WELCOME TO THE FIRST VIDEO
CONFERENCE OF THE NEW YEAR,
2011.
WE'RE HAPPY TO SEE YOU ALL THAT
MADE THRIET THE VARIOUS -- MADE
IT THROUGH THE VARIOUS BLIZZARDS
IN THE NORTHEAST AND MIDWEST.
WE HAVE A FEW -- A FEW TALKS
SCHEDULED SO FAR IN FEBRUARY,
DR. CARLOS OF SUNNII WILL BE
TAKING ABOUT DYNAMIC ASPECTS OF
DNA RECOGNITION, AND DR. U FROM
THE UNIVERSITY OF MICHIGAN WILL
SPEAK ABOUT THE ROLE OF HISTONE
OW BIC QUINN NATION AND DNA
DAMAGE RESPONSE.
WE STILL HAVE A FEW OPENINGS IN
THIS SERIES, SO PLEASE CONTACT
ME OR DR. BORE IF YOU HAVE
SUGGESTIONS FOR THAT.
AND ALSO BE SURE TO NOTE THESE
WITH THE E-MAIL LIST THAT YOU
SEND ME.
AND [INAUDIBLE] NIH.GOV YOU CAN
GET ON THE LIST.
TODAY WE'RE HAPPY TO HAVE A TALK
FROM -- COMING FROM THE AGING
INSTITUTE, BUT DR. SANDY CHANG
AT YALE WILL BE SPEAKING ABOUT
HOW TELEMERES, CHROMOSOME,
GAUGING THE DNA DAMAGE RESPONSE.
BE SURE EVERYONE ELSE MUTES AND
WE'LL LET THE INTRODUCTIONS FROM
BALTIMORE.
>> WHAT ABOUT COMMENTS ABOUT
LECTURES IN THE FUTURE?
HAVE PEOPLE DONE THEIR -- MADE
THEIR CONTRIBUTIONS AND
SUGGESTIONS?
WE NEED TO HAVE SPEAKER
SUGGESTIONS FROM ALL THE
DIFFERENT SITES.
REMEMBER THAT.
>> AND SO WE'RE VERY PLEASED TO
HAVE SANDY CHANG HERE WITH US
TODAY WHO IS GOING TO TALK ABOUT
HOW TELOMERES PROTECT CHROMOSOME
ENDS FROM ENGAGING THE DNA
DAMAGE RESPONSE.
SOMETHING WE'RE ALL INTERESTED
IN.
AND SANDY DID HIS MD Ph.D. AT
ROCKEFELLER UNIVERSITY AND DID A
POSTDOC WITH [INDISCERNIBLE] AND
THERE HE PUBLISHED A NUMBER OF
IMPORTANT PAPERS, BUT ONE WAS
PARTICULARLY IMPORTANT FOR US
INTERESTED WHICH WAS A VERY
SIGNIFICANT OBSERVATION ABOUT
THE WARNER'S MOUSE.
FROM THERE HE WENT TO HOUSTON,
MD ANDERSON.
WAS A PROFESSOR THERE FOR A
NUMBER OF YEARS, THIS JULY HE
ACCEPTED A PROFESSORSHIP AT YALE
AND HE HAS BEEN AROUND.
WE'RE PLEASED YOU COULD COME TO
TALK TO US TODAY, SANDY.
IF YOU DON'T --
>> THANK YOU.
FOR INVITING ME TO NIH.
I'M VERY HAPPY TO BE HERE AND
MEET UP WITH OLD FRIENDS.
AND I'M SORRY TO SAY, APOLOGIZE
IN ADVANCE THAT I BROUGHT SOME
BAD WEATHER FROM CONNECTICUT.
WE HAD 2 FEET OF SNOW.
I'M HOPING YOU DON'T SUFFER THE
SAME THING.
MY LAB WORK --
>> SOMEBODY IS NOT MUTED.
PLEASE MUTE THEIR MICROPHONES,
ASIDE FROM HERE, PLEASE.
TELOMERE ARE COMPOSEED -- THE
OVERHANG SHOWN HERE AND THIS
OVERHANG ACTUALLY ENGAGES IN
ENZYME TELOMERE NEEDED TO
MAINTAIN A LENGTH OF TELOMERE.
IN HUMAN CELLS, BECAUSE OF THE
REPLICATION PROBLEM, THE
CHROMOSOME ENDS CANNOT BE FULLY
REPLICATED AND TELOMERES SHORTEN
WITH EACH ROUND OF CELLULAR
REPLICATION.
WITHOUT THE END ZIMB TELOMERE,
THEY SHORT TO KNOW THE EXTECT
THEY BECOME DYSFUNCTIONAL.
WE KNOW THEY ENGAGE IN A DNA
DAMAGE RESPONSE.
THIS IS THE FOCUS OF MY TALK.
WE KNOW TELOMERES ARE BOUNDED BY
A NUMBER OF PRONES, AND --
PROTEINS.
IT SHELTERS FROM THE DAMAGE AND
IN PARTICULAR I'M GOING TO FOCUS
ON PROTEIN PART 1, PROTECTION OF
TELOMERE 1 AND THE PR2 COMPLEX
THAT WE COLLABORATE, UNIVERSITY
OF MICHIGAN, AND I'M GOING TO
TELL YOU A LITTLE BIT ABOUT
THAT.
SO I WAS ALSO WANTING TO TELL
YOU THAT TELOMERES DON'T EXIST
ALWAYS AS A LINEAR STRUCTURE.
THEY'RE LIKELY TO BE IN A LINEAR
FORM DURING REPLICATION WHEN THE
ENDS NEED TO BE REPLICATED, BUT
THE REPLICATION MACHINERY, BUT
THEY CAN ADOPT TO WHAT'S CALLED
A CLOSE CONFIRMATION OR T. LOOP
STRUCTURE IN WHICH THE OVERHANG
LOOPS BACK ON THE CELL.
[INAUDIBLE] AND FORM THIS LOOP
LIKE STRUCTURE.
AND THIS IS PARTIALLY A
PROTECTIVE MECHANISM TO EVADE
THE TELOMERE FROM BEING
RECOGNIZED BY DAMAGE BECAUSE YOU
CAN SEE THE ENDS ARE WELL
SEQUESTERED INTO THE DOUBLE
STRANDED REGION.
IF YOU UNCAP TELOMERES BUT
REMOVING THE SHELVING EXEENLT,
IF YOU HAVE -- EXCEPT, IF YOU
HAVE INSUFFICIENT QUANTITY OF
TELOMERE, THEY COULD BECOME
INACTIVATED, AND DEPENDING ON
THE STATUS OF THE P53, COULD
LEAD TO CELLULAR LINE
NECESSARIANCE, OR APOPTOSIS.
AND COULD BE A TUMOR PROMOTING
MECHANISM.
SO THIS IS WHAT HAPPENS IN A
TELOMERE KNOCKOUT MICE.
THESE ARE TUMORS WITH SH SHORT
TELOMERE.
THESE ARE CANCER NOMAS.
MANY OF THE CHROMOSOMES HAVE
JOINED.
THIS IS THE HALLMARK OF THE
FUNCTIONAL TELOMERES.
YOU CAN SEE THAT THESE
CHROMOSOMES FUSE AND IF YOU DO
SPECTRA CHARIO TYPING SHOWN IN
THIS PANEL, IT REVEALS MULTIPLE
COMS HAVE FUSED -- CHROMOSOMES
HAVE FUSED.
IT'S A CHARACTERISTIC FEATURE OF
MICE WITH TELOMERE DYSFUNCTION.
ALSO [INAUDIBLE] IN THIS CASE WE
REMOVE THE PROTEIN, HERE,
TELOMERES HAVE LIGATED END TO
END.
THE SEQUENCES ARE VERY ROBUST.
THESE ARE TELOMERE SEQUENCES
MARKED IN RED OR GREEN,
DEPENDING HOW WE LABEL THEM.
THEY BECOME DYSFUNCTIONED, EVEN
THOUGH THEY'RE VERY LONG.
THESE CHROMOSOMES MUCH NO
CAPPING FUNCTION BECAUSE TR2 IS
GONE.
ALL THE CHROMOSOMES HAVE ENGAGED
IN FUGS -- FUSION REACTION.
WHAT WE THINK NOW, AS YOU AGE OR
WITH INCREASED EPITHELIAL CELL
RENEWAL, MOST COMPARTMENTS LACK
AT THE LOCAL RACE, SO IT NEEDS
TO DYSFUNCTION WHICH COULD
ENGAGE IN CHROMOSOME REACTIONS,
IN THE BREAKING CYCLE THAT WAS
FIRST DISCOVERED BY BARBARA
McCLINTIC MANY YEARS  CYCLE, CHROMOSOME 
CAN BE
OPPOSITE ENDS OF THE SPINDLE
APPARATUS, AND RANDOM DOUBLE
STRAND BREAK THROUGH GENERATING
DAUGHTER CELLS.
AND IT COULD [INDISCERNIBLE] TO
THE FORMATION OF CELLS, SOME OF
WHICH I'VE SHOWN BEFORE IN THAT
TUMOR MOUSE MODEL.
DELETIONS OF, FOR EXAMPLE,
ONCOGENES, COULD ENGENDER A PRO
TUMOR GENIC PHENOTYPE THAT COULD
PROPAGATE TO CARCINOMA IN SITU,
AND REACTIVATION IN RARE
INDICATIONS CARCINOMA.
WE BELIEVE THIS HAPPENS WITH 90%
OF HUMAN CANCERS.
