULTISUBUNIT COMPLEX, MANY COMPONENTS INCLUDING DOUBT STRANDED DNA BINDINGS INCLUDING TRF 1 AND 2 EXPW PROTEIN POT 1. I WOULD SAY ABOUT 15 YEARS AGO, THIS COMPLEX REALLY EXPAND TO INCLUDE THIS NONCODING RNA TRANSCRIBING FROM ENDS REFERRED TO AS TELEMERRIC REPEAT CONTAINING RNA, AND MY LAB AND OTHER VS GONE ON TO SHOW THAT THIS LONG NONCODING RNA CONTRIBUTES TO THE DNA RESPONSE AT TELOMERES AND NOT SURPRISINGLY, WE NOTICED THAT THERE'S AN INCREASE IN THIS LONG NONCODING RNA TRANSCRIBED IN A SUBSET'VE CANCERS AND SO WHILE THIS HAS BECOME A FOCUS OF MY LAB, I WON'T BE TALKING ABOUT TARA TODAY, WHAT I'M MORE FOCUSED ON IS AS I MENTIONED THIS DNA DAMAGE RESPONSE TO TELOMERES AND SO, COLLECTIVE EFFORTS OF CHILDREN AND TARA, BEING TO PROTECT THE TELOMERES FROM MACHINE DAMAGE, AND MAINTENANCE AND PROMOTES THE ELONGATION OF TELOMERES VIA A NUMBER OF NECKANISMS AND SO THE MOST COMMONLY KNOWN MECHANISM RIGHT, BEING TELEMERACE AND THOSE ARE COMMON IN THE MAJORITY OF CANCERS AND PROMOTES VERMEN INFECTED VERSE TRANSCRIPTION. AND I WOULD SAY THAT THE--SORRY THE SLIDES WERE SLOW TO CHANGE THERE. SO IN ADDITION TO TELEMERACE, THIS ALTERNATIVE MECHANISM OF TELOMERE LENGTHENS THAT WE REFER TO AS ALT. THIS IS A PATHWAY FOR TELOMERE ELONGATION, SO WHILE THE VAST MAJORITY OF CANCERS AS A MENTIONED RELY ON THE TELEMERACE, SO ABOUT 85-95%--AND ACHIEVE REPLICATIVE IMMORTALITY. ABOUT 5-10% RELY ON THIS OLD MECHANISM OR ALTERNATIVE LENGTHENING OF TELOMERES, IT'S NOT DISTRIBUTED ACROSS ALL CANCER, SO BREAST CANCER DOESN'T HAVE 10%, LIVER DOESN'T HAVE 10%, THIS IS DISTRIBUTED QUITE UNEACHLY AND THE VAST MAJORITY OF CANCERS THAT RELY ON THEM ARE MEZENCAMALL, AND ENDOTHELIAL AND THESE PICTURES AS DEPICTED HERE ARE OFTIO SARCOMAS, GLUE MARIOUS O BLASTOMAS, AND ENDOCRINE TUMORS THESE CANCERS HAVE POOR 5 YEAR SURVIVAL RATES AND THE THERAPEUTIC MODALITIES FOR THEZ CANCERS HAVEN'T CHANGED IN 40 YEARS. SO I THINK IT WAS CLEAR EARLIER THAT HAVING A NEW REGULATED TELOMERE PATHWAY WOULD PROVIDE INSIGHT NOT ONLY INTO THE MECHANISM BUT ALSO IN THE OPPORTUNITY FOR THERAPEUTIC DEVELOPMENT AND SO ABOUT A 11 YEARS AGO, I THINK THE FIRST MUTATIONS WERE IDENTIFY INDEED THE CHROMATIN REMODELING GENES AND ATRX AND DAXX, THESE DISCOVERIES WERE MADE BY CHRIS [INDISCERNIBLE] IN THE LAB, HE'S DONE AN AMAZING JOB OF THESE COMPLEXES. DAXX, BINDS THE EFTHIMIOS OPEN VARIANT SPECIFICALLY H3 PBT 3 AND ATRX FACILITATES THE DEFINITION OF THESE REGIONS, INCLUDING TELOMERES, AND IN THE ABSENCE OR LOSS OF FUNCTION OF THESE PROTEINS, YOU GET LOSS OF H3.3 DEPOSITION, YOU GET CHROMATIN DECOMPACTION AND THIS IS BELIEVED TO PROMOTE MORE PERMISSIVE ENVIRONMENT FOR RECOMBINATION AT THE TELOMERES. SO WHEN I STARTED MY LAB, I WAS REALLY INTERESTED IN UNDERSTANDING THE BASIC MECHANISMS OF THE ALT PATHWAY, AND SO, AT THE TIME I STARTED MY LAB, THERE WERE VERY FEW RESOURCES AVAILABLE IN THE WORLD OF ALT. THERE WERE A FEW CELL LINES THAT WERE NOTE INDEED THE LITERATURE AND OF COURSE A FEW TUMORS BUT WE DIDN'T HAVE ANY OF OUR OWN AND SO I STARTED COLLECTING A NUMBER OF THESE CELL LINES AND TUMORS BECAUSE I WAS VERY CURIOUS NOT JUST ABOUT THE MECHANISM BUT ABOUT THE GENETICS UNDERLYING ALT. AND SO 1 OF THE THINGS THAT STRUCK ME WHEN I STARTED TO ACQUIRE THESE TUMORS IS I HAD NO IDEA WHETHER THEY WERE ALT OR TELEMERACE, AND SO I HAVEN'T LISTED ALL OF THEM HERE. THESE ARE THE 1S THAT ENDED UP BEING ALT POSITIVE. SO IN AN EFFORT TO CHARACTERIZE THESE TUMORS AND CELL LINES WE USE A NUMBER OF DIFFERENT ASSAYS, SO FIRST STARTING OFF WE CAN ANALYZE FOR TELE MERAISE ACE ACTIVITY AND WE CAN RULE OUT THAT THAT DO OR DO NOT HAVE THE ACTIVITY, WE LIKE TO CONFIRM ALT ACTIVITY AND WE HAVE TO DO THIS USING A DIFFERENT NUMBER OF ASSAYS AND THESE WILL BE RELEVANT THROUGHOUT THE TALK SO I HAVE JOUST OUTLINES THEM HERE. SO 1 OF THEM IS THE FORMATION OF 1 OF THESE WHAT WE CALL APB AND THESE ARE PML BODIES AND SO THAT IS WHEN THE TELEOR MERRIC PROTEINS CO LOCALIZE WITH THE LEUKEMIA PROTEIN, AND YOU CAN SEE, THEY GENERATE THESE COLOCALIZATION EVENTS AND THESE OCCUR IN ALT CELLS AND ALMOST NEVER IN TELEORDER OF MICRONSER ACE CELLS SO THIS IS A STRONG INDICATION OF ALT POSITIVITY IN A CELL OR TUMOR AND IN ADDITION TO THE APBs, I WOULD SAY THE GOLD STANDARD IN THE FIELD MAY BE IS TO ANALYZE THE COMA TELEO MERRIC REPEATS AND THESE ARE CIRCULAR SPECIES OF DNA, THESE 1S IN PARTICULAR ARE C-RICH AND THEY HAVE A PARTIAL DIEWP LEX REGION AND THIS DUPLEX REGION SERVES AS A PRIMER IF YOU USE THIS SUBSTRATE IN A ROLLING CIRCLE AMLIAISONIFICATION REACTION, CAN YOUASMLIFY THESE C-CIRCLE PRODUCTS AND IF YOU RUN THEM OUT IN A DOT BLOT AND PRESCRIBE, YOU