Tip:
Highlight text to annotate it
X
>> GOOD AFTERNOON, WELCOME TO
THE WEDNESDAY AFTERNOON LECTURE
SERIES AND OF COURSE FOR THE
NIH, I KNOW THE SHUT DOWN HAVOC
WITH OUR SCIENCE HERE WE WISH
WOULD HAVE NEVER HAPPEN BUT I
KNOW PEOPLE ARE HAPPY TO BE
BACK.
WE DID MISS FOUR OF THE LECTURES
DURING THE SHUTDOWN WE'RE NOT
SURE ABOUT RESCHEDULING YAM A
ANAKA BECAUSE HE'S IN JAPAN.
WATCH YOUR E-MAIL TO FIND OUT
WHEN THE OTHERS WILL BE TALKING
AGAIN.
BEFORE WE START OUR LECTURE
TODAY, I'D LIKE TO TELL YOU A
LITTLE BIT ABOUT GEORGE KHOURY
IN WHO'S THIS LECTURE IS NAMED.
WE APOLOGIZE FOR NO PROGRAM.
HE WAS THE CHIEF OF MOLECULAR
BIOLOGY AT NCI AND DIED AT 1987
AT THE YOUNG AGE OF 43 FROM
COMPLICATIONS WITH LIMB --
LYMPHOMA.
HE WAS AN ESTABLISHED SCIENTIST
WORKING ON SB 40, STUDY GENE
REGULATION.
.
THIS PARADIGM SHIFTING WORK
RESULTED IN HIS ELECTION BEFORE
HIS DEATH EVEN AT AGE 43 TO THE
NATIONAL ACADEMY OF SCIENCES.
GEORGE WAS ALSO KNOWN TO HIS
FRIENDS AND COLLEAGUES AND TO
HIS MANY TRAINEES AT A VERY
WONDERFUL MENTOR AND A PATIENT
TEACHER.
HE TAUGHT AND SHOWED HIS
COLLEAGUES AND TRAINEES HOW
ENORMOUS ENERGY COMBINED WITH A
MODEL SYSTEM WHICH WAS TRACTABLE
COULD YIELD INCREDIBLY USEFUL
INFORMATION ABOUT THE BIOLOGICAL
WORLD.
HE WAS ENTHUSIASTIC ABOUT
EVERYTHING AND EVERYONE, AND I
CAN STILL SEE HIM, HE USED TO
SIT IN THE FRONT ROW -- NOT MANY
PEOPLE DO -- AND HE WOULD SIT ON
THE EDGE OF HIS CHAIR TO ASK
QUESTIONS WHICH WERE CREATIVE.
HE HAD I78 PACT NOT ONLY AT THE
NIH WHERE HE TRAINED MANY PEOPLE
BUT ALSO AT PRINCETON WHICH WAS
HIS AL MAMA TA WHERE HE HELP
ESTABLISH THE DEPARTMENT OF
MOLECULAR BIOLOGY.
NOW ONE OF GEORGE'S CLOSE
COLLEAGUES DURING HIS TIME AT
THE NIH WAS TSHANG WHO USED TO
ORGANIZE THIS LECTURE SERIES
EVERY YEAR AND UNFORTUNATELY
DIED A LITTLE LESS THAN A YEAR
AGO.
T HIMSELF WAS A VERY
ACCOMPLISHED SCIENTIST IN POLLY
MATH, WORKED ON HGLB 1 AND
UNRAVELLED THE MECHANISM BY
WHICH THIS VIRUS HIJACK CELLAR
MECHANISMS TO ENABLE
TRANSFORMATION.
HE ALSO EPITOMIZED MANY
CHARACTERISTIC AND QUALITIES
THAT GEORGE, HIMSELF, AT, BEING
N AN YOUTH STANDING SCIENTIST
AND WONDERFUL MENTOR.
HE WAS THE KHOURY LECTURER LAST
YEAR JUST A A FEW WEEKS BEFORE
HIS DEATH.
WE'RE DELIGHTED THIS YEAR TO
HAVE CARL WU AS THIS YEAR'S
KHOURY LECTURER.
KARLS HAS BEEN FOR THE LAST YEAR
OR SO A SENIOR FELLOW AND LAB
HEAD AT THE --
CARL WU, Ph.D. SENIOR FELLOW AND
LABORATORY HEAD AT JANELIA FARM
RESEARCH CENTER, HOWARD HUGHES
MEDICAL INSTITUTE, NIH SCIENTIST
AND RECEIVED HIS P H HD FROM
HARVARD.
H HE CAME TO THE NIH AND
EVENTUALLY BECAME CHIEF OF THE
LABORATORY OF MOLECULAR CELL
BIOLOGY AND THEN CHIEF OF THE
LABORATORY OF BIOCHEMISTRY AND
MOLECULAR BIOLOGY AT THE NCI.
DR. WU IS KNOWN AS ONE OF OUR
FOREMOST CONTRIBUTORS TO
UNDERSTANDING HOW CHROMATIN
STRUCTURE EFFECTS POSITIONING OF
NUCLEOSOMES AND GENE
TRANSCRIPTION.
HE BEGAN HIS WORK BY DISCOVERING
THAT DNAs 1 HYPER SENSITIVITY
SITE -- 20 YEARS AGO, HE
DISCOVERED THAT
PROMOTER-SPECIFIC CHROMATIN
REMODELLING IS MEDIATED NOT ONLY
BY SKENS-SPECIFIC TRANSCRIPTION
FACTORS BUT ALSO SURPRISINGLY BY
AN ATP-DEPENDENT FACTOR KNOWN AS
NUCLEAR CELL E REMODELLING
FACTOR.
HE CHARACTERIZED YOOET
REMODELLING PROCESSES AN TEN
YEARS AGO DISCOVERED THAT THE
YEAST SWR1 COMPLEX UNIQUELY
CATALYZES THE EFFICIENT
REPLACEMENT OF CONVENTIONAL H
HISTONE H 2 A WITH HISTONE
H2A.Z, A VERY IMPORTANT HISTONE.
THIS WAS ANOTHER PARADIGM SHIFT
IN UNDERSTANDING TRANSCRIPTION
REGULATION BECAUSE HE WAS A
DOOEF YENT TYPE OF HIS WHICH
CHALLENGED THE IDEA THAT
NUCLEOSOMES WERE BASED ON
CERTAIN KINDS OF STRUCTURES.
FOR THIS WORK UNCOVERING THE
SCENTS OF HOW CHROMATIN
REGULATES GENE EXPRESSION, HE
WAS AELECTED TO NATIONAL -- FIVE
OF THE MAJOR HONORARY SCIENTIFIC
SOCIETIES IN THE WORLD-HIS TALK
TODAY IS ENTITLED CHROMATIN
STRUCTURE AND GENE EXPRESSION.
CARL.
[APPLAUSE]
THANK
>> THANK YOU, MICHAEL, THAT WAS
VERY KIND.
IT'S MY PREF H LEDGE TO FOLLOW
THE LIST OF SPEAKERS WHO HAVE
SPOKEN TO THE KHOURY LECTURE
INCLUDING CHANGE WHO I KNEW WHEN
I WAS HERE.
I'VE BEEN IN THE AUDIENCE MANY
TIMES LISTENING, SO IT'S A TREAT
FOR ME TO UP HERE TO PRESENT OUR
WORK IN JOURJ KHOURY'S HONOR.
I FIRST MET JOURJ MYSELF IN 1981
WHEN I WAS LOOKING IF FOR A JOB
AND I CAME DOWN TO BETHESDA TO
VISIT ANOTHER LAB AND I REMEMBER
I KNEW PEOPLE IN GEORGE'S LAB.
I REMEMBER WHACKING INTO THE OLD
BUILDING 41 AND SPEAKING TO
GEORGE AND PETER -- WHO WAS A
POST DOC AT THE TIME -- AND
LEARNING ABOUT WHAT THEY WERE
DOING AND WHAT WAS GOING ON.
IT WAS VERY EXCITING AND WAS
PART OF THE [INDISCERNIBLE]
ABOUT THE PROGRAM THAT I TRY
COME HERE IN THE FIRST PLACE.
