>>WELCOME TO THE DNA REPAIR
INTEREST GROUP MONTHLY LECTURES.
I WILL BE THE HOST TODAY, EVEN
THOUGH KEN KRAMER AND I ARE NO
LONGER THE LEADERS OF THE
INTEREST GROUP, THAT IS CHUN
YANG AND KAREN USTEN, NOT ABLE
TO BE PRESENT, ALTHOUGH CHUN IS
HERE AND WILL HELP ME WITH
LEADING THE QUESTIONS.
I WANT TO MENTION THAT THE NEXT
MEETING APRIL 11 IS CHRIS LORD,
OPTIMIZING USE OF PARP
INHIBITORS TO TREAT CANCER, AND
ON TUESDAY MAY 9, PETER CHOIKA
FROM SWITZERLAND, WE HAVE A
REALLY GOOD PROGRAM THIS YEAR.
AND WE'RE VERY PLEASED TO HAVE
JESSICA DOWNS SPEAK TO US HERE
TODAY NOW, AND HERE IS A PICTURE
OF HER.
AND I'M SHARING MY SCREEN HERE,
I HOPE THAT WORKS.
AND YOU CAN SEE JESSICA FROM
THE -- PROFESSOR OF EPIGENETICS
AND GENOME STABILITY, DEPUTY
HEAD DIVISION OF CANCER BIOLOGY
AT THE INSTITUTE OF CANCER
RESEARCH DOWNTOWN LONDON.
AND SHE HAS DONE A LOT OF VERY
INTERESTING WORK RELATING TO
EPIGENETICS CHROMATIN STRUCTURE
AND DNA REPAIR.
SHE RECEIVED HER Ph.D. FROM
DEPARTMENT OF ZOOLOGY,
UNIVERSITY OF CAMBRIDGE, AND
WORKED THERE WITH STEVE JACKSON,
AND THEN SHE CONTINUED IN
JACKSON'S LAB AT A POSTDOC FOR
SOME YEARS, AND THEN SHE WENT TO
UNIVERSITY OF CAMBRIDGE AND WAS
A GROUP LEADER THERE, LATER
JOINED THE UNIVERSITY OF SUSSEX,
PROFESSOR OF GENOME STABILITY,
WE HAVE MANY FRIENDS THERE AND
COLLEAGUES.
LATER MOVED TO LONDON, DEPUTY
HEAD OF DIVISION OF CANCER
BIOLOGY AT INSTITUTE OF CANCER
RESEARCH, SINCE 2019.
AS I MENTIONED PUBLISHED MANY
IMPORTANT PAPERS IN THE FIELD OF
DNA REPAIR.
SHE'S ALSO PART OF THE EDITORIAL
LEADERSHIP OF DNA REPAIR, THE
JOURNAL, AND MANY OTHER
JOURNALS.
SHE WILL TALK ABOUT THE SWI/SNF
CHROMATIN REMODELING COMPLEX
MAINTAINS GENOME STABILITY AT
CENTROMERES.
JESSICA, PLEASE TAKE IT OVER
FROM HERE.
>>THANK YOU VERY MUCH
FOR THE INVITATION
HERE, IT'S A GREAT PLEASURE.
I'D LIKE TO TELL YOU ABOUT THE
RECENT WORK ON THIS CHROMATIN
REMODELING COMPLEX PBAF AND
SOMEHOW WE FOUND A ROLE FOR IT
AS CENTROMERES THAT'S IMPORTANT
FOR MAINTAINING GENOME STABILITY
AT CENTROMERIC SEQUENCES.
THIS IS A SET OF RECENT WORKS
UNPUBLISHED WORKS I'M KEEN TO
GET PEOPLE'S FEEDBACK ON THIS.
OKAY.
SO, TO BEGIN, MY LAB, WE'VE
ALWAYS BEEN INTERESTED IN
UNDERSTANDING HOW CHROMATIN
IMPACTS ON GENE OWN INTEGRITY,
AND OBVIOUSLY YOU CAN SEE FROM
THIS SLIDE IT'S TRYING TO
REPRESENT HOW AS YOU ALL KNOW
THAT THERE ARE MULTIPLE
DIFFERENT WAYS THAT CHROMATIN
BIOLOGY CAN INFLUENCE GENOME
INTEGRITY.
IT'S DYNAMICALLY INVOLVED IN
ANYTHING TO DO WITH DNA
METABOLISM SO WE CAN HAVE
TRANSCRIPTIONAL REGULATION, DNA
REPLICATION, WHAT I REALLY --
THE MAIN FOCUS OF THE LAB IS ON
DOUBLE STRAND BREAK REPAIR AND
HOW CHROMATIN INFLUENCES THE
REPAIR OF THOSE BREAKS AND
SIGNALING AROUND THEM.
BUT THE STORY I'M GOING TO TELL
YOU ABOUT TODAY KIND OF STEMS
FROM WORK A NUMBER OF YEARS
LOOKING AT RELATIONSHIP BETWEEN
PBAF AND CHROMATIN ADHESION, AN
INFLUENCE ON CENTROMERE
STRUCTURE WHICH IS IMPORTANT FOR
MAINTAINING GENOME INTEGRITY AT
THOSE REGIONS, ALSO IN TERMS OF
FIDELITY OF CHROMOSOME
SEGREGATION.
WE'RE INTERESTED IN REMODELING
COMPLEX, MULTI-SUBUNIT COMPLEXES
THAT USE ENERGY DIRECT FROM ATP
HYDROLYSIS TO ORGANIZE THE
RELATIONSHIP BETWEEN DNA AND
HISTONES, OBVIOUSLY THAT CAN
LEAD TO ALL KINDS OF DOWNSTREAM
ACTIVITIES THAT ARE IMPORTANT
FOR DNA METABOLISM BASED EVENTS.
IN PARTICULAR, WE'RE FOCUSED ON
THIS MAMMALIAN GROUP OF
CHROMATIN REMODELING COMPLEXES.
AS YOU CAN SEE YOU CAN DIVIDE
SWI/SNF REMODELES INTO THREE
BASED ON SUBUNIT COMPLEXES, BAF,
PBAF OR NCBAF, SHARING A NUMBER
OF CORE SUBUNITS IN GRAY, WITH A
CATALYTIC SUBUNIT IN GREEN, I'LL
MENTION THIS SUBUNIT LATER IN
THE TALK.
WE'VE SPENT A LOT OF TIME
FOCUSING ON THIS PARTICULAR
SUBUNIT OF PBAF, I'M GOING TO
REFER TO AS INTERCHANGEABLY AS
PBRM 1 OR BAF180.
ONE THING THAT'S STRIKING ABOUT
THIS FAMILY OF REMODELERS HOW
FREQUENTLY THEY ARE MISREGULATED
IN CANCER.
YOU CAN SEE FROM THE HEAT MAP
WHERE WE'VE GOT THE DIFFERENT
SUBUNITS ACROSS THE BOTTOM OF
THE HEAT MAP, AND THE -- LOTS OF
CANCER TYPES ALONG THE SIDE OF
THE HEAT MAP.
THIS IS A PROJECT, IN FACT A
LOCKDOWN PROJECT THAT WE DID A
COUPLE YEARS AGO WHERE WE JUST
ANALYZED EVERYTHING THAT WAS
AVAILABLE IN BIOPORTAL AT THAT
TIME.
WHAT WAS PLOTTED IS FREQUENCE OF
DELETERIOUS CHANGES TO THESE
SUBUNITS OF SWI/SNF.
ACROSS A GREAT CHANGE OF CANCER
TYPES YOU SEE FREQUENT MUTATIONS
AND MISREGULATION OF SWI/SNF.
IF YOU ADD TOGETHER, YOU SEE
THAT AROUND 30% OF HUMAN CANCERS
HAVE MISREGULATION OF AT LEAST
ONE SUBUNIT OF SWI/SNF,
PROFOUNDLY MISREGULATED IN
CANCER BIOLOGY.
THERE'S ELEGANT WORK FROM OTHER
LABS SHOWING THIS IS A BONA FIDE
TUMOR SUPPRESSOR ACTIVITY.
ONCE YOU LOSE IT, YOU OPEN THE
DOOR TO THAT PROGRESSION TOWARDS
TUMORIGENESIS.
OUR LAB IS INTERESTED IN
UNDERSTANDING HOW CHROMATIN
BIOLOGY AND SWI/SNF IN
PARTICULAR CONTRIBUTES TO GENE
OWN INSTABILITY, MULTIPLE
CELLULAR ACTIVITIES COULD
POTENTIALLY BE IMPORTANT FOR
TUMORIGENESIS, I'VE LISTED THOSE
HERE, GENE EXPRESSION CHANGES,
NOT THE LEAST AMONG THEM.
