I'M THE DIRECTOR OF THE NATIONAL
INSTITUTE OF DIABETES AND
DIGESTIVE AND KIDNEY DISEASES.
I'M HAPPY TO WELCOME
DR. MITCHELL LAZAR TELL US ABOUT
NUCLEAR RECEPTORS AND THE
EPIGENOMIC CIRCADIAN RHYTHMS AND
METABOLISM.
HE IS A PROFESSOR OF MEDICINE IN
GENETICS, CHIEF OF DIVISION OF
ENDOCRINOLOGY, DIABETES AND
METABOLISM AND THE DIRECTOR OF
THE INSTITUTE FOR DIABETES,
OBESITY AND METABOLISM AT THE
UNIVERSITY OF PENNSYLVANIA.
HE RECEIVED HIS UNDERGRADUATE
DEGREE IN CHEMISTRY FROM THE
MASSACHUSETTS INSTITUTE OF
TECHNOLOGY AND THEN WENT ON TO
RECEIVE HIS Ph.D. IN
NEUROSCIENCE AND HIS MD BOTH
FROM STAMFORD UNIVERSITY.
HE TRAINED IN INTERNAL MEDICINE
AT THE BRIGHAM AND WOMEN'S
HOSPITAL IN BOSTON AND
ENDOCRINOLOGY FELLOWSHIP AT THE
MASSACHUSETTS GENERAL BEFORE
JOINING THE FACULTY AT THE
UNIVERSITY OF PENNSYLVANIA IN
1989.
RESEARCH CONDUCTED BY DR. LAZAR
IN HIS LAB, PARTICULARLY THE
STUDY OF THYROID HORMONE
RECEPTORS LED TO DISCOVERIES OF
NUCLEAR RECEPTOR MEDIATED
REPRESSION.
IN ADDITION, HIS LAB IS USING
GENOME-WIDE METHODOLOGIES TO
BETTER UNDERSTAND THE EFFECTS OF
DIET, OBESITY AND CIRCADIAN
RHYTHMS ON METABOLISM, AS YOU
WILL HEAR TODAY.
INCREASINGLY DIFFICULT TO FIND
LEADING SCIENTIFIC JOURNALS TO
WHICH DR. LAZAR HASN'T
CONTRIBUTED.
I WAS JUST LOOKING OVER JUST THE
FIRST THREE MONTHS OF THIS YEAR,
DR. LAZAR'S WORK HAS BEEN
PUBLISHED IN "SCIENCE,"
"MOLECULAR ENDOCRINOLOGY" "THE
JOURNAL OF NEUROSCIENCE" AND THE
JOURNAL OF IMMUNOLOGY."
HE HAS GIVEN LECTURES THROUGHOUT
THE WORLD AND IS A FORMER MEMBER
OF OUR INSTITUTE, OUR NIDDK'S
CONSULT, ADVISORY CONSULT.
HE HAS ALSO BEEN ASSOCIATE
EDITOR OF DIABETES AND IS ON THE
EDITORIAL BOARD OF AT LEAST A
HALF DOZEN PRESTIGIOUS
PUBLICATIONS.
IN ADDITION TO NIH, MANY
ORGANIZATIONS HAVE SINGLED HIM
OUT AND RECOGNIZED HIS SEMINOLE
WORK AND HIS OUTSTANDING
ACHIEVEMENT.
THESE INCLUDE THE AMERICAN
THYROID ASSOCIATION, ENDOCRINE
SOCIETY, AND THE AMERICAN
SOCIETY FOR CLINICAL
INVESTIGATION.
I COULD GO ON AND ON ABOUT
DR. LAZAR'S COMPISHMENTS BUT I
SUSPECT YOU LIKE ME, WOULD
PREFER TO HEAR HIS PRESENTATION
FOR THAT LET ME TURN OVER THE
PODIUM TO DR. MITCH LAZAR.
[APPLAUSE]
>> THANK YOU FOR THAT VERY
FLATTERING AND GREAT
INTRODUCTION.
IT'S REALLY A PLEASURE TO BE
HERE SEEING OLD FRIENDS AND
COLLEAGUES AS WELL AS MEETING
NEW PEOPLE AND THE NIDDK IN
PARTICULAR, AND NIH IS SUCH
SPECTACULAR PLACES IT'S GREAT TO
SEE THE BREATH AND DEPTH OF
RESEARCH HERE.
ALTHOUGH I'LL ONLY TALK
ABOUT -- I'LL TRY TO BE FOCUSED.
I'LL START WITH BACKGROUND AND
WILL HOPEFULLY HIT ON AREAS OF
INTERESTS TO A NUMBER OF PEOPLE
IN THE CROWD.
I THOUGHT I'D START WITH THIS
BECAUSE THIS REMINDS US OF
REALLY WHAT WE ARE TRYING TO
UNDERSTAND.