SO GEORGE KHOURY WAS ON ONE OF
THE DISCOVERIES OF ENHANCER
RELEVANT AND THIS WAS A REVIEW
THAT GEORGE AND PETER -- THE
DIRECTOR OF [INDISCERNIBLE] IN
GERMANY -- THEY WROTE A REVIEW
ABOUT ENHANCER ELEMENTS.
THE IMPORTANCE OF THAT WORK WAS
TO TELL US THAT THERE WAS JUST
OSHL ONLY A SHORT PIECE OF A
SEQUENCE -- WHICH COULD ENHANCE
TRANSCRIPTION WHEN IT WAS PUT IN
FRONT OF ANOTHER SEQUENCE.
THEY ALSO SHOWED SUBSEQUENTLY
THAT YOU COULD TAKE
[INDISCERNIBLE] MOVE IT AROUND
IN INDEPENDENT MANNER AS WELL AS
PUTTING IT DISTANCE AWAY FROM
THE TRANSCRIPTION START AND IT
WOULD STILL WORK IN A VERY
IMPLIED LEVEL.
OF COURSE WE NOW KNOW THAT
GENOMES ARE FULL OF ENHANCER
ELEMENTS, EVEN UP TO A MILLION
IN CASES OF THE HUMAN GENOME.
UH BUT IT ALL STARTED, REALLY,
HERE N BUILDING 41.
NOW THAT WORK NOT ONLY
[INDISCERNIBLE] GREAT ADVANCES
THAT HAVE HAPPENED IN STUDY OF
ADVANCERS, BUT ALSO THE
PROTEINS, PROTEIN FACTORS THAT
RECOGNIZE THE SEQUENCES OF
[INDISCERNIBLE] ENHANCE THEIR
ELEMENTS.
ABOUT TWO YEARS LATER A NUMBER
OF PEOPLE AND MOST IMPORTANTLY
[INDISCERNIBLE] LAB AT BERKELEY
IDENTIFIED THE FIRST EUKARYOTIC
TRANSCRIPTION FACTORS.
THIS WAS GOING ON CONCURRENTLY
WITH THE WORK GOING ON IN DON
BROWN'S LAB WHO IDENTIFY RAEG
LATORY ELEMENTS [INDISCERNIBLE]
EUKARYOTIC PROMOTERS AND
[INDISCERNIBLE] BY BOB RAIDER
THAT RECOGNIZE NOSE REGULATORY
ELEMENTS.
TX 3 A AND P 1S HERE WERE REALLY
THE FIRST TRANSCRIPTION FACTORS.
THE GC-ARE RICH ELEMENTS -- AP
FACTORS INVOLVED IN RECOGNIZING
[INDISCERNIBLE] -- THAT IN TURN,
OF COURSE, RESTAGED THE
EXPRESSION OF STUDY ON
TRANSCRIPTION FACTORS,
SEQUENCE-SPECIFIC FACTORS AND AT
THE LAST COUNT I THINK WE NOW
HAVE ROUGHLY ABOUT 6,000 HUMAN
TRANSCRIPTION FACTORS -- --
INCLUDING MASS REGULATORS.
THE TRANSCRIPTION FACTOR THAT
CAN SWITCH FIBERGLASS INTO THE
MUSCLE LINEAGE AND THE FAB FOUR
HERE ARE THE FOUR
[INDISCERNIBLE] FACTORS THAT CAN
REPROGRAM A FIBERGLASS INTO A
PLURIPOTENT STEM CELL.
OF COURSE ALL THESE OTHER ONES
THAT ARE COMING OUT OF THE WOOD
WORK NOW.
THIS IS ALL VERY WELL AND GOOD,
WE HAVE ENHANCERS, TRANSCRIPTION
FACTORS THAT RECOGNIZE
[INDISCERNIBLE] ENHANCERS, BUT
THE ONE MORE PROBLEMS WHICH IS
THAT THE DNA IS NOT ACCEPTABLE
AND MANY OF YOU KNOW THIS.
BUT WHEN DNA WRAPS IN THESE FUND
MEN PARTICLES CONTAIN EIGHT
HISTONES [INDISCERNIBLE] --
GIVING YOU A HIS-SELF TONE
[INDISCERNIBLE] WRAPPING AROUND
THE [INDISCERNIBLE] AND WHEN SIT
SO WRAPPED THE INFORMATION IS
NOT ACCEPTABLE.
TOOK A COUPLE [INDISCERNIBLE]
AND -- YOU CAN SEE THE MAJOR
GROOVES AND YOU CAN PUT YOUR
FINGER IN THESE GROOVES AND READ
OFF ABOUT FOUR OR FIVE SPACE
PAIRS BUT THEN YOU RUN INTO THE
HISTONES USE READ INTO THE PART
THAT'S FACING THE HISTONES.
A LOT OF THAT INFORMATION IS
LOST AND MANY FACTORS REALLY
HAVE A POOR RECOGNITION OF NEW
OWE SOEM MALL DNA WITH A FEW
EXCEPTIONS.
BY IN LARGE IN GENERAL MANY OF
THEM WOULD MUCH RATHER HAVE THE
DNA STRING.
AND SO WHAT MANY YEARS AGO WE
FOUND HERE IS WAS REALLY THAT
THERE WAS A -- EXCUSE ME
[INDISCERNIBLE] -- PROMOTERS AN
ENHANCERS THAT CREATES THE
STRETCH OF DNA THAT SHOWS UP DNA
HYPER SENSITIVE SITE -- SHOWN BY
THE OLD FASHIONED SOUTHERN BLOT
FOLLOWED BY PRESCRIPTION ENZYME
CLEAVAGE.
IF YOU ACCEPTED THE WIDTH,
BREADTH OF THE TARGET THAT'S
ACCESSIBLE BY DNASE 1.
AND HERE WE'VE TALK LICENSE AND
PUT TOGETHER THIS LIGHT STRUR
CHUR, BUT THIS DNA REAL PROMOTER
[INDISCERNIBLE] KIND OF WIGGLED
LITTLE LIKE THIS IS PART OF THE
[INDISCERNIBLE] ITSELF.
THE QUESTION THEN BECOMES WELL
WHAT CREATES THIS CHROMATIN.
THIS IS REALLY A SORT OF A
UNIVERSAL SITE CHROMATIN
ARCHITECTURE.
WE NOW KNOW THAT IT'S THE
CULMINATION OF TRANSCRIPTION
FACTORS THAT RECOGNIZE DNA, THAT
RECRUIT A WHOLE FAMILY OF COAL
REGULATORS, REMODELLING ENZYMES,
TWO CLASSES THOSE THAT COVALTLY
MODIFY THE [INDISCERNIBLE]
TAILS, AND THE SECOND CLASS ARE
THE ONES THAT USE THE NGPH
HYDROLYSIS TO MOBILIZE H
HISTONES AND CREATE THESE NEW
OWE SOEM FREE REGION.
NUCLEOSOME-FREE REGION.
IF YOU ADD UP THESE CLASSES
WE'RE COMING UP TO AGAIN ROUGHLY
A THOUSAND OR SO PROTEINS IN THE
HUMAN GENOME SO WE'RE TALKING
ROUGHLY 10-15% OF GENETIC
INFORMATION THAT'S DEVOTED TO
REGULATING GENE EXPRESSION ON
THE GENOME-WIDE BASIS.
SO NOWADAYS THIS IS SORT OF MORE
CONTEMPORARY VIEW MAPPING NEW
TROE SOEMS ON DNA.
[INDISCERNIBLE] WHICH ONE USED
AN ENZYME [INDISCERNIBLE] CHOKED
UP DNA BETWEEN NEW
NUCLEOSOMES -- MAP US BACK TO
THE GENOME, ITSELF.
THEN BECAUSE THEY'RE ALWAYS
WORKING WITH POPULATIONS ON
CELLS, THIS SHOWS YOU WHAT THE
ARRANGEMENT WOULD LOOK LIKE FOR
POPULATION ONE GENE.
SO IF THE NUCLEOSOME WAS
EXTREMELY POSITIONS -- THEN
EVERY CELL WOULD GENERATE A DNA
FRAGMENT OFF TO THIS SIDE
LOCATED LIKE SO AT THIS POSITION
HERE.