AND THERE'S NO DOUBT CELL TYPE
AND CONTEXT-SPECIFIC INFLUENCES
ON WHICH OR ANY OF THESE ARE
ACTUALLY IMPORTANT FOR
TUMORIGENESIS.
AND AGAIN JUST TO REMIND YOU
THIS PARTICULAR STORY I WANT TO
TELL YOU ABOUT CAME OUT OF SOME
WORK WE DID A FEW YEARS BACK
LOOKING AT HOW PBAF INFLUENCES
SISTER ADHESION.
I'LL SHOW ONE OF THE KEY
FINDINGS FROM THAT PROJECT THAT
WE HAD.
SO, THIS IS SOME WORK WE DID
LOOKING AT SISTER CHROMATID AT
HE'S IN ABSENCE OF THE SUBUNIT
OF PBAF.
WE'RE USE FISH PROBES, TO
CHROMOSOME ARMS OR TELOMERES,
WHAT WE FOUND WHEN WE COMPARED
THE CONTROL CELLS WHICH ARE IN
BLACK HERE TO THOSE WHERE WE
DEPLETED PBRM 1 OR BAF 180, YOU
LOOK AT THE READOUT.
YOU CAN SEE IN THE ABSENCE OR
WHEN PBRM 1 IS DEPLETED THE
DISTANCE INCREASES, OR THE
DISTRIBUTION INCREASES IN THIS
TOP PANEL WHEN WE'RE LOOKING
AFTER THE CELLS USING A
CENTROMERE-SPECIFIC PROBE.
BUT INTERESTINGLY WE DIDN'T SEE
THAT SAME DIFFERENCE WITH PROBES
TO CHROMOSOME ARMS OR TELOMERES.
THAT'S SOMETHING WE WANTED TO
DELVE INTO TO UNDERSTAND MORE
FULLY.
ONE OF THE OTHER THINGS WE'VE
BEEN DOING IN THE LAB IS TO
CREATE A WIDE PANEL OF ISOGENIC
CELL SIGNS, DISRUPTING WITH
CRISPR/CAS9.
WE'VE CREATED BETWEEN 3 AND 8
CLONALLY DERIVED PBRM1 DEFICIENT
LINES IN A NUMBER OF CELL LINE
BACKGROUNDS.
WE'VE GOT A FEW NON-CANCER, JUST
IMMORTALIZED CELL LINES SUCH AS
BECOMING WIDELY USED EPITHELIAL
CELL LINE AND A FEW
CANCER-DIRECT CELL LINES AS
WELL.
CREATED CELL LINES WAS TO DO
WHOLE PROTEOME ANALYSIS.
ONE THING WE WANTED TO ASK IS
WHETHER OR NOT WE COULD IDENTIFY
WHAT WE CALL CORE PATHWAYS THAT
ARE REGULATED BY SWI/SNF AND BY
PBRM1.
THERE WOULD BE PATHWAYS WHERE
MISREGULATION WAS APPARENT IN
ONE OR MAYBE A COUPLE CELL LINES
BECAUSE IT WAS VERY SPECIFIC
EITHER TO THAT TISSUE TYPE OR TO
THAT GENETIC CONTEXT BUT WE
MIGHT SEE PATHWAYS DISRUPTED OR
DYSREGULATED ACROSS THESE CELL
LINES GIVING INDICATION OF WHAT
SORT OF THINGS ARE SORT OF
BROADLY MISREGULATED WHEN PBRM1
IS DEFICIENT, TO THINK ABOUT
THIS FROM THE POINT OF VIEW OF
HOW WIDELY THIS IS MISREGULATED
IN CANCER, AND IF WE HAVE A
CANCER THAT HAS A MUTATION, LOSS
OF FUNCTION MUTATION IN PBRM1
ARE THERE PREDICTIONS WE COULD
MAKE ABOUT MISREGULATED PATHWAYS
OR PHENOTYPES ASSOCIATED WITH
THAT.
WE ANALYZED THESE DATA.
ONE OF THE THINGS WE WERE KEEN
TO LOOK AT BASED ON PREVIOUS
OBSERVATIONS AROUND SISTER
CHROMATID ADHESION AT
CENTROMERES WAS CENTROMERE
ASSOCIATED PROTEINS AND
PATHWAYS.
WE FOUND THAT FELL INTO ONE OF
THESE TYPE OF CATEGORIES,
BROADLY ASSOCIATED WITH PBRM1
LOSS, ALL OF THE CLONES IN ALL
CELL LINE BACKGROUNDS ARE THE
SAME CHANGES, AND THOSE ARE
REPRESENTED HERE.
SO, THIS IS -- THESE ARE DATA
PLOTTED FROM THE RPE1 CELL LINE,
I'M GOING TO PRIMARILY SHOW THAT
CELL LINE-BASED DATA UNLESS I
SPECIFY OTHERWISE.
WE'VE GOT LOG TWO FOLD CHANGE IN
KNOCKOUT COMPARED TO PARENTAL.
WE'VE DIVIDED THESE PROTEINS
BASED ON THEIR ORGANIZATION SORT
OF ROUGHLY ACCORDING TO THIS
SCHEMATIC IN THE BOTTOM, SO
COMING FROM THE MOST CLOSELY
ASSOCIATED MOVING OUTWARD TO
CONNECTING WITH MICROTUBE ALLS,
YOU CAN SEE OUTER CORE, INNER
CORE, OTHER
CENTROMERE-ASSOCIATED PROTEINS.
ACROSS THE BOARD WE DON'T HAVE
DRAMATIC DECREASE OF PROTEIN
LEVELS OF INDIVIDUAL PROTEINS
BUT THEY ARE ALL SIGNIFICANTLY
AND REPRODUCIBLY DOWN IN THE
KNOCKOUTS.
AS I SAY, THIS PATHWAY ON
ALTHOUGH IT DOESN'T LOOK LIKE
THE SAME PATTERN, THEY HAVE
DOWNREGULATION ACROSS THIS PANEL
OF PROTEINS.
SO, WE THOUGHT THIS WAS
EXTREMELY INTERESTING GIVEN WHAT
WE'VE SEEN PREVIOUSLY AND
MECHANISTICALLY THE FIRST THING
WE THOUGHT WAS MAYBE THE GENES
ARE ALL REGULATED BY PBAF, WHEN
WE KNOCK OUT ONE OF THE SUBUNITS
OF PBA, IF WE SEE DOWNREGULATION
OF PROTEINS.
SO, WE LOOKED TO SEE WHETHER OR
NOT WE HAD A SORT OF BROAD
REGULATION OF THE GENES THAT
ENCODE THESE DIFFERENT PROTEINS,
AND WE DID THIS AGAIN ACROSS THE
DIFFERENT CELL LINES AND IN A
COUPLE DIFFERENT WAYS BUT WHAT
I'LL SHOW HERE IS THE RNAseq
DATA FROM RPE1.
AGAIN, PLOTTED ON THE TOP THIS
IS THE PROTEOME DATA.
AND ON THE BOTTOM IS THE
RNAseq DATA, PLOTTED IN THE
SAME WAY, LOG TWO-FOLD CHANGE,
KNOCKOUT RELATIVE TO PARENTAL.
WHAT YOU CAN SKI HERE AGAIN
THESE ARE LINED UP IN THE SAME
ORDER, THERE'S A LITTLE BIT OF
DIFFERENCE BECAUSE OBVIOUSLY
THERE'S SOME THINGS WE COULD
PICK UP IN THE RNAseq WE
DIDN'T PICK UP IN THE PROTEOME
BUT GENERALLY SPEAKING YOU CAN
SEE THAT THERE IS NOT A GREAT
CORRELATION BETWEEN THE PROTEIN
LEVELS AND TRANSCRIPT LEVELS,
AND INDEED IF YOU LOOK AT SOME
OF THE TRANSCRIPT LEVELS
DECREASED IN THE PBRM1 KNOCKOUT
SUCH AS THIS HERE WE DON'T SEE A
GREAT CORRELATION WITH WHAT'S
HAPPENING AT THE PROTEIN LEVEL
WITH CENFAS.
WE DID A CUT AND RUN MAPPING OF
THE CATALYTIC SUBUNIT OF PBAF TO
SEE GLOBALLY WHERE THIS IS
BOUND.
AND OF COURSE WE CAN MAP THAT TO
GENE PROMOTERS AND ENHANCERS AND
WHAT WE FIND IS THERE'S A
LIMITED ASSOCIATION OF SMARCA4.
WE DON'T SEE STRONG EVIDENCE
DOWNREGULATION IS DOWN TO
MISREGULATION OF THE GENES OF
TRANSCRIPTION OF THOSE GENES.
SO WERE DO WE SEE
DOWNREGULATION?
