>> GOOD AFTERNOON EVERYONE.
I WANT TO GET STARTED WITH THE
ANITA B. ROBERTS.
THE ANITA B. ROBERTS LECTURE
SERIES WAS ESTABLISHED BY THE
WOMEN SCIENTISTS ADVISORS
COMMITTEE IN 2006, AND THE
SERIES DEDICATED TO THE MEMORY
OF DR. ANITA ROBERTS WHO WAS THE
CHIEF OF THE LABORATORY OF CELL
REGULATION AND -- AT THE
NATIONAL CANCER INSTITUTE.
IN MARCH 2004 DR. ROBERTS WAS
DIAGNOSED CAN GASTRIC CANCER
WHICH SHE FOUGHT UNTIL HER
DEATH.
PRIOR TO THIS, DR. ROBERTS WAS
AT THE NIH AS A STRONG AWE WARD
WINNING INVESTIGATOR AND
RESEARCH LEADER.
SHE ARRIVED AT NCI IN 1976 AFTER
COMPLETING HER PH.D. IN BIO
CHEMISTRY AT THE UNIVERSITY OF
WISCONSIN, AND AFTER THAT SHE
WENT ON TO A POST DOC TROUBLE
FELLOWSHIP.
HER PIONEERING WORK AT NIH
CHARACTERIZED TRANSFORMING AND
GROWTH FACTOR BETA, AND
ESTABLISHED A SIGNIFY'S AS A
CRITICAL CELLULAR PROCESSES OF
HEALING AND REGULATION OF CANCER
PARTICULARLY METASTASIS.
NOTE PUBLISHED WORK FROM 1982 TO
TOY WAS AMONG THE TOP 50 MOST
CITED AND SHE WAS THE MOST CITED
FEMALE SCIENTIST IN THE WORLD.
WHILE DR. ROBERTS WAS THE LEADER
IN THE SCIENTIFIC FIELD OF
CARCINOGENESIS DISEASE AND WOUND
HEALING, HAS SERVED AS AN
EXAMPLE OF A PROFESSIONAL THIS
BALANCED FAMILY LIFE WITH WORK
LIFE AND WAS REMEMBERED AS A
WARM ENTHUSIASTIC SUPPORTED
MEMBER OF THE NIH COMMUNITY.
THIS LECTURE SERIES GOES TO
HONOR HER TO RECOGNIZE HER AWE
CHEAVMENTD OF OUTSTANDING WOMEN
SCIENTISTS IN THE HIH INTRAMURAL
PROGRAM.
SO TODAY'S HONORARY WILL BE
DR. KEIKO OZATO FROM NICHD AND I
WILL TURN THE FLOOR OVER TO
DR. JUDY -- FROM NICH.
THANK YOU.
>> IT'S MY FLESH TO INTRODUCE
KEIKO OZATO WHO IS THE
INVESTIGATOR IN THE PROGRAM OF
GENOMICS DIFFERENTIATION AT
NIHD.
KEIKO GREW UP IN JAPAN AND
PURSUED HER POSITION AND
OBTAINED A MASTERS IN SCIENCE
AND A PH.D. IN DEVELOPMENTAL
BIOLOGY AT KYOTO UNIVERSITY IN
1973.
SHE TRAVEL TO THE U.S. TO THE
CARNEGIE INSTITUTE IN
WASHINGTON, AND BECAME HEAD OF
THE UNIT ON MOLECULAR GENETICS
OF IMMUNITY POSITION SHE STILL
HOLDS TODAY.
THROUGHOUT HER CAREER, KEIKO
PURSUED EXPERIENCE ADDRESS IS
FUNDAMENTAL BIOLOGICAL QUESTIONS
OF THE IMMUNE SYSTEM AND FIRMLY
BELIEVES THAT EXPERIMENTS IN
BASIC SCIENCE LEAD TO OUR
UNDERSTANDING OF THE
COMPLEXITIES OF LIFE AND
ULTIMATELY ENABLE US TO ACHIEVE
BETTER HEALTH FOR ALL PEOPLE.
KEIKO'S MANY ACHIEVEMENTS ARE
DOCUMENTED IN THE PROGRAM, AND I
JUST WOULD LIKE TO HIGHLIGHT TWO
THINGS.
ONE IS THAT SHE'S THE 2004
RECIPIENT OF THE MILLSTEIN AWARD
RECOGNIZING THE HIGHEST
ACHIEVEMENT IN THE CYTOKINE
INTERFERON RESEARCH FIELD.
AND AMONG HER NUMEROUS COMMUNITY
SERVICE, SHE IS THE PRESIDENT OF
THE NIH JAPANESE SCIENTISTS
ASSOCIATION AND SHE MADE A MAJOR
CONTRIBUTION TO INTRAMURAL
NIH-WIDE EFFORT TO ASSIST SIGH
ADVERTISES AFFECTED BY THE 2011
EARTHQUAKE.
SO WOULD YOU PLEASE ALL JOIN ME
IN WELCOMING KOIKO TO THE
PODIUM.
[APPLAUSE] 
>> NICE INTRODUCTION.
I'M HONORED TO SPEAK HERE.