SOME OF THE CELLS HAVE
NUCLEOSOMES RIGHT NEXT TO IT BUT
OTHER CELLS HAVE [INDISCERNIBLE]
WELL THEN DAY BUN DANCE OF
RESISTING FRAGMENTS WOULD DROP
AND [INDISCERNIBLE] SEE THAT IT
DIDN'T SCRAMBLED, EACH CELL
WOULD GIVE YOU A DIFFERENT
PATTERN AND IF YOU THEN ADD THEM
ALL UP YOU GET SOMETHING LOOKING
VERY SCRAMBLED.
YOU CAN PICK DATA AND IN THIS
CASE YOU'VE TAKEN DATA IN THAT
CAME FROM DAVID CLARK LAB,
PUBLISHED IN 2011 FOR THE YEAST
GENOME.
YOU'RE LOOKING AT ABOUT FOUR
THOUSAND FIVE HUNDRED GENE HERE,
EACH MINE REPRESENTS JOOIN AND
WE'VE ALIGNED ALL GENES
ACCORDING TO TRANSCRIPTION
START.
WHAT YOU'LL SEE AS YOU GO DOWN
IS THESE ARE A ARRANGED BY PETER
LABORATORY IN A WAY SUCH THAT
SOME GENES HAVE ZERO
NUCLEOSOME-FREE REGION [LOW
AUDIO] AND OTHERS HAVE VERY LONG
ONES [INDISCERNIBLE].
AS YOU LOOK THROUGH THESE SET OF
GENES YOU CAN ARRANGE THEM IN
ORDER OF THE INCREASING LENGTH
OF NUCLEOSOME FREE REGION AND
THIS IS WHAT [INDISCERNIBLE]
WHAT'S STRIKING IS INDICATES
BUDDING YEAST THERE'S THIS BIG
GAP THAT JUST BASICALLY SEEN
THROUGHOUT THE GENOME WITH
EXCEPTION OF A SMALL GROUP OF
GENES UP HERE WHICH HAVE NO GAP,
THESE ARE THE ONES THAT ACTUALLY
INDUCIBLE GENES AND IN FACT
REQUIRES SIGNALING TO TURN THE
GENES ON AND CREATE [LOW AUDIO]
AND YOU CAN SEE VERY NICE
[INDISCERNIBLE] ONE, TWO, THREE,
THE IF YOU KEEP GOING DOWN TO
THE END OF THE GENE THINGS STOP
BEING SCRAMBLED.
THE KIND OF ARCHITECTURER IS A
UNIVERSAL ONE.
HERE'S A SUMMARY OF ALL THE DATA
SETS THAT CAME FROM TWO LABS AND
WE'RE LOOKING AT NOW THIS THIS
IS THE [INDISCERNIBLE] SHOWING
AGAIN THE ALIGNMENT IS TO THE
TRANSCRIPTION START SITE AND
GOING RIGHT UP STREAM OF THAT IS
[INDISCERNIBLE].
THIS ONE LOOKS VERY NICE, BUT
HERE'S THE WORM AND AGAIN IT
DROPS HERE.
HERE'S [INDISCERNIBLE] HUMAN
BLINDFOLD SYNOPSIS
[INDISCERNIBLE] EVEN AR KAY
YEAH, AN ORGANISM THAT LIVES IN
HOT SPRINGS AND THEY HAVE AR KAY
YEAH HISTONES.
EVEN THEIR START SIGHT RIGHT
UPSTREAM HAVE A REGION FREE OF
[INDISCERNIBLE].
THIS SEEMS TO BE A UNIVERSAL
ARCHITECTURE OF CHROMATIN.
ONE UNTRADITIONAL ASPECT OF THIS
ARCHITECTURE WHICH IS THE
SUBJECT OF MY TALK TODAY IS THAT
IS HISTONE AIR JEBT CALLED H 2
A.D AND THAT'S ALSO FOUND AT IS
SO-CALLED PLUS ONE NUCLEOSOME A
ALONG WITH MODIFICATIONS.
WHAT IS HISTONE H2A.Z?
IS A VARIANT HISTONE, IT IS
LOCATED HERE AT THE PLUS ONE, A
LITTLE BIT AT THE MINUS ONE.
THE STRUCTURE [INDISCERNIBLE]
WHICH YOU CAN BARELY SEE, IS
VERY MUCH LIKE THE
[INDISCERNIBLE] ONE.
THEY'RE VERY SUBTLE DIFFERENCES
IN THE OVERALL SHAPE HOWEVER
YOU'LL SEE THERE'S INTERACTIVE
MATTER A A LOT AND THOSE ARE THE
ONES WHICH MAKES A FUNCTIONAL
DIFFERENCE IN CONFERRING ALL THE
PROPERTIES [INDISCERNIBLE].
IF YOU WERE TO LOOK AT THE
DISTRIBUTION OF H2A.Z
[INDISCERNIBLE] -- THIS IS WHAT
YOU SEE THAT THERE IS ENRICHMENT
OF THE H 2 A REGION AT THE PLUS
ONE THUK OWE SOEM DOWN STREAM TO
THE NUKE OWE-FREE REGION.
H2A.Z WAS DISCOVERED AT NCI BY
MICHAEL AND BILL.
BILL IS STILL HERE IN BUILDING
37.
WHAT BILL KIS COVERED WAS REALLY
A WHOLE FAMILY OF HISTONE H 2 A
LIFE PROTEINS AND HE DISCOVERED
THIS BY A SERIES OF
MANIPULATIONS OF GEL CONDITIONS
WHICH WAS THE ONLY WAY ONE COULD
USE ONE COULD USE TO DISTINGUISH
PROTEINS THAT WERE CLOSELY
RELATED BACK THEN.
WHAT HE DID WAS PURIFY WHAT WE
ALL THOUGHT WAS A HOMOGENEOUS
POPULATION OF HISTONES AND RAN
THEM UNDER THESE TURN GELS AND
HE DISCOVERS THERE WERE WHOLE
NUMBER OF VARIATIONS ON THESE
PROTEINS THAT SHOWED UP AS
DISCREET VARIANT AND THE SECOND
VARIANT HE CALLED H2A.Z, H 2 AX
THE OTHER ONE HE DISCOVERED, H 2
A-Z COMPRISED ABOUT 4% OF 4SHGS
2 A IN MOUSE CELLS AND FRACTION
OF EACH OF THOSE HISTONES IS
COMBINED AND THE LOWEST SUM
PROXIMATE MOLD AT H 4 AND OTHER
HISTONES.
THERE'S ANOTHER VARIANT THAT
BILL DID NOT DISCOVER CALLED
SCENT A, BUT THAT'S WHAT HISTONE
34SHGS VARIANT.
THE MAIN HISTONE VARIANTS ARE
REALLY H 2 .Z
[INDISCERNIBLE] -- ANYWAY S THIS
WAS PUBLISHED IN 1980 AND I
CONFESS I WANT TO SAY IT'S
SOMETHING WE ALL PAID ATTENTION
TO UH BUT THAT'S CHANGED BUT
CHANGED BECAUSE IN THE 90s AND
EARLY TO LATE 90s KNOCKOUT
TECHNOLOGIES WERE APPLIED TO THE
HISTONES AND BECAUSE H2A.Z WAS A
[INDISCERNIBLE] WANTS TO KNOCK
IT OUT AND THAT'S WHEN THINGS
COMPLETELY CHANGED.
IT TURNS OUT THAT THIS WAS A
VERY IMPORTANT IS TONE,
FUNCTIONALLY REQUIRED, IT IS
CONSERVED AND IF WE KNOCK IT OUT
IN A FLY, WORM OR MAMMALIAN
ORGANISM LIKE MOUSE, THEY
BASICALLY DON'T SURPRISE AND THE
TIME OF DEATH IN EARLY
DEVELOPMENT.
MOLECULAR STUDIES MIXED WITH LAB
ESPECIALLY DISCOVERED THAT THIS
WAS IN FACT THIS WAS FOUND AT
THE PROMOTERS.