CAN SEE THAT THE ALT CELLS HAVE A DRAMATIC INCREASE IN THE PREVALENCE OF THESE CIRCLES AND THIS IS IMAGED AND SORT OF A GOLD STANDARD OF ALT ACTIVITY IN CELLS AND TUMORS. AND THEN FINALLY, WE HAVE A TENDENCY TO ANALYZE THE ACTUAL EXCHANGE EVENTS WHICH HAPPEN AT TELOMERE EVENTS AND WE CALL THESE EXSCHAINCHS AND WE DO THIS BY PREPARING ENDS AND WE CAN ANALYZE THE ENDS USING PROBES, SO RED OR GREEN PROBES THAT RECOGNIZE THE ARM OF THE CHROMOSOME AND YOU CAN SEE WHERE THERE'S INDEPENDENT RED AND GREEN SIGNAL BUT WHEN THERE'S A REON COMBINATION, YOU GET AN OVERLAP OF THE PROBES AND YOU CAN SEE THESE ARE VISUALIZED AND THEY ARE OVERLAP NOTHING THAT COLOR SIGNAL SO ONCE I ESTABLISHED A PANEL OF ALT TUMORS AND CELL LINES IN THE LAB, I WAS CURIOUS ABOUT THE GENETICS SOY INITIATED A SIGNIFYQUENCING CAMPAIGN OF THESE ALT POSITIVE SAMPLES TO SEE IF WE COULD DEFINE ANY ADDITIONAL MUTATIONS THAT MAY BE CONTRIBUTING TO THE--PERCENT OF THESE TUMORS AND CELL LINES HAD THE MUTATIONS IN ATX. SO THERE'S A VARIETY OF DIFFERENT MUTATIONS AND BECAUSE OFTIO SARCOMAS ARE HIGHLY REARRANGED, SOME OF THEM WERE DEEP DELETIONS, INVERSION MUTATIONS BUT AGAIN 50% RIGHT OFF THE TOP HAD MUTATIONS ATRX. --PUBLISHED IN THE LITERATURE TO BE WILD TYPE FOR ATRX AND DAXX. BUT WHEN WE DID OUR INITIAL ANALYSIS, WHAT WE NOTICE SIDE LOOKING AT THOSE STRUCTURAL REARANKMENTS, WE COULD SEE A STRONG OR SIGNIFICANT TRANSLOCATION BETWEEN DAXX ON FIFTH YEAR AND KIPS-ON 16, SO WE LOOK TO CONFIRM THIS BY USING CARIO TYPING AND I THINK THIS IS IMEAN A BEAUTIFUL VISUAL REPRESENTATION NOT ONLY THE CHANGES IN EMPLOYEDY BUT THE INCREASES IN STRUCTURAL REARRANGEMENTS THAT ARE PREVALENT THROUGHOUT THESE OFTIO SARCOMA TUMORS AND WE CAN HIGHLIGHT HERE THE TRANSLOCATION WE IECIALIZED IN OUR SLOTS WHERE WE SEE THIS TRANSLOCATION BETWEEN CHROMOSOME 6 AND THE PINK AND CHROMOSOME 16 IN THE BLUE. SO WHEN WE TOOK A REAL BIT OF A DEEPER DIVE INTO THAT STRUCTURAL REARRANGEMENT, WE COULD LOOK AT THE DNA LEVEL AND WE COULD SEE THAT THIS DAXXKIFC3, HAD A BREAK POINT THAT WAS IN THE PRIME POINT OF DAXX, AND WHEN I PUBLISHED BEFORE THESE WERE WILD TYPE IN THE PREVIOUS CELLS, A LOT OF THESE WERE USING WHOLE EXOME SEQUENCING TO ANALYZE THESE TUMORS AND THIS WAS A LESSON IN WHY THIS IS DIFFICULT IN OFTIO SARCOMA, SOMETHING THAT'S HIGHLY REARRANGED BECAUSE IT WAS FULLY INTACT AND WHILED TYPE DAX JEANS AND THERE WAS NO EFFECT TO THEAXONS THEMSELVES BUT WHEN WE WENT TO ANALYZE THE RNA WE FOUND THAT IN FACT, THIS FUSION RESULTED IN A TRANSCRIPT THAT WAS ALTERNATIVELY SPLICED AND AS A CONSEQUENCE SPLICED OUT AXON 8. SO FOLLOWING AXON 7, DAXX WAS FUSED TOAXON 10 OF KIFC3. SO THIS TOLD US WE HAD A KHIHIGH MERRIC RIGHT GENE PRODUCT BUT LIKELY IF THERE WERE TRANSLATED WE MIGHT ALSO HAVE A KHIHIGH MERRIC PROTEIN AND THAT'S WHAT WE SAW SO IF WE LOOK AT THESE CONTROL CELLS WE CAN SEE WILD TYPE DAX PROTEIN HERE WE CAN OF COURSE KNOCK DOWN WITH SIRNA TO DAXX, AND YOU CAN ALSO SEE THIS HIGHER DAKS AND IF YOU USE DAKS WE CAN ALSO KNOCK DOWN THIS FIEWGDZ PRODUCT SO THIS SUGGESTED TO US THAT NOT ONLY WAS THERE A TRANSWELL ONICATION BUT IT MAINTAINS EXPRESSION OF A PROTEIN PRODUCT SO OF COURSE THE QUESTION BECOMES WELL, WHAT'S THE FUNCTION OF THIS PROTEIN OR IS IT A LOSS OF FUNCTION FOR DAXX. AND THAT DAXX, C-TERMINUS IS CRITICAL FOR A NUMBER OF DAXX FUNCTIONS, SO SPECIFICALLY REGIONS IN THAT AXON 8 WHICH IS ELIMINATED FROM THE TRANSCRIPT AND NOT PRODUCED AS PART OF THE PROTEIN. SO 1 THING WE LOOKED AT RIGHT WAIRKS WAS THE IDEA OF DAXX INTERACTIONS SO IT'S KNOWN TO INTERACT WITH THE PML. AND AS I MENTIONED IN THE PREVALENCE OF CAPSERS OR IN THE COLOCALIZATION OF TELEO PLEERS. SO WHEN WE LOOK AT THESE G2 FINE 2 CELLS, AND WE STAIN WITH A DAXX ANTIBODY WITH THE ENDOGENOUS PROTEIN, WE SEE LITTLE SIGNAL AND THE ANTIBODY DOES IN FACT RECOGNIZE A PART OF THE PROTEIN IT EXPRESSED, IT DOESN'T RECOGNIZE THE C-TERMINUS, SO WE SEE ALMOST NO SIGNAL FROM DAXX, BUT WE DO SEE COLOCALIZATION AS WE WOULD EXPECT TO INDICATE THE PRESENCE OF APB, SO WE DECIDED, CAN WE RESCUE THIS ALT STATUS OF THIS CELL AND REPRESS APB BY EXPRESSING WILD TYPE DAXX, SO WE DID THAT AGAIN BY EXPRESSING THIS FLAG TAGGED WILD TYPE DAXXFOR EITHER 2 OR SEX DAYS AND WHAT WE COULD SEE IS NOT ONLY DO WE START TO ACCUMULATE THE EXPRESSION OF THIS PROTEIN AND THESE, BUT YOU CAN SEE THEY ACCUMULATE WITH PML AS WOULD BE PREDICTED FOR NORMAL DAXXSPAY FUNCTION AND THESE FOCI NO LONGER CO LOCALIZE WITH THESE TELOMERES SUGGESTS THAT APB AND THESE WERE QUANTIFIED ON THE RIGHT AND THIS SUGGESTED THAT IN THE FACT IN THE CELL LINES, THIS DAXX KIFC3 FUNCTIONALLY ENACTIVATES DAXX. AND UNDERSTANDING