I DIDN'T KNOW ANITA ROBERTS --
RESCOTCH ON TGF-BETA AND TWO
YEARS THROUGH HER CANCER WHICH
SHE DISCLOSED IN HER BLOG -- FOR
HUMANITY HAVE BEEN VERY POWERFUL
AND IS A LEGACY FOR ME.
I WOULD LIKE TO THANK -- FOR
INNOVATING THE FEMALE SCIENTISTS
AT NIH -- YEARS AGO NIH WAS --
FEMALE SCIENTISTS HAD TO --
RECOGNITION.
IT WAS TO MEN WITH SIMILAR
QUALIFICATIONS.
THIS IS PUBLISHED IN 1990 IN
WASHINGTON POST DEPECKING THE
LABORATORY CORRIDOR, A FEMALE
SCIENTIST IS WALKING THROUGH AND
SENIOR INVESTIGATORS --
LAUGHING -- LOOKS CAN BE A VERY
SCARY PLACE.
BUT THINGS HAVE CHANGED
DRAMATICALLY SINCE THEN PARTLY
THANKS TO WSA'S INITIATIVE.
SO WE ENJOY EQUAL STATUS NOW.
HOWEVER, IN A GLOBAL
PERSPECTIVE, WOMEN HAVE SOME
WAYS TO GO.
IN MANY PARTS OF THE WORLD, WILL
STILL BEAR A GREATER SOCIAL AND
ECONOMIC BALANCE.
IN THIS RESPECT -- I'M THE FIRST
AGENT TO SPEAK IN THIS CITY.
NOW HISTORICALLY SCIENCE HAS
BEEN IN THE DOMAIN OF WESTERN
CIVILIZATION, ESTABLISHED
RESULT -- IT IS ONLY LATE 20TH
CENTURY WHEN MOSTLY MEN BEGAN TO
PARTICIPATE IN OUR FIELD.
HOWEVER WOMEN LAGGED BEHIND
BECAUSE WE HAD MORE OBSTACLES TO
OVERCOME.
BUT THE SITUATION CHANGED VERY
FAST.
CURRENTLY THERE ARE MANY FEMALE
SCIENTISTS IN NIH CONSTITUTING
CONSIDERABLE WORK FORCE MAKING
THE PROGRESS IN OUR SCIENCE.
BECAUSE WE ARE THE LAST TO
ARRIVE.
WE ARE THE -- GROUP IN NIH.
HOWEVER, WE THINK THAT AS TIME
GOES ON, MORE ASIANS WILL COME
INTO THE FIELD AND MAKE
SUBSTANTIAL CONTRIBUTIONS.
SO PLEASE BE AWARE OF OUR GROUP.
NOW TODAY -- OVER MY MOST WORK
INTERFERON AND THE TRANSCRIPTION
FACTOR BUT I HAVE TO SPEND MUCH
OF THE TIME ON OUR ONGOING
RESEARCH ON TRANSCRIPTION
INNOVATION AND EXCHANGE THAT
RELATES TO IMMUNITY -- YOU CAN
IMAGINE PHAGES -- IMMUNITY.
THIS IS THE THROUGH VARIOUS
ASSISTANCE UNDEVELOPED
ANTI-VIRAL AND ANTI-MICROBIAL
RESPONSE.
NUMEROUS CYTOKINE AND --
IMMUNITY.
NOW AMONG CYTOKINES INTERFERENCE
ARE REAL THE CHAMPION OF INNATE
IMMUNITIES.
FOR EXAMPLE THIS IS INTERFERON
BETA, TYPE ONE INTERFERON.
THERE ARE MANY TYPE ONE
INTERFERENCE THAT ARE SIMILAR.
SMALL SECRETED MOLECULES.
THEY DO MANY THINGS THEY PROVIDE
INNATE RESISTANCE AGAINST
PATHOGENS, IMMUNE -- CONTROL
CANCER GROWTH AND IMMUNITY AND
INFLAMMATORY CONDITIONS.
FOR THIS REASON, INTERFERON, WHY
DO WE USE TO THE -- VIRAL
INFECTIONS, CANCER AND MULTIPLE
SCLEROSIS.
THERE ARE TWO OTHER INTERFERENCE
THAT ARE DISCOVERED MORE
RECENTLY.
TYPE THREE INTERFERON AND TYPE
TWO INTERFERON.
TYPE TWO INTERFERON IS A
INTERFERON -- ALSO CALLED IMMUNE
INTERFERON.
ALSO IN A SIMILAR WAY AS TYPE
ONE INTERFERON.
INTERFERON HAS ADDITIONAL
PROPERTIES TO REGULATE.
LYMPHOCYTES AND MACROPHAGES --
ACTIVATE MACROPHAGES TO FARTHER
BOOST INNATE IMMUNITY.
TIME THREE INTERFERON DISCOVERED
IN THIS CENTURY -- IN RESPONSE
TO TYPE ONE INTERFERON.
SO TYPE THREE INTERFERON IS
INTERFERON-INDUCED PROTEIN.
AND ACTIVITY IS SOMEWHAT SIMILAR
TO TYPE ONE INTERFERON.
NOW INTERFERON DEPENDS ON --
FAMILY IN TERMS OF THEIR
PRODUCTION.
IN TERMS OF THEIR VIRAL
ACTIVITIES.