WE ARE TRYING TO UNDERSTAND WHAT
OBESITY, WHAT CAUSES OBESITY AND
THEN WHAT OR HOW IT CAUSES
MORBIDITY AND MORTALITY THAT IS
ASSOCIATED WITH.
AND THEN WHAT TO DO ABOUT THOSE
THINGS.
AND ONE OF THE MAJOR THINGS THAT
WE THINK ABOUT, WE THINK ABOUT
THE MORBIDITY ASSOCIATED WITH
OBESITY, CAN BE BOILED DOWN TO
THIS NOTION OF THE METABOLIC
SYNDROME.
JERRY, ONE OF MY MENTORS AT
STAMFORD IS THE PERSON WHO
PIONEERED THE CONCEPT OF
SYNDROME X, AND I THINK THAT
FROM HIS POINT OF VIEW, AND THIS
IS INTERESTING TOPIC WE CAN
DISCUSS, THIS IS REALLY INSULIN
RESISTENCE WHICH IS OTHERWISE
THE CRITERIA THAT GETS YOU INTO
THE SYNDROME OF METABOLIC
SYNDROME.
TO THIS DAY HE WOULD ARGUE THIS
IS INSULIN RESISTENCE AND IT IS
INSULIN RESISTENCE DRIVING ALL
THE OTHER BULLET POINTS ON HERE.
AS THE METABOLIC SYNDROME HAS
BEEN DEFINED, INCLUDES THIS
DYSMETABOLLIC PARAMETERS OF
CENTRAL OBESITY, HIGH BLOOD
PRESSURE, HIGH TRIGLYCERIDES AND
LOW HDL CHOLESTEROL AND ALL OF
WHICH ARE RISK FACTORS FOR
CARDIOVASCULAR DISEASE.
AND OF COURSE INSULIN RESISTENCE
IS KNOWN OF TYPE II DIABETES.
TYPE II DIABETES ALSO IS
EPIDEMIC, LIKE OBESITY, AND THE
LEADING CAUSE OF DEATH FROM TYPE
II DIABETES AND CARDIOVASCULAR
DISEASE, MACRO VASCULAR
COMPLICATIONS ASSOCIATED WITH
INSULIN RESISTENCE, AS WELL AS
THESE OTHER FACTORS.
WHETHER OR NOT THEY ARE CAUSED
BY INSULIN RESISTENCE OR PART OF
THIS CONSTELLATION.
I'M LARGELY GOING TO FOCUS ON
TODAY TO KEEP A PARTICULAR FOCUS
OF THE TALK, IS ANOTHER ASPECT
OF THE METABOLIC SYNDROME THAT
IS ONLY RELEVANTLY RECENTLY
RECOGNIZED AS MORE THAN JUST A
CURIOSITY AND THAT'S THE FATTY
LIVER.
NOT TOO LONG AGO, ALTHOUGH IT
WAS KNOWN THAT DIABETICS AND
OBESE PATIENTS HAD FATTY LIVERS,
IT WAS THOUGHT THAT IT WAS
REALLY IKNOCKIOUS.
THE FATTY LIVER IS REALLY A
PRECURSOR OF A SEVERE PROBLEM.
AND THE PROBLEM CAN BE
CONSIDERED AS ON THIS SLIDE,
WHERE THE FATTY LIVER CAN BECOME
INFLAMED AND LEAD TO SO-CALLED
NONALCOHOLIC EP TIGHTIS.
THAT COULD LEAD TO FIBROSIS
WHICH COULD LEAD TO CIRRHOSIS
AND INDEED, FATTY LIVER AND
LIVER DISEASE ASSOCIATED WITH
FATTY LIVER IS IN CREASINGLY
BECOMING A CAUSE FOR CONCERN AND
THE CIRRHOSIS CAUSED BY IT IS
ONE OF THE LEADING CAUSES, I
BELIEVE IT'S NOW SECOND TO
ALCOHOLIC DISEASE.
MORE WIDE, HEPATITIS C IS UP
HIGH ON THE LIST.
CIRRHOSIS IS A PRECURSOR OF
HEPATOCELLULAR CARCINOMA AND WE
REALIZE THAT OBESITY IS A MAJOR
RISK FACTOR FOR MANY TYPES OF
CANCER, ONE OF THE LEADING ONES
IS LIVER CANCER.
SO THE AFFECTS OF THIS
DYSMETABOLLIC SITUATION ON THE
LIVER IS OF INCREASING CONCERN.
NOW, IF YOU GO ON THE ENTREES
INTERNET, YOU CAN BUY THIS
STUFF.
METABOLIC SYNDROME FIGHTER.
I AM HAPPY TO SEE ONE OF THE
EXCITING THINGS THAT DR. COLLINS
IS SPEARHEADING IS INTERACTION
WITH THE FDA AT NIH AND MAYBE
THE FDA WILL PLAY A ROLE IN
PRODUCTS LIKE THIS.