THE MUTATIONAL PROFILE, BUT IF WE WANT TO DIG FURTHER AND CHARACTERIZE SOME OF THESE OTHER UNKNOWNS THAT WERE SORT OF GLARING AT THE TIME TO US, WE NEEDED TO REALLY DIG INTO THE MECHANISTIC DETAILS OF THE PATHWAY. AND SO I MENTIONED TO YOU EARLIER THAT THE ALT PATHWAY RELYS ON RECOMBINATION, THIS EVENT IS NOT UNLIKE OTHER TYPES OF RECOMBINATION FOR EXAMPLE IN A DOUBLE STRAND BREAK, WHERE YOU HAVE PROCESS THAGOREAN CREATES THIS 3 PRIME SINGLE STRANDED OVERHANG AND THIS 3 PRIME OVERHANG CAN INITIATE STRAND INVASION AND HOMOLOGYY SEARCH AND CAPTURE, IT DOESN'T HAVE TO BE A HOMOLOGOUS CHROMOSOME, ANY CHROMOSOME THAT HAS TELEOR MERRIC SEQUENCE, AND SOME OF THOSE PREETS I MENTIONED EARLIER. SO IN THESE INSTANCES YOU CAN ENVISION, YOU GET THE SMALLER CAPTURE AND EXTENSION AND TELOMERE SYNTHESIS, BUT WHAT I DIDN'T MENTION IS THAT IN ADDITION TO THE CANNONICLE GG8, THAT ARE PREULENT AT THE TELOMERE ENDS, WE'VE COME TO LEARN THERE IS THE INCORPORATION OF DEGENERATE OR VARIANT REPEATS AND SO INSTEAD OF TTA, GGG, THESE MAY CONSIST OF TTA, GGG, SO A SLIGHT VARIATION IN THE NUCLEOTIDE SEQUENCE, AND SO YOU CAN ENVISION NOW, IF YOU HAVE INITIATION OF THIS HOMOLOGY SEARCH AND CAPTURE AND A REGION OF HOMOLOGY THAT HAS DEGENERATE OR REPEATS DOWN STREAM, YOU CAN SEE HOW EXTENSION DNA SYNTHESIS AND MIGHT THROUGH THIS REGION MIGHT INCORPORATE VARIANT REPEATS THROUGHOUT THAT SEQUENCE. AND SO WHY DOES THAT MATTER? IT MATTERS BECAUSE AS I TOLD YOU BEFORE, THE SHELTER AND PROTEIN COMPLEX BINDS TO THESE WITH THESE SPECIFICITIES AND THESE BINDINGS PROTEINS, AND THESE VARIANT REPEATS LEAD TO DECREASE IN THE EFFICIENCY OF CHILDREN BINDING IT AT THESE ALT TELEOR MERES AND IF THIS IS THE CASE, YOU WOULD IMAGINE GIVEN THE ABNORMALITIES SENSZ OF SHELTERIN, THAT THIS WOULD INDUCE SOME LEVEL OF REPLICATION STRESS AND ULTIMATELY A DNA DAMAGE RESPONSE. AND SO THIS LED TO OUR FIRST QUESTION WHICH WAS DOES CHRONIC REPLICATION STRESS PERPETUATE ALT ACTIVITY. AND SO, WE SORT OF RELIED ON SOME PUBLISHED DATA FROM DAVID CORTEZ'S GROUP WHERE E IDENTIFY THAD THIS WAS 1 OF THE MOST ABUNDANT PROTEINS BOUND OF SITES STALLED BY REPLICATION OR REPPLICATION STRESS. THIS IS A KNEELING HELIX CASE AND WE WANTED TO KNOW WHETHER SMARCAL LOCALIZED TO TELOMERES. SO ON OUR INITIAL PASS AND THROUGHOUT THE TALK I WILL USE A NUMBER OF THESE ALT CELL LINES THAT WE'VE GATHERED OVER THE YEARS AND THIS PARTICULAR INSTANCE, THERE ARE OFTENNAN OITATED AS SO BEING ALT. AND O IN THESE 2 ALT CELL LINES WE CAN SEE THESE REALLY BEAUTIFUL FOCI THAT CO LOCALIZE IN TELOMERES IN THE ALT POSITIVE CELL LINES AND WE SEE MINIMAL COLOCALIZATION BETWEEN SMARCAL, AND TELEOR MERES THESE ARE REGULATED BY TELEORDER OF MICRONS RACE. SO WE CAN FURTHER SUPPORT THIS DATA USING CHP, USING SMARCALL, IN THIS INSTANCE AND WE IPsING IT HERE OR AS A POSITIVE CONTROL TRF2, AND YOU CAN SEE IT ASSOCIATES BOTH WITH TELOMERES AND ALT-POSITIVE TELOMERES, THIS IS SIGNIFICANTLY RICHED IN THE ALT TELEO MERRIC SAMPLES, SUGGESTING OR HIGHLIGHTING THE PREVALENCE ALT 1 TELOMERES. SO MORE RECENTLY WE'VE BEEN PUT NOTHING A CONCERTED EXPOSURE TO RADIATION TO CHARACTERIZE THE DO PAIN IN AMERICAS THAT ARE RESPONSIBLE FOR THE RECRUELTIMENT OF SMARCALL, 2 SITES OF TELEMERRIC STRESS AND THOSE HAVE BEEN IDENTIFIED IN THE LITERATURE. SO STRUCTURALLY THIS IS THE SMARCALL 1 PROTEIN WHICH CONTAINS A BINDING DOMAIN THE EPPED TERMINUS, CONTAINS 2 SEQUENTIAL HEART DOMAINS THAT WERE THOUGHT TO MEDIATE ACTION WITH DNA AND AS I MENTIONED IT'S A SWITCH TO CHROMATIN REMODELING DESIGN AND CONTAINS A SNF 2. DELETION OF THE RNA BINDING DOMAIN MAIN, OR WE CREATED POINT MUTATIONS WITHIN THE INITIAL HARP DOMAIN, THE SECOND HARP DOMAIN AND OF COURSE THE COMBINED HARP DOMAINS AND FINALLY, THIS ENACTIVATES THE ATP ACE OF SMACALL AND WE CAN SEE BY THE IMAGE --IMAGES, WE EXPRESS TELOMERES. AND THE SAME COULD BE SAID FOR THE DELETION OF THE RNA BINDING DOMAIN AS TO SOME EXTENT IN THE MUTANTS AND THE RDQ. AND THE ONLY 1 THAT'S--WAS THIS DUAL HARP DOMAIN MUTANT. AND SO WE CAN QUANTIFY THIS HERE AGAIN WHERE WE SEE THAT EVEN THE [INDISCERNIBLE] INTERACTING WITH THE TELOMERES, IT'S THESE DUAL HARP DOMAIN MUTANTS THAT SEEM TO INCREASE SMARCALL 1 BINDING OF TELOMERES. THIS SAN INTERESTING FINDING, SPECIFICALLY BECAUSE IN THE LITERATURE SMARCALL IS MENTIONED TO BIND WITH RFA, AND THIS RPA BINDING DOMAIN IS REQUIRED FOR THE LOCAL SITES OF DNA DAMAGE WIDE AND IT SUGGESTS THAT PERHAPS THESE HARP DOMAINS ARE REQUIRED FOR TELEO MERRIC DNA AND THIS IS ONGOING IN THE LAB AND WE HOPE TO BE FOLLOWING UP ON THIS SOON. BUT I THINK IT SUGGESTED TO US IN GENERAL, WAS THAT IF OR AT LEAST IT LED