IT'S VERY MINOR ACTION 4% ON
ITSELF TURNS OUT TO BE THE MOST
4% BECAUSE THAT'S WHERE THE SAKS
TOUCHING PROMOTERS.
THE SINCELATORS IN THE FACT THAT
THAT -- INSULATORS IN EVERY
ORGANISM [LOW AUDIO].
THIS IS QUITE A SIGNATURE.
QUITE A BIT OF WORK DONE
[INDISCERNIBLE] IT AFFECTS THE
ACCESSIBILITY OF THE FACTORS AND
ACID DI OF H 2 A.Z, AFFECTS
DYNAMICS.
IT'S EXCHANGED A LOT MUCH H MORE
THAN OTHER NEW TROE SOEM ALONG
THE GENE.
THERE'S OTHER BIOCHEMISTRY THAT
SHOWS IT AFFECTS GENETIC
TRANSCRIPTION AND OTHER
TRANSCRIPTION.
THIS TURNED OUT TO BE A VERY
IMPORTANT HISTONE.
HOW DID WE GET INTO THIS?
BY ACCIDENT.
SO THE QUESTION BECOMES, HOW IS
THIS HISTONE PUT INTO THE PLUS
ONE NUCLEOSOME.
WE'VE BEEN STUDYING THIS WHOLE
FAMILY AND SYSTEMATICALLY
PURIFYING AND CHARACTERIZING
COMPONENTS AND WE SWITCHED FROM
[INDISCERNIBLE] TO BEING TO WORK
WITH [INDISCERNIBLE] WHICH WAS
GENETICALLY AND BIOCHEMICALLY
ACCESSIBLE.
IN PURIFYING ONE OF THESE
ENZYMES CALLED THE SWR1
ENZYME -- I ARE REFER TO THIS AS
SWR1, YOU GET A WHOLE BUNCH OF
FANS IN A GEL WHICH WAS FINE AND
IN THOSE DAYS, WHICH WAS BEFORE
MOLECULAR PROTEOMICS REALLY TOOK
OFF ONE HAD TO CUT OFF THE GEL
AND SEND THEM TO MAS SEC WHICH
WAS WHAT WE DID.
ONE OF THE POST DOCS WORKING ON
THE PROJECTS DID A COMPLETE --
HE CUT OFF THE BOTTOM OF THE GEL
BECAUSE USUALLY THAT'S STUFF YOU
THROW AWAY, UNFORTUNATELY, HE
DIDN'T DO THIS.
WHEN WE SEQUENCED THE BOTTOM OF
THE GEL NOT ONLY DID YOU FIND
HISTONES BUT HE FOUND THERE WAS
A H2A.Z THAT WAS DISCOVERED LONG
AGO WHICH WAS HIGHLY REPRESENTED
IN THAT POPULATION AND IT WAS
THE SLOW ONE PURIFICATION
AMONGST THE EIGHT OR NINE
ENZYMES PURR FIED THAT HAS THE
ENRICH AREMENT OF THE H2A.Z SO
IT SUGGESTED TO US THAT SWR1 HAD
TO BE CONNECTED TO THE
BIOCHEMISTRY OF THE H2A.Z AND
THE SIMPLEST INFORMATION WOULD
BE IS THAT IT COULD HAVE BEEN
IMPORTANT FOR DEPOSITING THAT 42
A
A.Z.
THIS IS TO SHOW AMONG ALL THE
MEMORIES OF THE MODELLING ENZYME
FAMILY ALL OF WHICH CONTAIN THIS
IS SO-CALLED SS 2 FAMILY OF DNA
TRANSLOCATIONS.
THIS IS THE ONE THAT IS INVOLVED
IN ALG RITMENTES AND FOUND
UNIQUE TO HUMAN SLIDES.
-- ALGORITHMS.
THIS IS HOW WE SAW IT.
THEN A POST DOC IN THE LAB SPENT
A COUPLE OF YEARS WORKING ON HOW
THIS ENZYME IS ASSEMBLED BY A
COMBINATION OF GENETICS AN
CHEMISTRY PAY BASICALLY FIGURING
OUT WHAT WERE UH TRUE SUB UNITS
AND HOW DID THEY ASSEMBLE WHICH
WERE THE ONES WERE MOST
IMPORTANT, WHICH WERES LESS
IMPORTANT.
DON'T WERE -- WORRY ABOUT ANY
OF THESE NAMES.
IT APPEARS NOT ONLY AT THE
CATALYTIC [INDISCERNIBLE].
FROM 2009 -- EVEN BEFORE THAT --
WE WERE IN COLLABORATION TO
SOLVE THIS STRUCTURE.
THIS ENZYME HAS 14 SUB UNITS AND
FINALLY IN 2013, THIS YEAR, OUR
COLLABORATORS AT HARVARD
PRESENTED THIS SUBJECT TO US
WHICH IS ANOTHER VIEW OF THE
ENZYME AND I'LL COME BACK TO
THIS MORE, BUT BASICALLY IT GAVE
US AT LEAST SOME IDEA OF WHAT IT
LOOKS LIKE PHYSICALLY AND
ALLOWED US TO NOW BEGIN TO HANG
SOME OF THESE MODULES ON TO THE
STRUCTURE ITSELF.
I'D LIKE TO POINT OUT THIS IS
THE FIRST VIEW I CAN'T EVEN TELL
YOU WHAT THE RESOLUTION IS
BECAUSE IT DOESN'T MAKE IT THAT
MEANINGFUL JUST SAFES YOU THE
ANALYSIS BUT AT LEAST IT'S
SOMETHING FOR US TO LOOK AT.
AND THE STRUCTURE TELLS US THAT
THERE ARE IN FACT CERTAIN
MODULES THAT MIRRORER THE SDWRE
NE TICK MODULES WE SAW BEFORE.
THIS REGION HERE CONTAINS
[INDISCERNIBLE] PROTEINS AND
THERE ARE SIX OF THEM FORMING
STRUCTURES AND THEN THIS REGION
HERE THE BLUE ONE IS CATALYTIC
AGENT.
THIS MODULE HERE CONTAINS SIX
PROTEINS SO CAN'T EVEN
DISTINGUISH THE SUB UNITS YET,
AND THIS MODULE CONTAINS THESE
FROUR PROTEINS.
WHAT IS INTERESTING -- AND I
HAVE NO EXPLANATION FOR THIS NOW
IS THAT THERE ARE SIX SUB UNITS
THAT BIND TO REGION D.
THIS IS AN AT PSHGSZ HIGH
PROIZING ENZYME BUT WHY DO WE
NEED SIX SUB UNITS, IS UNCLEAR
AT THE MOMENT.
THE BASIC REACTION THAT THE WORK
IS SHOWN HERE, YOU TAKE THE
ENZYMES, TAKE THE H2A.Z, COMES
ALONG WITH ITS OWN CHAPERON
[INDISCERNIBLE] AND WE HOPE TO
GIVE IT A NEW TROE SOEM
CONTAINED.
YOU ADD THESE TOGETHER AND WHAT
HAPPENS IS THE [INDISCERNIBLE]
AND THE Z GETS PUT INTO THE
[INDISCERNIBLE].
THIS IS SHOWN WHERE Z HAS A FLAG
TAG ON IT SO WE CAN FOLLOW THE
H2A.Z.
WE CAN DO THIS AFTER WE'VE DONE
THE REACTION SOME RAYS CAN BE
COOLED DOWN ON THE DINER AND SEE
A ACCUMULATION OF THIS FLAG ON
THE D OR WHEN YOU HAVE IF IF
ENZYMES AND ATP PRESENT AND
[INDISCERNIBLE] PRIME.
ALL OF THE ABOVE GIVES YOU
REPLACEMENT SIGNAL.
THIS IS DEPLETION FROM THE
[INDISCERNIBLE] HERE.
IF YOU ADD ANY OF THESE
INHIBITORS THE REACTION IS STILL
[INDISCERNIBLE] AND MOST
IMPORTANTLY, IF YOU LIVE
MUTATION IS THE UNIQUE BINDING
POCKET OF THE # 1 CATALYTIC PUB
UNIT.