AND THEY ARE LINED UP HERE ALL
OVER THEM ACCORDING TO DISCOVERY
ACTUALLY AND THE RED PART SHOWS
YOU THE DNA BINDING DOMAIN AND
THE BLUE INDICATES THE --
DOMAIN.
AND THEY FIND COMMON DNA
ELEMENT, ISRE AND THE REGULAR --
SHOWN HERE.
SOME YEARS AGO, WE ISOLATED IRS8
AND SINCE THEN WE'VE BEEN
WATCHING THIS FACTOR VERY HARD.
IRS8 IS AN IMMUNE
SYSTEM-SPECIFIC MEMBER OF THE
IRS FAMILY.
AND THERE IS ANOTHER MEMBER THAT
HAS THESE PROPERTIES, NAMELY
IRS4.
IRS78 IS EXPRESSED IN
MACROPHAGES -- THE ASPECT WE
FOCUSED ON.
BUT IRS8 ALSO IS EXPRESSED IN --
AND IN NIAID HAS WATCHED THIS
ASPECT EXTENSIVELY AND FOUND
MANY INTERESTING OBSERVATIONS.
NOW BY STUDYING IRS8 -- WE FOUND
THAT IRS8 PREVENT MACROPHAGE
DEVELOPMENT.
MACROPHAGE CAN DEVELOP -- CAN
BECOME A NEUTROPHIL OR
MACROPHAGES.
WE FOUND THAT IRS8, THIS AXIS OF
DEVELOPMENT IT'S INTERESTING
WITH THE NEW AXIS.
SO IRS78 MICE DON'T HAVE AS MANY
MACROPHAGES AND THEY ARE
FUNCTIONALLY DEFECTIVE.
THEY DON'T PRODUCE RIGHT SO
MANIES AND ENZYMES -- INDUCE A
NUMBER OF GENES THAT ARE
IMPORTANT FOR US IN THE INNATE
IMMUNITY.
THE DEVELOPMENT ASPECT OF IRS8
IN MACROPHAGES ARE BEING STUDIED
BY THE -- WHO USED TO WORK IN
OUR LABORATORY BUT NOW PROFESSOR
IN JAPAN AND ISRAEL.
WE FOUND THAT IRS8 IS ALSO
CRITICAL FOR THE DEVELOPMENT OF
THE -- AMONG FOUR MAJOR
DISEASE -- IRS8 FOUND TO BE
ESSENTIAL FOR -- THAT PRODUCE
LARGE AMOUNT OF IO   1 2, THE
CYTOKINE NECESSARY FOR
INDUCING -- IN ADDITION IRS8 IS
REQUIRED FOR THE DEVELOPMENT OF
THE -- POSITIVE -- DC THAT ARE
SPECIALIZED IN PRODUCING LARGE
AMOUNT OF TYPE ONE INTERFERON.
INTERESTINGLY THE MEMBER IRS4 IS
ESSENTIAL FOR THE DEVELOPMENT OF
THE REMAINING DISEASE UPSET,
CD -- DC.
NOW HERE WE COULD ESTABLISH DC
PREPROGENITOR CELLS BY SIMPLY --
IRS8 -- IN THE PRESENCE OF 3A.
UPON IRS8 TRANSFER
DIFFERENTIATED INTO IMMATERIAL
DCS RIGHT AWAY THAT ARE BECOMING
ACTIVE MATERIAL DC AFTER
STIMULATION BY -- CPG AND
PRODUCED COPIOUS AMOUNT OF TYPE
ONE INTERFERON AS WELL AS
IO12P40 SIMILAR TO IO TYPE 12.
THE SITUATION HAS BEEN FOUND FOR
MACROPHAGES SO WE COULD GENERATE
MACROPHAGE PROGENITORS FROM
MICE.
NOW THE DIFFERENTIATION
PROMOTING PROPERTY -- THE VERY
IMPORTANT GROWTH -- YOU CAN SEE
THAT AS SOON AS IRS8 TRANSFERRED
INTO THESE DC PROGENITORS, THEY
STOPPED -- THAT MEANS IRS8 IS A
CHEMO SUPPRESSOR.
THE MICE DEVELOPED -- SYNDROME.
YOU CAN SEE THE PROGENITORS AND
THE -- IN THE RAT.
THIS WAS MADE IN THE
OBSERVATIONS, MADE IN THE
MOUSE -- INTEREST AND THESE
STUDIES THAT ARE COMING FROM
OTHER LABORATORIES HAVE SHOWN
THAT INDEED IRS78 IS DOWN
REGULATED IN -- LEUKEMIA AND
TREATMENT WITH INTERFERON WHICH
OFTEN -- IRS8 CORRELATES WITH
THE 
THE APPEAL ORATION.
IN A SIMILAR CONTEXT, IRS8 IS
FOUND TO BE CRITICAL FOR THE
DEFENSE IN HUMANS.
THESE INVESTIGATORS FOUND
INFANTS HAVING MUTATION IN IRS8.
THESE MUATIONS ARE FOUND IN THE
DNA BINDING DOMAIN AND THE-FANTS
WERE HIGHLY SUSCEPTIBLE TO
VARIOUS INFECTIONS.
INCLUDING BCD VACCINATIONS AND
THEY DIDN'T PRODUCE IR12P40 AND
INTERFERON.