TOLET HYPOTHESIS THAT IF IN GENERAL SMARCALL WAS REQUIRED TO MEDIATE REPLICATION STRESS THROUGH THESE CHALLENGING REGIONS IT MIGHT ALSO CONTRIBUTE TO THE PROMOTION OF TELOMERE STABILITY. SO WE ASKED IF WE ELIMINATE SMARCALL, AND INCREASED REPLICATION FORKS WOULD THEY BE SUSCEPTIBLE TO CLEAVAGE THAT PROMOTE DOUBLE STRAND BREAKS BY THESE ENZYMES? AND THESE COULD BEING RESECTED TO PROMOTE THE RECOMBINATION INDICATIVE OF DNA DAMAGE REPAIR. AND SO, THE FIRST QUESTION WAS IF WE GET RID OF SMARCALL, DO WE SEE AN INCREASE IN DOUBLE STRAND BREAKS AND INITIALLY WE LOOKEDDA THE IN THE CONTEXT OF GAMAH 2 X AND WE COULD SEE THAT IN THE ABSENCE OF SMARCALL, WE SEE THE FOCI, BUT THEY CO-LEGALIZE WITH THE TELOMERES AND THIS IS INCREASED WITH THE CONTROL CELLS, THAT LACKS AND LIKEWISE WE SEE A SIMILAR THING IN THE ABSENCE OF SMARCALL, AND WE CAN SEE THAT WE ALSO GET AN INCREASE IN THE RECRUITMENT OF RAD 51 TO THESE SITES. AND I HOPE IT'S NOT LOST ON ALL OF YOU THAT IN LOOKING AT THESE IMAGES YOU CAN GET A SENSE THAT THESE TELEOR MERE FOCI IN THE ABSENCE OF SMARCALL, ARE GIANT. AND IN THE LAB WE WOULD REFER TO THEM AS THE GIANT TELOMERE FOCI, BECAUSE WE DIDN'T KNOW WHY THEY WERE SO LARGE AND WE WANTED TO QUANTIFY THEM. SO WE FOUND THESE TELEMERRIC FOCI, IN THE ABNORMALITIES SENSZ OF SMARCALL WERE GREATER IF FOLD AND SIZE THAN THE OTHER TELOMERES THROUGHOUT THE CELL OR IN THE CONTROL CELLS. SO THERE WAS REALLY THE SIGNIFICANT INCREASE IN THE TELOMERE FOCI, AND SO ROGER GREEN BERG'S LAB HAD DEMONSTRATED THAT IN FACT, THIS--THIS TELOMERE CLUSTERING WAS REALLY PREVALENT IN ALT CELLS AND THAT IN FACT THIS WAS DRIVEN BY RAD 51 SO YOU CAN ENVISION THAT IN THE PRESCRIBING ESTIMATE THAD RESEARCH A DOUBLE STRAND BREAK AND YOU SEE THE FORMATION, YOU COULD PROMOTE THE CAPTURE, SEARCH AND CAPTURE OF THE HOMOLOGOUS SEQUENCE TO PROMOTE DNA DOUBLE STRAND REPAIR AND THIS CONTEXT THE TELOMERE AND WE REFER TO THESE AS CLUSTERING EVENT WHERE IS THERE'S AN EVENT WHERE 1 IS INVADING ANOTHER AND THERE'S MULTIPLE TELEOR MERES COALESCING IN 1 REGION AND SO THIS IS NOT UNIQUE TO THOSE TYPE 2 CELLS, IN FACT WHEN WE KNOCK DOWN THE GOALS AND THE CELL LINES WE SEE THESE GIANT FOCI AND THE AVERAGE ABUNDANCE OF THESE IS BETWEEN 20 AND 25% OF CELLS CONTAIN THESE REALLY GIANT TELOMERE FOCI. AND SO IN THIS FACT THAT'S A PRODUCT OF THE MECHANISM, WHERE IT'S MEDIATING THIS SEARCH AND CAPTURE TO PROMOTE RECOMBINATION OF TELOMERE ENDS AND IF YOU GET RID OF RAD 51 YOU COULD ELIMINATE THESE CLUSTERING EVENTS AND THESE GIANT F OCI AND WE DID THAT AND IMKED IT DOWN AND IN THE ABSENCE OF SMARCALL, YOU GET A PERCENTAGE OF INCREASE WITH THESE FOCI, BUT WHEN YOU KNOCK DOWN RAD 51 YOU CAN RESCUE THE PHENOTYPE. AND SO LIKEWISE IF WE TAKE A STEP BACKWARDS WE CAN ENVISION THAT IF YOU NEVER INDUCED THE NUCLEOLYTIC CLEAVEERAGE THAT PROMOTES THE DOUBLE STRAND BREAK, YOU WOULD NEVER ACHIEVE THE CLUSTERING EVENTS THAT WE'RE OBSERVING IN THESE CELLS. SO WE ASKED IF YOU ELIMINATE NUCLEOLYTIC CLEAVAGE DO YOU ELIMINATE THESE CLUSTERING EVENTS AS WELL, SO SIMILAR TO WHAT WE DESCRIBED BEFORE, IN THE ABSENCE OF SMARCALL, YOU SEE THE INCREASE FOR THESE GIANT TELEO MERRIC FOCI AND IF YOU COMBINE THIS WITH DELETION OR LOSS OF MOSS 81 AND [INDISCERNIBLE] AND THOSE PROMOTE THE STALLED REPLICATION FORKS YOU CAN RESCUE THE PHENOTYPE. AND SO TO FURTHER SUPPORT THE IDEA THAT IF FACT IN THE ABSENCE OF SMARCALL, YOU ARE PROMOTING RECOMBINATION. WE STARTED TO LOOK AT SPREADS FOR KNOCK DOWN, AND WE CAN SEE IN THE CONTROL CELLS WE HAVE THESE REALLY DISCREET--CAN APPRECIATE THIS ON THE SCREEN. A NUMBER OF THESE REALLY UNIQUE FUSION EVENTS AND IF WE CAN BLOW THESE UP A BIT ON THE RIGHT HERE, YOU CAN SEE THAT THESE FUSION EVENTS ARE ACTUALLY WITHIN THE TELOMERES AND THEIR TELOMERE BRIDGING. AND SO AS I MENTIONED BEFORE, 1 OF THE GOLD STANDARDS IN THE FIELD IN LOOKING AT TELOMERE RECOMBINATION IS THE ANALYSIS OF C-CIRCLES AND THESE ARE GENERALLY THOUGHT TO BE A BYPRODUCT OF THOSE RECOMBINATION EVENTS AND SO IF YOU HAVE HEIGHTENED RECOMBINATION IN THESE CELLS YOU CAN ENVISION YOU WOULD HAVE AN INCREASE IN THESE CIRCLES AND IN FACT WHEN WE KNOCK DOWN SMARCALL, WE SEE AN INCREASE IN THE C-CIRCLE ABUNDANCE IN THESE CELLS AGAIN SUGGESTING THAT WE'RE REALLY PUSHING THE RECOMBINATION EFFECTS IN THE ABSENCE OF SMARCALL. SO WHAT ARE THE CONSEQUENCES OF THESE RECOMBINATION EEIVETS? I THINK WE NOTICED VERY EARLY ON THAT IN THESE CELLS SPECIFICALLY WE WOULD START TO SEE A LOT OF CELL DEATH IN THE ABSENCE OF SMARCALL AND WE WERE CURIOUS WHETHER THEY WERE CONSISTENT ACROSS ALL OF OUR CELL LINES AND USING A PANEL OF THESE CELL LINES WOO WE'VE KNOCKED