WE STARTED THINKING ABOUT IT THE
OTHER WAY AROUND, IT WAS A
CONSEQUENCE OF A FAILURE TO
ASSEMBLE INTO THE PROTEIN
COMPLEX.
SO EVERYONE WILL BE FAMILIAR, I
PUT PBAF HERE, WHEN WE MAKE A
KNOCK OUTCELL LINE OR DEPLETE BY
S RNA WE USE STABILITY.
LEVEL OF PROTEINS DOWN ACROSS
THIS ASSOCIATED WITH CENTROMERE
MIGHT BE A READOUT OF FAILURE TO
ASSEMBLE INTO THE COMPLEXES
WHICH THEN WOULD STABILIZE THE
PROTEIN LEVELS GLOBALLY.
AND THAT'S ACTUALLY NOT
UNREASONABLE THOUGHT WHEN YOU
THINK THAT THE CHROMATIN
STRUCTURE AT CENTROMERES IS
OBVIOUSLY VERY COMPLICATED AND
WHAT YOU NEED TO DO IS
REORGANIZE FIRST THAT AT THIS
INDIVIDUAL LEVEL REORGANIZE
NUCLEOSOMES TO PREVENT ASSEMBLY
OF VARIOUS ASSOCIATED PROTEINS
AND THEN BUILD THIS COMPLEX
OUTWARD TOWARD MICROTUBULES AND
WE HAD A DEFECT IN SISTER
CHROMATID ADHESION AND WE KNOW
FROM A LOT OF PEOPLE'S WORK
THERE ARE THREE DIMENSIONAL
ORGANIZATION OF CENTROMERES AND
PERICENTROMERES THAT INVOLVES
LOOPING.
A FAILURE OF CHROMATIN REMODELER
TO DOUGH TO CENTROMERES AND
ORGANIZE THIS CHROMATIN COULD
POTENTIALLY LEAD TO A SLIGHT
DECREASE IN THE EFFICIENCY OR
ABILITY OF THESE PROTEIN
COMPLEXES TO ASSEMBLE IN THE
APPROPRIATE WAY.
SO, WE WANTED TO SEE WHETHER OR
NOT THERE WAS ANY EVIDENCE THAT
EITHER THE STRUCTURE OR
INTEGRITY OF THESE SEQUENCES WAS
IMPAIRED IN ANY WAY IN THE
ABSENCE OF PBRM1.
I'LL SHOW YOU A FEW EXAMPLES OF
THOSE DATA.
IN THIS EXPERIMENT WHAT WE DID
WAS JUST TO SIMPLY USE FISH
PROBES AGAINST CENTROMERIC
SEQUENCES IN CHROMOSOME 2 OR 10.
AND VISUALIZE THOSE
MICROSCOPICALLY.
HOPEFULLY YOU CAN SEE
SUBJECTIVELY IN THE IMAGES IS
THAT THE INTENSITY AND SIZE OF
THE SIGNALS THAT WE SEE IN THE
KNOCKOUT CELLS IN THE BOTTOM TWO
ROWS IS GREATER THAN THAT WE SEE
IN THE PARENTAL.
THIS IS QUANTIFIED ON THE OTHER
SIDE OF THE SLIDE.
AND I PUT IN HERE THESE CHANGES
ARE OBSERVED IN ALL OF THE CELL
LINES WE'VE TESTED SO FAR,
SUGGESTING THAT THESE
CENTROMERES ARE NOT CREATING
THIS THREE DIMENSIONAL STRUCTURE
IN THE SAME WAY IN THE KNOCKOUTS
AS IN PARENTAL, AT LEAST THAT'S
ONE INTERPRETATION OF THESE
DATA.
IT'S WORTH NOTING THIS IS A
FEATURE OF PBRM1 KNOCKOUT IN THE
ABSENCE OF PERTURBATION.
THIS IS SIMPLY HOW THEY LOOK
UNDER NORMAL GROWTH CONDITIONS.
THIS IS THE CELL LINES WE'VE
LOOKED AT SO FAR INCLUDING
CANCER DIRECT AND NONCANCER
DIRECT.
WHAT ABOUT THE INTEGRITY OF
THESE CENTROMERIC REPEATS?
WE WANTED TO LOOK AT THAT AND WE
DECIDED TO USE AN ASSAY THAT WAS
DEVELOPED WHERE THEY WERE
LOOKING AT THE PROTECTIVE
ABILITY OF THE CENP-A, AS WELL
AS ASSOCIATED PROTEIN CENP-C AND
CENP-T.
AND WHAT THEY DID HERE WAS TO
USE BRD, BRDC LABELING TO
INCORPORATE INTO THE DNA, AND
THEN THEY COULD PROCESS THAT
WITH NUCLEASE DIGESTION IN ORDER
TO GIVE YOU A STRAND-SPECIFIC
LABELING, USING -- YOU CAN SEE
FLUORESCENTLY LABELED PROBES
AGAINST THE CENP DNA.
IF YOU DO THAT IN NORMAL CELLS,
WHAT YOU GET IS THIS VERY LOVELY
SIGNAL WHERE ONE SISTER
CHROMATIN LIGHTS UP WITH RED,
ONE WITH GREEN.
BUT IF YOU'VE GOT INAPPROPRIATE
RECOMBINATION OR REARRANGEMENT
BETWEEN THESE REPETITIVE
SEQUENCES IN THE CENTROMERES YOU
START TO SEE THESE ABERRANT
SIGNALS WHERE YOU GET DIFFERENT
PATTERNS OF RED AND GREEN
ASSOCIATED WITH SISTER
CHROMATIDS, AND IMAGES SHOWN ON
THE BOTTOM, YOU CAN SEE IN THE
CONTROL CELL LINE YOU'VE GOT
REALLY LOVELY GREEN AND RED
SIGNALS, AND THEN WHEN THEY
DEPLETE CENP-A, SIGNALS BECOME
NO LONGER SORT OF BEAUTIFULLY
PATTERNED ACCORDING TO SISTER
CHROME SIDS.
WE SET THIS UP TO SEE WHETHER
LOSS OF PBRM 1 WOULD HAVE IMPACT
ON INTEGRITY OF CENTROMERIC
SEQUENCES.
DATA THAT WE GOT ARE SHOWN HERE.
SO THIS IS -- YOU CAN SEE THIS
IS A SET OF CHROMOSOMES LABELED
IN THE WAY DESCRIBED PREVIOUSLY.
YOU GET REALLY NICE ONE RED
SIGNAL, ONE GREEN WITH SISTER
CHROMATIDS.
POSITIVE CONTROL, DELETED
CENP-A, THAT LEADS TO A DECREASE
IN THE NUMBER OF NORMAL SIGNALS
ASSOCIATED WITH SISTER
CHROMATIDS.
AND THESE ARE THE DATA WITH THE
CONTROL.
SO THIS IS WHAT WE FOUND IN --
WHEN WE QUANTITATED NUMBER OF
ABNORMAL MITOSIS, THIS IS WHAT
WE SAW.
WHEN KEY DEPLETE CENP-A THERE'S
INCREASE IN NORMAL MITOSIS, AS
WE WOULD EXPECT.
WHEN WE LOOKED AT PARENTAL CELL
LINE AND KNOCKOUTS WE SAW
SOMETHING SIMILAR.
THIS AGAIN IS THE PARENTAL,
WHICH LOOKS LIKE NON-TARGETING
CONTROL WITH S RNA, AND THEN
THESE ARE THE TWO PBRM1 CLONES,
THE RPE CELL LINE.
WE SAW SOMETHING THAT WAS FAIRLY
SIMILAR TO WHAT THEY WERE SEEING
WITH CENP-A DEPLETION, LOSING
ABILITY OR LOSING PROTECTION OF
THESE CENTROMERIC SEQUENCE WHEN
PBRM1 IS MISSING.
SO, I MENTIONED THAT WE FOUND
THIS BY SORT OF LOOKING AT THE
PROTEOME ACROSS OUR DIFFERENT
CELL LINE MODELS WE GENERATED IN
THE LAB, WHICH ARE GREAT BECAUSE
THEY ARE ISOGENIC, BUT THE
ORIGINAL HERE WAS THIS IS
SOMETHING THAT MIGHT BE
GENERALLY TRUE, IN LOTS OF
DIFFERENT CELLS LACKING PBRM1,
MORE BROADLY THINK FROM A CANCER
POINT OF VIEW.
SO, ONE OF THE THINGS WE DID WAS
TO DOWNLOAD SOME DATA FROM THE
STUDIES WHERE THEY DID THE
CANCER CELL LINE ENCYCLOPEDIA,
AND IF YOU REMEMBER WE CAN'T SEE
THIS CHANGE IN CENTROMERE AT THE
TRANSCRIPT LEVEL, SO WE
DOWNLOADED THE DATA WHERE THERE
WERE FULL PROTEOMES, NOT LOOKING
AT ISOGENIC PAIRS, WHAT WE DID
WAS ORDER BASED ON PBRM1
EXPRESSION FROM PROTEOMIC DATA,
SHOWN HERE.