BECAUSE IN 90% AT THE --
[I
I'M GOING TO TALK ABOUT THE
SHELTERING COMPLEX.
THE TELOMERE ONE 1 PROTEIN, THIS
IS A PROTEIN THAT WAS FIRST
DISCOVERED BY -- AND CLONED BY
TOM'S LAB FROM PALM BAY, AND
THIS PROTEIN IS VERY INTERESTING
BECAUSE IT IS THE ONLY COMPONENT
OF THE SHELTERING COMPLEX THAT
BINDS TO THE SINGLE STRANDED
TELOMERE UNIT.
AND IT COMPLEXES WITH THE OTHER
PROTEIN CALLED TPP1 AND FORMS A
HETERODIMER.
THIS REGULATES THE ACCESS TO.
THE ELOM eRA SE.
IT COULD BE INHIBITED IF PERIOD
OF POT 1
IS DETERMINED TO BE THE
OVERHANG.
ON THE FLIP SIDE OF THIS,
[TECHNICAL DIFFICULTIES]
[INDISCERNIBLE] NUCLEOTYPE
BINDING DOMAINS, BINDS TO THE
SINGLE STRANDED AND CRYSTAL
STRUCTURE IS SHOWN HERE.
IN BLUE IS 1, RED IS T YOU CAN
SEE THAT THE SINGLE STRANDED DNA
ADOPTS A 90-DEGREE KINK WHEN IT
BINDS.
IT'S VERY INTERESTING, WHY THE
GENOME HAS EVOLVED TO POT 1,
WHERE THE HUMAN ONLY HAS ONE.
WE'RE INTERESTED IN ANSWERING
THIS QUESTION.
SO AS APPROACH WE USE THE
CONDITIONAL KNOCKOUT APPROACH TO
UNDERSTAND THE FUNCTION OF PART
ONE.
THIS IS -- THIS WAS PUBLISHED A
NUMBER OF YEARS AGO.
WE [INDISCERNIBLE] TO ENABLE
CONDITIONAL DELETION OF THIS
GENE.
AND REMOFNL OF THE POT 1 GENE
REACTS IN THE ACTIVATION OF
DAMAGE RESPONSE, TELOMERES.
YOU SEE HERE IN PANEL A -- MY
CURSER KEEPS DISAPPEARING.
ON PANEL A, 2F MARVEGERS OF
DAMAGE.
YOU CAN SEE THAT.
IF YOU LOOK AT 53, AND STYL
TELES COLOCALIZE VERY WELL.
P351.
SO WE UNDERSTAND NOW THE -- IT'S
A PROTECTOR OF TELOMERE FROM
ENGAGING THE DAMAGE RESPONSE,
BECAUSE IF YOU REMOVE PART 1A,
YOU GIT MIGRATION OF DNA MARKERS
BUT ALSO ACTIVATION OF
DOWNSTREAM, FOR EXAMPLE, FOS
PHOSPHORYLATION, CHECK ONE AND
CHECK 2.
LOSS OF [INDISCERNIBLE], ALSO
ISLIST STTS A CHROMOSOME FUSION
PHENOTYPE.
THESE ARE DUE TO THE FORMATION
OF CHROMOSOME BEING PULLED.
HERE YOU CAN SEE NORTH INDEED 2
CHROMOSOMES.
HERE ARE 3 THAT ARE FUSED.
WE GET FUSION THAT INVOLVES ENDS
THAT DON'T HAVE TELOMERE
SIGNALS, WHICH IS QUITE
INTERESTING TO US.
IT APPEARS THAT SOME OF THE
TELOMERES ARE DELETED.
IN DATA I WON'T SHOW YOU, IF YOU
LOSE PERIOD OF POT 1, YOU GET TELOMERES
COMBINED.
AND DELETE SEQUENCES AND GET
FUSIONS WITHOUT ANY TELOMERES
[NO AUDIO]
>> WE'RE LOSING THE VOLUME.
WE CAN'T HEAR HIM NOW.
>> CAN YOU HEAR ME NOW?
IT'S A LITTLE BETTER.
YOU HAVE TO KEEP TALKING.
>> IS THIS BETTER?
>> NOW IT'S MUCH BETTER.
THANKS.
>> KEEP IT TO MY HOW?
>> IF YOU HAVE FINE BROVMENT
BLASTS, THESE TUMOR ADAPT MANY
CHARACTERISTICS OF TELOMERE
DYSFUNCTION SHOWN BY KERO
TYPING.
WE RECENTLY GENERATED A
CONDITIONAL DELETION OF PART 1
IN THE BREAST EPITHELIUM SO WE
FOUND THAT YOU COULD -- LOSS OF
PART 1 PROMOTES CANCER IN A
DEPENDENT MANNER, THAT IS, IF
YOU DO NOT DELETE PART ONE OR
P53, THESE MICE ARE PERFECTLY
FINE.
THEY DON'T DEVELOP
[INDISCERNIBLE] OF THE BREAST.
THESE MICE GET TUMORS IN 15 OR
SO MONTHS OF AGE.
HOWEVER, IF YOU DELETE BOTH
ALLYMS OF PART 1A AND ALSO
CONDITIONALLY DELETE P53, THESE
GET TUMORS IN 9 MONTHS.
THESE ENCOMPASS SINGLE LESIONS,
MOSTLY IN MAMMARY GRAND NO. 4.
AND THE TUMORS ALL HAVE
DELETIONS OF THE POT 1A. GENE
WHRAVMENT WE FOUND INTERESTING
IS THAT TUMOR CELLS IN ALMOST
VERY VARABLY PRODUCE THE HUGE
AMPLIFICATIONS.
YOU CAN SEE HERE CHROMOSOMES,
HUGE TRACKS OF TELOMERE SIGNALS,
ALL 4 DEPENDENT TUMORS ON THE
BREAST.
AND RIGHT NOW WE DON'T
UNDERSTAND EXACTLY WHAT THE
MECHANISM BEHIND THE GENERATION
OF THESE RAMPANT TELOMERIC
AMPLIFICATION, WE THINK IT COULD
BE DUE TO A RECOMBINATION
PHENOTYPE.
SO TO SUMMARIZE THIS PART OF THE
TALK, SHOWING THE PART 1A IS
VERY IMPORTANT TO PROTECT
TELOMERES ENGAGING IN A DAMAGE
RESPONSE IN THAT IF YOU REMOVE
POT 1A CONINGSALLY, YOU'RE GOING
TO -- CONDITIONALLY, YOU'RE
GOING TO GENERATE A
DYSFUNCTIONAL PHENOTYPE MARKED
BY DAMAGED -- DAMAGED DNA.
YOU CAN GET FOCI FORMATION.
IN THE SETTING OF DEFICIENCY,
IT'S TUMOR PROMOTING.
I DON'T HAVE TIME TO SHOW YOU
POT 1A IS A REPRESSER OF
TELOMERES, AND THAT'S WHY WE
THINK THIS PHENOTYPE COULD BE A
CONSEQUENCE OF POT 1A FUNCTION.
LOSS OF FUNCTION IN REPRESSING
HOE MODELGOUS FUNCTION --
HOMOLOGOUS FUNCTION.
POT 1B IS COMPLETELY VISIBLE
VIABLE AT BIRTH.
SO IT TELLS YOU THESE 2 PROTEINS
MAY BE DOING SOMETHING
DIFFERENT.
WHAT WE DID WAS GENERATED
ALLELES THAT ARE DIFFERENT IN
POT 1B AND TELOMERASE.
IT ACTS AS A TELOMERASE TO ENDS
SO WE THOUGHT IT WOULD BE
INTERESTING TO ALSO LOOK AT THE
ABSENCE OF TELOMERASE IN A
SETTING OF POT 1B DEFICIENCY.
SO WE LOOKED AT THESE MICE.
WE FOUND IF YOU DELETE POT 1B
THEY'RE FAIRLY NORMAL IN THE
FIRST YEAR OR SO OF LIFE.
SO THEY DON'T DIE.
BUT IF YOU DELETE POT 1B AND YOU
DELETE TELOMERASE, THESE MICE
ARE DEAD IN ABOUT 10 DAYS.
IF YOU DELETE POT 1B AND HAVING
50% TELOMERASE, THE MICE DO
BETTER BUT THEY DIE AROUND 6
MONTHS OF AGE.
WE WANTED TO KNOW WHAT THESE
MICE ARE DYING FROM.
WHAT WE FOUND, VERY STRIKING,
WAS THAT THE POT 1B TELOMERASE
KNOCKOUT MICE ARE DEAD IN 10
DAYS.
THEY NEVER FORM BONE MARROW.
THESE ARE THE FEMURS FROM THE
DOUBLE KNOCKOUT MICE.
YOU CAN SEE THERE WAS NO BONE
MARROW FORMATION AT ALL.
IF YOU LOOK AT THE POT 1B
HETEROZYGOUS ANIMALS, AT EARLY
AGE, THERE IS FULL COMPLEMENT OF
BONE MARROW CELLS.
AROUND 6 MONTHS OR SO OF AGE YOU
SAW THEM LOSING BONE MARROW.
ACTUALLY, THEY HAVE [NO AUDIO]
>> WE LOST THE SOUND AGAIN.
WE DON'T HAVE ANY SOUND NOW.
>> HERE WE GO.
>> CAN YOU HEAR ME NOW?
NOW YOU'RE BACK.
YES.
>> WE'RE BACK.
OKAY.