IT SAYS IT MAY ASSIST IN
OVERCOMING INSULIN RESISTENCE,
WITH AN ASTERISK.
IT'S TRUTH IN ADVERTISING.
OF COURSE WITH THIS DISEASE, I
WOULDN'T BE TELLING YOU ABOUT
THIS STORY.
THE OTHER POINT I WANT TO BRING
IN AS PART OF THE INTRODUCTION
IS THE INCREASING -- HOW
INCREASINGLY CLEAR IT IS THAT
CIRCADIAN RHYTHMS PLAY A ROLE IN
METABOLISM.
ENDOCRINOLOGISTS RECOGNIZE THAT
MOST ENDOCRINE SYSTEMS OBEY A
CIRCADIAN PATTERN, THAT COULD
CONTRIBUTE TO THE DYSMETABOLISM
OF METABOLIC SYNDROME AND THE
ISSUES WE ARE DEALING WITH.
JUST TO RUN THROUGH THE MANY
ASPECTS OF INTERRELATIONSHIP
BETWEEN CIRCADIAN RHYTHM AND
METABOLISM, MANY HORMONES HAVE
CIRCADIAN RHYTHMS.
THERE ARE OBVIOUS LINKS BETWEEN
CIRCADIAN RHYTHMS AND METABOLIC
PHYSIOLOGY, WHETHER YOU'RE
NOCTURNAL BIKE A MOUSE OR NOT
LIKE A HUMAN, YOU TEND TO EAT
WHEN YOU'RE AWAKE, NOT WHEN
YOU'RE SLEEPING.
GLUCOSE AND LIPID METABOLISM
HAVE CIRCADIAN RHYTHMS AND
SEVERAL RISK FACTORS, INCLUDING
THROMBOSIS, POTENTIAL OF THE
BLOOD, HAVE CIRCADIAN RHYTHMS
SUCH THAT MOST MYOCARDIAL
INFARCTIONS OCCUR EARLY IN THE
MORNING.
GOOD NEWS AND BAD NEWS AS WITH
ANY SICKLY PROCESS.
THE GOOD NEWS IS WE MADE IT
THROUGH THE MORNING AND WE HAVE
ABOUT A 75% LOWER RISK OF HAVING
A HEART ATTACK IN THE MORNING
BUT TOMORROW IS ANOTHER DAY.
OBESE PEOPLE HAVE SLEEP
DISORDERS AND SLEEP DEPRIVATION
ARE ASSOCIATED WITH METABOLIC
DISTURBANCES.
THAT IS SOMETHING THAT I THINK
WE ALWAYS ARE ASPECTS OF PEOPLE
THAT YOU KNEW OR SHIFT WORKERS
YOU KNEW.
SOMETIMES NURSES IN HOSPITALS
WORKING THE NIGHT SHIFT, A SENSE
THEY WERE METABOLIC DISTURBANCES
BUT NOW IN HAS BEEN PUT INTO A
MORE PHYSIOLOGICAL CONTEXT
LARGELY BY THE LARGE FIELD OF
CIRCADIAN BIOLOGIST.
THIS IS A FIGURE I TOOK FROM THE
LITERATURE THAT TRIES TO
ILLUSTRATE THE CONCEPT OF A
DYSOR MISALIGNMENT BETWEEN WHEN
THE BODY WOULD LIKE TO HAVE ITS
BIOLOGICAL AND CHEMICAL RHYTHMS
AND HOW THAT IS DIFFERENT FROM
WHEN IT IS SLEEPING AND WHEN THE
PERSON IS EATING AND HOW THIS
LEADS TO THIS MISALIGNMENT AND
MALL ADAPTION.
THERE ARE A NUMBER OF STUDIES
FROM CHUCK AND HIS COLLEAGUES,
AT BRIGHAM, WHICH HAVE, AND MANY
OTHERS, WHICH HAVE MADE THIS
CLEAR IN A CONTROLLED LABORATORY
SETTING.
BUT EVEN OBSERVATIONALLY, THERE
IS MORE AND MORE STUDIES LIKE
THIS IN THE OCCUPATIONAL AND
ENVIRONMENTAL MEDICINE JOURNAL,
WHICH I PERSONALLY DON'T READ
VERY OFTEN.
BUT WE ARE STARTING TO SEE MORE
AND MORE OF THESE TYPES OF
STUDIES THAT MAKE THIS
ASSOCIATION.
THESE ARE PEOPLE WHO WERE ALL ON
THE DAY SHIFT AND SOME WENT TO
THE NIGHT SHIFT AND THEN THIS IS
THE INCIDENCE OF METABOLIC
SYNDROME OVER THE YEARS AS THEY
STAYED ON THE DAY SHIFT OR
SWITCHED TO THE NIGHT.
AND THERE IS REALLY A DRAMATIC
INCREASE.