[INDISCERNIBLE].
THE ENZYME IS THERE.
THIS ESTABLISHED BASIC REACTION
YOU COULD RECAPITULATE IN VITRO
UNDER REL TA LIVE PHYSIOLOGICAL
CONDITIONS AND OF COURSE IN VIVO
WE AND OTHERS SHOW THAT IF YOU
KNOCK OUT ANY ONE OF THESE
COMPONENTS THE ENZYME DOES NOT
WORK IN CELLS AND IS NOT
DEPOSITED AT THE PLUS ONE
MUTATION.
SO OVER THE LAST COUPLE OF
YEARS, WE'VE NOW BEEN GOING
THROUGH THE SECOND ROUND OF
ANALYSIS TO THIS REACTION, AND
WHAT WE SEE IS IF YOU THE CYCLE
WHERE YOU HAVE THE ENZYMES, IT
HAS NUCLEUS SUB STRAIGHT AND HAS
TO BIND TO IT, IT HAS H 2 A
POINT D YOU BE STRAIGHT -- THERE
ARE TWO SUB UNITS THAT BIND IN
TWO LOCATIONS.
YOU HAVE ALL THREE SUB STRAIGHTS
YOU HAVE ACTIVATION OF THE ATP P
A WHICH IS HERE AND THIS IS
ACTIVATION, WHAT ABOUTS IS THE
LOSS OF 1 H 2 A AND THE
INSERTION OF 1 H 2 AB.
WE'RE LOOKING AT MODERN
EUKARYOTIC -- NUCLEOSOME NOW.
YOU GET ONE H # A AND REALLY SEE
AN UP SHIFT IN THIS NATIVE GEL
IS THAT THE D FLAG CONFERS [LOW
AUDIO] WHEN YOU HAVE TWO FLAGS
YOU SHIFT AGAIN.
YOU CAN SEE GOING FIRST TO THE
ONE AND THEN TO THE DOUBLE
SUBSTITUTION THAT'S NEEDED IS
THE FINAL PRODUCT.
SO THE WAY WE THINK ABOUT THIS
IS THAT IF YOU LOOK AT ONE STEP,
THIS INTERMEDIATE THIS
ASSOCIATED FANTASTICALLY IS
REBINDED BUT NOT TO THE A FRAME.
THIS MECHANISM TERMINATING THE
REA WHEN YOU END UP IN A DOUBLE
C.
WHAT DO E WE NEED TO KNOW ABOUT
THIS REACTION?
THIS IS HOW WE KNOW THE SYSTEM
IS WORKING.
NEXT QUESTION IS, SO NOW WE HAVE
THE ENZYME, HAVE THE SUB
STRAIGHTS, HAVE WORK, IN VIVO,
HOW DO THE ENZYMES KNOW HOW TO
FIND IT'S TARGET.
THE TARGET IS ERE PROMOTER.
ALL THE NEW OWE SOEM FREE
REGIONS ARE EITHER PROMOTERS OR
ORIGINS OF APPLICATION.
FIRST IT COULD BE SEQUENCE OF
FACTORS.
THERE'S AN [INDISCERNIBLE] -- WE
FELT THIS WAS NOT A GOOD WAY TO
DESIGN THE SYSTEM BECAUSE THEY
WOULD HAVE TO HAVE LOTS OF
TRANSCRIPTION FACTORS ALL
CARRYING SOME DOMAIN THAT
ATTRACTS THE SAME ENZYME.
ANOTHER POSSIBILITY IS THAT THE
ENZYME IS TRACKING THE UNIVERSAL
STATE OF THE PROMOTER
ARCHITECTURE WHICH IS THEY'RE
SIMPLE MODIFICATIONS OCCURRING
AT PROMOTERS IN GENERAL AND IN
THE FACT OF GENETIC EVIDENCE
THAT THERE WAS A SIDE THROUGH
MATTER TO SOME EXTENT FOR THE
RECRUITMENT OF [INDISCERNIBLE]
BUT THE POSSIBILITY IS THAT THE
NUCLEAR FREE REGION ITSELF WHICH
IS THE ARCHITECTURE THAT HAS
BEEN RECOGNIZE BID THE STOLEN
ENZYME TURNS OUT THAT THIS TURNS
OUT TO BE THE MOST DOMINANT
MECHANISM AND THERE'S NO
EVIDENCE IN AN UNBIASSED SCREEN
BY OTHER LABS WHICH HAVE LOOKED
FOR ENZYME ASSOCIATING WITH ANY
TRANSCRIPTION FACTORS, NOTHING
TO SHOW THEM.
LET ME TAKE YOU TO EXPERIMENTS
WHICH WERE CONDUCTED AND
PUBLISHED LAST MONTH.
I WANTED TO TEST THIS DIRECTLY
BY CONSTITUTING -- AS A SNAPSHOT
THAT IS TO SAY -- FROM A
TRANSIENT POINT OF VIEW OF A
SNAPSHOT THIS IS LIKELY TO EXIST
AND WE CAN THEN FREEZE THIS
SNAPSHOT BY MAKING OUR
ARTIFICIAL CONSTANT.
YOU TAKE THE NATURAL GENE
PROMOTER WHICH IS HAS
[INDISCERNIBLE] CENTER AND THEN
TWO CONVINCES WHICH ARE
EXTREMELY HIGH POSSESSIONING
NUCLEOSOMES.
WITH THESE FLANKING THE
PROMOTERS, ONE CAN GO THROUGH
THE STANDARD DIALYSIS PROCEDURE
AND GENERATE A SERIES OF
RECONSTITUTES.
WHAT YOU'VE SEEN IS THREE DNA,
DNA WITH ONE, TWO NUCLEOSOME AND
THREE NUCLEOSOMES.
THE TRICK IS TO PUT
[INDISCERNIBLE] DNA TOGETHER
WITH [INDISCERNIBLE] SO THAT
COMPETES AWAY FOR ALL THE STUFF
YOU REALLY DON'T WANT.
UNDER THESE CONDITIONS, ONLY THE
[INDISCERNIBLE] THAT CONTAIN THE
DINING CONCERN YOU CAN PURIFY
THIS ON A SUPER GRADIENT AND
IMAGE THIS AND SEE THESE
[INDISCERNIBLE] CONSISTING OF
VALUE SYSTEMS.
YOU CAN NOW TAKE THIS
PREPARATION, ENABLE THAT WITH
TWO DIFFERENT COLORS.
WE CALL THIS A LONG LINKER AND
THEN THE CONTROL IS THE
NUCLEOSOME THAT [INDISCERNIBLE]
IN GREEN.
IN THE SAME MIXTURE YOU CAN HAVE
OUT OF THE ENZYME
[INDISCERNIBLE] DOES IT PREFER
ONE OTHER THE OTHER.
IT'S QUITE CLEAR THAT THE ENZYME
MUCH PREFERS THE RED LONG
[INDISCERNIBLE] OVER THE GREEN
SHORT LENGTH AND EVEN THE
BINDING.
THIS BINDING THEN HAS A
FUNCTIONAL CONSEQUENCE BECAUSE
YOU CAN SEE THAT THE LONG LENGTH
OF NEW TRI ARE CELLS IN THIS NOW
THIS IS A FUNCTIONAL ASPECT
LOOKING AT THE TWO S PRODUCTS
SHOWING THAT WHEN YOU HAVE LONG
LENGTH, THE REACTION GOES MUCH
MORE EFFICIENTLY THAN THE SHORT
LINGER NUCLEOSOME.
THERE'S THE BINDING AGAIN.
SO UH NOW YOU CAN TAKE A LOOK AT
THE IMAGE THE ENZYME ALONG WITH
THE PROMOTERS.
[INDISCERNIBLE] THEY HAVE A LOT
OF FREE UNBOUND NUCLEOSOMES AND
ONLY A LITTLE OF THE UNBOUND.