IF YOU LOOK AT THE POPULATION OF
THE PART 1B HETEROZYGOUS MICE,
YOU CAN SEE THAT THE POPULATION
LOOKING AT 1 MARKER AND CD
[INDISCERNIBLE] B IS ACTUALLY
ABOUT 4 FOLD, SO YOU SEE A RAPID
DELETION OF THE GRANTOCYTE
COMPARTMENT.
FURTHER ANALYSIS HAVE SHOWN US
THAT IT IS THE PROGENITOR STEM
CELL COMPARTMENTS OF THE PART
ONE B HETEROZYGOUS MICE THAT ARE
BEING DEPLETED.
IF YOU LOOK AT WILDTYPE MICE,
AND LOOK AT SKY 1 POSITIVE, THEN
NEGATIVE POPULATIONS YOU CAN SEE
AROUND 6 MONTHS OF AGE THERE IS
ABOUT .23% OF THESE CELLS, VERY
ROBUST T-BONE MARROW LOOKS --
BONE MARROW FROM A FEMUR SECTION
LOOKS VERY GOOD.
AS EARLY AS 2 MONTHS OF AGE IN
THE POT 1B, YOU CAN SEE A DELETE
OF THIS.
BUT THE BONE MARROW CELL LOOK
FINE.
6 MONTHS OF AGE THERE IS BARELY
INTO EXISTENCE OF THESE
THEMSELVES AND YOU CAN SEE A
HIGHLY COMPROMISED APLASTIC BONE
MARROW.
IT'S THE PROGENITOR CELLS BEING
EFFECTED IN THIS PARTICULAR
GENETIC BACKGROUND.
AND WHAT WE NOW ANALYST IN
GREAT -- ANALYZE IN GREATER
DETAIL, BY LOOKING AT THE
VARIOUS HEMATOPOWIC LINEAGES, WE
FOUND THIS PERIOD OF TIME 1*B,
THIS WAS A DEPLETION.
WE FOUND A SIGNIFICANT INCREASE
IN DYSPLASTIC CELLS THAT LOOK
LIKE GLASS.
THESE ARE ILLUSTRATED HERE.
THESE ALMOST HAVE A MDS LIKE
PHENOTYPE OR AML IN THESE
ANIMALS.
AND THIS PHENOTYPE COUPLED WITH
A MASSIVE APOPTOSIS THAT'S
OBSERVED IN THE [INDISCERNIBLE],
YOU CAN SEE THE CELLS
ENCOMPASSING 71% OF THE CASE IN
THE MICE, ABOUT 9% OF THESE
 CASE.
SO WE SEE A MASSIVE LOSS OF THE
STEM CELL COMPARTMENTS DUE TO A
HUGE INCREASE IN APOPTOSIS.
WE ALSO NOTICE THAT VERY
INTEREST, TRY ADPHENOTYPE OF
SKIN PIGMENTATION.
IF YOU LOOK AT THE CAUSE OF THE
TELOMERASE HETEROZYGOUS NICE,
THEY HAD VERY DARK PAWS, WE USED
THIS TO GENOTYPE OUR SMS.
WE DON'T HAVE TO DO PCR ANY NOR
BECAUSE THE PAWS PIGMENTATION
ACTUALLY ENABLES US TO TELL THE
ANIMALS APART FROM WILDTYPE OR
HETEROGIGIS.
AND WE LOOKED AT THE NAILS OF
THESE MICE.
MANY OF THEM ARE KREURLD CURLED
UP, HAVE A VERY STRANGE
APPEARANCE.
NOTHING LIKE WILDTYPE CONTROLS.
THIS TRIAC, THIS RESEMBLED A
HUMAN DISEASE AND THIS IS A RARE
DISEASE OF TELOMERE METAB BLIMP,
CHARACTERIZED BY NORMAL SKIN
PIGMENTATION, WHITE PLAQUES IN
THE ORAL MUCOSA MALE DYSTROPHY
THAT YOU CAN SEE HERE, THESE
PATIENTS GET BONE MARROW FAILURE
AROUND 40 YEARS OF AGE IF THIS
IS A TELOMERE DISEASE, IF YOU
LOOK AT THE PATIENTS, ALL OF
THEM HAVE DEFECTS IN THE
MAINTENANCE OF TELOMERES.
FOR EXAMPLE, THE FIRST CASES IS
THE PROTEIN CALLED
[INDISCERNIBLE] WHICH IS THE
[INDISCERNIBLE] AND IT'S
INVOLVED IN THE MODIFICATION OF
RNAs AND NEEDED TO -- FOR THE
MATURATION FOR THE RNA AND
TELOMERASE.
IF YOU HAVE MUTATIONS, YOU
ACTUALLY INHERIT AND X FORM LINK
OF THIS DISEASE.
THE CHARACTERISTIC IS ALL THE
TRIALS WE TALKED ABOUT BEFORE
COUPLED WITH BONE MARROW
FAILURE, PULMONARY FBI ROSIS,
THE GUNG SHOWS DEFECTS AND THEY
HAVE TELOMERES.
LATER ON IT WAS DEMONSTRATED
THAT THE GENE ITSELF, THE RNA
COMPONENT COULD ALSO BE
MUTALLETED TO GENERATE
[INDISCERNIBLE] AUTO SOMAL
FORMS.
AND RECENTLY, A PROTEIN CALLED
KNOCK 10 INVOLVED IN THE
MATURATION OF THE RAP COMPONENTS
IS ALSO MUTATED.
SO WHAT'S VERY INTERESTING, THE
MUTATION IN THE SHELTERING
COMPONENT, THIS IS THE FIRST
TIME THAT PROTEINS INVOLVED IN
TELOMERE PROTECTION AND NOT
THROM RATION, WHICH HAVE BEEN
SHOWN TO BE IN PATIENTS WHICH IS
CONGENITAL PHENOTYPE AND THESE
PATIENTS GET THESE AS EARLY AS
10 YEARS OF AIM.
VERY, VERY SEVERE DISEASE.
WE THINK THAT OUR MOUSE MODEL IS
A MODEL FOR THIS.
IT'S ACTUALLY THE BEST MODEL.
IF YOU KNOCK OUT [INDISCERNIBLE]
ITSELF YOU DON'T GET THE
PHENOTYPE THAT WE SEE IN OUR
MOUSE MODEL.
SO IF THIS IS TRUE, WE SHOULD
SEE A TELOMERE DEFECT IN THIS
MODEL AND THIS IS WHAT WE SEE.
I'M GOING TO POINT YOU TO THIS
PANEL.
IF YOU TAKE POT 1B AND MASSAGE
IT OVER TIME IN VITRO, YOU CAN
SEE THE SINGLE STRAND OVERHANG
IS SHORTENED.
TELOMERE LENGTH IS SHORTENED.
COUPLED WITH INCREASED INFUSION
PRODUCTS, YOU GET
[INDISCERNIBLE].
THIS IS AGAIN ILLUSTRATED HERE.
WE'VE USED QUANTITATIVE TELOMERE
FISH TO LOOK AT TELOMERE STATUS
OF MOUSE IMBRO OI FIBROBLASTS.
YOU CAN SEE A LOT OF CHROMOSOMES
ARE FUSED IN THE HETEROGENEOUS
CASE.
THE TELOMERES ARE SHOTER
SHORTENED.
YOU CAN MESHTER LENGTH, ALMOST
50% SHORTER.
AND ISOLATE DIRECTLY FROM THESE
ANIMALS, CHROMOSOMES ARE FUSED
WITH LOTS OF UNCAPPING OF
TELOMERES.
FINALLY, WE LOOK AT THESE MICE,
THE EMBRYO, THE DAMAGE RESPONSE.
WE FOUND THE TELOMERE
HETEROGIGIS ACTUALLY SHOW A VERY
HIGH DEGREE OF DNA DAMAGE.
THIS IS AGAIN WITH THE ASSAY,
YOU CAN SEE ABOUT ACCUMULATION
OF DAMAGED FOCI IN THIS
PARTICULAR GENOTYPE.
AND DATA I'M NOT GOING TO SHOW
YOU, I'M GOING TO TELL YOU THIS
IS AA TR -- AN ATR DEPENDENT
PROCESS.
WHAT YOU GET IS -- IF YOU
REPLICATE THE LEADING STRAPPED
OF TELOMERES, YOU GET A BLUNT
END.
IF YOU REPLICATE THE LIGAND
STRANDS YOU GET A SMALL
OVERHANG.
WHAT ARE NEEDED IS PROCESSING
THAT GENERATE THE LONG SINGLE
STRANDED OVERHANG THAT TELOMERE
NEEDS TO MAINTAIN THE ENDS.
POT 1B WE THINK ACTUALLY
SUPPRESSES OR MODULATES THIS
ACTIVITY.
IF YOU'RE MISSING POT 1B.
YOU GET A VERY LONG TELEMERIC
OVERHANG.
WE THINK THAT THIS OVERHANG IS
INSUFFICIENT TO BE PROTECTED BY
THE REMAINING POT 1A SO WHAT
HAPPENED, THERE IS DNA DAMAGE
RESPONSES GENERATED, LEADING TO
INCREASED DNA DAMAGE RESPONSE
AND DEPENDENT APOPTOSIS WITH YOU
WE SEE IN OUR MOUSE
COMPARTMENTS.
OKAY.
SO I'M GOING TO SWITCH GEARS AND
TALK ABOUT A STORY THAT WE JUST
PUBLISHED.
IN THAT WE WANTED TO KNOW THE
ROLE OF ANOTHER EXCEPT CALLED
WRATH 1.
REPRESSER ACTIVATED PROTEIN 1.