ONE OF THE THINGS, WHICH I WON'T
TALK ABOUT IN THE LAB
TODAY -- ABOUT TODAY, WE WORK ON
IN MY LAB AND I THINK IT MAY BE
ONE OF MY EDUCATIONAL OBVIOUSLY
OBJECTIVES.
SO I'LL GET IT OVER WITH HERE,
IT HAS TO DO WITH THE
RELATIONSHIP BETWEEN THE FAT
CELL AND INSULIN RESISTENCE.
ONE OF THE MOLECULES WHICH WAS
DISCOVERED IN MY LABORATORY IS
RESISTANT.
I WON'T GO INTO DETAIL OTHER
THAN TO SAY THAT MAYBE TIEING
THIS OC WOIN MY LAB TOGETHER
WITH THIS LAB IS THIS
OBSERVATION THAT NIGHT-SHIFT
WORKERS HAVE INCREASE TO
RESISTENTS.
AND NOW I WANT TO SHOW YOU AN
OBSERVATION THAT WAS MADE ALMOST
40 YEARS AGO THAT REALLY DOESN'T
HAVE A OR HAS NOT HAD A
MOLECULAR EXPLANATION AND I LIKE
TO SUGGEST WHEN THAT WE HAVE A
PLAUSIBLE EXPLANATION FOR THIS
PHENOMENON RELATED TO CIRCADIAN
RHYTHM AND METABOLISM.
HERE IS MICE EITHER WILD TYPE
MICE OR OBOB GENETICALLY MORE
MORBIDLY OBESE MICE ON THE UPPER
PART OF THE GRAPH HERE AND I'LL
FOCUS ON THE LOWER PART BUT
REALLY THE OBOB AN EXAGGERATION.
AND ALSO EMBEDDED ON THIS SLIDE,
THIS IS THE ARTICLE IT COMES
FROM, IS ADIPOSE TISSUE, SIMILAR
TO LIVER IN THIS REGARD, BUT
SINCE I'M GOING TO FOCUS ON
LIVER, I'M GOING TO SHOW YOU THE
LIVER DATA.
YOU CAN SEE WHEN THE MICE NORMAL
MICE ARE IN THIS CASE, ARE IN
THE DARK, SO THEY ARE ACTIVE IN
FEEDING, THAT'S THEY HAVE THEIR
INCREASE IN FATTY ACID SYNTHESIS
OR LYPOGENESIS.
SO DID NOVO LYPOGENESIS, FATTY
ACID SYNTHESIS BY THE LIVER HAS
A CIRCADIAN RHYTHM WE HAVE KNOWN
ABOUT A LONG TIME BUT THE
MECHANISM HAS NOT BEEN
ESTABLISHED.
I'M GOING TO SUGGEST ONE FOR IF
YOU THIS LECTURE.
TO INTRODUCE THE PLAYERS I'D
LIKE TO SUGGEST IS NUCLEAR
RECEPTORS.
I WANT TO BRING UP THE WHOLE
NOTION OF WHETHER THIS IS
RELATED TO GENES OR VIRUS.
WHEN YOU LOOK AT PEOPLE LIKE
THIS, WHO PROBABLY HAVE
METABOLIC SYNDROME BUT ARE OBESE
AND GENETICALLY IDENTICAL, WE
KNOW FROM THE WORK OF MICKEY AT
THE UNIVERSITY OF PENNSYLVANIA
AND MANY OTHERS, IDENTICAL TWIN
STUDIES, TWINS READER APART,
SHOWING THERE IS A TREMENDOUS
CONCORDANCE IN OBESITY AND TYPE
II DIABETES IN IDENTICAL TWINS.
IT'S A GENETIC COMPONENT.
AND YET THE OBESITY IN DIABETES
EPIDEMIC THAT WE ARE SO AWARE OF
THAT REALLY BECAME OBVIOUS IN
THE 50's AND ACCELERATED IN
THE 70's, 80's, 90's, AND
SHOWED NO SIGN OF SLOWING DOWN
IN THIS MILLENNIUM, THESE
EPIDEMICS OCCURRED DURING A TIME
IN THE ENVIRONMENT CHANGED
DRAMATICALLY.
THERE WAS SO MUCH EASIER TO GET
FAST-FOOD, HIGH CALORIC FOOD AT
LOW COST.
YOU COULD DRIVE TO WORK RATHER
THAN WALK.
AND THE LIFESTYLES ALL CHANGED.
AND YET THIS TIMEFRAME IS TOO
SHORT TO BRING ANY MEANINGFUL
GENETIC CHANGES TO EXPLAIN THE
EXPLOSION OF METABOLIC DISEASE.
SO THERE HAS TO BE AN
ENVIRONMENT GOING AND WE KNOW
THERE IS A GENETIC COMPONENT.
AND I THINK MANY IN THE AUDIENCE
ARE ALREADY THINKING THIS WAY.
BUT IT'S THE SCIENCE OF
EPIGENOMICS THAT RATHER THAN
HAVING PEOPLE GET INTO FIGHTS AT
COCKTAIL PARTIES.