YOU CAN SEE IN THE FIELD IS
YOU'VE GOT SOME OF THESE
DUMBBELLS YOU SAW BEFORE UNBOUND
AND NOW HERE YOU SEE A BIG BLOB
AND TURNS OUT TO BE ENZYME
[INDISCERNIBLE] AND WE CAN
MEASURE THEM AND TURNS OUT THAT
IN FACT IF TO YOU THIS FOR THE
WHOLE POPULATION THERE'S A VERY
CLEAR ENGAGEMENT OF THE
[INDISCERNIBLE] THAT GIVES YOU A
MUCH SHORTER PREDNA TIER.
THEN IF YOU SEND THIS UP TO THE
MICROSCOPE IF WE CAN MEASURE
THESE PARTICLES AND THAT
MEASUREMENT COMES TO 1.2
MILLIGRAMS, 1.0 IS 2 CALCULATED
MASS OF THE ENZYME ITSELF AND.2
COMES P FROM THE NEW TRA CELL IS
SO IT'S VERY CONSISTENT THAT THE
RATIO OF ONE ENZYME TO ONE NEW
TROE SOEM IN THE COMPLEX OVER
THERE.
SO NOW BIOCHEMICALLY YOU CAN SEE
THIS IS GETTING BETTER AND YOU
CAN GIVE THIS ENZYME THREE
DIFFERENT SUB STRAIGHT, THREE
DNA A PARTICLE THAT HAS NO LINK
TO DNA AT ALL AND NOW
[INDISCERNIBLE] AND THE ENZYME
PREFERRED FOR THE FACT THIS SUB
STRAIGHT MUCH BETTER THAN
PARTICLE OR THE DNA ALONG SO YOU
NEED ONE NEW TRA CELL WITH A
LONG LENGTH AND THEN YOU CAN GO
THROUGH THE WHOLE SERIES OF
LINKAGE AND BEGIN ON WORK AND IT
TEPIDS TO BE A A BIG JOB ONCE
YOU BUILD UP [INDISCERNIBLE].
52 SHOULD BE A MAGIC NUMBER THAT
THE ENZYME PREFER AND FORGET
ABOUT H HISTONES, JUST GIVE IT
THE DNA 'CUZ YOU HAVE A LADDER,
YOU KNOW COMMERCIAL DNA LADDER.
THIS IS A DNA BINDING PROTEIN
[INDISCERNIBLE].
THEN WE MEASURE THIS AND SEE
WHAT THE PREFERENCES ARE.
IT MUCH REFER THINGS THAT ARE AT
LEAST 50 AND 60 COMPARED TO 40,
30, AND 20.
ENZYMES CAN READ THE LENS OF THE
NUCLEOSOME-FREE REGION AND WILL
INTERACT WITH THAT STRUCTURE.
THAT'S JUST FOR RECOGNITION, FOR
RECRUITMENT.
ONCE YOU'VE RECRUITED THE ENZYME
DO YOU REALLY NEED THE LONG
LENGTH?
WE GAVE THE ENZYME ARTICLE BY
ITSELF WITH NO DNA AND IN THIS
SECOND GENERATION
[INDISCERNIBLE] YOU CAN SEE
THESE INCORPORATING ON TO THAT
NUCLEOSOME QUITE WELL WITHOUT
ANY LINKING TO DNA AT ALL.
THIS IS ONLY FOR RECRUITMENT NOT
FOR ACTIVATION.
COMING BACK TO ENZYME OR
RECRUITMENT IS LIKELY TO HAVE
STRETCH OF DNA FLAGGED BY UH NEW
TA CELLS.
WE WENT INTO THE LITERATURE AND
COMPARED THIS IS WHAT IT SHOWED
BEFORE THAT DATASETS SHOWING
THIS DISTRIBUTION OF YEAST
[INDISCERNIBLE] NUCLEOSOME-FREE
REGION HERE AND NOW THE SAME
ORDER OF GENES ARE USED TO
ATRAINING DATA SETS FROM ONE LAB
AND WE'RE NOW LOOKING AT
CHROMATIN AND PRECIPITATION OF
THE SWOLLEN ENZYMES ON THESE
PROMOTERS IN ORDER TO
NUCLEOSOME-FREE REGION.
WHAT YOU CAN SEE WAS THAT IT
TAKES A GREAT CORRELATION AN
INCREASE THE OF NFR AND ENZYME
UP TO ABOUT 20 BASES THEN THE
STATUE DROPS.
IT TURNS OUT THAT THE WAY WE
NORMALLY SNAP NEW TRA CELLS ARE
JUST IT, OVERSTRINGENT.
NEWARK O--
IF YOU DON'T EXHAUST
[INDISCERNIBLE] YOU GET THE SAME
KIND OF ARCHITECTURE BUT WHERE
YOU NOW HAVE BIG GAPS HERE, IN
FACT IN MA JIEL NUCLEOSOMES, IT
WAS STARTING TO [INDISCERNIBLE]
THAT I CAN EXPLAIN WHY THERE'S A
DROP HERE.
NUCLEOSOME-CREATED REGION ARE NO
LONGER [INDISCERNIBLE] -- BUT
THEY'RE FRAGILE.
THIS IS INTERESTING, JUST ONE
ENZYME THAT TOOK IN A BUNCH OF
OTHER ENZYMES AND CORONATED
ANALYSIS AND FOR EXAMPLE ANOTHER
REVOLVING ENZYME CALLED RICK
HAVE NO -- IN TERMS OF
DISTRIBUTION IT'S RELATION IN
ABUNDANCE TO THE [INDISCERNIBLE]
BUT INTERESTINGLY COMPONENT OF
THE PRESTATION COMPLEX HAVE VERY
GOOD CORRELATION IN TB P P TO
CELL WHICH IS IMPLY AT LEAST THE
POSSIBILITY THAT IN FACT A LOT
OF OTHER ENZYMES OUT THERE USE
THE LENGTH OF THE NEW TRA
CELL-FREE REGION AS ONE NECK
NICHL OF RECOGNIZE WHERE IT
SHOULD BE RECRUITED.
WHO'S RESPONSIBLE?
14 SUB UNITS, WHICH OF THESE ARE
IMPART TO RECOGNIZE THAT LONG
[INDISCERNIBLE] NEXT TO THE NUKE
NUCLEUS.
ONE SUB UNIT IN PINK HERE ARE
MOST REWARDING ONE.
WE KNEW YOU COULD CUT THE ENZYME
IN HALF AND LIMITS THAT
STRUCTURE AND THE ENZYMES WOULD
NOT FALL APART.
THESE SUB UNITS WOULD GO ALONG
WITH THE N PART AND THESE SUB
UNITS WOULD COME TO THE C PART.
THERE WE CAN HAVE WHO'S
RESPONSIBLE FOR RECOGNIZING
NUCLEOSOME FREE REGION.
HERE THESE LONG LENGTH OF
NUCLEOSOMES.
HERE'S THE END PART P THAT GIVES
THE TRUTH DOES A GOOD JOB OF
BINDING.
OF COURSE THE STABILITY OF
CHANGE BECAUSE YOU'VE GOT HALF
THE ENSOEM.
IF YOU JUST KNOCKED OUT THE PINK
SUB UNIT [LOW AUDIO].
THIS TOLD US THIS WAS A VERY
IMPORTANT COMPONENT OF
RECOGNITION AND IF YOU MAKE US
AN OFFER, YOU ALSO CAN SHOW THAT
THE BINDING OF THE ENZYME EMSS
IS IN HERE [INDISCERNIBLE]
PRECIPITATION EXPERIMENT SHOWING
ENRICHMENT ALL FOR FOUR OR FIVE
GENES IN THE GENOME AND WHICH IS
WILD TYPE SIGNAL AND THESE THEN
THESE ARE THE [INDISCERNIBLE] SO
IN VIVO IS FOR RECOGNITION.
WE LOOK OVER THREE PRO TOE CELLS
WHICH WAS A WIDE THING.
[INDISCERNIBLE].
NOW WER COMING TO THE NEXT
MECHANISM -- UP 'TIL NOW IT
SEEMS LIKE DNA BINDING IS THE
MOST IMPORTANT IS IMPORTANT BUT
IS IT MORE IMPORTANT OR LESS
IMPORTANT THAN [INDISCERNIBLE]
ACETYLATION.
IT TURNS UH OUT ACETYLATION IS
LESS IMPORTANT COMPARED TO DNA
BINDING.