WE'VE GOT EACH COLUMN HERE
REPRESENTS A CELL LINE WITH THE
ENCYCLOPEDIA WITH PROTEOMIC DATA
AVAILABLE.
BELOW IT IN THE HEAT MAP SHOWING
RELATIVE SCALED ABUNDANCE OF THE
CENTROMERE-ASSOCIATED PROTEINS
IN PROTEOMES IN THE
CENTROMERE-ASSOCIATED PROTEINS,
IN THE DATA.
AND WHAT YOU CAN SEE RED IS
HIGH, BLUE IS LOW, AS YOU GO
FROM HIGH TO LOW LEVELS YOU SEE
A GENERAL CHANGE IN THE
CORRELATION AND ABUNDANCE OF
CENTROMERE-ASSOCIATED PROTEINS,
SOMETHING MORE BROADLY TRUE WHEN
YOU LOSE PBRM1 YOU GET DECREASE
IN CENTROMERE-SPECIFIC PROTEINS.
WHAT ABOUT IMPACT ON FRAGILITY
OR INSTABILITY?
EVEN IN THE ABSENCE OF
PERTURBATION WE WERE SEE
REARRANGEMENTS OF CENTROMERES IN
THE SYSTEM.
PREVIOUSLY WHEN WE FOUND THIS
DEFECT IN SISTER CHROMATID
COHESION WE LOOKED AT
ANEUPLOIDY, THERE WAS AN
INCREASED LEVEL OF ANEUPLOIDY IN
WHOLE CHROMOSOME INSTABILITY
WITH LOSS OF PBRM1.
IS THIS TRUE IN CANCER SAMPLES?
DO YOU SEE EITHER WHOLE ARM OR
WHOLE CHROMOSOME INSTABILITY
ASSOCIATED WITH PBRM1 LOSS?
FORTUNATELY FOR US A BEAUTIFUL
STUDY CAME OUT FROM FLORIAN
MARKOWITZ'S LAB LAST YEAR
LOOKING AT CHROMOSOME
INSTABILITY BROADLY, USING --
LOOKING PAN-CANCER, TRYING TO
UNDERSTAND SIGNATURES, THAT WERE
APPARENT IN CANCER SAMPLES.
AND THEY DID THAT, AND THEY
IDENTIFIED 17 SIGNATURES
ASSOCIATED WITH CHROMOSOME
INSTABILITY BASED ON DIFFERENT
FEATURES AND LOOKED TO SEE
WHETHER THERE WERE GENETIC
DETERMINANTS, THIS IS ONE OF THE
PAPERS SHOWING THAT, YOU CAN SEE
THE 17 17 SIGNATURE SIGNATURES,
LOSS WAS ASSOCIATED WITH
SIGNATURE 1.
SIGNATURE 1 WHEN THEY WERE
DESCRIBING THESE SIGNATURES AND
BASED ON MOLECULAR
CHARACTERISTICS OF WHAT THEY
WERE SEEING IS ASSOCIATED WITH
DEFECTS IN MITOSIS, AND
ASSOCIATED WITH EITHER WHOLE ARM
OR WHOLE CHROMOSOME CHANGES.
THIS BEAUTIFULLY FIT WITH WHAT
WE WERE FINDING IN OUR CELL LINE
MODELS, SUGGESTING THIS IS
ACTUALLY PERHAPS MORE BROADLY
ASSOCIATED WITH CANCER SAMPLES
AND SOMETHING THAT'S RELEVANT IN
CANCER BIOLOGY.
SO, WHAT WE WANTED TO DO WAS
PUSH THIS SYSTEM.
SO FAR WE'VE JUST BEEN MAPPING
AND CHARACTERIZING WHAT'S
HAPPENING IN THE ABSENCE OFPBRM1
UNDER NORMAL CONDITIONS IN THE
CELLS.
HERE WE'VE SEEN WHETHER OR NOT
THEY SHOW HYPERSENSITIVITY TO
MITOTIC PERTURBATION.
WHAT I'M GOING TO SHOW IS AN
EXPERIMENT THAT WE DID WHERE WE
JUST TREATED THE CELLS WITH CDK
1 INHIBITOR FOR 24 HOURS,
REPORTED IN LITERATURE, CELLS
WITH PROBLEMS AT CENTROMERES ARE
PARTICULARLY SENSITIVE TO.
YOU CAN SEE THE SCHEME WE USED,
SHOWN HERE, TREATED WITH CDK1
INHIBITOR FOR 24 HOURS, WASHED
IT OFF AND ALLOWED CELLS TO
REENTER CELL CYCLE AND MONITORED
AT 24 AND 48 HOURS POST I GUESS
RELEASE FROMMED DRUG.
AND WE JUST MONITORED THEM
VISUALLY.
WE WERE LOOKING AT THEIR NUCLEI
AND SCORING THEM AS TO WHETHER
OR NOT THEY LOOKED NORMAL OR
ABNORMAL.
OF COURSE, WE REALIZE THE ONES
WE'RE SCORING AS NORMAL COULD
STILL HAVE GONE THROUGH AN
ABNORMAL MITOSIS BUT WE'RE JUST
LOOKING AT THIS MORE GROSS
OUTCOME OF MITOTIC PERTURBATION.
AND PUT THEM INTO THESE VARIOUS
CATEGORIES FROM FAIRLY MILD TO
WHAT IS QUITE CLEARLY NOT A
SUCCESSFUL MITOSIS EVENT AT THE
END HERE.
AND WHAT YOU CAN SEE IN THE
PARENTAL CELL LINE IS THAT IN
UNTREATED CONDITIONS, AS YOU
EXPECT, VAST MAJORITY OF THE
CELLS LOOK NORMAL.
IF YOU TREAT THEM FOR 24 HOURS
WITH CDK1 INHIBITOR AND RELEASE
YOU CAN SEE INCREASE IN THINGS
THAT HAVE GONE AWRY IN THESE
CELLS.
THEY ARE STARTING TO RECOVER A
LITTLE BIT BY 48 HOURS BUT
THERE'S STILL SOME THINGS THAT
HAVE GONE WRONG THERE.
WHEN WE DO THE SAME THING IN THE
KNOCKOUT CLONES IN THE CELL LINE
BACKGROUND, IN THE UNTREATED
CELLS AGAIN WE'RE NOT SEEING A
MASSIVE IMPACT AT THIS STAGE, SO
NORMALLY DURING MITOSIS THEY ARE
PROGRESSING APPROPRIATELY.
EVEN THOUGH AS WE KNOW THEY HAVE
ACTUALLY GOT SORT OF BIGGER
CENTROMERES AND THEY ARE
RECOMBINING MORE THAN THE
PARENTAL.
BUT WHEN WE CREED WITH CDK1
INHIBITOR TO PUSH THINGS WE SEE
FOR CELLS WITH PROBLEMS AS THEY
GO THROUGH MITOSIS COMPARED TO
PARENTAL, CONSISTENT WITH IMPACT
ON CENTROMERE BIOLOGY.
WE DID ANOTHER EXPERIMENT, IN A
LOT OF DIFFERENT CELL LINES BUT
I'M SHOWING THIS BECAUSE HERE
WE'RE NOT USING ISOGENIC CELL
LINE MODELS.
WE LOOKED AT A PANEL OF RENAL
CANCER-DERIVED CELL LINES, AND
THIS IS WHERE YOU SEE VERY
FREQUENT PBRM1 MUTATION, LOSS OF
FUNCTION MUTATIONS THAT RESULT
IN LOSS OF PROTEIN EXPRESSION.
AND YOU CAN SEE, AGAIN, WE'VE
GOT THREE THAT HAVEPBRM1
EXPRESSION, NOW DEPLETING TO
PROMOTE MITOTIC TRANSITION,
USING TWO siRNAs AGAINST
CYCLIN B1 THOSE LACKING BPRM 1
ARE MORE SENSITIVE TO DEPLETION
THAN THOSE THAT DON'T LACK
PBRM1.
THIS SUGGESTED WE MIGHT HAVE
SOMETHING THAT WE COULD ACTUALLY
MAKE USE OF IN TERMS OF CLINICAL
POTENTIAL.
SO, WHAT WE NOTED THERE WAS VERY
LITTLE, AS I SHOWED WITH THE CDK
1 INHIBITOR DATA WHEN WE DON'T
PROCURE THINGS THERE ARE CHANGES
WE CAN DETECT BUT OVERALL
FITNESS OF CELLS LOOKS OKAY AND
VAST MAJORITY GO THROUGH MITOSIS
NOR OR LESS NORMALLY.