I THINK EPIGENOMICS ARE WHAT
LINK THE ENVIRONMENT TO GENE
EXPRESSION.
AND HERE I'M TALKING ABOUT
MODIFICATIONS, DNA MODIFICATION
THAT RESULT IN CHANGING IN GENE
EXPRESSION AND FUNCTION WITHOUT
A CORRESPONDING ALTERATION IN
DNA SEQUENCE.
NOTICE I'M NOT IN MY DEFINITION
OF EPIGENOMICS SAYING ANYTHING
ABOUT HERITABILITY IN THE
EPIGENETICS COMMUNITY AND IN THE
EPIGENETICS WORLD, BECAUSE OF
THE PART OF THE TERM GENETICS, I
THINK IT'S REASONABLE TO
CONSIDER AND AS MANY WILL
REQUIRE, HERITABILITY, WHETHER
IT IS FROM MOTHER TO PROGENY OR
FROM MOTHER CELL TO DAUGHTER
CELL, REQUIRE HERITABILITY OF
THE CHANGE.
EPIGENOMICS AND THE EPIGENOME
TO, IN THIS DEFINITION, REALLY
REFERS TO THE WAY THAT THE
GENOME, WHICH IS MORE OR LESS
EVERY CELL EXCEPT FOR VERY
SPECIAL ISLANDS CELLS OF COURSE,
IS CONFIGURED IN THE NUCLEUS.
-- SPECIALIZED CELLS.
THAT INVOLVES THE NUCLEOSOME,
HISTONE OPT MERES AND THESE
HISTONE TAILS WHICH GET MODIFIED
AND IT'S THOSE MODIFICATIONS
THAT I'LL FOCUS ON.
AND THE REASON THAT THIS IS HOW
THE ENVIRONMENT INTERACTS IS
THAT IT IMPACTS AND CHANGES THE
EPIGENOME.
AND I'M DIVIDING HERE THE
ENVIRONMENT INTO THE INTERNAL
ENVIRONMENT AND THE EXTERNAL
ENVIRONMENT.
THE INTERNAL ENVIRONMENT IN PART
AS A HOMAGE TO CLUB BERNARD, THE
FAMOUS FRENCH ENDOCRINOLOGIST
AND THE FIRST ENDOCRINOLOGYGIST
THAT POSTULATED THE ROLE OF
HORMONES AND METABOLITES IN
REGULATING THEIN CERTAINLY
ENVIRONMENT.
-- INTERNAL ENVIRONMENT.
THE EXTERNAL ENVIRONMENT IS
NOTABLE FOR THE DIET DOWN UP
NORTH WHERE I COME FROM, IN
PHILADELPHIA, THIS IS THE GREAT
STUFF WE LOVE TO EAT AND WHICH
IS CONTRIBUTING TO THE PROBLEM,
BUT OF COURSE IT ALSO
CONTRIBUTES NUTRIENTS AND
VITAMINS YOU CAN'T COMPLETELY GO
WITHOUT A DIET.
ANOTHER ENVIRONMENTAL FACTOR,
WHICH I ALREADY INTRODUCED, IS
THE CIRCADIAN RHYTHM AND THE
SLEEP-WAKE CYCLE AND SO FORTH.
SO NUCLEAR RECEPTORS, WHICH WE
HAVE BEEN STUDYING SINCE I WAS A
POSTDOC WITH BILL CHIN IN THE
80's AND MY OWN LAB AT PEN FOR
22 YEARS.
THERE ARE MANY FANTASTIC GROUPS
HERE AT NIH WHO ARE ALSO NUCLEAR
RECEPTORS, CAN BE THOUGHT OF IN
ONE SENSE AND I'LL PROPOSE TO
YOU THAT THEY ARE REALLY
TRANSDUCERS OF ENVIRONMENTAL
SIGNALS INTO CHANGES IN GENE
EXPRESSION, YES, BUT THROUGH
EPIGENOMIC CHANGE.
SO THE MODEL FOR NUCLEAR
RECEPTORS IS THAT THEY ARE
TRANSCRIPTION FACTORS THAT BIND
TO SMALL MOLECULES TO REGULATE
GENE EXPRESSION AND THESE SMALL
MOLECULES AREIN VARIABLY DERIVED
FROM THE ENVIRONMENT.
THE INTERNAL ENVIRONMENT, THE
HORMONES AND METABOLITES IS
WHERE THE NUCLEAR RECEPTORS,
WHICH ARE ORIGINALLY KNOWN AS
THE NUCLEAR HORMONE RECEPTORS
BECAUSE THE FIRST MODULATORS
WERE THE THYROID HORMONES,
STEROID HORMONES,
GLUCOCORTICOIDS COIDS AND
ESTROGENS AND SO FORTH, AS WELL
AS METABOLITES AND MORE RECENTLY
THE METABOLITES SUCH AS FATTY
ACIDS.