AND THEY HAD THIS DRAMA NUMBER,
ALL THE MONOCYTE AND DC BUT --
DISEASE CONDITIONS VERY SIMILAR
FOR IRS78 IN MICE.
SO ENDING THIS PART, I LIKE TO
EMPHASIZE THAT IRS8 IS A
FUNCTION -- TO THE ACTIVITY OF
INTERFERON AND ALL THREE TYPES
OF INTERFERON.
TYPE ONE, TYPE TWO AND TYPE
THREE INTERFERON.
IN ANY CASE THE QUESTION WE HAVE
TO ASK IS HOW DO INTERFERENCE --
INNATE IMMUNITY.
NOW THIS IS A DIAGRAM OF
STUDYING THE INTERFERON
SIGNALING PATHWAY.
NOW INTERFERENCE BIND TO THE
RECEPT ACTIVATING THE PATHWAY
AND ASSEMBLING TRANSLATION
FACTORS -- ONE, TWO AND IRS9 --
ISRE.
AND THIS LEADS TO TRANSCRIPTION
DEVELOPING IN INDUCTIONS OF THE
MODERN 1600 GENES THAT
COLLECTIVELY PRODUCE INTERFERON
ACTIVITIES REPEATING AGAIN
PROVIDING ANTI-VIRUS,
ANTI-MICROBIAL ACTIVITY,
BOOSTING THE IMMUNE FUNCTION AND
CONTROLLING THE -- NOW IN THIS
MATT WAY, THIS TRANSCRIPTION IS
THE FINAL DESTINATION AND THE
END POINT OF SIGNALING.
FOR THIS REASON.
THE MAJORITY OF STUDIES HAS
FOCUSED ON TO STUDYING EVENT IN
THESE EXTREME REGIONS.
OUR AIM HAS BEEN TO INVOLVE
DEEPER BEYOND THE END POINT AND
ASK WHAT IS THE MECHANISM OF THE
CURRENT STAGE.
AND WE ARRIVED AT TRANSCRIPTION
AND CHROMATIN EXCHANGE.
LET ME START WITH SOME
BACKGROUND.
RNA02 IS -- SITE IN MANY ACTIVE
GENES -- IS BOUND TO THE PAUSING
COMPLEX NELF/DSIF.
PRODUCTIVE ELONGATION STARTS
WHEN PTEFB AND OTHER ELONGATION
FACTORS ARE RECRUITED.
NOW THE NEXT THING ELONGATION TO
CHROMATIN IT'S BEEN SHOWN THAT
SOME INNOVATION FACTORS SUCH AS
SPT6 -- ARE REQUIRED FOR THE
MAINTENANCE OF NUKE -- IS SHOWN
TO BE PRESENT PREDOMINANTLY IN
ACTIVITY.
NOW LET ME TALK ABOUT THAT HERE
PAUSE THIS IS OUR CENTRAL
INTEREST.
NOW AS WE KNOW, DNA GENOMIC DNA
IS ORGANIZED INTO CHROMATINS
THROUGH BEING DNA -- AND EACH
NUCLEOSOME IS SUPPOSED OF H3,
H4, H2AB.
NOW H3 THAT WE ARE INTERESTED IN
IS DISTANT ONLY A FEW AMINO
ACIDS FROM THE STANDARD HISTONE
H3, 3.1 AND 3.2.
THE DIFFERENCE IN THIS SMALL
HELIX IN THE HISTONE.
IT'S 3.3 IN EUKARYOTE AND IT'S
HELD TO BE EVENTUAL FOR MOUSE
DEVELOPMENT.
ALTHOUGH STRUCTURALLY SIMILAR
THE 3.3 IS DIFFERENT FROM THE
STANDARD HISTONE IN A RADICAL
WAY IN TERMS OF ITS BEHAVIOR.
NAMELY IT'S 3.3 IS EXPRESSING
NON-S PHASE AND INCORPORATED IN
THE GENOME ALONG WITH
TRANSCRIPTION.
IN CONTRAST THE STANDARD HISTONE
ARE EXPRESSED IN THE S PHASE AND
IS DEPOSITED TO THE DNA
REPLICATE.
NOW IT'S BEEN SHOWN -- THAT IT'S
3.3 IS PRESENT IN
TRANSCRIPTIONALLY AFTER LESIONS
OF THE GENOME -- AND IN ESO AND
IS IMPLATED IN EPIGENETIC
REGULATION.
HOWEVER, OVERALL IT IS
UNDERSTOOD ABOUT H3.3 IN TERMS
OF HOW IT WORKS AND THE MEANING
OF THIS HISTONE.
THE UNDERSTANDING OF H3.3 REALLY
COMES FROM THE TECHNICAL --
STUDYING THIS WHICH IS AT LEAST
PARTLY ATTRIBUTED TO THE FACT
THAT THERE IS NO SPECIFIC
ANTIBODY.
NOW, AS YOU WILL SEE THAT
HISTONE IS 3.3'S ACTIVITY IS
CLOSELY TO ELONGATION AND MUST
INTRODUCE BRD4 HERE.
TODAY IN THE LAB ISOLATED THIS
PROTEIN IN 2000.
AND BRD4 -- DOMAINS CARRYING
TWO -- DOMAINS.