PERHAPS THE REASON IS BECAUSE
THEY HAVE GOT INTACT SPINDLE
ASSEMBLY CHECKPOINT, AND THAT
THEY MIGHT BE RELIANT ON THAT.
AND THIS OF COURSE IS A USEFUL
THING TO UNDERSTAND BECAUSE
INHIBITORS OF THE SPINDLE
ASSEMBLY CHECKPOINTS IN THE
CLINIC COULD BE A GOOD WAY OF
TREATING PBRM1 DEFICIENT CANCERS
IS HE WE WANTED TO ASK WHETHER
CELLS WITH SENSITIVE TO NPS
INHIBITORS, THIS IS SHOWING
RPE1-BASED SYSTEM, RED LINES ARE
THE TWO CLONES, BLACK LINE IS
THE PARENTAL, WE'RE USING THREE
DRUGS HERE.
SO WE'VE GOT AN ASTRAZENECA DRUG
INHIBITOR, AND ONE THAT WAS
GENERATED IN THE ICR, THIS BOS
NPS1 INHIBITOR, YOU CAN SEE TO
VARYING DEGREES KNOCKOUT LINES
ARE MORE SENSITIVE TO NPS1
INHIBITORS THAN PARENTAL.
SO THIS SEEMED SORT OF
ENCOURAGING THING TO LOOK AT IN
MORE DETAIL, WHAT WE WANTED TO
DO WAS GO TO A MOUSE MODEL AND
SEE WHETHER OR NOT TREATING
CELLS WITH NPS 1 INHIBITORS IN
MICE COULD HAVE IMPACT ON
DIFFERENTIAL SENSITIVITY.
WE GENERATED KNOCKOUTS IN A
MOUSE CELL LINE.
THIS IS THE MOUSE B16 CELL LINE,
A MELANOMA LINE, USEFUL, GOOD
FOR DOING MOUSE XENOGRAFT WORK,
AND BY DOING IT IN MOUSE CELL
LINE AT ALLOWED TO GO INTO
IMMUNOCOMPETENT MOUSE MODEL,
RELATED TO ANOTHER PROJECT IN
THE LAB WHERE WE SEE CHANGES IN
INNATE IMMUNE SIGNALING WHEN WE
LOSE PBRM1 1 AND WANTED TO KILL
TWO BIRDS WITH ONE STONE.
WE NEEDED TO SEE WHETHER OR NOT
WE GOT THE SAME IMPACT ON
CENTROMERES, AS WE DID IN THE
MOUSE CELL LINE, AS WE DID IN
THE HUMAN CELL LINES.
THE FIRST THING DID WAS TO LOOK
AT PROTEOME ANALYSIS, AND AGAIN
I'M PLOTTING THE SAME DATA, SORT
OF LOG TWO-FOLD CHANGE VERSUS
PARENTAL, VERY SATISFYINGLY AS
YOU CAN SEE ACROSS THESE VARIOUS
DIFFERENT COMPLEXES ASSOCIATED
WITH CENTROMERES WE CAN SEE
REDUCTION ACROSS THE BOARD OF
THESE PROTEIN LEVELS.
ARE THE CELLS SENSITIVE TO NPS1
INHIBITORS?
AGAIN THE ANSWER IS YES.
WE'VE GOT THE ICR DIRECT
COMPOUND AND ASTRAZENECA
GENERATED COMPOUNDS, WE SEE
SENSITIVITY TO BOTH.
TWO THINGS ABOUT THE MOUSE CELL
WORK THAT WAS PERHAPS SLIGHTLY
SUBOPTIMAL, ONE IS THAT OUR
COLLABORATORS WERE RESTRICTED TO
USING THIS ICR-GENERATED MPS1
INHIBITOR, WE SEE BIGGER EFFECTS
WITH ASTRAZENECA ONE, SOMETHING
WE WANTED TO COME BACK TO, TO
UNDERSTAND BETTER.
SECOND THING, THIS IS THE
SCHEMATIC OF THE APPROACH.
THE THE STUDY ENDED, THE MOUSE
HOUSE WAS BEING CLOSED FOR
REFURBISHMENT SO WE HAD TO END
THE EXPERIMENT.
WHAT WE DID WAS EITHER TAKE THE
PARENTAL B16 LINE OR TWO
KNOCKOUTS OF PBRM1, PUT THEM
INTO GROUPS OF MICE WHERE THEY
WERE TREATED WITH THE VEHICLE OR
WITH THE MPS1 INHIBITOR AND LOOK
AT SURVIVAL AND TUMOR GROWTH.
THIS IS THE SURVIVAL CURVE ON
THIS SIDE, THE PARENTAL.
YOU CAN SEE MPS1 INHIBITOR IS
LIMITED AND WASN'T STATISTICALLY
SIGNIFICANT.
THE OTHER TWO PANELS ARE
KNOCKOUT LINES, IMPACT ON
SURVIVAL, IN THIS CASE WITH THE
B25 WAS SIGNIFICANT INCREASE IN
SURVIVAL, DIDN'T REACH
SIGNIFICANCE WITH THE OTHER
CLONE BUT WHAT YOU'LL ALSO NOTE
IS THAT EVERYTHING ENDS ABRUPTLY
AT DAY 42.
IF YOU LOOK AT THE NUMBER OF
MICE THAT WERE STILL ALIVE AT
DAY 42 YOU SEE THERE ARE A
REASONABLE PERCENTAGE IN MOUSE
GROUPS FROM THE KNOCKOUT THAT
HAVE BEEN TREATED WITH MPS1
INHIBITOR, SOMEBODY WE WANTED TO
DIG INTO AND LOOK AT A BIT MORE
BECAUSE WE THINK
PRELIMINARYARIAL AT LEAST THIS
IS GIVING A GOOD INDICATION THAT
COULD BE SOMETHING YOU CONSIDER
USING WITH PBRM1 CANCERS IN THE
CLINIC.
GETTING BACK TO MECHANISMS, SO
OUR HYPOTHESIS IS THAT PBRM1 AND
PBAF REMODELING COMPLEX IS
WORKING DIRECTLY AT CENTROMERES
IN ORDER TO CREATE A SUBSTRATE
WHICH IS ACTUALLY A GOOD
PLATFORM FOR ASSEMBLY FOR THESE
PROTEINS AND THEY CAN ASSEMBLE
IN THE ABSENCE OF PBRM1 BUT NOT
AS EFFICIENTLY IN THE PRESENCE.
LOSS OF PROTEINS LEADS TO
PROBLEMS AND VULNERABLE TO
MITOTIC STRESS.
SO, PREDICTION THEREFORE IS THAT
WE SHOULD BE ABLE TO SEE PBRM1
AND PBAF COMPLEX OR CENTROMERIC
OR PERICENTROMERIC CHROMATIN.
THIS IS REPETITIVE ELEMENTS
ASSOCIATED WITH CENTROMERES FROM
THE LATEST TELOMERE-TO-TELOMERE
ANALYSIS OF HUMAN CHROMOSOMES
LOOKING AT REPETITIVE DNA IN
PRETTY AMAZING DETAIL.
WE USED THIS T-TO-T GENOME GENES
TO ALIGN MAPPING DATA, USING CUT
AND RUN TO MAP SMARC4 TO THE
GENOME TO SEE WHERE IT WAS
ALIGNING, AND WE CENP TO LOOK AT
THINGS BINDING TO REPETITIVE
SEQUENCES, TO SEE IF SMARC4 IS
BINDING.
WE CHOSE THAT BECAUSE WE COULD
THEN LOOK IN THEPBRM1 KNOCKOUT
TO DESEE -- TO SEE IF IT'S
ASSOCIATING ON CENTROMERIC
ALLIANCES.
THIS IS A SCREEN SHOT OF THE OF
ASSOCIATION OF THESE PROTEINS
WITH CENTROMERES.
I WANTED TO SHOW THE PATTERN
ACROSS THE CENTROMERIC REGION.
WE'VE ANNOTATED IN BLACK,
REPEATS WHICH CONTAIN ALPHA
SATELLITE SEQUENCES IN RED,
WE'VE GOT PERIPHERALLY TO THAT
MORE DIVERGENT ALPHA SATELLITE
SEQUENCES AND OTHER REPETITIVE
SEQUENCES SUCH AS HSAT 2 AND 3,
AND IN GRAY TRANSITION ARMS
WHICH HAVE THESE, AGAIN,
REPETITIVE SEQUENCES BUT NOW
WE'RE LOOKING AT SOME
INTERSPERSED ACTIVE GENES AND SO
FORTH.
SO WHERE ARE THINGS BINDING?
WE DID CENP-B, YOU CAN SEE
ENRICH.
ACROSS HIGHER ORDER OF REPEATS,
WHERE YOU EXPECT TO SEE B
ASSOCIATION, THAT ENRICHMENT,
COMPARED TO IgG NEGATIVE
CONTROL.