THE FAT SOLUABLE VITAMINS, A AND
D, GET CONVERTED INTO NUCLEAR
RECEPTOR LIGANDS, THE RETINOIC
ACID RECEPTOR AND TARGETED AND
THEN THERE IS A
FASCINATING -- FRANKLIN IS ONE
OF THE LEADERS IN THIS AREA, IN
WHICH THE NUCLEAR RECEPTORS ARE
A SUBSET OF NUCLEAR RECEPTORS
ARE SENTENCING IN A VERY
PRIMITIVE IMMUNE DEFENSE WAY IF
YOU WILL, DRUGS OR NONNATURAL
COMPOUNDS THAT ARE THEN DRIVING
THE TRANSCRIPTION AND SYNTHESIS
OF THE SYSTEM IN THE LIVER THAT
DETOXIFIES THE CHEMICALS THAT.
IS SOMETHING THAT FRANK TALKED
ABOUT HERE MANY TIMES.
HERE JUST MAKES THE POINT IN MY
ARGUMENT THAT THE FAMILY OF A
WHOLE HAS POTENTIALLY HAS IN
COMMON THIS ROLE OF RESPONDING
TO THE ENVIRONMENT.
SO, WHAT DO THEY DO?
THEY REGULATE TRANSCRIPTION AND
BIND TO A WHOLE SET OF GENES AND
I LIKE TO USE THE TERM, ASSIST
STROME BECAUSE OF THE OHMICS ERA
--- CISTROMES -- IT SUBITUTES
FOR SAYING, ALL OF THE BINDING
SITES IN THE GENOME OCCUPIED BY
FACTOR X.
YOU COULD JUST SAY THE CISTROMES
OF FACTOR X.
IT'S A USEFUL TERM.
NOW THE NUCLEAR RECEPTORS BIND
TO THESE SITES AND THEY RECRUIT
CO-REGULATOR MOLECULES AND THERE
WERE TWO FLAVORS OF THESE.
LOTS OF SPECIFICS.
BUT THE MAJOR ONES ARE
CO-REPRESSORS THAT REPRESS
TRANSCRIPTION AND UPON ABSENCE
OF THE LIGAND OR CO-ACTIVATORS
THAT ACTIVATE TRANSCRIPTION IN
THE PRESENCE OF THE
ENVIRONMENTAL DERIVED SIGNAL.
AND THEY DO SO BY ACTING AS
PLATFORMS THAT THE LIVER, MANY
ENZYMES, LOCALLY, TO THESE
REGIONS OF THE GENOME.
THIS IS IN COMPLETE LIST I'D
LIKE TO FOCUS ON THE MOST FAMOUS
ONES WHICH OF THE HISTONE
DEACETYLASES AND TRANSFERASES,
MODULATE THE ACETYLATION OF
THESE RESIDUES IN THE TAILS OF
THE HISTONE MOLECULES MAKING UP
THE NUCLEOSOME AND CHANGE THE
EPIGENOMIC STRUCTURE IN WAYS
THAT EVENTUALLY LEADS TO
REGULATION OF GENE EXPRESSION.
SO THAT IS THE LINK THAT I REFER
TO HERE.
AND I'D LIKE TO, IN THE REST OF
THE TALK S TO START FOCUSING
SORT OF MORE AND MORE INTENSELY
AND I'M GOING START WITH THE
HISTONE DEACETYLASES.
NOW THE WHOLE FAMILY OF HISTONE
DEACETYLASES SHOWN HERE, I DON'T
KNOW HOW WELL YOU CAN SEE IT,
CLASS ONE, CLASS TWO AND CLASS 3
AND THEN THE NAD DEPENDENT
MOLECULES ARE ALSO BECOMING
INCREASINGLY FAMOUS FOR THEIR
POTENTIAL ROLE IN CALORIC
RESTRICTION, LONGEVITY AND
METABOLISM.
INHIBITORS OF THE NONHISTONE
DEACETYLASE INHIBITORS, FORMED
THE BASIS OF EPIGENETIC THERAPY
OF A NUMBER OF CANCERS.
I'M SHOWING YOU A LIST HERE.
IN CLINICAL USE TODAY, ARE A
NUMBER OF MOLECULES THAT HAVE
LOTS OF OTHER AFFECTS.
I REALLY SHOULDN'T EMPHASIZE
THAT AND THEY COME UP IN THE
QUESTION AND ANSWER PERIOD.
BUT THEY INHIBIT HISTONE DESET
LAYSES AND ESPECIALLY THE CLASS
ONE AND OFTEN THE CLAYS 2.
 CLASS 2.
AND I'M GOING TO FOCUS ON A
PARTICULAR CLASS 1 HISTONE
DEACETYLASE CALLED HDAC THREE.
AND AT THIS POINT, NOT ALL THE
HDACS ARE CREATED EQUAL.