THIS IS A VERY INTERESTING
PROTEIN, BECAUSE THIS [NO AUDIO]
>> WE LOST YOUR SOUND AGAIN.
SO RAP 1, YOU FIND IT IN YEAST
AND ALL YOUR EUCARYOTICS.
BUT WE KNOW THAT RAP 1 IS
INVOLVED IN PROTECTING
TELOMERES, NON MOM -- HOME
MODELGOUS AND JOINING REACTIONS.
IT EXISTS HERE AS A HETERODIMOIR
RAP 1.
SO ALL THE STUDIES, FOR EXAMPLE,
THESE STUDIES IN WHICH HE
DELETED OR MUTATED TRF2, THEY
FOUND IT'S VERY VERY IMPORTANT
IN TERMS OF TELOMERE AND
PROTECTION.
WE NEVER KNEW WHAT WAS HAPPENING
WITH RAP 1.
WE DON'T KNOW WHETHER THE
INCREDIBLE CHROMOSOME FUSION
PHENOTYPE IN THE DNA DAMAGE
RESPONSE THAT WE GET, IS THAT
DUE TO TRF2 ALONE OR AND RAP 1
FUNCTION.
SO WE WANT TO KNOW WHAT THE
FUNCTION IS IN TERMS OF TELOMERE
AND PROTECTION.
SO THERE ARE SEVERAL WAYS TO GET
RID OF RAP 1.
ONE, YOU 
THIS WAS DONE BY USING shRNA
THAT DOES NOT WORK VERY WELL IN
GETTING RID OF RAP 1.
WE COULD NEVER DELETE THIS
PROTEIN BY MORE THAN 60 OR 70%.
AND WE NEVER FEEL LIKE WE'RE
GETTING ENOUGH REMOVAL OF IT.
WE THOUGHT ABOUT GENERATING A
CONDITION, A KNOCK OUT BUT WE
DIDN'T DO THAT BECAUSE RAP 1 IS
VERY CLOSE TO A VERY ESSENTIAL
GENE, ACTUALLY SHARES THE GENE'S
LAST EXON, IN BETWEEN RAP 1'S
NON CODING REGION.
WE WORRY IF WE PERTURB THIS GENE
WE COULD GENERATE A DEAD MOUSE,
NOTHING TO DO WITH RAP 1
FUNCTION.
SO WE USED A THIRD APPROACH,
OUTLINED HERE IN THAT WE THOUGHT
WHAT IF WE COULD RECONSTITUTE
CELLS WITH A MUTANT THAT CANNOT
BIND RAP 1.
INTRODUCE A MUTATION INTO TRF2.
WE COULD HAVE 2 FUNCTIONS AT
TELOMERE BUT NOT RAP 1
FUNCTIONS.
SO THIS WILL UNCOUPLE RAP 1s
ABILITY TO BIND TRF2, AND COULD
DISSECT THE FUNCTION INDEPENDENT
OF RAP -- AND THAT WAS THE GOAL.
THE GOAL IS COUPLED WITH THE
IDEA THAT WE COULD DELETE TRF2
VERY EFFICIENTLY FROM CELLS, AND
THIS IS SHOWN HERE.
W50E6 A VERY ROBUST shRNA
FACTOR THAT COULD DELETE IT
WITHIN ABOUT 72 HOURS.
CHROMOSOMES ARE FUSING WITH ONE
ANOTHER.
BY 120 HOURS, EVERY ONE HAS
FUSED.
AND WE CAN ACTUALLY RESCUE THIS
PHENOTYPE INTRODUCING A FULL
LENGTH TRF2.
YOU CAN SEE INTRODUCTION OF THIS
 shRNA TO COMPLETE YOUR
SCREWS OF CHROMOSOME FUSION.
SO WE THOUGHT THAT, YOU KNOW,
COULD INTRODUCE TRF2 WITH
MUTATIONS, TO ASK THE QUESTION
WHAT HAPPENS TO TELOMERES IN THE
ABSENCE OF RAP 1 BINDING TO
TELOMERES.  SO WE COLLABORATED
ON THE PROJECT.
THIS IS A VERY FRUITFUL
COLLABORATION.
DEFINED THE BINDING DOMAINS
BETWEEN RAP 1 AND TRF1.
AND THE BINDING MOTIF.
RBM.
SO WE TOOK THESE 2 LITTLE
PEPTIDES, ACTUALLY USING
FILTRATION, THEY BIND TO EACH
OTHER.
IT SHOWS THESE 2 PEPTIDES AS
PURIFIED PROTEINS, AND HE WOULD
ACTUALLY MAKE THE CRYSTAL
STRUCTURE OF THE TRF2
[INDISCERNIBLE].
SO THIS IS THE CRYSTAL STRUCTURE
, FOCUSED YOU HERE.
THE YELLOW OF ALPHA HE WILL
LICKS ARE THE END DOMAIN, FORM
TO A CHANNEL IN THE RTC DOMAIN.
IT KIND OF HOLDS THE 2 HE HELP --
HELIXES IN HIS FINGERS, IF YOU
LOOK AT WHAT'S HIGHLIGHTED HERE,
THE RAP 1 HIDE DROVMENT PHOBIC
RESIDUES ARE OUTSIDE LINED HERE.
HERE IS TRF1 POSITIONED RIGHT
ABOVE THE POCKET.
WE'RE NOT ABLE TO -- TO PROMOTE
INTERACTION [NO AUDIO]
>> WE ARE AWAKE BUT WE CAN'T
HEAR YOU.
NO SOUND YET.
>> ARE WE GO NOW.
>> IT'S BACK.
>> OKAY.
SO.
>> NOW IT'S GONE AGAIN.
CAN YOU HEAR ME NOW?
>> YES.
>> SO T.F.2 BINDS TO RAP 1, AND
THIS  RESIDUE IS VERY
IMPORTANT.
WE WANT TO VERIFY THE CRYSTAL
RESULTS WITH IN VIVO DATA.
WE MADE PROTEINS THAT ARE --
THAT CONTAIN MUTATIONS IN THE
RAP 1 BINDING DOMAIN, FOR
EXAMPLE, WE MADE
[INDISCERNIBLE].
OR MUTATED LYSINE IN.
THER F2 DOMAIN.
SO WE CORRELATED THIS WITH NVL
DATA, LOOKING AT THE EXACT
MUTATIONS.
YOU CAN SEE THAT TRF2 MUTATIONS
ABOLISH THE INTERACTION OF THE
PROTEINSABILITY TO PRODUCE
ACTIVITY.
SO THESE DATA SUGGEST THE
COMPACTED MUTATIONS SOLVED BY
CRYSTAL STRUCTURE ARE
RESPONSIBLE FOR THE RAP 1 WITH
TRF2 NVLG.
WE DID -- NVL.
WE DID THIS IN FINE BROVMENT
BLASTS, AND WITH COIP
EXPERIMENTS.
WE DEMONSTRATED THAT THESE
MUTATIONS IN RAP 1 PREVENTED
BINDING TO WILDTYPE, TRF2.
AND HERE IS A CORRESPONDING
MUTATION IN TRF2 PREVENTED
BINDING OF RAP 1.
SO I THINK WE HAVE DEMONSTRATED
THE FACT THAT THESE 2 PROTEINS
INTERACT WITH EACH OTHER WITH
SPECIFIC RESIDUES, AND NOW WE
CAN SEE WHAT HAPPENS WITH
TELOMERES.
DOES THIS REQUIRE BINDING TO
TRF2.
TO ANSWER THIS QUESTION, WE
DISSECTED FULL LENGTH RAP 1 INTO
MOUSE EMBRYO FIBROBLAST, THESE
ARE HH, COLOCALIZED TO TELOMERES
100%.
WHAT ABOUT MUTANT RAP 1 THAT
CANNOT BIND TO TRF2.
NEITHER OF THESE WERE ABLE TO
[INDISCERNIBLE] INDICATING THAT
THIS BINDING OF RAP 1 TO TRF2 IS
CRITICALLY IMPORTANT.
WHAT ABOUT THE OTHER WAY AROUND?
SO HERE IS FULL LENGTH TRF2,
LOCALIZES TO TELOMERES.
HERE IS THE TRF2 WITH THE
MUTATION THAT COULD BIND RAP 1,
STILL GOES TO TELOMERES.
THIS IS NOT SURPRISING.
WE KNOW THAT TRF2 BINDING
REQUIRES A MID DOMAIN, SO THIS
DOMAIN, THE RBM DOMAIN THAT
BINDS TO RAP 1 IS NOT REQUIRED
FOR LOCALIZED TO TELOMERE.
WE CAN ASK WHAT HAPPENS IF WE
GET RID OF RAP 1 IN THIS
SETTING?
SO THIS IS SHOWN HERE.
SO IF YOU GET RID OF TRF2 IN THE
CELL, AGAIN, CELLS THAT UNDERGO
A VERY POTENT DAMAGE RESPONSE IS
SHOWN HERE, ALL COLOCALIZED WITH
TELOMERES.
IF YOU PUT IN FULL LENGTH
WILDTYPE, BUT IF YOU PUT IN FULL
LECT TRF2 WITH THE MUTATION THAT
CANNOT BIND RAP 1, THE DAMAGE
RESPONSE IS STILL ABROGATED.
THIS MEANS THAT RAP 1 IS NOT
REQUIRED TO REPRESS THE DAMAGE
TO TELOMERES.  WHAT DOES RAP 1
DO?
WHEN WE LOOK AT CHROMOSOMES IN
THE ABSENCE OF RAP 1, WHAT WE
FOUND WAS THAT THERE WAS A
HYPERRECOMBINATION.