AND THROUGH THESE DOMAINS BRD4
BINDS -- HISTONES H3 AND H4.
THIS IS VERY UBIQUITOUS PROSEEN.
BRD4 REMAINS ON CHROMOSOMES IN
MITOSIS.
IN THIS SENSE THROUGH -- WE ARE
ABLE TO SHOW THAT WE ARE BRD4
 IN
AN ELONGATION -- BECAUSE OF
THESE PROPERTIES BRD4 IS A STRAP
SCRIPTION OF THE MEMORY ACROSS
REGIONS.
-- IN NCI HAS SHOWN THAT IN
ADDITION BRD4 CAN REGULATE --
NCI IS ALSO FINDING NEW FUNCTION
ACTIVITY ASSOCIATED WITH BRD4
WHICH I HOPE THEY HAVE THE
OPPORTUNITY TO PRESENT TO US
SOON.
IN ANY CASE, WE ASK WHAT HAPPENS
TO BRD4 WHEN HE'S STIMULATED BY
INTERFERON.
NOW INTERFERENCE CAN STIMULATE
MANY CELLS TO INDUCE INNATE
IMMUNE GENES, INCLUDING -- WE
LOOKED AT -- WHICH IS AN
INTERFERON STIMULATING GENE
CALLED ISG.
AND YOU CAN SEE THAT AFTER
STIMULATION -- AND SPEAKING
ABOUT 4 TO 6 HOURS AND THEN COME
DOWN.
BINDING BRD4, I TRIED DIFFERENT
REGIONS.
WE'VE SEEN THE ONE GENE WHICH IS
ABOUT 9 KILO BITES LONG.
IT'S AFTER INTERFERON
STIMULATION -- BRD4 BINDING
WAS -- IN OTHER PARTS -- MADE
FOR OTHER INTERFERON-STIMULATED
GENES OBSERVED EXCEPT GAPDH
WHICH IS A GENE NOT INDUCED BY
INTERFERON.
NOW WE THINK THAT THIS
RECRUITMENT IS PARTLY DUE TO
RAPID INCREASE IN HISTONE
ACCELERATION -- H4.
NOW BECAUSE OF IT THE SPEAK NOW
COMES INTO THE ISG1 GENE -- PSS
VERY RAPIDLY.
ALSO FOUND IN THE GENE BODY --
PHOSPHORYLATED -- ANIMATING
FORM -- AND MOVES THROUGH THE
GENE TO REACH THE END.
NOW WE ALSO FOUND THAT THE
QUALITY COMPLEX PROTEIN AND THE
DSIS -- ONLY AFTER INTERFERON
STIMULATION.
THESE RESULTS HAVE
INTERESTING -- FOR OWE LONG
GAITION -- BUT WE CAN SKIP THAT
ASPECT TODAY.
DSIF THIS IS A BONE FIDE
ELONGATION FACTOR TO MOVE TO
REACH THE END OF THE GENE ALSO.
NOW -- IS ANOTHER ELONGATION
FACTOR THAT ALSO ACTS AS A
HIGGINS TONE -- HAVING A ROLE IN
NUCLEAR MAINTENANCE WAS ALSO
RECRUITED AFTER INTERFERON
STIMULATION AND MOVED THROUGH
THE GENE DURING THE
TRANSCRIPTIONS.
SO THE ELONGATION FACTORS ARE A
PLACE IN ISGs AFTER INTERFERON
STIMULATION.
IN ORDER TO DELINEATE THE
HIERARCHY OF ELONGATION FACTOR
RECRUITMENT, WE TESTED, RECENTLY
DEVELOPED  SIMILAR MOLECULE THAT
TARGETED AND SPECIFICALLY
INTERFERES WITH THE BINDING AND
HISTONE TWO, THE FAMILY OF THE
DOMAIN WHICH IS RIGHT HERE IN
THIS DOMAIN.
AND AS YOU MIGHT EXPECT, THIS
DRUG BUT NOT ISOMER COMPOUND --
RECRUITMENT WITH THE RD4 AFTER
INTERFERON STIMULATION TO THE --
1 GENE.
ALSO YOU HAVE RECRUITMENT
INHIBITED WHICH YOU WILL SEE
LATER ON.
BUT MORE IMPORTANTLY, ALL THE
OTHER ELONGATION FACTORS ARE
ALSO INHIBITED AND BEING
RECRUITED.
IT'S INTERESTING THAT BRD4 IS
THE HIERARCHY.
WHAT YOU MIGHT EXPECT BECAUSE OF
THIS, THIS INHIBITED EXPRESSED
ALL THE OTHER ISD WE'VE TESTED
BUT NOT THE CONTROL OF SPRT.
NOT INDUCED BY INTERFERON.
SO INTERFERON STIMULATION IS
RECRUITMENT OF BRD4 TO THE NEWLY
STIMULATED ISG PRANG SCRIPTION
SITE.
BRD4 RECRUITMENT IS THE PRIMARY
EVENT THAT INITIATES A CASCADE
OF  ELONGATION FACTOR RECRUITMENT
WHICH LEADS TO ELONGATION.
SO WE ENVISION THAT ASSEMBLES
MANY FACTORS BEFORE ELONGATION
STARTS AND WHY SOME FACTORS
REMAIN AFTER -- MANY FACTORS
MOVE -- THROUGH THE GENE.