DOWN SMARCALL USING THE COMBINATION OF THESE TOOLS--ALSO FUNCTION TO REPRESS TELOMERE COMBINATION. SO THE QUESTION CAME IF SMARCALL WAS LIKE ATRX, WAS IT POSSIBLE THAT IT WAS LOST IN THE ALT POSITIVE CANCERS AND SO WHAT WE FOUND IS IN FACT, YES, THAT IN A NUMBER OF THESE CELL LINES WE HAD DAMAGING MUTATIONS IN SMARCALL, THAT LED TO A ALMOST COMPLETE LOSS IN THE CELLS. SO CAN YOU GET THE PICTURE THAT WE'RE FILLING IN THE GAPS WE HAVE IN THE SAMPLES WE HAVE UNDERSTANDING THE MUTATION PROFILE OF THESE ALT POSITIVE TUMORS AND SINCE SOME OF THESE DISCOVERIES, WE'VE ACTUALLY BEEN ABLE TO SEE THESE COME OUT IN THE LITERATURE, SO, ATRX, DAX AND SMARCALL HAVE BEEN IDENTIFIED NOT JUST IN AFTIO SARCOMAS, SUBSETS OF LIVER CANCERS, AND THESE ARE IN THE CONTEXT OF ALT AND THESE WERE SOME OF THE FIRST INDKAIGDZS THAT THESE WERE THE CRITICAL PLAYERS IN IN PATHWAY. AND SO AS SATISFYING AS IT WAS TO SEE THESE MUTATIONS, IT'S CLEAR, ESPECIALLY IN THE TUMOR SAMPLES THAT WE WEREN'T THERE IN THE BEDDING OF THE GENETICS OF ALT, AND WE WANTED TO DIG DEEPER INTO THE NECKANISM. SO 1 OF THE THINGS THAT WE EALZED AT THE TIME, THERE HAD BEEN SEMINOLE PAPERS IN THE FIELD FROM ROGER GREEN BERG AND HILDA PICKET'S LAB WHICH LAID THE FOUNDATION FOR UNDERSTANDING OF TELOMERE ELONGATION AND ALT AND THAT THIS IS A PROCESS MEDIATED BY BREAK INDUCED REPLICATION, AND SO FOLLOWING FORK COLLAPSE OR JUST AT CRITICALLY SHORT TELOMERES THESE GROUPS COLLECTIVELY DEMONSTRATED THAT THAT THE BTR COMPLEX OR BLOOM TAUPE 3 AND RMI 1 AND 2, ACTUALLY REGULATE DNA SYNTHESIS ASK TELEOR MERE ELONGATION AND DISSOLUTION. AND IN AN OPPOSING AND PARALLEL PATHWAY THE SMX OR SLX 1 AND 4 AND 81 COMPLEX REGULATES THESE CROSS OVER EVENTS WITHOUT ELONGATION WHICH WE REFER TO AS THE TELOMERE ASHES CYSTED EVENTS. SO I THINK WHAT WAS LEFT LINGERING WHEN THESE PAPERS CAME OUT WAS HAQUE REGULATES THESE PATHWAYS HOW ARE THEY BALANCED? SO AT THIS TIME WE WERE LOOKING INTO THESE TUMORS WE HAD THAT WE KNEW WERE ALT AND WE NOTICED THESE INTERESTING MUTATIONS IN A PROTEIN CALLED SLICKS 4 IP AND SO WHEN WE CROSS CHECKED OUR GENETIC DATA WE COULD SEE THERE WERE THESE SIGNIFICANT DAMAGE OF MUTATIONS, FOR SLICKSS 4 IP AND SO AT THE TIME IT WAS KNOWN THAT SLICKs 4 IP INTERACTED WITH SLICKS 4 BUT IT WASN'T CLEAR THAT CONTRIBUTED TO THE DNA RESPONSE AND WHAT WAS IT DOING AT TELOMERES AND SO AROUND THE TIME OF THIS DISCOVERY FOR US WE WERE CHATTING WITH SIMON FULTON'S LAB AT FRANCIS CREEK AND THEY STARTED TO HOW IP TRIBUTES TO THE MECHANISM OF ALT SO IN A COLLABORATION WITH THEIR GROUP SIMON WAS AND 1 OF THE CRITICAL REGULATORS OF THE BALANCE OF THESE 2 ARMS OF THIS BREAK INDUCED REPLICATION PATHWAY. WHERE SLX4 AND BLM TO TOLERATE AND BALANCE THIS REPLICATION THROUGH BLOOM AND DNA SYNTHESIS AND TELOMERE ELONGATION AND DISSOLUTION, BALANCING THAT WITH THE CLEAVAGE AND CROSS OVER EVENTS THAT ARE IN THE EXCHANGES AND SO SLX 4 IP BECAME A CRITICAL REGULATOR OF THE BALANCE OF THIS PATHWAY. IT WAS AT THIS TIME THAT WE REALLY BECAME VERY INTERESTED IN UNDERSTANDING THE MECHANISM THAT IS REQUIRED TO PROMOTE THESE DNA SYNTHESIS EEIVETS SPECIFICALLY THROUGH BLM, AND PART OF THIS CURIOSITY CAME FROM SOME OF THE MECHANISTIC UNDERSTANDING WE HAD FROM PREVIOUS STUDIES THAT DEMONSTRATED THAT THESE VARIANT REPEATS THAT I MENTIONED BEFORE ARE REALLY PREVALENT THROUGHOUT THE TELOMERE AND WHAT'S INTERESTING ABOUT THIS IS THAT AS I HAD MENTIONED WHEN YOU HAVE STRAND INVASION AND HOMOLOGY SEARCH FOR THIS HOMOLOGOUS SEQUENCE IN A NEIGHBORING CHROMOSOME, YOU CAN ENVISION A SCENARIO THAT ARE BY BUT NOT ASENTIOUS FICIENT FOR HETEROGENEOUSERROLOGIOUS EXTEBDERS SO HOW WERE THESE BLOOMING BEHIND THESE MIGRATION EVENTS GIVEN THAT BLM IS NOT AN EFFICIENTLE HELIOS POSITIVE EXCITATORY CASE OVER THIS I HAVEROLOGY, SO WE KNEW THAT RAD 4 WAS THE MOST WELL CHARACTERIZED THAT REGULATES MIGRATION THROUGH HETEROGENEOUSROLOGY. SO WE ASKED DOES RAD54 FACILITATE BREAK INDUCED REPLICATION AT ALT TELOMERES? AND SO IN QUEST QUEE STARTED OFF OF COURSE LOOKING ACROSS OUR PANEL OF ALT TELEOR MERES AND WE CAN SEE THAT RAD 54 BUT THESE CO LOCALIZE WITH THE TELOMERE CELL LINES AND THESE ARE OFTEN ENRICHED AS COMPARED TO NONALT TELOMERES AND WE CAN EXACERBATE THIS PHENOTYPE BY TAKING ADVANTAGE OF A CELL LINE THAT HAD BEEN PREVIOUSLY PUBLISHED IN THE LITERATURE, A UTO AN S CELL LINE THAT HAS A FUSION PROBLEM AND IT HAS THE ENZYME THAT CAN PROMOTE CLEAVE LAGE OF DOUBLE STRANDED DNA AND THIS TRF 1 AND FOK 1 IS DRIVEN BY TAMOXIFEN AND FOLLOWING EXPRESSION--FOLLOWING INDUCTION WITH TAMOXIFEN, YOU CAN SEE EXPRESSION OF NOT JUST THE WILD TYPE BUT ALSO AN ENZYMEATICALLY INACTIVE