WE CAN USE CHROMOSOME
ORIENTATION [INDISCERNIBLE],
INVENTED BY SUSAN BAILEY'S
GROUP.
WE CAN USE OUR RED COLOR SEA
PROBES OR T. RICH PROBE TO LOOK
AT STRANDS OF TELOMERES.
IF THERE IS A COME BIP NATION
WHAT YOU GET IS A YELLOW SIGNAL.
THIS IS A BLOWN UP OF THIS
SITUATION.
YOU CAN SEE NORMAL CHROMOSOMES
SHOULD HAVE A RED DOT AND GREEN
DOT.
BUT WE SEE WITH
HYPERRECOMBINATION, IT'S THE
RECOMBINATION.
IT'S A GREEN OVER RED SO YOU GET
A YELLOW DOT.
SO THIS IS ACTUALLY INCREASED
ABOUT 4 FOLD WHEN YOU GET RID OF
RAP 1.
SO WHAT WE CAN SAY IS THAT NOW
WE HAVE THE ABILITY TO SAY WHAT
HAPPENED IF YOU YOU GET RID OF
RAP 1.
RAP 1 WE THINK IS IMPORTANT IN
PROTECTING TELOMERES FROM
ENGAGES IN A HOME MOWINGGOUS
COMBINATION PROCESSES, IF YOU
GET OF RAP 1, YOU HAVE MINIMAL
DNA DAMAGE RESPONSE.
YOU HAVE VERY FEW CHROMOSOME
FUSIONS BECAUSE TRF2 IS STILL
THERE, IS MAIN PROTECTOR OF
CHROMOSOME ENDS FROM CLASSIC
[INDISCERNIBLE], CHROMOSOME
FUSIONS, BUT YOU GET TONY LA
RUSSA HR, THE -- TELOMERE HR.
SO FINALLY, I WANT TO END MY
TALK BY SAYING A FEW WORDS ABOUT
DNA REPAIR IN THE ABSENCE OF
TELOMERE END PROTECTION FACTORS.
WE AND OTHERS HAVE SHOWN THAT
THE COMFLEX IS VERY IMPORTANT IN
PROCESSING TELOMERE ENDS DEVOID
OF TRF2.
THE NUCLEUS ACTIVITY IS
GENERATING SUBSTRATE THAT ARE
AMENABLE FOR REPAIR.
SO [INDISCERNIBLE] HAS SHOWN
THAT THE PROTEIN, 53B IS VERY
IMPORTANT TO LIGATE TELOMERES
WITHOUT TRF2.
HER LAB SHOWS IN -- IT HONES IN
ON TELOMERE CAN DEVOID OF TRF2,
SO THEY CAN DEFINE ONE ANOTHER
IN A TIMELY FASHION.
I THOUGHT THAT WAS INFERSING.
THIS REQUIRED 4, BECAUSE THIS IS
THE END JOINING REACTION.
BUT WE HAVE ALSO SHOWN THAT
RAP -- PART ONE AND TRF2 HAVE
DISTINCTION MECHANISM TO REPRESS
THE DNA DAMAGE RESPONSE.
WE'VE SHOWN THAT PART ONE
ACTUALLY REPRESSES ATR
ACTIVATION.
RAP 1 REPRESSES ATM.
THIS IS VERY IMPORTANT BECAUSE
ATR MEDIATED DAMAGE IS USUALLY
TRIGGERED BY THE SINGLE STRANDED
DNA.
WE THINK BECAUSE PART 1 ONLY
SERVES TO PROTECT SINGLE TRAINED
DNA, WHAT'S HAPPENING IS THAT
PART 1 IS PREVENTING THE
RECRUITMENT OF OUR
[INDISCERNIBLE] TO THE SINGLE
STRANDED DNA.
AND ACTUALLY WE HAVE EVIDENCE TO
SHOW THAT THIS IS TRUE IN
THE:RICATION WITH
WITH OCOLLABORATION, BUT I'M NOT
GOING TO TALK ABOUT THAT TODAY.
WE THOUGHT MAYBE THE REPAIR
FUNCTIONS GENERATED BY TELOMERES
DEVOID OF RAP 1 OR TRF2 MAY BE
DIFFERENT.
WE WANT TO ADDRESS THIS BY
LOOKING AT HOW TELOMERES ARE
REPAIRED, FOR EXAMPLE, IN
ABSENCE OF TRT 2 AND POT 1.
SO WE DID THIS IN THE FIRST CASE
WITH TRF2, DOING THIS IN A
SETTING OF GENETIC DELETIONS OF
CLASSIC FACTORS, IN THE SETTING
OF CELLS WITHOUT [INDISCERNIBLE]
OR DNA LIGASE 4.
WHAT WE FOUND IN ALL INDICATIONS
WAS THAT IF YOU DELETE ANY OF
THESE COMPONENTS OF THE CLASSIC
PATHWAY WAY, CHROMOSOME IS
COMPLETELY ABOLISHED IF YOU
REMOVE IT.
SO TRF2, LOSS OF IT, REPAIR
THAT'S GENERATED IS ALL DONE
THROUGH THE CLASSIC PATHWAY.
SO OTHER QUESTION IS WHAT
HAPPENS TO TELOMERES DEVOID OF
POT 1?
SINCE IT GOES THROUGH ATR, IS
THE CLASSIC PATHWAY REQUIRED?
WE DID THE SAME 
EXPERIMENTS, REMOVED PART 1A IN
THE SETTING OF 53B
[INDISCERNIBLE], ALL THE CLASSIC
FACTORS ARE REQUIRED FOR DOUBLE
STRAND DNA BREAKS.
WE SAW SOMETHING VERY
INTERESTING.
IF YOU DELETE PART 1A IN THE
SETTING OF ANY OF THESE -- LOSS
OF ANY OF THESE CLASSIC FACTORS,
FUGS ARE STILL RAMPANT.
IF YOU LOOSE PART 1A, THE FUSION
DOES NOT REQUIRE A CLASSIC
PATHWAY.
HERE WE'RE GETTING RID AFTER POT
1A USING A DOMINANT NEGATIVE OF
PPP1 WHICH REMOVES BOTH POT 1A
AND B, AND YOU GIVE RAMPANT
CHROMOSOME FUSIONS.
SO THIS IS IN THE ABSENCE, IT'S
A CLASSIC NHG FACTOR, THIS TYPE
OF FUSION CANNOT BE DEPENDENT ON
CLASSIC PATHWAY.
WE DID THE SAME WAY WITH
[INDISCERNIBLE].
AGAIN, CHROMOSOME FUSIONS IN
ABSENCE OF LIGASE 4.
JUST TO BE MORE ROBUST, WE DID
THIS IN CUE, GET THE SAME
PHENOTYPE.
SO OBVIOUSLY, WE MOVING
[INDISCERNIBLE] FROM TELOMERES
IS GENERATING A SUBSTRATE NOT
AMENABLE FROM LIGRATION.
WHAT IS THE PATHWAY THAT COULD
BE DOING THIS?
SO WE THOUGHT THAT IT COULD BE
THE ALTERNATIVE PATH, A PATH
THAT HAS BEEN INVOLVED RECENTLY,
INVOLVED IN LIGATING A LOT OF
SUBSTRATE THANK REQUIRES -- THAT
REQUIRES MICROHOMOLOGY AT THE
SITE OF DNA BREAKS.
WE KNOW THAT IT REQUIRES
COMPLEX, CTRP AND REQUIRES THE
DISSECTION OF THE BREAKS THAT
GENERATE LONG OVERHANGS TO
MEDIATE ALTERNATIVE LIGASE 3
DEPENDENT JOINING REACTIONS.
AND THIS AS YOU MENTIONED, A
REACTION LINKED TO THE PRESENCE
OF THE NOTICEABLE
TRANSLOCATIONS.
SO WE THOUGHT THAT SINCE
TELOMERES HAVE NATURAL 3 PLIM
OVERHANGS, MAYBE THIS IS THE WAY
THAT SUBSTRATES DEVOID OF POT 1A
AND B ARE BEING JOINED.
WE EXPLORED THIS FURTHER BECAUSE
WE WANTED TO KNOW WHETHER
[INDISCERNIBLE] IS IMPORTANT FOR
THIS.
WE KNOW THAT THE ALTERNATIVE
PATHWAY IS CRITICALLY DEPENDENT
ON THIS PROTEIN, PRESUMABLY TO
BE A NUCLEI ACE, FACILITATES
RESECTION OF THE DOUBLE STRANDED
BREAK THAT GENERATES
[INDISCERNIBLE].
SO WE DID THIS EXPERIMENT.
WE HAVE GENERATED shRNA THAT'S
VERY GOOD AT DELETING, AND THE
ABSENCE OF POT 1A AND B IS
COMPLETELY ABOLISHES, SUGGESTING
THAT THE FUSION WE SEE IN
ABSENCE OF POT 1A AND B REQUIRED
THE PRESENCE.
SO WHAT HAPPENS?
THE TELOMERES, IF YOU GET RID OF
TELOMERASE.
WHAT HAPPENS TO THOSE TELOMERES?
SO WE GENERATED A MOUSE IN WHICH
THE TELOMERES ARE SH SHORT.
THIS IS A GENERATION OF KNOCKOUT
MICE, IN THE 53PB1 DEFICIENCY,
OR [INDISCERNIBLE], AND AGAIN
IT'S A CLASSIC FACTOR THAT IS
REQUIRED FOR CLASSIC
[INDISCERNIBLE], AND IF IT'S
REQUIRED TO JOIN NOT TO SHORTEN
TELOMERES WE SHOULDN'T BE ANY
FUSIONS IN THE SETTING OF THE G4
STLOM RACE KNOCKOUT MICE.