NOW WITH THIS, WE CAN NOW TALK
ABOUT INCORPORATION OF HISTONE
H3.3.
IN INTERFERON STIMULATED GENE,
WHICH WE STUDIED IN 3D3 CELLS
EXPRESSING WHY 3.3 AND WITH
CONTROL WE TESTED 3.3 EXPRESSING
YFP STANDARD HISTONE AT 3.1.
AS YOU CAN SEE, HISTONE 3.3
RESIDES PREDOMINANTLY IN THE
NUCLEUS -- CHROMATIN INDICATING
THAT 3.3 MUST BE AUTHORIZED TO
THE TRANSCRIPTION REACTIVE
CHROMATIN WHEREAS ON HISTONE 3.1
CAN DISTRIBUTE THE NUCLEUS
INCLUDING HISTONE CHROMATINS
WHERE GENES ARE SILENT.
NOW HERE WE TESTED INCORPORATION
OF 3.3IC1 GENE WHICH YOU HAVE
ALREADY SEEN AT THREE DIFFERENT
REGIONS.
TRANSCRIPTION SITE BODY AND THE
END OF THE GENE.
AND WE FOUND THAT 3.3 HAS
INCORPORATED DRAMATICALLY
INDUCED AFTER INTERFERON
STIMULATION.
AND THIS HAPPENS FOR THE
STANDARD HISTONE 3.1.
IT REMAINED AT THE BACKGROUND,
AND IN AXIS IS ENLARGED --
ASPECT IN THIS RESULT.
NUMBER ONE, IT'S 3.3
INCORPORATION KEPT RISING FOR
TWO DAYS, 48 HOURS, LONG
AFTER -- SYNTHESIS ENDED.
YOU CAN SEE BY THIS MRNA CARD.
NUMBER TWO, THE H3.3
INCORPORATION, THE MORE DOMINANT
OF THE GENE END AND GENE BODIES
SHOWED PROMINENT INCREASE.
HOWEVER -- IT'S 3.3 WAS NOT
OBSERVED.
AND THIS CAN BE SEEN EVEN BETTER
FOR -- THE AMOUNT OF 3.3 IS
REDUCED AFTER INTERFERON
STIMULATION.
BASICALLY THE SAME RESULTS ARE
OBSERVED FOR ALL OTHER IS GOOD
THAT WE'VE TESTED.
TO OUR KNOWLEDGE THIS CONDITION
PROLONGED 3.3 INCORPORATION IN
RESPONSE TO TRANSCRIPTION
ACTIVATION HAS NOT BEEN OBSERVED
BEFORE.
NOW, IN ORDER TO BEGIN ASKING
THE MEANING OF THE 3.3
INCORPORATION, WE TESTED 3.3 --
HERE WE USED SHRNA WHICH
PRODUCED 3.3RNA BY MORE THAN
75%.
WE -- THE STANDARD IS 3.1.
AND WE FOUND THAT 3.3 KNOCK DOWN
EXPRESSED -- MUCH MORE
SUFFICIENTLY.
AND THE CONTROL SHRNAS YOU CAN
SEE.
BASICALLY THE SAME RESULTS WERE
OBSERVED AGAIN FOR ALL OTHER
ISGs THAT WE'VE TESTED.
INDICATING THAT H3.3
INCORPORATION SOMEHOW
CONTRIBUTES TO ISD TRANSCRIPTION
ITSELF.
NOW, IN ORDER TO GAIN SOME
MECHANISTIC PROOF, WE LOOKED FOR
MOLECULAR EVENT THAT MIGHT
CORRELATE AT 3.3 INCORPORATION.
AND THERE'S ONLY ONE THING THAT
WE FOUND THAT WAS H3K36
METHYLATION.
IT WENT UP SHARPLY AFTER
INTERFERON STIMULATION.
AND REMAINED HIGH AFTER
TRANSCRIPTION ENDED.
FURTHERMORE, THIS INCREASE WAS A
CONDITION DEPENDENT, THE HIGHEST
AT THE END -- IN THE GENE BODY
AND NO INCREASE AT THE PSS.
THIS AT BEST IS THE FACTORS --
METHYLATION MAY ALSO REGULATE
3.3 INCORPORATION.
NOW -- IN JAPAN RECENTLY SHOWN
THAT WSSC1 IS A SYSTEM
TRANSFERASE -- PROTEIN WITH
INTERESTING DOMAINS INCLUDING
THE FIFTH DOMAIN.
NOW HERE THE GROUP MARKED OUT
THESE GENES AND SHOWED WSC1 IS
IMPORTANT FOR -- DEVELOPMENT.
WHSC1 IS ALSO SHOWN TO BE
TRANSLOCATED -- IN SOME MYELOMA.
NOW -- IN COLLABORATION WITH --
LAB TESTED THE INCORPORATION OF
3.3, USING WSC1 -- AND TO OUR
SURPRISE WE FOUND THAT THERE WAS
BASICALLY NO 3.3 INCORPORATION
IN THE CELLS EVEN THOUGH WHILE
THE -- DIFFERENT THAN 3.3
INCORPORATION.
SO WSC1 SOMEHOW  MEDIATES
INTERFERON 3.3 DEPOSITION.