NOW WE GO BACK TO THE BINDING.
ACETYLATION BINDING NEW TROE
SOEM SHOWING THAT THE ENZYME IS
IMPORTANT FOR THIS REACTION AND
PURIFIED ENZYME TO STUDY AND
ACETYLATED TO BE NEW TROE SOEMS
HERE AND THEN IN RED THE CONTROL
WHICH HAS NOT BEEN ACETYLATED
AND NOW THE SAME REACTION WE
HAVE ONE ENZYME PREFER OVER THE
OTHER AND YOU CAN SEE NOTHING
HAS BEEN ACETYLATED BUT NOW IF
YOU ADD ON ACETYLATION, YOU CAN
IMPROVE THE BINDING.
SO ASSCETYLATION IS IMPORTANT BUT
NOW YOU ADD WHO'S MORER
IMPORTANT AND TAKE THAT ENZYME
AND CUT IN HALF.
THIS HALF CONTAINS A DNA BINDING
COMPONENT AND THIS HALF CONTAINS
PROTEIN CALLED BBF 1 WHICH HAS
DOMAINS THAT RECOGNIZE
ACETYLATED HISTONES.
THIS PIECE AND THIS PIECE BOTH
HAVE 15 [INDISCERNIBLE] OF
CONCENTRATION.
THEN CAN SHOW THE WEAK BINDING
GIVING HERE NOTHING QUITE LIKE
THE COMPLEXES.
SO BOTTOM LINE IS THAT THE DNA
BINDING NOMINATES OVER THE
HISTONE BINDING AND WE THINK
THIS IS RELEVANT, GENERALLY
SPEAKING, BECAUSE THERE'S BEEN A
LOT OF DISCUSSIONS IN THE
COMMUNITY ABOUT WHETHER GENE
REGULATION IS BASICALLY
NOMINATED BY HISS TONE
MODIFICATION SIGNALS OR
NON-NATED BY SEQUENCE OF RED
TRANSCRIPTION FACTORS.
I THINK THIS WAS PROBABLY THE
FIRST DRAWING THAT ONE CAN
ACTUALLY MEASURE THE RELATIVE
REFERENCE DIRECTLY IN VITRO
WITHOUT INTERFERING.
BASICALLY WITH THE CORPORATION
BETWEEN THE DNA BINDING AND THE
ACETYLATION HISTONE BINDING UH
BUT IT'S A HIGHER BINDING.
ONE GENERAL LESSON WE'VE TAKEN
AWAY.
THE SECOND IS THAT YES FOR MANY
YEARS WE'VE BEEN TALKING ABOUT
N
NEWT ROE SOEM-FREE REGIONS.
NOT ONLY IS IT ASSISTING AND
SHUT BUT IT'S THE DEGREE OF
BEING OPEN THAT ALSO SEEMS TO
PLAY A ROLE.
I WAS JUST ALTERING THE
STRUCTURE WITHOUT MOVING IT
AROUND [LOW AUDIO].
ALL THE WAY TO CREATING A FREE
REGION THAT'S COMPLETELY DEVOID
OF NEW TROE SOEMS.
I THINK THIS MAY WELL BE
[INDISCERNIBLE] FOR OTHER
CHROMATIN-BASED REACTION.
SO IN THE CASE OF TURN DEN ONE,
THE TWO MOST IMPORTANT
COMPONENTS, DNA BINDING COME
PRESENTS AND THAT'S THE ONE THAT
CAN SENSE THE 60 BASE OR MORE
REGION NEXT TO ONE NUCLEOSOME --
TOGETHER I THINK THEY'RE
RESPONSIBLE FOR BRINGING THIS
ENZYME TO THE PLUS ONE LOCATION
IN THE CASE OF GENE PROMOTIONS.
THERE ARE HUMAN VERSIONS OF
THIS, OF COURSE, AND I WOULD
IMAGINE THAT SUCH PRINCIPLES ARE
ALSO IMPLIED WITH THE ADDITION
THAT IN THE CASE OF MAMMALIAN --
THE TWO MECHANISMS ARE NOT
MUTUALLY EXCLUSIVE.
LET ME TELL YOU WHERE WE ARE
NOW.
WE HAVE A GOOD STRUCTURE FOR
THIS AND THAT AND AS I MENTIONED
THIS STRUCTURE WAS SOLVED.
THE NEXT CHALLENGE TO GET THE
STRUCTURE OF THE [INDISCERNIBLE]
SUB STRAIGHT HANDS ON TO REACH
ANALOG AND SEE WHAT THE
STRUCTURE OF THAT MIGHT LOOK
LIKE, BUT WE REALLY WANT TO KNOW
UH HOW THE ENZYME MANAGES TO
PULL APART AND PULL OUT THE H 2
A AND PUT IN THE H 2 A.Z.
A LOT OF THIS IS GOING TO
INVOLVE HARD-CORE BIOCHEMISTRY
AND MOLECULE AN GET A HOLD OF
GENETICS OF THESE REACTIONS.
ONE OF THE APPROACHES WE'RE
BEGINNING TO WORK ON NOW IS
BEGINNING TO LOOK AT THE
DYNAMICS OF THE H2A.Z HISTONES
IN LIVE CELLS AND TO USE IS A
PRIORITY ARE OF MICRO SKOP TICK
TECHNIQUES TO BEGIN TO ADDRESS
ISSUES ABOUT HOW LONG DOES IT
TAKE TO GET A H 2 A INTO A NEW
TROE SOEM AND OUT OF A
NUCLEOSOME.
[INDISCERNIBLE] -- MOLECULE
IMAGING H 2 AD THAT ARE SHOWING
UP AND THIS PARTICULAR MICRO
SOEM OPERATING -- WHAT IT CAN DO
IS TAKE NINE SEKS -- YOU DON'T
HAVE TO GO THROUGH AND THEN GO
BACK AND GENERATE A A SERIES.
IT'S ALL DONE IN ONE SHOT AND
THAT'S PARTICULARLY USEFUL TO
WANT TO CAPTURE DYNAMICS OF
INDIVIDUAL PARTICLES.
I HAVE TO SAY THIS IS DONE BY A
LAB.
THIS HASN'TEN MADE IT TO OUR LAB
MEETING SO IT'S RAW DATA AND
COMPLETELY UNFILTERED BUT I JUST
WANTED TO SHOW THIS TO YOU TO
GIVE YOU THE FLAVOR OF WHAT WE
WOULD LIKE TO DO WHICH IS TO TRY
TO IMAGE THE INDIVIDUAL
MOLECULES IN VIVO.
WHAT ONE DOZ IS THIS IS A
LIFESTYLE AND ONE TAKE MISS MORE
[INDISCERNIBLE] [LOW AUDIO] AND
THAT GETS MAZ COMPUTATIONAL
INVOLVEMENT TO GENERATE
BASICALLY LOCATIONS OF THESE
PARTICLES AND SINCE WE GOT SHUT
DOWN SO WE DON'T HAVE ALL OF
THAT FOR YOU TODAY, BUT WE DO
HAVE THE OTHER HISTONE
[INDISCERNIBLE] AND WHAT YOU'RE
LOOKING AT ARE SINGLE MOLECULES
OF SET A WHICH ARE ARRANGED
LOCATED AT THE CENTRAL
[INDISCERNIBLE] -- WE'RE LOOKING
AT INDIVIDUAL [INDISCERNIBLE]
AND WHAT BASICALLY WHAT WE FIND
REALLY AMAZING ABOUT THE
TECHNOLOGY IS THAT WITHIN 20
NANO METERS, YOU CAN LOCALIZE
THE POSITIONS OF THE INDIVIDUALS
OF HISTONES.
SO AGAIN, THIS IS NOT DYNAMIC,
BUT IT'S SIMPLY THE FOUNDATION
OF WHERE I THINK WE WOULD LIKE
TO TAKE FOUR PROJECTS [LOW
AUDIO].
LET ME TRY TO WRAP UP.