WHAT WE SAW WAS THE COMPLETE
OPPOSITE.
IN THE BACKGROUND, CHROMOSOME
FUSION STILL PERSISTS, THIS
MEANS THAT THE FUSION'S REACTION
IS NOT DEPENDENT AND IT'S LIKELY
DEPENDENT ON THE ALTERNATIVE
PATHWAY, SOMETHING WE'RE
CURRENTLY PURSUING RIGHT NOW.
SO WHAT WE -- WHAT I WANT TO
SUMMARIZE IS THAT THERE ARE
DISTINCTIVE PATHWAYS THAT
TELOMERES NEED TO PROTECT TO
GUARD OR PROTECT AGAINST AND
THESE CAN BE ENGAGED
DIFFERENTLY.
I SHOULD MENTION THIS IS A VERY
SPECIFIC WAY TO TRIGGER DAMAGE
RESPONSES.
SO WHAT YOU TRIGGER IS AN
INDEPENDENT DAMAGE RESPONSE,
ACTIVATING A CLASSIC END JOINING
PATHWAY, DEPENDENT ON
[INDISCERNIBLE].
IF YOU DELETE PART 1A, OTHER
THAN, [INDISCERNIBLE] WHICH  ALTERNATIVE
 TYPE OF
REPAIR MECHANISMAN, [INDISCERNIBLE] WHIC
H  ALTERNATIVE TYPE OF
REPAIR MECHANISM NATURAL ATTRITION
UTILIZES THIS PATH FOR REPAIR.
WE THINK THAT THERE IS SOMETHING
VERY INTERESTING ABOUT WHY
TELOMERES NEED TO GUARD AGAINST
CLASSIC AND ALTERNATIVE PATHS.
ONE REASON COULD BE BECAUSE
TELOMERES ARE HIGHLY RECOME BIP
GENETIC.
A AND GUARDING AGAINST A LOT OF
PATHWAYS SIMILARITIES TO
HOMOLOGOUS, MAY HAVE GUARDING AGAINST A 
LOT OF
PATHWAYS SIMILARITIES TO
HOMOLOGOUS, MAY HAVE POT 1 IS
DEFINITELY A PROTEIN THAT DOES
THIS.
SO [NO AUDIO]
>> WE LOST THE VOLUME AGAIN.
>> CAN YOU HEAR US NOW?
>> YES, WE CAN.
>> FOR ACKNOWLEDGMENT I WANT TO
THANK [INDISCERNIBLE].
WE'RE CONTINUING TO DO THIS TYPE
OF WORK BECAUSE ERIC HAS FOUND
VERY INTERESTING STEM CELL
PHENOTYPING.
THE RAP 1 WAS COLLABORATION WITH
THE UNIVERSITY OF MICHIGAN, AND
NHEJ WAS DONE WITH COLLABORATION
WITH PHIL CARPETENER.
AND I WANT TO THANK MY FUNDING
SOURCES.
THANK YOU FOR YOUR ATTENTION.
[APPLAUSE]
>> THANKS VERY MUCH FOR AN
EXCELLENT LECTURE.
AND KEN, ARE YOU GOING TO DO THE
ROUNDS?
>> IF YOU'D LIKE.
>> I'M NOT SURE WHO IS ON RIGHT
NOW.
SO WHY DON'T YOU DO IT?
SO EACH PLACE GETS AN
OPPORTUNITY TO ASK ONE QUESTION.
AND THEN WHEN THE ROUND IS DONE,
WE GO BACK AND SEE IF THERE ARE
MORE QUESTIONS.
THEN WE ASK QUESTIONS HERE
SANDY, IS THAT OKAY?
>> SURE.
>> OKAY.
>> WHY DON'T WE GO TO BROOKHAVEN
TO START WITH.
BE SURE TO MUTE EVERYWHERE ELSE.
>> VERY NICE TALK.
I DON'T THINK WE HAVE ANY
QUESTIONS HERE RIGHT NOW.
I'LL WAIT FOR SOMEBODY FROM
CHAPEL HILL TO ASK SOME
QUESTIONS.
>> OKAY.
>> OKAY.
ANYONE FROM CHAPEL HILL THAT
WANTS TO ASK A QUESTION?
I THINK CHAPEL HILL MAY HAVE
GOTTEN SNOWED OUT.
LET'S GO TO KENTUCKY.
HELLO?
>> THIS IS DAVE WARREN, SANDY,
THAT WAS A GREAT TALK.
>> HOW ARE YOU.
>> I'M GOOD.
WE'RE ACTUALLY HAVING RAIN SO
WE'RE SAFE FOR THE MEANTIME.
ANYWAY, YOU NICELY SHOWED THAT
ALL [INDISCERNIBLE] WAS PROBABLY
RESPONSIBLE FOR FUSIONS IN THE
ABSENCE OF, I GUESS, POT 1A.
HOWEVER, ARE THERE ANY --
EXPERIENCE SHOWS THAT IT'S NOT
JUST OVOAKED IN THE ABSENCE OF
NHGGA.
IT'S A SECONDARY PATHWAY ANYWAY?
MAYBE YOU'RE CTIP EXPERIMENTS
SHOWED IT DIDN'T QUITE GET
THOSE.
MAYBE YOU COULD CLARIFY?
>> I THINK THE IDEA IN THE
FIELD, THIS PATHWAY COULD BE A
BACKUP PATHWAY.
A LOT OF PEOPLE CALL IT THAT.
IT COULD BE A BACKUP PATHWAY,
ONLY MANIFESTS WHEN YOU DELETE
COMPONENTS OF THE CLASSIC
PATHWAY.
BUT I THINK THAT THERE IS
ACTUALLY A MECHANISM AT
TELOMERES, ACTUALLY REPRESS HR
BASED TYPE RECOMBINATION, AND IT
COULD BE ONE OF THESE, IN THAT
IF YOU GET RID OF CTIP YOU CAN
CLEARLY SEEN CHROMOSOME FUSIONS
ARE ABOLISHED, IF YOU GET RID OF
PART 1A, SUGGESTING THAT THESE
PATHWAY IS ACTUALLY ROBUST.
YOU HAVE TO PROTECT AGAINST THIS
IN A CELL, WHEN THE CLASSIC
COMPONENTS ARE INTACT.
I THINK THAT'S THE GIST OF THIS
DATA EXPERIMENT.
NOW, YOU COULD STILL OBSERVE THE
CONSEQUENCES OF THE ALTERNATIVE
WITH A SETTING OF IN TACT
COMPONENTS.
IF YOU GET RID OF POT 1A AND TP1
AT TELOMERES.
>> OKAY.
THANKS AGAIN.
>> THANK YOU.
>> OKAY.
THANK YOU.
AND KENTUCKY, LET'S MOVE TO
STONEY BROOK.
HI SANDY.
IT'S ORLANDO.
>> HI.
>> ONE QUESTION REGARDING YOUR
[INDISCERNIBLE] SEEMS TO BE VERY
SIMILAR TO SINGLE STRANDED
[INDISCERNIBLE] PATHWAY.
CAN YOU TELL ME IN WHAT ASPECTS
2 PATHWAYS ARE DIFFERENT?
>> THAT'S A GREAT QUESTION.
SO SINGLE STRANDED
[INDISCERNIBLE] I THINK REQUIRES
[INDISCERNIBLE] IF I'M NOT
MISTAKEN.
IS THAT CORRECT?
52.
52.
IS THAT RIGHT?
RAP 52.
WE HAVEN'T EXPLORED THE SINGLE
STRANDED IN ANY DIFFERENCE
BETWEEN -- AT ALL IN GREAT
DETAIL.
WHAT I COULD TELL YOU, IN OUR
PATHWAY, IF YOU GET RID OF RAP
52 YOU ALSO ABOLISH THIS
REACTION.
RAP 52 IS SHARED.
IF YOU GET RID OF RAP 51, THERE
IS NO EFFECT.
SO IN THE ABSENCE OF RAP 1W5 1,
CHROMOSOME FUSIONS STILL OCCUR.
SO IT'S SUGGESTING IT'S PROBABLY
NOT A THROMGOUS COMBINATION
BASED SYSTEM.
IT COULD BE SINGLE STRANDED
LINEAL, THE DISTINCTION BETWEEN
THOSE WE HAVE NOD MADE. -- NOT
MADE.
PETER BOUGHMAN HAS WORK
SUGGESTING IF YOU GET RID OF POT
1A IT IS A SINGLE STRANDED
MECHANISM THAT'S ACTIVATED, AT
LEAST IN THE SYSTEM.
>> DOES IT REQUIRE
[INDISCERNIBLE]
>> I DON'T THINK HE TESTED THAT
YET.
THAT'S A GOOD QUESTION.
WE HAVEN'T TESTED OURSELVES.
>> THANK YOU.
>> OKAY.
THANK YOU.
LET'S MOVE TO RESEARCH TRIANGLE,
NIEHS.
>> MATT LANGLEY.
WONDERFUL TALK.
>> THANK YOU.
>> I WAS THINKING ABOUT HOW YOU
HAVE 1 POT [INDISCERNIBLE] IN
HUMANS, 2 IN MICE.
ARE THERE ANY KNOWN MUTATIONS
THAT WOULD BE INFORMATIVE TO THE
DIFFERENCE BETWEEN POT 1 AND POT
1A AND POT 1B FUNCTION?