ALSO TO OUR SURPRISE WSSC1 --
EXPRESSED VERY LITTLE INTERFERON
STIMULATED GENES.
AS YOU CAN SEE IN THIS SHRNA
INDICATING THAT WHSC1 HAS A ROLE
IN -- TRANSCRIPTION.
OTHER ISGs SHOWED SIMILAR
RESULTS SUPPORTING THESE
INTERPRETATIONS.
NOW, ALSO FOUND THAT WSC1
RADIATES ELONGATION AS WELL.
HERE YOU CAN SEE THAT HOW DO WE
RECRUIT -- ELONGATION FACTOR
HIGHLY RECRUITED TO WSC1 CELLS.
THE ACTIVITY WERE -- ALSO WHSC1
CELLS DID NOT HAVE A FINDING
THAT CHROMATIN ASSEMBLY FACTOR
SPECIFIC FOR 3.3, NAMELY HIGHER.
FOR WHSC1 IS REALLY REALLY
IMPORTANT FOR 3.3 INCORPORATION.
NOW, IN ORDER TO FIGURE OUT THE
RELATIONSHIP OF BRD4 AND WSC1
WE'VE TESTED AGAIN THE DOMAIN
SMALLER -- AND AS YOU EXPECT IT
AGAIN INHIBITED RECRUITMENT OF
RBD4.
BUT IN APPEAR DITION G --
RECRUITED WHSC1 WHICH OCCURRED
AFTER INTERFERON STIMULATION.
EVEN MORE IMPORTANTLY JG1 -- 3.3
DEPOSITION HERE.
WE ALSO FOUND THAT OTHER ISGs
ARE SHOWN WITHOUT A DOUBT.
THIS DATA INDICATES THAT BRD4
VERY DIRECT WSC1 RECRUITMENT AND
3.3 DEPOSITION.
SO IN CONCLUSION, INTERFERON
STIMULATION WITH 3.3 DEPOSITION
IN ISG.
AND 3.3 DEPOSITION IS POSITION
DEPENDENT, NAMELY END OF GENE
BIAS AND CONTINUES AFTER
TRANSCRIPTION.
AND ALSO 3.3 DEPOSITION DEPENDS
ON THE BRD4WSC1 CONNECTED -- SO
WE THINK THAT THE 3.3 IS NOT
PRESENT IN ISGs BEFORE
STIMULATION AND AFTER
ELONGATION, 3.3 IS INCORPORATED
INTO THE GENES LIKE THIS
CONTINUES THEREAFTER.
MECHANISMS ASIDE, OUR DATA
CLEARLY SHOWS THAT THE 3.36
REMAINS LONG AFTER ISG
TRANSCRIPTION.
THIS IS FROM TRANSCRIPTION
MEMORY.
DATA ALSO CLEARLY SHOWS THAT
IT'S 3.3 DEPOSITION ISN'T
IMPORTANT HALF OF THE ISG
TRANSCRIPTION.
IT WILL BE IMPORTANT TO STUDY
THE BIOLOGICAL MEANING OVER THIS
OBSERVATION.
IN THE LAST I DRAFT -- PEOPLE
WHO WORKED IN OUR LABORATORY --
PAST INVESTIGATORS -- PEOPLE WHO
CONTRIBUTED TO WHAT I MENTIONED.
I ALSO HAVE TO MENTION -- FOR
WORKED IN OUR LABORATORY BUT IS
NOW WATCHING A DAY IN PRODUCTIVE
MEMBER OF THE NIH COMMUNITY AS
WELL.
I ALSO THANK A NUMBER OF THE
COLLABORATORS WE HAVE HAD OVER
THE YEARS IN NIH AND ELSEWHERE.
I AND WOULD LIKE TO THANK --
PEOPLE ALL OVER THE WORLD.
THANK YOU FOR YOUR ATTENTION.
[APPLAUSE] 
>> HI, YES.
>> GREAT TALK.
I HAVE ONE QUESTION REGARDING
THE FUNCTION OF 3.3.
YOU SUGGESTED 3.3 IS DEPOSITED
AFTER THE ELONGATION.
SO THEN WHAT'S THE FUNCTION.
>> SO SOMEHOW I THINK THAT IT
MIGHT BE RELATING TO --
NUCLEOSOME IN THE ISG.
BECAUSE MOST ISGs ARE -- AND
OTHER TRANSCRIPTIONS ELONGATIONS
GO ON.
NUCLEOSOME STRUCTURE WILL BE
SOMEWHAT DISORGANIZED SO TO
SPEAK.
AND YOU HAVE TO EITHER
PREPARE -- THAT MIGHT BE
NECESSARY TO CONTINUE
TRANSCRIPTION.
THAT'S MY SPECULATION.
DO YOU HAVE ANY IDEA?
>> NO.
I'M JUST ASKING.
BECAUSE THE TRANSCRIPTION LEVEL
APPEARS TO ALREADY DECREASE
AFTER SIX HOURS.
>> THAT'S CORRECT.
>> BUT THE 3.3 IS MAINTAINED
OVER 50 HOURS.
>> THAT'S RIGHT.
>> SO IF YOU'RE -- AFTER SIX
HOURS, DO YOU THINK THE
EXPRESSION OR THE ACTIVATION OF
THE GENE GOES FASTER.