GENE REGULATION EUKARYOTIC
TEXTBOOK MODEL WE STUDIED
[INDISCERNIBLE] -- YES, THAT
DOES OPERATE ALSO IN EUKARYOTIC
AND THERE'S A LOT MORE OF THOSE
FACTORS AS I MENTIONED IN THE
MAMMALIAN GENOME BUT THERE'S A
WHOLE LAYER THAT'S PUT ON TOP OF
THAT WHICH PLAYS INTRINSICICALLY
IMPORTANT ROLE IN REGULATING
GENES.
THE ENZYMES THAT MOBILIZE
SYSTEMS, ENZYMES THAT PUT IN
CHEMICAL SIGNALS ON TO THE
HISTONE TAILS T VARIANTS OF THE
HISTONES, THE [INDISCERNIBLE],
THE CHAPERONS OF THE HISTONES, I
DIDN'T HAVE TIME TO GET INTO
THOSE WITH THE EXCEPTION OF THE
G 1 THAT I MENTIONED BRIEF HLY.
THE WHOLE REALM OF NON-HISTONES
ARCHITECTURAL PROTEINS ALSO ARE
WORKING FOR EUKARYOTIC
REGULATION.
THEN WE MOVE ON TO D H NA
METHYLATION THEY HAVE IMPORTANT
ROLES FOR MAIN [INDISCERNIBLE]
BUT NOT ALL OF THE FOUNDING
YEAST, BUT STILL VERY IMPORTANT.
AN THEN LASTLY THE WHOLE
MACHINERY THAT BROUGHT DOWN TO
THE CHROMATINS TO HELP IN
REGULATING.
SO IT'S A LOT MORE COMPLICATED
BUT I THINK MAMMALIAN
[INDISCERNIBLE] IS NOT TOO
SURPRISING BUT IT DOES SAY WHEN
WE LOOK BACK AND THINK ABOUT
TRANSCRIPTION FACTORS AND THE
ONES THAT ARE NOW VERY PASSION
NABL THAT IF YOU EVER GET THEM
TO WORK PROBABLY, IT'S IMPORTANT
TO UNDERSTAND THE TEXTURE YOU
SHOULD WHICH THEY OPERATE AND
AGAIN ALL THOSE PRINCIPLES SHOW
UP AGAIN HERE [LOW AUDIO].
LET ME THEN THANK THE PEOPLE
WHO'VE BEEN CONTRIBUTING TO OUR
WORK FOR THE PAST TEN YEARS FOR
H 2 A.Z.
MANY OF THEM HAVE GONE TO OTHER
LABS, MOVED ON TO THEIR OWN
LABS.
THE CURRENT CREW [INDISCERNIBLE]
I MENTIONED TO YOU HAS BEEN A
POST DOC FOR A NUMBER OF YEARS
IN THE LAB.
[LOW AUDIO].
[INDISCERNIBLE] BASICALLY WAS A
COLLABORATION BETWEEN HIM,
RESEARCH ASSISTANT, D
[INDISCERNIBLE] AND
[INDISCERNIBLE] AND THESE
STARTED H2A.Z PROJECT.
[INDISCERNIBLE] SHOWED A LITTLE
BIT OF HIS WORK.
COLLABORATORS, FITZGERALD, ALL
THE BIOINFORMATICS.
CHRIS [INDISCERNIBLE] WHO WAS
ONE OF THE TWO LAB TECH WHO
DISCOVERED NUCLEOSOMES IN 1970s
GOT CHRIS OUT OF RETIREMENT.
[LOW AUDIO] -- NOW RUNNING THEIR
OWN LABS.
.
COLLABORATORS AT HARVARD.
GERALD AT NCI.
MAX BEEN WORKING ON THE
[INDISCERNIBLE] CLOLLABORATING
WITH THEM.
SO THANK UH YOU VERY MUCH.
[APPLAUSE]
>> I'M SURE CARL WILL BE HAPPY
TO ANSWER ANY QUESTIONS.
>> CARL, I HAVE A QUICK
QUESTION.
SO THE FIRST LIGHT IT A APPEARS
THE YEAST THE FUNCTION SLIGHT
DIFFERENCE FROM MEDIUM BECAUSE
THE MAMMALIAN [INDISCERNIBLE]
REQUIRED TO MAINTAIN
[INDISCERNIBLE], BUT THE YEAST
PROMOTER IT APPEARS FLOOR
EXPLOIT THE NUCLEOSOME FREER
REGION SO THAT MEANS THAT
NUCLEOSOME THAT HAS STABILITY
THAT ARE NOT REQUIRE H2A.Z; HOW
DO YOU EXPLAIN THE EVENT?
WHAT MAINTAINS THE NUCLEOSOME-F
FREE REGION.
>> THAT'S A GOOD QUESTION.
I DON'T THINK THE YEAST PEOPLE
HAVE LOOKED AT THE ACCESSIBILITY
IN WHICH THIS WAS THE SAME WAY
AND SO IT'S PROBABLY WORTH WHILE
TO REDO THOSE EXPERIMENTS.
ASIDE FROM THAT, THE POINT -- I
THANK YOU FOR THE QUESTION --
WHICH IS THAT THE YEAST AND
ENZYMES WANT TO TAKE ADVANTAGE
OF THE NUCLEOSOME-FREE REGION.
WHO MAKES THAT?
PROBABLY OTHER TRANSCRIPTION
FACTORS AND OTHER
[INDISCERNIBLE] AND IN THE CASE
OF THE ONES I SHOW EED YOU WE
KNOW REV 1 AND RIFF ACTUALLY
PLAY AN IMPORTANT ROLE IN
CREATING NUCLEOSOME-FREE REGION
FIRST.
I SUSPECT THAT SO THAT THE
SEQUENCE OF EVENTS ARE ARE
PROBABLY GOING BE GENERAL.
>> [LOW AUDIO].
>> CAN YOU REPEAT THAT 'CUZ I
DIDN'T CATCH IT.
>> [LOW AUDIO].
>> [INDISCERNIBLE].
>> [LOW AUDIO].
>> I'M NOT SURE I FOLLOW THAT.
[INDISCERNIBLE] -- MAY BE THE
BEST THING TO DO.
>> YOU SHOWED VERY CLEARLY THAT
[INDISCERNIBLE] RECOGNIZED DNA
AND ASSIMILATION COULD HELP IN
BINDING, WHAT ABOUT H 2 A, H 2 A
POINT Z CHANGE?
>> ALSO I DIDN'T SHOW THE DATA
BUT IN FACT IT DOES HAVE --
[CROSS TALK].
WHAT WE HAVE NOT DONE AND MAYBE
YOU CAN CORRECT ME WE HAVE NOT
DONE THE EXPERIMENT WHICH BYPASS
THE PERSON COMPLETELY.
>> [LOW AUDIO].
>> AND JUST SEE HOW IT PUBLISH
IN SECOND REACTION THAT MAY HAVE
BEEN DONE, MAYBE NOT.
IF NOT IT SHOULD BE DONE.
>> RIGHT.
'CUZ IT SHOULD BE THE STUB
STRAIGHT [LOW AUDIO].
BEYOND THE BINDING SHOULD BE
FOUR NOT H2B -- [LOW AUDIO].
>> THAT'S A GOOD QUESTION.
>> WE WILL HAVE A RECEPTION IN
THE LIBRARY SPONSORED BY THE FAS
WHERE YOU CAN ASK CARL MORE
QUESTIONS.
NEXT WEEK, WEDNESDAY, THURSDAY,
AND FRIDAY, WE HAVE RESCHEDULED
THE RESEARCH FESTIVAL SO I HOPE
YOU CAN ALL ATTEND THOSE EVENTS.
THAT WILL INCLUDE NEXT WEEK'S
WEDNESDAY AFTERNOON LECTURE
SERIES, NEXT WEDNESDAY AT 3
IMBED INTO THE RESEARCH
FESTIVAL.
END OF NEXT WEEK, EXPECT TO HAVE
A LOT OF GREAT TIME.
THANK UH YOU ALL AND WE'LL SEE
YOU AT THE RECEPTION.
[APPLAUSE]
ALSO NEXT WEEK, RANDY SHEKMAN
WILL BE GIVING A TALK ON
NOVEMBER 4TH AT 11:00 A.M. HERE.