>> THAT'S A GREAT QUESTION.
RIGHT.
SO THAT HAS BEEN DONE AND I WAS
TOLD THAT THERE WAS AN EFFORT TO
SEQUENCE THE POT 1 GENE IN
PATIENTS WITH SEVERE ANEMIA OR
BONE MARROW FAILURE, WITH NO
OTHER MUTATIONS.
AND SO FAR NO MUTATIONS IN THE
POT GENE HAS BEEN FOUND.
ONLY THE [INDISCERNIBLE]
MUTATION.
SO THE WAY I LOOK AT THIS, IT'S
EITHER NOT ENOUGH PATIENTS HAVE
BEEN SEQUENCED, OR THAT
MUTATIONS IN THE POT 1 GENE MAY
BE SO LETHAL.
THAT YOU MAY NOT EVEN GET A
HYPERMORPH.
WHEREAS 10-2, YOU GET
HYPERMORPHED MUTATIONS ON A VERY
SEVERE PHENOTYPE.
I THINK THE JURY MAY STILL BE
OUT ON THAT.
>> THANK YOU FOR THE ANSWER.
>> THANK YOU.
>> OKAY.
THANK YOU.
LET'S MOVE TO ANN HARBOR,
MICHIGAN.
>> HI.
DAVE FERGUSON HERE.
>> HOW ARE YOU?
>> GOOD.
GOOD.
QUESTION ABOUT ATR.
IN YOUR SLIDE YOU HAVE MRN,
UPSTREAM OF ATR.
I'VE ALWAYS ENVISIONED THE GOAL
OF MRN JUST BEING NUCLEARCALLYIS
ACTIVITY GENERATING SINGLE
STRAND DNA.
POT 1 ADDITIONFY, YOU ALREADY
HAVE A SINGLE STRANDED DNA
THERE.
DO YOU NEED [INDISCERNIBLE]
ACTIVITY IN THAT CONTEXT FOR ATR
ACTIVATION?
>> WE'RE DOING THAT EXPERIMENT
WITH YOURSELF.
SO WE'RE ACTUALLY USING MRE11
NUCLEAR DEAD CELLS.
SO WHEN WE GET THE DATA, I WILL
TELL YOU.
I DON'T HAVE THE DATA YET.
BUT THATS ALLEGATIONS OF ABUSE A
GREAT QUESTION -- THAT'S A GREAT
QUESTION.
>> I THINK IT MIGHT BE
DISTINCTION --
>> I CAN'T HEAR YOU.
>> I WOULD THINK IT MIGHT BE
DISTINCT FROM A CLEAN DOUBLE
STRAND BREAK.
CAN YOU HEAR ME NOW?
>> YES.
YES.
>> OKAY.
>> ONE OTHER QUESTION. --
>> WE TAKE ONE QUESTION A SITE.
>> WE JUST --
>> LET'S GO ONE QUESTION PER
SITE.
>> OKAY.
THAT'S FINE.
THANK YOU.
>> LET'S GO TO PORTLAND, OREGON.
>> HI.
LORAL EARLY FROM PORTLAND,
OREGON.
I WAS CURIOUS, IN THE PLUMBER'S
NOEL CANCER CELLS, DO YOU THINK
IT'S POSSIBLE THAT POT 1 MIGHT
BE INVOLVED IN THEIR HOMOLOGOUS
COMBINATION TO EXTEND THEIR
TELOMERE LENGTH.
>> THAT'S A GREAT QUESTION.
WE'RE APPROACHING THAT QUESTION
USING OUR MOUSE BREAST TUMOR
MODEL TO LOOK AT THAT.
WE HAVEN'T LOOKED AT ACTUALLY
HUMAN SAMES YET. -- SAMPLES.
FROM WHAT WE'RE FINDING IN OUR
POT 1A DELETION, THE DELETION OF
POT 1A IS LIKELY REQUIRED FOR
THE GENERATION OF THESE CRAZY
CHROMOSOME FUSIONS AND THE
ABILITY WE SEE THAT ACCELERATES
BREAST CANCER TUMOR GENESIS.
>> THANK YOU.
>> OKAY.
DR. -- I'LL ASK A QUESTION.
DOCTORS ALTAR AND SAVAGE HERE AT
NCI ARE STUDYING [INDISCERNIBLE]
AND ACTUALLY DR. ALTER GAVE A
TALK IN THIS SERIES EARLIER THIS
YEAR.
AND ONE OF THE POINTS THAT SHE
MADE WAS THAT THERE ARE MANY
PATIENTS WITH CLINICAL
DISKEROTOSIS CONGENITA, WHO'S
MUTATIONS HAVE NOT BEEN
IDENTIFIED.
AND SO COULD YOU REVIEW WITH US
WHICH OF THESE PROTEINS ACTUALLY
ARE ASSOCIATED WITH HUMAN
DISEASES?
THE PROTEINS YOU'VE MENTIONED?
WHICH DO YOU THINK ARE
CANDIDATES?
>> THE ALTERNATIVE TO THIS
SUGGESTION THAT YOU HAD IS THAT
THESE -- ONE ANSWER IS THAT THE
PROTEINS ARE SO IMPORTANT FOR
LIFE THAT IF YOU HAVE DAMAGED
THEM YOU'RE DEAD.
THE FLIP SIDE OF THE COIN IS THE
CHANGES MAKE SUCH MINIMAL
CHANGES THAT YOU MAY NOT DETECT
IT.
WE COME AGAINST THIS.
>> ABSOLUTELY.
SO THE PROTEINS THAT WE KNOW
THAT ARE INVOLVED --
[INDISCERNIBLE]
>> TUMOR DISEASES AND WHICH DO
YOU THINK ARE CANDIDATES THAT
MIGHT BE GOOD ONES TO LOOK FOR
FOR THESE PATIENTS WHERE THEY
HAVE CLINICAL DISEASE BUT NO
MUTATIONS YET?
>> RIGHT, SO THE PROTEIN THAT WE
KNOW ASSOCIATED WITH THESE,
ACTUALLY FORM MUTATIONS.
THE AUTOSOMAL FORMS AND
COMPONENTS OF TELOMERE, LIKE
TURK, THE RNA COMPONENT, RNA,
GENES ENCODING THE PROTEIN TROM
RAILS AND THIS -- TELOMERASE AND
THE CODE 10 PROTEIN.
SO THE NEW PLAYERS, I THINK
DR. SAVAGE AND ALTAR HAVE SHOWN
IN THEIR NICE PAPERS THAT KIDS
WITH VERY SEVERE FORMS OF DC
ACTUALLY POSSESS MUTATIONS IN
10-2.
THE FIRST DISCOVERY THAT THE
SHELTERING COMPONENT ACTUALLY
PROTECTIVE AGAINST DC --
AGAINST, THE SHELTERING
COMPONENTS DO NOT HAVE, YOU
KNOW, A DIRECT ROLE IN TELOMERE
LENGTH REGULATION OTHER THAN THE
FACT THAT POT 1 REGULATES
TELOMERE ACCESS WHICH COULD
CONTROL TELOMERE LENGTH.
SO I THINK THAT OBSERVATION,
THAT THE 10-2 MUTATIONS CAUSE
SUCH A SEVERE [INDISCERNIBLE]
ARE EXCITING, LED TO THE
PROPOSAL, ALSO IMPLEMENTATION OF
SEQUENCING ALL THE SHELTERING
COMPONENTS.
I THINK THE JURY MIGHT BE OUT
BUT AS FAR AS I KNOW, THE PART 1
MUTATIONS HAVE NOT BEEN FOUND IN
PATIENTS WITH CLINICALLY
RELEVANT DC.
AS FAR AS I KNOW.
AGAIN, AS I SAY YOU'RE RIGHT,
THAT IT COULD BE BECAUSE THE
MUTATIONS ARE SO MILD THAT WE
DON'T SEE.  THAT SO IN EITHER
CASE WE DON'T KNOW.
BUT I THINK OUR MOUSE MODEL
SUGGESTIONS THAT AT LEAST IN
TERMS OF MUTATIONS IN PART 1B,
IN THAT LOSS OF PART 1B, IT'S
VERY DIFFERENT FROM LOSS OF PART
1A.
IN THAT LOSS OUGHT PART 1B GIVES
YOU A PHENOTYPE.
BUT IF YOU LOOSE PART 1A IT
GIVES YOU A CHROMOSOME
INSTABILITY PHENO TYPE.
SO WE KNOW THAT IN HUMANS, THE
HUMAN ON HAS ONE PART GENE,
BUT THE HUMAN PART GENE HAS PART
1A AND PART 1B FUNCTIONS.  WHAT
WE'RE TRYING TO UNDERSTAND NOW
IS THAT WHAT EXACTLY IN HUMAN
PART 1 IS RESPONSIBLE FOR THE
PART 1B LIKE PHENOTYPE.
THAT'S WHAT WE'RE DOING IN THE
LAB.
SO MAYBE THAT CAN HELP US PIN
POINT MUTATIONS IN PART 1 THAT
MAY NEED TO ARISE, FOR EXAMPLE,
YOU NEED TO DELETE OR MUTATE THE
HUMAN PART 1'S FUNCTION AS
ANALOGOUS TO MOUSE PART 1B.
>> THANK YOU.
ARE THERE QUESTIONS THAT ARE IN
BALTIMORE?
>> LET ME ASK ONE QUESTION HERE,
WHICH IS HOW SPECIFIC ARE THESE
FACTORS, THE SHELTERING PROTEINS
AND TELOMERE FACTORS FOR THE
TELEMERIC PROCESSING?