AND I WOULD FOCUS ON THIS
BECAUSE WE CONTINUING TELLS US A
LOT, PER SE AND I HOPE YOU'LL
FIND THE STORY INTERESTING.
AND I ALSO THINK IT IS
PARADIEMATIC FOR HOW WE HAVE TO
BEEN MOLECULES ONE BY ONE, NOT
JUST AS A CLASS.
AND MOST OF THESE DRUGS ARE
REALLY DOING A LOT MORE THAN
POTENTIALLY WHAT THEY NEED TO DO
FOR THEIR THERAPEUTIC EFFECT.
SO, WHY DO WE FOCUS ON HDAC3?
THE REASON IS BECAUSE YEARS AGO,
IN THE COURSE OF THE SERIES OF
STUDIES IN MY LAB AND ALSO
AROUND THE WORLD, THAT WE ARE
TRYING TO UNDERSTAND HOW NUCLEAR
RECEPTORS WORK AND LED TO THE
DISCOVERY OF THE CO-REPRESSORS,
IN PARTICULAR, NCOR AND THE
SMART, AND THEN ALMOST
IMMEDIATELY THAT WAS THE ERA
THAT THE AUDIENCE PROBABLY
REMEMBER WHEN WE WERE FIRST
STARTING TO REALIZE HOW
IMPORTANT PROTEIN-PROTEIN
INTERACTIONS WERE AND THE FIRST
THING PEOPLE DID WAS TO TAKE
EITHER PIECES OF THESE MOLECULES
OR THE WHOLE MOLECULE AND LOOK
FOR POTENTIAL INTERACTIONS IN
THINGS LIKE YEAST 2 HYBRID
SCREENS WHICH WERE EXTREMELY
VALUABLE AND LED TO GREAT
INSIGHTS BUT ALSO LED TO A LOT
OF FALSE POSITIVES IF YOU WILL.
AND WE WENT VERY QUICKLY BECAUSE
THESE ARE VERY LARGE MOLECULES.
ABOUT 2,500 AMINO ACIDS EACH.
IT WENT FROM A SHORT PERIOD OF
TIME WHERE THERE WAS NO CLUE
WHAT THEY DID TO HAVING HUNDREDS
OF MOLECULES THEY COULD
POTENTIALLY INTERACT WITH AND
WHAT THAT DID -- THIS IS OUR
LAB'S VERSION OF SLIDING THROUGH
HOW THE COREPRESSOR INTERACTS
WITH THE NUCLEAR RECEPTOR
THROUGH THE CORNER PEPTIDES AND
THEN THE COMPLEX.
AND THAT'S WHAT YOU'LL FIND IN
THIS COM PENDIUM OF PAPERS.
IT WAS MAT'S WORK THAT I WILL
HIGHLIGHTS NOW.
WHAT MAT DID WAS TO PURIFY THE
COMPLEX OF SMART, ONE OF THESE
TWO COREPRESSOR MOW TEENS WHAT
HE FOUND WAS A VERY TIGHT
COMPLEX WE REFER TO AS THE CORE
COMPLEX CONTAINING A COUPLE OF
INTERESTING MOLECULES I WON'T GO
INTO IN DETAIL.
BUT CONTAINED A SINGLE ACETYLASE
AND SPECIFICALLY HISTONE
ACETYLASE 3 AND IN ABUNDANCE.
AS I ALREADY MENTIONED, THIS IS
THE REGULATOR OF EPIGENOMIC
STRUCTURE AND EVENTUALLY GENE
EXPRESSION.
AND ANOTHER THING THAT MATTE
DISCOVERED SURPRISED US BECAUSE
WE WERE THINKING ALL ALONG, I
THINK IT'S LARGELY TRUE THE
DOCKING PROTEINS, COREPRESSORS
SERVE AS DOCKING PROTEINS THAT
DELIVER THESE ENZYMES AND OTHER
INTERESTING AND IMPORTANT
MOLECULES TO TARGET THE GENOME T
TURNED OUT THAT ACTUALLY HDAC3,
THE POLYPEPTIDE HDAC3 DID NOT
HAVE OPENER HAS INTRINSIC
ACTIVITY BUT YOU COULDN'T SEE
ITS ACTIVITY IF YOU PURIFIED IT
FROM E.COLI OR FROM INSECT CELLS
OR MADE IT.
YOU DIDN'T SEE ANY ENZYME
ACTIVITY UNLESS YOU INCUBATED IT
WITH THE CO-REPRESSOR AND THAT
WAS ABLE TO REDUCE THIS TO A 90
AMINO ACID SEGMENT THAT CONTAINS
THE SO-CALLED MOTIF WHICH SOME
IN THE AUDIENCE KNOW WHAT IT IS.
IT IS A MOTIF FOUND IN A LOT OF
TRANSCRIPTION FACTORS, 50 AMINO
ACIDS.
IT'S NECESSARY BUT NOT SAFE.