WE IS SMARCA4 WITH A KNOCKOUT
CELL LINE, INSTEADING USING
IgG TO USE THE ANTIBODY
AGAINST SMARC4 IN CELLS THAT
DON'T CONTAIN SMARCA4.
KNOCKOUT NEGATIVE CONTROL IN
GRAY, AND ASSOCIATION OF SMARCA4
WITH CHROMATIN IN THE PARENTAL
CELLS IS IN GREEN.
WHAT YOU CAN SEE WE GET A NICE
ENRICHMENT OF SMARCA4 ACROSS THE
SAME REGION OF THE CENTROMERE WE
SEE CENP-B ENRICHMENT AND ACROSS
THE CENTROMERES.
AS WE WERE ANALYZING THIS WE
WANTED TO GET A FULLER PICTURE,
FROM THIS IT WAS REASONABLE TO
CONCLUDE WE HAVE ENRICHMENT OF
SMARCA4 AT CENTROMERES BUT WE'RE
RUNNING INTO A COUPLE ISSUES TO
SEE WHERE IT IS BINDING, ONE YOU
CAN SEE FROM TWO NEGATIVE
CONTROLS YOU GET SOME BACKGROUND
READS FROM REPETITIVE SEQUENCES
THAT ARE A LITTLE BIT HIGHER
THAN FROM NON-REPETITIVE
SEQUENCES.
THAT'S ACTUALLY BEEN REPORTED IN
THE LITERATURE.
WE CAN LOOK AT WHAT'S ENRICHED
ABOVE THAT.
THE OTHER CHALLENGING ISSUE IS
MAPPING READS, INTERESTED TO
UNDERSTAND WHERE THINGS COULD
MAP MULTIPLE PLACES LOSING
ENRICHMENT TO TELL US IT WAS
BOUND AT A SICK SEQUENCE.
IN ORDER TO GET A MORE -- I
GUESS A FULLER PICTURE EXACTLY
WHERE SMARCA4 IS BINDING, WE
DECIDED TO USE THIS ANALYSIS, A
SEQUENCE OF K AS A SUBSEQUENCE
OF SOME SORT OF LARGER READ THAT
YOU'RE LOOKING AT.
LOOKING AT 50 K-MERS, AND THIS
WE SORT OF FOLLOWED THE APPROACH
THAT WAS TAKEN BY THE STRAIGHT
LAB, USING MAPPING OF CENP-A TO
IDENTIFY SEQUENCES ENRICHED,
K-MERS ENRICHED IN THE CENP-A
READS TO GET A SENSE OF
CENTROMERIC SEQUENCES.
WE FOLLOWED THAT APPROACH TO ASK
THE QUESTION OF PATTERN OF
SEQUENCES BOUND BY SMARCA4?
I WILL BRIEFLY GO THROUGH THIS
FOR THE SAKE OF TIME BUT HAPPY
TO SPEAK WITH PEOPLE LATER ABOUT
IT IF THEY ARE INTERESTED,
ESSENTIALLY WE TOOK THOSE CUT
AND RUN READS ASSOCIATED WITH
THE CENTROMERIC REGION INCLUDING
TRANSICS A LITTLER, NORMALIZE
K-MER COUNT AND THE KEY THING
CALCULATING THEM, THE FOLD
CHANGES, ADJUSTED P-VALUES OF
K-MERS BETWEEN OUR POSITIVE
CONTROL OR NOT POSITIVE
CONTROLS, THE CENP-B AND
NEGATIVE CONTROL.
YOU'RE LOOKING AT ENRICHED K
MERS ASSOCIATED WITH CENP-B,
WHAT TYPE OF CENTROMERE SEQUENCE
ARE THESE ASSOCIATED WITH RATHER
THAN WHAT POSITION ALONG HIGHER
ORDER REPEAT DOES IT BIND TO.
AND AS A NEGATIVE -- EXTRA
NEGATIVE CONTROL WE TOOK THE
ENRICHED K-MERS, GOT A LOT FOR
CENP-B
FOR SMARC4s, IF YOU HAD THEM
RANDOMLY ASSOCIATED WITH GENOME
JUST TO SORT OF GIVE OURSELVES
EXTRA CONFIDENCE THAT ANY
PATTERN WE WERE SEEING WAS REAL
AND NOT A CONSEQUENCE OF
DIFFERENT PROPORTIONS OF DNA
THAT WAS OCCUPIED BY THE VARIOUS
TYPES OF CENTROMERE REPEATS.
I HOPE THAT MAKES SENSE.
OKAY.
WE DID THAT.
AS I SAY WE DID THIS SHUFFLED
AND AS WE ANTICIPATED, AS WE
HOPED, WHICH IS TO SAY IT WAS A
VERY FLAT ASSOCIATION, SO THE
NUMBER OF K-MERS WHEN YOU
SHUFFLE THE LOCATIONS THAT ARE
RANDOMLY ASSOCIATED WITH THESE
DIFFERENT TYPES OF REPEATS
PRETTY MUCH CORRESPONDS TO HOW
MUCH DNA SEQUENCE THAT OCCUPIES
IN ANY GIVEN CENTROMERE.
WE DID IT WITH CENP-B, MAPPING
BACK PRIMARILY TO THE HIGHER
ORDER REPEATS, SO THE ACTIVE
HIGHER ORDER REPEATS ASSOCIATED
WITH CENTROMERES, WHAT YOU WOULD
PREDICT AND DID THIS WITH SMARC4
AND GOT A PATTERN LIKELY,
BIGGEST ENRICHMENT IN HIGHEST
ORDER REPEATS FOLLOWED BY HSAT 1
AND 2, AND LOW LEVEL WITH
TRANSITION ARMS, WE STILL NEED
TO DO MORE WORK BUT ALMOST A
REASONABLE INTERPRETATION, VERY
SPECIFIC POINTS ASSOCIATED WITH
GENES IN THOSE TRANSITION ARMS.
BUT REALLY WHAT WE'RE GETTING IS
A BIG ENRICHMENT OF REPETITIVE
SEQUENCES ASSOCIATED WITH HIGHER
ORDER REPEATS.
SO THIS IS GREAT.
BASICALLY FROM BOTH WE CAN SAY
WITH CONFIDENCE SMARCA4 IS
ASSOCIATED WITH CENTROMERES AND
PARTICULARLY WITH HIGHER ORDER
REPEATS IN CENTROMERES.
WHAT HAPPENS WITH THE SAME THING
IN THE PBRM1 KNOCKOUT?
SHUFFLE LOOKS THE SAME.
CENP-B LOOKS THE SAME.
WE CAN CONCLUDE CENP-B
ASSOCIATION TO PARTICULAR
SEQUENCES, THE ABILITY TO FIND
THE SEQUENCES AND PATTERN OF
BINDING IS UNAFFECTED BY THE
ABSENCE OF PBRM1.
WHAT HAPPEN WAS SMARCA4?
IF WE LAYER THE DATA, THERE WERE
NONE.
WE FOUND NO K-MERS ENRICHED IN
THE SMARCA4 CUT AND RUN DATASET
IN THE ABSENCE OF PBRM1 COMPARED
TO NEGATIVE CONTROLS.
SO THE ABILITY OF SMARCA4 TO GET
TO THOSE PARTICULAR SITES IN
CENTROMERES IS DEPENDENT ON
PBRM1.
SOAK.
WHAT IS PBAF DOING AT THESE
REGIONS IN THE CENTROMERE IN
ORDER TO PROMOTE ASSEMBLY OF THE
CENTROMERE-ASSOCIATED PROTEINS
AND OF COURSE PREVENT
INAPPROPRIATE RECOMBINATION OF
SO FORTH?
AN OBVIOUS THING TO LOOK AT,
TRANSCRIPTION.
WE'VE GOT A CHROMATIN REMODELER
WHICH WE KNOW ASSOCIATES WITH
HIGHER ORDER REPEATS, IMPORTANT
FOR CENTROMERE STRUCTURE AND
FUNCTION, AND WORK FROM LABS
SHOWS THERE'S A LOW LEVEL OF
CENTROMERE-DERIVED TRANSCRIPTS
IMPORTANT FOR ASSEMBLY OF THESE
PROTEINS AT CENTROMERES AND FOR
KINETIC ATTACHMENT.
THIS SEEMED LIKE A REASONABLE
HYPOTHESIS THAT WE'VE GOT
SOMETHING THAT'S ACTUALLY BEING
TRANSCRIPTIONALLY MISREGULATED
IN ABSENCE OF PBAF, AND WHAT
PEOPLE HAVE SHOWN, THIS IS
IMPORTANT FOR THE NEXT SLIDE, IS
THAT EITHER TOO MUCH OR TOO
LITTLE TRANSCRIPTION FROM THESE
CENTROMERIC SEQUENCES IS ENOUGH
TO IMPAIR CENTROMERE ASSEMBLY,
CENTROMERE ASSEMBLY AND CORE
ATTACHMENTS IN CENTROMERE
FUNCTION.