D450 A, TRF FAK 1, AND WE CAN SEE WHEN WE INDUCE WITH TAMOXIFEN, YOU GET THE INCREASE IN RAD 54 FOCI FORMATION AND THESE FOCI CO LOCALIZE WITH TELOMERES ONLY WHEN YOU HAVE WILD TYPE FAK 1 AND NOT WITH THE D450 A SO WHEN WE QUANTIFY THIS, WE CAN SEE THIS AS AN INCREASE IN THE PERCENTAGE OF RAD 54 COLOCALIZATIONS PER SE, AGAIN ONLY IN THE WILD TYPE FAK WILD TYPE SPRIPPING CELLS AND NOT ONLY THAT BUT WE NOTICED WE HAD A SIGNIFICANT INCREASE IN THE PERCENTAGE OF CELLS THAT HAD FAR MORE RAD 54 TELEMERRIC FOCI THAN WE HAD SEEN IN THE UNTREATED CELLS. SO IT WAS NOT ONLY THE PERCENTAGE OF CELLS WITH THIS HIGH FREQUENCY OF COLOCALIZATION. AND SO, THERE HAD BEEN A LOT OF RESEARCH IN THE FIELD ON RAD 54 GIVING BOTH PRESYNAPTIC AND POST SYNAPTIC FUNCTION OR ASSIGNING BOTH OF THESE FUNCTIONS TO RAD 54 AND THERE WAS A LOT OF DATA SWIRLING AROUND THE REGULATION OF RAD51 AND MEDIATED STRAND INVASION AND THAT IT MIGHT BE REQUIRED FOR THIS FUNCTION AND SO WE STARTED OFF BY ASKING WHETHER RAD54 WAS IN FACT REQUIRED FOR RAD51 FUNCTION AND SO, WHILE OUR INITIAL ANALYSIS REALLY STARTED WITH LOOKING AT RAD 51 AND SO I DON'T HAVE ALL OF THE DATA, I'M NOT GOING TO PRESENT THIS HERE TODAY, BUT WHAT WE CAN SEER IN THESE CELLS IS THAT IN BOTH OF THESE ALT CELL LINES YOU CAN SEE RAD51 FOCI THAT CO LOCALIZE IN TELOMERES WE SEE NO STATISTICALLY SIGNIFICANT CHANGE IN THE NUMBER OF PREVALENCE OF THESE RAD51 FOCI AT THAT CO LOCALIZE AT THE TELEO MERENDS AND THIS IS NOT ACTUALLY SO WHILE IT DOESN'T XCTLY GET THE AT IDEA WHETHER RAD IS REQUIRED FOR FILAMENT FORMATION, WE THOUGHT WE WOULD FOLLOW THIS UP BY SAYING THAT IF WE ANALYZE THESE SYNAPTIC STRUCTURES, YOU WOULD IMAGINE IF THE NUCLEOFILAMENT WAS DISRUPTED, YOU MIGHT ALSO SEE A DECREASE IN THE RECRUITMENT OF SUBSEQUENT ENZYMES THAT ARE REQUIRED FOR THIS BREAK INDUCED REPLICATION PROCESS. AND SOME OF THOSE WOULD BE PC NA AND THE POLYMERASES THEMSELVES RESPONSIBLE FOR BREAK INDUCED REPLICATION SO I DON'T HAVE THE PC NA DATA HERE BUT WE NOTICED THAT PC NA LOOKS SIMILAR TO ANALYSIS OF POLA AND POLB, ARE THE 2 THAT HAVE BEEN IMPLICATED IN BREAK REPLICATION, IN THE TELOMERES SO USING A THESE WE CAN SEE THE FOCI REGULATION AND THESE CO LOCALIZE WITH TELOMERES. WHEN YOU KNOCK DOWN RAD54 WE DIDN'T SEE ANY CHANGE OF POL 8A, FROM THIS ANALIS COMBINED WITH THE RAD 51 THAT IT DOESN'T SEEM LIKE RAD54 IS CONTRIBUTING TO ANY PRESYNAPTIC FUNCTIONS OF THE BREAK INDUCED REPLICATION PROCESS. AND SO WHAT ABOUT MATURATION OF THESE D-LOOP STRUCTURES, AND HOW DO WE ANALYZE THOSE? AND SO, MATURATION OF THESE D-LOOPS REALLY INVOLVES NOT JUST RECRUITMENT OF THESE ENZYMES BUT ALSO PRODUCTIVITY OF THESE ENZYMES, SO FOR EXAMPLE, CAN WE MONITOR EDITORROR DNA SYNTHESIS FROM THESE D-LOOP STRUCTURES. AND SO WE CAN DO THIS BY TREATING CELLS WITH THE THYMA DINE ANALOGUE EDU AND USING CLICK CHEMIST RADIOY TO DETECT EDU OR THE FISH PROBES TO DETECT TELOMERES AND WE CAN MONITOR EDU CORPORATION IN TELOMERES IN NONS-PHASE CELLS AND YOU CAN APPRECIATE THAT WE GET THESE FOCI EDU POSITIVE CELLS THAT CO LOCALIZE WITH TELOMERES INDICATIVE OF ALT TERTELOMERES AND WE SAW IN BOTH OF THESE CELL LINES WHEN YOU KNOCK DOWN RAD 51, NOT ONLY DO YOU SEE A DECREASE IN THE NUMBER OF EDU POSITIVE TELOMERES BUT WE ACTUALLY SEE A DECREASE IN THE FLUORESCENTS INTENSITY AND THE SIGNAL FROM THIS EDU, SUGGESTING IN FACT, THAT THERE IS A DEFECT IN THE ACCUMULATION OF EDU AT ALT TELOMERES. AND SO RAD 54 IS A MONITOR MERSOLUTION AND OLIGMERRIZES IN THE PREPS ENSEL OF DNA AND SO WE THOUGHT WE MAY BE BETTER ABLE TO CHARACTERIZE RAD 54 FUNCTION AT ALT TELOMERES BY LOOKINGA THE THESE PREVIOUSLY CHARACTERIZED MUTANTS IN THE FIELD. SO THIS OLIGMERRIZATION IS ACTUALLY ANTAGONIZED BY PHOSPHORYLATION AT SERIES POINTSINE 49, AND SO IN THE PRESENCE OF PHOSPHORYLATION, THESE MONOMERS OLIGMERRIZE AND NO LONGER MAINTAIN BRANCH MIGRATION ACTIVITIES. SO WE ASKED, CAN WE RESCUE THIS DEFECT IN DNA SYNTHESIS AT ALT TELOMERES WITH WILD TYPE RAD 54 OR A K-R TRANSLO CASE CASE MUSEUM TABT OR A VERSION OF RAD 54 WHERE WE MAKE SERIES POINTSINE 49 CONSTITTATIVELY PHOSPHORALATED USING AN SDE MUTANT. SO WHAT WE FOWBD FROM THIS ANALIS WAS THAT IN FACT, AGAIN AS WE SAW BEFORE, WHEN WHEN YOU GET RID OF RAD 54, YOU SEE AIAN DECREASE IN THE TELOMERES WHEN RECONSITUTE WITH RAD 54 YOU CAN RESCUE THAT FEIGNEE TYPE BUT NEITHER THE TRANSLOCATION LOCATE A 9 MUTANT NOR THE--BUT IN FACT, THE BRANCH MIGRATION ACTIVITY OF RAD 54 IS CRITICAL FOR THIS FUNCTION. AND SO, IF IN FACT, ALT RELIES ON BREAK INDUCED REPLICATION, AND RAD54 IS MEDIATING THIS PATHWAY, POTENTIALLY IN CONJUNCTION WITH TD BTR INDUCED REPLICATION, YOU CAN