SO THERE HAD BEEN REPORTS THAT
PR2 CAN GO TO OTHER SITES, SO DO
YOU THINK THAT WE'RE TALKING
ABOUT A SPECIFIC DNA REPAIR
PROCESS ONLY HAPPENING IN
TELOMERES?
OR ARE THESE FACTORS
[INDISCERNIBLE] ALL DNA REPAIR
FACTORS [INDISCERNIBLE]
>> RIGHT.
WHAT DID YOU THINK?
>> I THINK THAT'S A GREAT
QUESTION.
I THINK THERE IS EMERGING DATA
THAT SUGGESTS THAT THESE
SHELTERS EXIENTS MAY NOT BE
AS -- COMPONENTS MAY NOT BE AS
SPECIFIC AS WE THINK.
THIS ARISES FROM THE
EXPERIMENTS, AND THAT IONIZING
[INDISCERNIBLE].
AND THE PAPER THAT SUCCEEDED IN
KNOCKING OUT RAP 1.
IN HER CASE, SHE FOUND A RAP 1
NOT ONLY HAS A ROLE IN TELOMERE
END PROTECTION, BUT LOSS OF RAP
1 MESSES UP THE TRANSCRIPTION
MACHINERY OF MANY, MANY GENES,
THIS TELLS US THAT MAYBE RAP 1
IS DOING A LOT OF OTHER THINGS.
MAYBE IT HAS A ROLL IN TRNGS
WHICH I THINK -- TRANSCRIPTION,
WHICH WILL BE VERY INTERESTING.
WE THOUGHT THAT THAT FUNCTION
MIGHT BE LOST IN THE MAMMALIAN
SYSTEM, IN THAT IT COULD BE A
PROTEIN THAT DIMERIZES TO
PROTECT TELOMERES, BUT MAYBE
NOT.
>> BUT [INDISCERNIBLE] VERY
SPECIFIC --
>> POT 1 IS VERY SPECIFIC BUT WE
HAVE A PAPER COMING OUT WITH --
IN COLLABORATION WITH LEE THAT
SHOWS THAT ACTUALLY PART 1s
ROLE IS TO REPRESS BINDING OF
ATR TO THE SINGLE STRANDED
OVERHANG.
IF YOU GET RID OF POT 1 --
SORRY, RAP 1 TO ACTIVATE ATR.
IF YOU GET RID OF POT 1, IT
BIPEDS VERY NICELY TO ACTIVATE.
BUT RPA CAN BE KICKED OUT BY
OTHER PROTEINS, LIKE AK AND RMP1
TO MEDIATE A CYCLE.
BUT THIS PROTEIN CANNOT KICK OUT
PART 1.
SO PART 1 HAS A PERIPHERAL
LOCALIZATION -- PREFERENTIAL
LOCALIZATION.
IT'S NOT EASILY DISPLAYED BY THE
PROTEINS IN THE COMPLEX.
IT'S A COMPETITION OF TELOMERES
BETWEEN PART 1 AND
[INDISCERNIBLE].
SO MAYBE THIS WHOLE THOUGHT THAT
SHELTERING IN EXCLUSIVE TO
TELOMERES MAY NEED SOME
REVISION.
THERE IS AGAIN INCREASED
EVIDENCE.
ALSO THE ROLE THAT TELOMERASE
PLAYS HAS BEEN CHALLENGED BY
STEVE'S WORK, SUGGESTING THAT
TELOMERASE, YOU KNOW, COUPLES
WITH THE WINNING PATH TO
MAINTAIN STEM CELL ABILITY
INDEPENDENT OF THE TELOMERES.
SO MAYBE THESE PLAY A FACTOR
ELSE WHERE.
>> OKAY.
THANK YOU.
WE'LL GET ONE MORE SHOT.
IF ANY SITE HAS AN ADDITIONAL
QUESTION, JUST UNMUTE AND JUMP
IN.
MY.
THIS IS DAVE AGAIN FROM
KENTUCKY.
IN YOUR EXPERIMENTS WHERE YOU --
NOT KNOCKOUT MICE FOR POT 1B AND
TURK HETEROGIG THE, I WAS
SURPRISED ABOUT WHY THEY SHOT
THE SAME DC TYPE OF DEFECTS
AFTER 6 MONTHS.
WHAT IS YOUR MODEL FOR HOW -- IF
YOU JUST NOT SUFFICIENT
TELOMERASE EXPRESSION OR IS
THERE SOME MODEL FOR THE
COMBINATION OF REDUCED TELOMERE
PLUS POT 1B THAT'S CONTRIBUTING
TO THE INABILITY TO REPLERCHISH
OR KEEP UP STEM CELL
COMPARTMENTS?
>> THAT'S A GREAT QUESTION.
WE WERE [INDISCERNIBLE] BY
THAT -- INTRIGUED BY THAT
QUESTION.
WHY IS THE PHENOTYPE NOT
IMMEDIATE?
WE UNDERSTAND WHY YOU GET AN
EARLY LETHAL [INDISCERNIBLE].
YOU DON'T MAKE Y BONE MARROW
IN THE FIRST  DIE AT 10 DAYS OF
AIMING.
WHY DO THESE MICE DIE AT 6
MONTHS, IF YOU ONLY HAVE 50% OF
TELOMERASE?
WHAT WE THINK, WITH OUR LATER
WORK WITH ERIC LANG, WE NEED A
PERIOD OF TIME FOR TELOMERES TO
SHORTEN.
SO IF YOU LOSE POT 1 ACUTELY,
TEM TELES ARE PROTECTED BUT
DON'T SHOW IT IMMEDIATELY.
FOR EXAMPLE, YOU HAVE TO PASSAGE
THAT OVER SEVERAL GENERATIONS TO
ENGENDER THE SUFFICIENT TELOMERE
LOSS TO TRIGGER A DAMAGE
RESPONSE.
SO IF YOU LOOK AT MOUSE EMBRYO
FIBROBLAST, ONE DAY OF AGE, YOU
DON'T SEE AGE DEPENDENT FOCI.
BUT YOU PASSAGE THOSE MOUSE
EMBRYO FIBROBLASTS, 30 OR 40
POPULATIONS, I THINK WE NEED A
PERIOD OF TIME FOR TELOMERES TO
BECOME SHORTENED TO A SUFFICIENT
WAY TO RECRUIT DAMAGE RESPONSE.
SO LOSS OF POT 1B IS NOT
SUFFICIENT AT THE GET GO TO
ELICIT THE DAMAGE RESPONSE.
SOMETHING ELSE HATCHES TO
TELOMERES AS -- HAPPENS TO
HELHELS AS THEY SHORTEN.
AND ALSO YOU HAVE THIS INCREASE
AT OVERHANG GENERATION IN THE
ABSENCE OF POT 1B AND HAPPEN
HAPPEN OF -- HAPLOINSUFFICIENTCY
IN TELOMERASE.
SO IT'S GENERATING A LARGE
SINGLE STRAND DNA SUBSTRATE THAT
CAN NO LONGER BE PROTECTED BY
RNA THAT'S STILL THERE.
SO THAT DAMAGE RESPONSE, THAT
REQUIRES MULTIPLE PASSAGES, YOU
KNOW, FOR THE ORGANIZE EU678 TO
AGE OVER -- ORGANIZISM TO AGE.
>> I HAVE A SHORT FOLLOW UP
QUESTION.
[INDISCERNIBLE] TELOMERE
HETEROZYGOUS MICE BY
TRANSPLANTING IN BONE MARROW
FROM WILDTYPE?
>> SO WE DID THAT -- WE DID A
CONVERSE EXPERIMENT.
WE TOOK THE PART 1B NOLE
TELOMERE BONE MARROW, AND PUT
THAT INTO -- THESE ARE MIXED
GENETIC BACKGROUND.
WE PUT THOSE BONE MARROW INTO A
MOUSE.
THAT WAS NOT ABLE TO RESCUE, SO
WE DID THE OTHER WAY, WHICH
WE'RE GOING TO DO THE OTHER WAY.
WE CAN'T DO IT UNTIL WE GET THE
MICE INTO ISO GENIC BACKGROUND.
SO WE'RE GOING TO DO THAT
EXPERIMENT.
BUT OUR THINKING IS THAT YES, IT
COULD.
WE WOULD LOVE TO DO THAT
EXPERIMENT.
THAT WOULD BE A WAY TO CORRECT
THE DEFECT.
>> ANOTHER QUESTION HERE FROM
STONEY BROOK.
AND IT'S RELATED TO THE PREVIOUS
ONE.
WHY DO YOU THINK THAT POT 1B
DEFECT IS SPECIFIC TO BONE
MARROW AND DOESN'T EFFECT OTHER
PROLIFERATING TISSUES?
>> I'M SORRY.
I DID NOT [INDISCERNIBLE] SO
IT'S A GLOBAL STEM CELL DEFECT.
WE SEE DEFECTS AT THE GERM CELLS
AND IN THE INTESTINAL STEM CELLS
AS WELL.
IT'S A GLOBAL DEFECT.
NOT JUST BONE MARROW.
I JUST SHOWED YOU DATA FROM THE
BONE MARROW.
I DON'T MEAN TO IMPLY IT'S ONLY
BONE MARROW.
>> THANKS.
>> OKAY.
ALL RIGHT.
I THINK WE'RE DONE WITH THE
VIDEO CONFERENCE.
THANKS TO EVERY ONE FOR
PARTICIPATING.
[APPLAUSE]
>> THANK YOU.
AND ...