WE LOOKED AT TRANSCRIPTION, WE
DID THIS A COUPLE WAYS.
HERE IS DATA USING PRIMER PAIRS
AGAINST ALPHA SATELLITE
SEQUENCES PRIMARILY BUT WE ALSO
GOT AN HSAT 2 SET OF SEQUENCES.
THE PARENTAL IS SHOWN IN BLACK.
TWO PBRM1 KNOCKOUTS IN BLUE, IN
TWO CELL LINES, AND WE GET
MISREGULATION OF TRANSCRIPTION
BUT IN FACT IT'S UPREGULATED IN
THE KNOCKOUTS AND WE SEE THE
SAME THING WHEN WE LOOK AT
RNAseq DATASETS, LOOKING INTO
THIS TO SEE IF WE CAN RELATE TO
K-MERS AND POSITION OF PBAF
BINDING.
TO PULL INTO A MODEL, TO FINISH,
WHAT WE KNOW NOW IS THAT PBAF IS
RECRUITED -- IS ASSOCIATED WITH
CENTROMERES, WITH OTHER
REPETITIVE ELEMENTS, IN THE
ABSENCE PBAF FAILS TO GET TO
CHROMATIN, AS A CONSEQUENCE WE
THINK THAT THAT -- THAT LEADS TO
INAPPROPRIATE REGULATION OF THE
CENTROMERE-SPECIFIC
TRANSCRIPTIONS AND THAT IS SHOWN
TO BE IMPORTANT FOR COMPLEX
ASSEMBLY.
IF YOU DON'T HAVE SAY
APPROPRIATE COMPLEX ASSEMBLY
THAT LINKS US INTO ALL THESE
PHENOTYPES WE'RE SEEING,
DECREASE IN
CENTROMERE-ASSOCIATED COHESION,
INCREASED CENTROMERE FRAGILITY,
WHOLE ARM AND WHOLE CHROMATIN
INSTABILITY.
AND WITH THAT I'LL BRIEFLY
MENTION ALL OF THE WONDERFUL
PEOPLE WHO ARE INVOLVED.
IN MY LAB, KAREN LANE IS A
FANTASTIC POSTDOC WHO HAS DRIVEN
THIS PROJECT AND DONE CELL
MOLECULAR BIOLOGY, WITH A LOT OF
INPUT FROM JEFFREY FUNG, WHO HAS
RECENTLY LEFT TO DO POSTDOC AT
THE CRICK.
LILLIAN WU DID THE CUT AND RUN
ASSAYS WITH SMARCA4 AND ALLISON
DID A FORMAL JOB WITH --
PHENOMENAL JOB WITH K-MER
ANALYSIS.
PROTEOMEIC ANALYSIS IS DONE WITH
GREAT COLLABORATORS WITH JYOTI.
I'LL STOP AND TAKE QUESTIONS.
THANK YOU.
>>THANK YOU VERY MUCH FOR AN
EXCELLENT TALK.
REALLY BEAUTIFUL DATA AND VERY
INTERESTING AND CHALLENGING.
I'M WAITING FOR SOME CHAT
MESSAGE QUESTIONS TO COME HERE.
AND I GUESS I WOULD LIKE TO ASK
ABOUT THE RELATION TO DNA DAMAGE
AND REPAIR MAYBE HERE BECAUSE
YOU SAY THAT DISRUPTING
CENTROMERES WITH PBAF AND PBRM1
DEPLETION CAUSES CHANGES IN
SURVIVAL, IN TRANSCRIPTIONAL
THINGS AND STATUS.
DOES IT CHANGE DNA DAMAGE IN
RELATION, DOES IT CHANGE DNA
REPAIR?
>>YEAH, THAT IS AN EXCELLENT
QUESTION.
SO, THERE WAS A RECENT -- WELL,
A NUMBER OF PAPERS AROUND DNA
DAMAGE RESPONSES IN CENTROMERES
BUT RECENT PAPER SHOWED THERE
ARE DOUBLE-STRAND BREAKS
NORMALLY ASSOCIATED WITH THESE
CENTROMERIC SEQUENCES, AND SO
ONE REALLY ATTRACTIVE
POSSIBILITY IS THAT BECAUSE WE
KNOW THAT PBAF PLAYS A ROLE AT
DOUBLE-STRAND BREAKS, AND SO ONE
ATTRACTIVE POSSIBILITY WE'RE
LOOK BOOK IS IS THERE'S A
RELATIONSHIP BETWEEN PBAF
TRANSCRIPTION AND THE
DOUBLE-STRAND BREAKS, AND OF
COURSE WHICH WAY AROUND IT GOES
IS NOT ENTIRELY CLEAR BUT ONE
WAY YOU CAN IMAGINE IS THAT WE
KNOW PBAF ASSOCIATES IS
SEQUENCES TO RESTRAIN
TRANSCRIPTION, IT COULD BE TOO
MUCH TRANSCRIPTION BRINGS IN THE
SORT OF ACTIVITIES LIKE
TOPOISOMERASES WHICH CREATE
BREAKS THAT LEAD TO FRAGILITY
WE'RE SEEING.
THERE MIGHT BE A REALLY STRONG
ASSOCIATION, THAT'S SOMETHING WE
WANT TO LOOK INTO TO SEE WHETHER
OR NOT NUMBER AND LOCATION OF
DOUBLE-STRAND BREAKS IS
DIFFERENT IN PRESENCE OR ABSENCE
OF PBRM1.
>>RELATING TO ACCESSIBILITY
ISSUE, AND I GUESS THE SURVIVAL
DEFICIENCY THAT YOU SEE, IS THAT
THEN POSSIBLY RELATED TO
DOUBLE-STRAND BREAKS?
>>YEAH, I THINK IT COULD BE.
WE SEE -- IT'S REALLY DIFFICULT
TO DISENTANGLE THESE THINGS
BECAUSE WE ALSO SEE A DECREASE
IN SURVIVAL WHEN WE USE
REPLICATION STRESS.
AND SO, YOU KNOW, WHAT WE WANT
TO TRY TO GET AT IS WHETHER OR
NOT WE CAN LOOK AT THE FATE OF
THE CELLS AFTER WE TREAT THEM
WITH, SAY, REPLICATION STRESS
VERSUS NPS1 INHIBITOR TO ASK
EXACTLY HOW THEY DIED, WAS IT
DOWN TO THE DNA DAMAGE AND
FAILURE OR INAPPROPRIATE
RECOMBINATION OF FAILURE TO DEAL
WITH THINGS AT CENTROMERES, YOU
KNOW, AS A CONSEQUENCE OF TAKING
AWAY THE CHECKPOINT ACTIVITY.
>>A QUESTION, GREAT TALK, GREAT
ANALYSIS.
ONE QUESTION, IS THE CENP
COHESION PHENOTYPE DEPENDENT ON
ACTIVITY OF PBAF?
>>FANTASTIC.
HELLO AND THANK YOU VERY MUCH.
YEAH, IT'S A GREAT QUESTION.
YES, WE DID THE EXPERIMENT A
COUPLE WAYS, WE USED ATPase
MUTANTS OF SMARCA4 BUT NOW
WHAT'S WONDERFUL FOR US THERE
ARE BOTH ATPase INHIBITORS,
AND WE REPEATED A LOT OF
EXPERIMENTS AND LOOKED AT THE
RESPONSES TO THE PROTACs AND
ATPase INHIBITOR, YOU NEED
REMODELING TO GET ADHESION AND
PREVENT THESE SORTS OF ISSUES.
>>DO YOU WANT TO READ THE NEXT
QUESTION?
>>YES.
SO WE HAVE A QUESTION FROM
ALEXANDER.
HAVE YOU LOOKED AT THE COHESION
LEVEL BY CUT AND RUN IN THE
ABSENCE OF PBMR1?
DO YOU THINK THIS IS BECAUSE OF
TRANSCRIPTION EFFECTS OR
PROBLEMS WITH COHESION LOADING?
>>THAT'S A GREAT QUESTION.
IN FACT, INTERESTINGLY, THE
PHENOTYPES FROM YEAST WERE WHAT
MADE US LOOK AT SISTER CHROMATID
COHESION IN THE MAMMALIAN SYSTEM
TO SEE WHETHER THAT WAS
CONSERVED, SO, YEAH.
WHAT WE FIND IS THERE'S NO
DEFECT IN TRANSCRIPTION OF ANY
COHESION-ASSOCIATED -- THE
PATHWAY, TRANSCRIPT IS NORMAL IN
ABSENCE OF PBRM1.