IMAGINE IF YOU ELIMINATE YOU WOULD SHIFT THE BALANCE FAVORING CLEAVAGE AND CROSS OVER PRODUCT FORMATION VIA THIS SMX PATHWAY. SOY THE FIRST QUESTION QUEE ASK IS IF YOU GET RID OF RAD 54 IN CELLS, AND IN THIS PARTICULAR INSTANCE WE USE MUS81 AS A SURROGATE FOR THE SMX COMPLEX AND YOU CAN APPRECIATE IN THESE IMAGES THAT IN THE ABSENCE OF RAD54 WE GET A SIGNIFICANT INCREASE AND THEY CO LOCALIZE WITH TELOMERE ENDS SUGGESTING THAT WE ARE FORCING RESOLUTION WITH A LOOK AT THESE TELOMERE EXCHANGES I DESCRIBED EARLIER. IF IN FACT, YOU ARE FORCING CLEAVAGE AND CROSS OVER EVENTS YOU MENTD ENVISION YOU HAVE A INCREASE IN TSCE IN THE CELLS AND IN FACT, AGAIN IF WE TAKE THESE SAME ALT POSITIVE CELLS AND WE KNOCKED DOWN RAD54, CAN YOU SEE IN THE CONTROL SAMPLES YOU HAVE THIS NICE STAINING OF TELOMERE ENDS INDEPENDENTLY OF 1 ANOTHER. AND IN THE ABSENCE OF RAD54, YOU SEE AN INCREASE OF THESE RECOMBINATION EVENTS AND OVERLAPPING SIGNAL AT THESE ENDS, SO AGAIN, THIS WAS WAS INDICATIVE TO US THAT IN THE ABSENCE OF RAD54, YOU ARE PROMOTING THESE CROSS OVER EVENTS AT TELOMERE ENDS. AND SO IF YOU GET RID OF THE BTR COMPLEX AND THE SMS COMPLEX, CAN YOU IMAGINE THAT IN THE END YOU TAKE OUT BOGHT PATHWAYS YOU COULD PROMOTE THESE RECOMBINATION ISHT MEDIATES AND THIS IS ACTUALLY VISUALIZE AS WHAT WE REFER TO AS BRIDGES AND SO WE ASKED IF IN THE ABSENCE OF RAD 54 AND THE IN ABSENCE OF THE SMX PATHWAY DO WE SEE INCREASES IN ULTRA FIND ANAPHASE BRIDGES AND THE WAY WE ANALYZE THIS IS USING THE PROTEIN PITCH WHICH SPECIFICALLY BINDS THE ULTRA FINE BRIDGES AND THESE ARE ANAPHRASE PREPARATIONS WHERE WE'VE STAINED FOR PITCH AND YOU CAN SEE THAT IN THE CONTROL SAMPLES IF YOU GET RID OF BLOO BLM AND SLX 4, YOU GET THE BEAUTIFUL ANAPHASE BRIDGES CAN YOU VISUALIZE WITH PITCH. AND LIKEWISE, IF YOU GET RID OF SLX 4 AND RAD54, YOU CAN SEE AN INCREASE IN THE THESE BRIDGES SO DATA I'M NOT SHOWING HERE IS THAT IN ADDITION IT SEEING AN INCREASE IN CELLS, THAT HAVE THESE ANAPHASE BRIDGES, I THINK YOU CAN APPRECIATE, WE SEE AN INCREASE IN THE NUMBER OF ANAPHASE BRIDGES BETWEEN THESE CELLS. SO 1 MORE POINT ABOUT THIS ANALYSIS WAS THAT IF WE ACTUALLY COMBINED BLM, SLX 4 AND RAD54 KNOCK DOWN, WE DON'T SEE A FURTHER INCREASE WHEN WE QUANTIFY HERE ON THE RIGHT. WE DON'T SEE A FURTHER INCREASE SUGGESTING THAT BLM AND RAD 54 ARE IN FACT EPISTATIC. AND SO, THAT WAS INTERESTING DATA TO US BUT IN FACT, RIGHT, PITCH IS A GENERIC PROTEIN THAT MIGHT MARK ANY ANAPHASE BRIDGE AND WE WANTED TO NOTE WHETHER THESE BRIDGES WERE TELOMERES AND A PRODUCT OF TELOMERE DYSFUNCTION. SO INSTEAD OF USING PITCH, WE DECIDED WE WERE GOING TO ANALYZE THESE BRIDGES USING THE TELOMERE BINDING PROTEIN TRF 2, WHICH HAS BEEN ANOTHER PROTEIN THAT'S BEEN ESTABLISHED TO STAIN THESE BRIDGES AND SPECIFICALLY FOR TELOMERIC SEQUENCES AND SO, WE'RE IN CONTROL CELLS OR SINGLE DEPLETION ALONE, WE DON'T REALLY SEE ANY CHANGES IN THE FORMATION OF THESE UMENT RAFINE BRIDGES AND SPECIFICALLY 1S THAT CONTAIN TELOMERES WHEN WE KNOCK DOWN RAD 54 AND SLX 4 TOGETHER, AGAIN WE SEE THIS INCREASE IN THESE ULTRA FINE BRIDGES THAT ARE STAINED WITH THE TELOMERE BINDING PROTEIN SUGGESTING THAT IN FACT, THESE ULTRA FINE BRIDGES ARE ACTUALLY TELOMERE BRIDGES. AND SO IF THESE ULTRA FINE BRIDGES ACTUALLY PERSIST THROUGH TELEO PHASE, THEY WOULD CAUSE FRAGMENTATION AND YOU WOULD GET THE MICRONUCLEI AND THESE MAY CONTAIN TELEO MERRIC SEQUENCE IF THE BRIDGES ARISE FROM TELOMERE DYSFUNCTION AND SO, HERE WHAT WE ASK WAS, DO WE SEE AN INCREASE IN THE PERCENTAGE OF MICRONUCLEI THAT CONTAIN TELEO MERRIC SEQUENCE IN THE ABSENCE OF COMBINED DEPLETION OF RAD 54 AND SLX 4. SO POTENTIALLY DIFFICULT TO SEE ON THE SCREEN BUT HERE'S AN EXAMPLE OF THE MICRO NUCLEI WE'RE COUNTING AND THE SIGNAL INSIDE OF THAT OF TELOMERES AND WHEN WE DO THAT IN THE COMBINED DEPLETION DO WE SEE A COMBINED INCREASE OF CELLS MICRONUCLEI SUGGESTING THAT IN FACT, LOSS OF RAD54 AND SLX 4 PATHWAYS IS LEADING TO FRAGMENTATION OF THE DNA OR THE TELEO MERRIC DNA, AND CREATING THESE MICRODNA. SO I HOPE FROM THIS DATA I'VE GIVEN YOU A SENSE THAT RAD54 ACTUALLY FUNCTIONS TO PROMOTE BREAK INDUCED REPLICATION BY PROCESSING OF THESE D-LOOP STRUCTURES AND WORKS COORDINATELY WITH BLM AND TOP1311 TWIN STUDIES 2 TO PROMOTE BRANCH MIGRATION, AND DNA SYNTHESIS AND ULTIMATELY TELOMERE ELONGATION AND THAT IN THE ABSENCE OF RAD 54, THIS PATHWAY IS FORCED TO RELY ON RESOLUTION OF THESE STRUBLGHTURES THROUGH CLEAVAGE AND CHROMA TID EXCHANGES. SO WHILE I QUISH I COULD SAY THAT THE FINAL SAMPLE THAT LINGERS IN OUR MIND IN OUR LAB WAS IN FACT MUTATED--LAB LOOKING TO DEFINE THE GENETICS OF THESE ALT TUMORS, --I LIKE