>> WHAT IS INTERESTING IS THAT
IT'S 3.3 -- RNA FILMED THE
BEGINNING VERY EARLY ON -- SO I
THINK AFTER TRANSCRIPTION IS
OCCURRING AS THE RECYCLING --
THAT AS IF HE CAN IS -- BUT YOU
ARE RIGHT IN THAT TO CONTINUE
INCORPORATION AFTER
TRANSCRIPTION ENDED, I THINK
IT'S IMPORTANT.
I DON'T THINK THE PEOPLE KNOW
WHAT THAT MEANS.
>> SO WE HAD -- LAB 3.3.
SO IT SHOWED THAT THE
INCORPORATION OF BOTH -- THE
NUCLEOSOME AND THEN PROPOSED TO
FACILITATE THE REMOVAL OF THE
NUCLEOSOME TO ALLOW TO BIND THE
PROMOTER REGION.
ALL OF THOSE -- BINDING APPEARS
TO BE LOWER FOR THE PROMOTER.
SO MAYBE IT HAD ANOTHER FUNCTION
IN THE INITIATION.
>> THAT'S RIGHT.
I THIRTY 3.3 INCORPORATION AFTER
TRANSCRIPTION SITE MEANS -- I
HAVE LYMPHOCYTES THAT INCREASE
THE INCORPORATION AT THE END
BECAUSE I THINK THAT THERE IS
SOMETHING VERY NEW ABOUT VERY
INTERESTING ABOUT IT.
BUT I THINK THAT REDUCE
INCORPORATION MEANS SOMETHING AS
WELL.
>> THANK YOU.
>> THANK YOU FOR YOUR LECTURE.
DO YOU THINK THE H3.3 IS MORE
RESISTANT TO OXIDATIVE STRESS
THAT WOULD INDUCE THE CELLS.
>> WE SAW SIMILAR END BIASED
INCORPORATION OF 3.3 IN RESPONSE
TO UV STIMULATION.
AND BECAUSE OF THIS, WE THINK
THAT THIS IS NOT TO INTERFERON
STIMULATED RESPONSE.
BUT THIS HAD SOMETHING TO DO
WITH OXIDATIVE STRESS AND WITH
3.3 IS MORE -- TO OXIDATIVE
STRESS, WE HAVE NOT STUDIED.
BUT I THINK THERE IS A
POSSIBILITY.
>> YOUR KNOCK DOWN CELLS WOULD
BE A NICE TOOL THEN TO SCREEN.
THE H3.3 KNOCKOUT CELLS SHOULD
BE MORE SUSCEPTIBLE THEN.
>> THAT'S CORRECT.
WE HAVE NOT EXAMINED THAT.
I'LL ASK YOU ALSO, DID YOU SHOW
US THE LEVELS OF K36 TRY
METHYLATION IN THE ISG'S BEFORE
STIMULATION.
>> YES.
BEFORE STIMULATION, IT'S ALL
ACROSS THE BOARD NAMELY AT ALL
THREE POINTS TESTED.
THIS WAS IN JUST ROUGH.
SO K36 STIMULATION REALLY GOES
UP AFTER STIMULATION.
AND AFTER -- METHYLATION AND
THEY FOUND THAT THE TRI
METHYLATION IS THE HIGHEST -- IN
THE ACTIVITY EXPRESSED GENE.
>> I GUESS WE WERE SORT OF
ASKING THE SAME QUESTION.
LIKE WHY WHSC1 IS IMPORTANT
REALLY AS EARLY STEPS ONLY SIX
HOURS WHEN MOST OF THE CHROMATIN
CHANGES SEEM TO HAPPEN LATER IN
THE PROCESS.
>> MY FEELING IS THAT WHSC1
REALLY CONTROLS ELONGATION,
FIRST OF ALL, AMONG -- FUNCTION.
AND WHEN ELONGATION IS BLOCKED,
EVERYTHING AFTER, SUBSEQUENT TO
ELONGATION STOPS AND THAT'S THE
REASON WHY WE ARE NOT SEEING
H3.3 INCORPORATION AND THE
RECRUITMENT OF ELONGATION
FACTORS.
AND STOPPED BEING ISG INDUCTION.
DID I ANSWER YOUR QUESTION?
>> [INDISCERNIBLE]
>> YES.
I'M AFRAID WE DON'T HAVE AN
ANSWER, BUT IT IS POSSIBLE THAT
THIS MARK IS A MARK TO SAY THAT
THIS TBEAN WASN'T PREVIOUSLY
EXPRESSED AND WE CAN RESPOND TO
THE SECOND STIMULATION
DIFFERENTLY.
SO THIS WE CALL INTERFERON
MEMORY.
SO IF WE STUDY INTERFERON MEMORY
CONDITION AND STIMULATING FOR
THE SECOND TIME WE MIGHT SEE
SOMETHING VERY DIFFERENT.
AND I THINK THAT THAT'S THE KIND
OF THING WE WOULD LIKE TO ASK IN
TERMS OF MEMORY AND
TRANSCRIPTION OF MEMORY -- WAS
STUDIED.
ANY OTHER ISSUES TO DISCUSS? 
[APPLAUSE]