BACK WHEN WE FOUND THE DEFECT WE
ACTUALLY TRIED LOOKING AT
PROTEIN LEVELS GLOBALLY AND WE
TRIED LOOKING AT PROTEIN LEVELS
ASSOCIATED WITH CHROMATIN, AND
WE NEVER FOUND ANYTHING THAT WE
THOUGHT WAS CONVINCINGLY
DEFECTIVE.
SO, WE WOULD HAVE SAID IT LOOKS
LIKE COHESION ASSOCIATION AND
LOADING ARE UNAFFECTED BY THE
ABSENCE OF PBRM1.
HAVING SAID THAT I THINK WE'VE
GOT BETTER TOOLS NOW THAT WE CAN
USE, AS I JUST MENTIONED WITH
INHIBITORS AND PROTACs, AND
THESE KNOCKOUT LINES INSTEAD OF
USING DEPLETION, AND SO WE'VE
JUST STARTED TO REVISIT THAT
QUESTION.
SO I STILL THINK THAT'S ON THE
TABLE AS A POSSIBILITY THAT
YOU'RE FAILING TO LOAD COHESION
TO APPROPRIATE PLACES IN AND
AROUND THE CENTROMERE.
>>GREAT.
THERE'S A QUESTION FROM IRENE,
WONDERFUL WORK, WHAT DO YOU
THINK PBAF IS DOING IN REPEATED
REGIONS SURROUNDING THE
CENTROMERE?
>>OH, YEAH, THANKS, IRENE.
GREAT QUESTION.
AND I HAVE NO IDEA.
SO, THIS IS PART OF THE REASON
WE WANTED TO DO THAT K-MER
ANALYSIS TO GET AT THE QUESTION
WHAT IS BINDING AND WHEN.
SO, ONE OF THE THINGS THAT WE
FOUND WHICH WE WANT TO SORT OF
DIG INTO A LITTLE BIT MORE, IS
THAT THERE'S AN ASSOCIATION
BETWEEN PBAF BINDING AND
STRUCTURED DNAs.
AND SO ONE OF THE QUESTIONS IS
WHETHER THIS IS ACTUALLY AROUND
THE SEQUENCES PER SE, OR WHETHER
IT'S THEIR PROPENSITY TO FORM
DIFFERENT DNA STRUCTURES.
AND WHETHER OR NOT THAT'S
ACTUALLY DIFFERENT BETWEEN THE
DIFFERENT REPEATS BECAUSE
OBVIOUSLY THEY ALL HAVE
DIFFERENT PROPERTIES.
SO, YEAH, IT'S EARLY DAYS.
I DON'T KNOW THE ANSWER TO THAT
QUESTION BUT IT'S DEFINITELY
SOMETHING WE WANT TO GET VASTLY
MORE INTO AND I WOULD LOVE TO
HEAR YOUR VIEWS ON IT.
>>AND WALT SAYS THANK YOU.
SO, I WANTED TO MAYBE JUST ASK
ABOUT THE -- YOU STARTED WITH
TALKING ABOUT DIFFERENT CANCERS,
ROLE OF DIFFERENT CANCERS, HOW
DO YOU SEE THAT?
CAN YOU EXPLORE THIS A LITTLE
MORE?
HOW DO WE CONNECT THIS WHOLE
THING AND WHAT CANCERS IN
PARTICULAR SHOULD BE OF
INTEREST?
>>YEAH, YEAH, THAT'S A REALLY
GOOD QUESTION.
AND SOMETHING THAT WE'VE BEEN
THINKING A LOT ABOUT RECENTLY.
SO, PBRM1 IS LOST IN ABOUT 40%
OF RENAL CANCERS, WHICH IS PART
OF THE REASON WE USED THAT PANEL
OF RENAL CANCER CELL LINES.
HAVING SAID THAT, WE ACTUALLY
DON'T SEE THAT THIS IS A RENAL
CANCER-SPECIFIC PHENOTYPE.
>>IT'S VERY BROAD.
>>YEAH.
SEEMS MORE BROAD.
SO, BUT YOU PROBABLY WILL HAVE
ALSO NOTICED THE DEGREE OF
SENSITIVITY THAT WE'RE SEEING TO
EITHER CDK-IS, BETTER IS THE NPS
INHIBITORS, IT'S NOT HUGELY
DRAMATIC.
SO I FEEL LIKE IN TERMS OF
THINKING ABOUT THE SORT OF
IMPACTING CANCER, IT WOULD BE
GREAT IF WE COULD COME UP WITH
SOMETHING THAT WAS
THERAPEUTICALLY, YOU KNOW,
SOMETHING THAT WOULD GO AND GIVE
US A HUGE THERAPEUTIC INDEX BUT
I THINK WHAT THIS MIGHT BE
TELLING US MORE IS EVOLUTION OF
CANCERS, IT'S ALMOST LIKE THE
RECOMBINATION AT THE CENTROMERES
HAPPENING EVEN UNDER NORMAL
CONDITIONS, SEEING THEM GROW
UNDER NON-STRESSED CONDITIONS,
YOU'RE GETTING REARRANGEMENTS
AND YOU'RE GETTING
RECOMBINATION.
SO, YOU KNOW, THE GENOMES ARE --
IT'S VERY PLASTIC.
AND SO YOU COULD IMAGINE
ACTUALLY THIS IS ONE OF THE
REASONS WHY THESE THINGS ARE
ACTUALLY SO INSIDIOUS AS TUMOR
SUPPRESSORS, THAT ONCE YOU LOSE
THEM, THEY GO THROUGH MITOSIS,
BUT THINGS ARE REARRANGING IN A
WAY THAT'S NOT APPROPRIATE.
IN ANOTHER STUDY WE FOUND LOSS
OF SWI/SNF UPREGULATES PATHWAYS
THAT ALLOWS YOU TO TOLERATE
ANEUPLOIDY BETTER SO IT'S A TERM
COMBINATION.
THEY SAME TIME YOU'RE MISSING
WITH FIDELITY OF CHROMOSOME
SEGREGATION YOU'RE ALSO TAKING
AWAY SOME OF THE BREAKS THAT THE
CELL HAS THAT WOULD NORMALLY
ACTUALLY MAKE THAT A LESS-FIT
CELL.
SO, I MEAN IT'S EXACTLY THE
RIGHT COMBINATION FOR A CANCER
CELL, A DANGEROUS COMBINATION BY
LOSING THE ONE PROTEIN COMPLEX?
>>THERE'S A LOT OF DNA REPAIR
CONNECTIONS TO RENAL.
>>YEAH, I THINK THAT RENAL
CANCER IN A WAY IS -- IT'S
NOT -- THERE'S NOT A SORT OF
BIOLOGICAL REASON IN TERMS
OFPBRM1.
RENAL CELLS LOSE ONE COPE OF
CHROMOSOME 3P, WELCOME
HAPLOINSUFFICIENT, WHERE PBRM1
SITS, ALL ON 3P, NOW
HAPLOINSUFFICIENT, AND SOME
REALLY NICE WORK FROM A COUPLE
LABS DOING MOUSE STUDIES SHOWED
YOU COULD JUST GET RID OF VHL
AND PBRM1, ENOUGH TO DRIVE RENAL
CANCER.
SO IT'S -- THE OTHER GENES
OBVIOUSLY COULD PLAY A ROLE SO
IT'S KIND OF JUST BAD LUCK IN
RENAL CANCER THAT YOU ARE
ALREADY HAPLOINSUFFICIENT, IT
JUST TAKES THAT ONE HIT.
YOU SEE LOSS AT LOWER LEVELS IN
OTHER CANCERS, I SUSPECT A LOT
OF PHENOTYPES ARE ALSO IMPORTANT
THERE.
IT'S JUST THE FREQUENCY IS SO
MUCH LOWER BECAUSE YOU NEED THE
TWO HITS, THAN WHAT YOU SEE IN
RENAL CANCER.
>>INTERESTING.
CHUN, DO YOU HAVE ANY QUESTIONS?
>>NO.
THANK YOU.
>> IRENE SAYS GREAT, I'LL FOLLW
UP.
>>GREAT.
THANK YOU.
>>WE'RE OUT OF TIME.
THANK YOU SO MUCH FOR A REALLY
ELEGANT LECTURE, GREAT QUESTIONS
ANSWERED.
AND WE WILL BE IN TOUCH WITH YOU
ABOUT POTENTIALLY POSTING THE
TALK AT OUR WEBSITE, IF THAT'S
OKAY WITH YOU.
>>ABSOLUTELY.
THANK YOU VERY MUCH.
>>THANK YOU TO THE ATTENDEES
FOR LISTENING.
SEE YOU IN A MONTH AT THE NEXT
DNA REPAIR INTEREST GROUP
SEMINAR.
>>THANK YOU.