TO SAY I THINK WE HAVE FIGURED OUT WITH THIS MUTATION, WHAT IT IS, BUT WE'RE IN THE EARLY STAGES. SO AS AN EARLY TEASER, I HOPE I CAN SHARE THAT DATA SOON, WITH THAT I WOULD LIKE TO THANK THE PEOPLE WHO DID THE WORK IN THE LAB, THIS IS REALLY A KIEWMRATIVE EFFORT OF CURRENT AND FORMER STUDENTS INCLUDING KELLEY, MASON-OSANN WHO DID A QUITE A BIT OF WORK, SUPPORTED BY A NUMBER OF PEOPLE AT THE LAB, INCLUDING HIMABINDU, GA, IR, AND ADAM LABADORF AND çNGEL DAI, AND SOME OF THE SMARCALL WORK WAS DONE AT THE UNIVERSITY OF SHERBROOK, AND THE SLX COLLABORATION WAS IN COLLABORATION WITH COLLEAGUES AT THE FRANCIS CRICK INSTITUTE. WITH THAT I WILL BE HAPPY TO TAKE ANY QUESTIONS. >> THANK YOU VERY MUCH FOR THIS EXCELLENT TALK. DR. BOHR AND I WILL TAKE TURNS ASKING THE QUESTIONS. THERE YOU ARE, DO YOU WANT TO GO FIRST? >> YES, YEAH. THANK YOU FOR THAT VERY INTERESTING TALK AND I HAVE ANOTHER QUESTION, BUT FIRST OF ALL, READ THE 1 HERE FROM GORDON AND BASED ON YEAST STUDIES LONG STRETCHES OF B-RICH BIR INVADING STRAND CAN STAY SINGLE STRANDED FOR A SIGNIFICANT TIME. THIS WILL MAKE IT PRONE TO FORMING G-GAWR AT THE TIMES WHICH MAY CAUSE A BIR BREAKAGE IN THE SUBSEQUENT REPLICATIONS. SHOULD THIS REALLY BE HAPPENING? WOULD IT BE VISIBLE IN YOUR ASSAY? >> OOH. THAT'S A GOOD QUESTION. WHEN WE THINK ABOUT THE CONTRIBUTIONS FOR G-QUADRUP LEX FORMATION ON RUPLICATION STRESS, IT'S NOT SOMETHING WE HAVE CEREBELLUMS PETTER EASE VISUALIZING, IT'S NOT SOMETHING WE LOOK AT. WOULD WE BE ABLE TO VISUALIZE IN THE ASSAY, I WOULD SAY PROBABLY NOT. >> OKAY, THANK YOU. MIKE AT LICHTON, ASKED THIS QUESTION, SHARED FUNCTION BETWEEN BLM AND RAD54 AND THE KNOCK DOWN FOR EITHER IS SYNERGISTIC WITH XLX4 KNOCK DOWN FOR UPF FORMATION. HAVE YOU EXAMINED A PHENOTYPE OF A BLM RAD54 DOUBLE KNOCK DOWN? >> JUST IN THE ULTRA FINE BRIDGE ANALYSIS WHERE WE SEE THAT COMBINED DEPLETION OF SLX 4 AND RAD54 BLM ARE IN THAT FORMATION, SO OTHER THAN THAT WHEN WE KNOCK DOWN JUST BLM AND RAD54 ALONE WE DON'T SEE ANY ADDITIONAL PHENOTYPES. >> GOOD DEAL. >> FROM JIM, YEAST BIR ALSO DEPENDS ON RAD54'S COULD YOU SAYIN RDH54, RAD54 B, WHAT ABOUT THAT IN YOUR CELLS? >> YEAH, GOOD QUESTION. WE DIDN'T ANALYZE RAD54 B BUT IT'S SOMETHING WE'RE REALLY INTERESTED IN. >> SO MICHAELLICATEON SAYS, HE MEANT SLX 4 BUT I THINK THAT WAS--KEN READ THAT. >> YEAH, I UNDERSTAND. SNIMENT --I WANT TO ASK YOU ABOUT THESE CELLS AND FOLLOWING THEM ALT. YOU SAY YOU USE THESE DIFFERENT 3 APPROACHES LOOKING AT ALT BODIES AND CIRCLES AND TELEO MERRIC SYSTEM CHROMA TID EXCHANGES TO WHAT EXTENT DO ALL THESE CELLS HAVE THESE CRITERIA OR SOME CELLS HAVE THEM AND SOME OF THEM NOT, AND OTHERS HOW AND DOES THAT AFFECT WHAT YOU CALL ALL CELLS AND HOW YOU CAN COMPARE THEM? >> YEAH, GOOD QUESTION. I WOULD SAY IN GENERAL THERE'S CERTAIN REQUIREMENTS OR THRESHOLDS THAT WE USE TO DEFINE ALT STATUS AND SO, FROM THE 1S THAT I SHOWED TODAY, THEY ALL HAVE ALL OF THOSE FEATURES, SO FOR EXAMPLE, THEY ALL HAVE C-CIRCLES, SOME MAY HAVE MORE THAN OTHER AND IN FACT, I THINK CELLS THAT ARE DEFICIENT FOR ATRX AND DAXX, DEMONSTRATE HIGHER LEVELS OF C-CIRCLES THAN THE OTHER MUTANTS BUT THEY ALL HAVE THEM. APBs AND TELOMERE SISTER CHROMA TID EXCHANGE ANALYSIS, BUT IT WOULD BE IMPOSSIBLE, NOT IMPOSSIBLE BUT VERY DIFFICULT TO DO ON A TUMOR SAMPLE SO THAT'S LIMITED TO CELL LINES. >> A COMMENT, I WAS PLEASED TO SEE THAT THE--YOU ARE DOING LOTS OF CARIO TYPES, THESE DAYS WHEN WE'RE LOOKING FOR DISEASE PHENOTYPES AND GENETICS, THEY JUST DO EXOME SEQUENCING, AND YOU CLEARLY POINTED OUT, YOU WILL--YOU WILL MISS TRANSLOCATIONS WHEN YOU DO THAT WHICH MAY BE A VERY IMPORTANT AND CARIO TYPING SEEMS TO BE NEGLECTED IN GENERAL WHEN THEY'RE LOOKING FOR GENETIC SYSTEMS, WE WORK WITH PATIENTS WITH 0 PIG MATOSA, AND THE VARIANT AND IN POL8A, AND IF YOU LOOKED AT AND WHAT WOULD BE THE EFFECT OF DEFICIENCIES IN POL8A IN YOUR ALT TELOMERES. >> YEAH, I THINK THOSE STUDIES HAVE BEEN DONE AND I WOULD NEED TO THINK ABOUT OR RECALL WHAT THE EXACT RESULT WAS BUT THE PRIMING EVENTS ARE THOUGHT TO BE--I THINK THE PRIMING EVENTS ARE THOUGHT TO BE REGULATED BY POLA BUT IT'S REGULATED BY POL D, BUT WE DON'T KNOW ENOUGH OF THE RECRUITMENT OF THOSE POLYMERASES TELEO MERRIC, TO KNOW WHAT THE CONSEQUENCE OF LOSS OF 1 ON OTHER. SO I THINK THOSE STUDIES HAVEN'T SPECIFICALLY BEEN DONE. >> YEAH, WE DON'T PARTICULARLY SEE STUDY GENETIC ABNORMALITIES IN THE PATIENTS, OR MAYBE WE HAVEN'T LOOKED HARD ENOUGH AS THE POSSIBILITY. YES. >> ANYMORE QUESTIONS, WILL? >> I'M LOOKING AT IF THERE ARE ANY MORE IN THE LIST HERE? IT'S REALLY GREAT, THANK YOU. >> OKAY, GREAT. THANK YOU SO MUCH. IT WAS A GREAT TALK.