>>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 ÁNGEL
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.