THERE IS -- SUFFICIENT -- THIS
IS ABOUT A 90 AMINO ACID
PEPTIDE, MUCH SMALLER THAN THE
2,500 IN THE COREPRESSOR.
AND UT THIS 90 SENECESSARY AND
SUFFICIENT FOR BIND HAVE GONE
HDAC3 AND FOR IMPARTING AND
ACTIVATING CATALYTIC ACTIVITIES.
SO IN OTHER WORDS, HDAC1 WAS
DISCOVERED AND BY INSILL COAND
OTHER MOLECULAR TECHNOLOGY, THEY
SAY IT WAS FOUND BY THE SEQUENCE
HOMOLOGY.
BUT IF WE PURIFIED HDAC3 IN THE
CLASSIC WAY, WE WOULD HAVE
PURIFIED THIS COMPLEX.
BECAUSE THE POLYPEPTIDE BY
ITSELF IS REALLY EXACTLY.
SO, WE TOOK ADVANTAGE OF THIS BY
STARTING TO STUDY WHAT HAPPENS
IF NCOR CANNOT INTERACT WITH
HDAC3.
THIS IS 3 PEOPLE IN THE LAB WHO
DID ALL THE CELL WORK AND THEN
KNOCKED IN THE MUTATION THAT WAS
FOUND TO ABROGATE THE BINDING
ACTIVATION OF HDAC THREE INTO
THESE MICE THAT CAN UNDERSTAND
IN-VIVO IMPORTANCE OF THIS
PROTEIN-PROTEIN PARTNERSHIP.
SO WHAT WAS FOUND WAS A POINT
MUTATION IN THAT MOTIF WITHIN
THIS DOMAIN WE CALLED THE
DEACETYLASE ACTIVATING DOMAIN.
IT'S SUFFICIENT NOT ONLY TO
BLOCK HDAC3 INTERACTION, WHICH
I'M NOT EVEN SHOWING YOU ON THIS
SLIDE, BUT NOW WE ARE LOOKING AT
THE HISTONE DOE ACETYLASE ENZYME
ACTIVITY OF A COMPLEX CONTAINING
WILD TYPE NCOR AND THIS MUTANT
EXPRESSED IN EQUAL AMOUNTS.
AND YOU CAN SEE THERE IS ALMOST
BASELINE AMOUNTS SUGGESTING THAT
NOT ONLY IS THIS NOT BINDING
HDAC3 BUT THAT IT IS BY FAR THE
MOST IMPORTANT HISTONE ACETYLASE
IN THIS COMPLEX.
SO THERESA MADE MICE WHICH VERY
IMPORTANT FOR US TO DO SO
BECAUSE THE NCOR NULL MUTATION
IS EM BRIE ONICALLY DONE BY JEFF
AND HIS COLLEAGUES.
AND THESE MICE IN THE CT7 PLAQUE
6 BACKGROUND, THEY LOOKED
COMPLETELY NORMAL.
AND WHAT THAT TOLD US WAS THAT
NCOR IS REQUIRED FOR LIFE, AND
SO IS HDAC3 AS WE HAVE SHOWN,
THEY ARE ALL BOTH REQUIRED THAT.
SAYS THAT IT IS COMPLICATED AND
THAT THERE IS OTHER
PARTNERSHIPS.
TRACKS WE KNOW THAT HDAC3
INTERACTS WITH SMART.
AND WE NONE THIS EXPERIMENT, IT
MUST BE THAT THESE OTHER DOMAINS
SERVED TO CROSS SPECIES ARE
CRITICAL FOR THE SURVIVAL.
IN MUTANT MICE, THE NCOR IS
EXPRESSED AT NORMAL LEVELS AND
DOESN'T INTERACT WITH HDAC3 AND
THAT AFFORDED US THE OPPORTUNITY
TO ASK THE QUESTION FOR THE
FIRST TIME, IS THERE A ROLE OF
THIS COREPRESSOR COMPLEX?
BECAUSE THIS IS A MUTATION OF
NCOR BUT HERE IT SAY MORE
SPECIFIC QUESTION.
IT'S THE HDAC3 INTERACTION AND
SHE FOUND THERE WAS A AFFECT ON
JUST -- I'M JUST GOING TO
SUMMARIZE THE HIGHLIGHTS AT THE
DESCRIPTIVE LEVEL OF THE PAPER
SHOWN HERE SO I CAN GET INTO
MORE NEW DIRECTIONS AND START
SLAYING SOME OF THE EARLIER
TERMINOLOGY IN CIRCADIAN BIOLOGY
AS I MENTIONED.
WHAT SHE FOUND IN THIS
EXPERIMENT WAS SHE TOOK OUT THE
INTERACTIONS SPECIFICALLY
BETWEEN NCOR AND THE HDAC3 AND
CHANGES IN BIOLOGICAL RHYTHMMS.
BIOLOGICAL, THE CHANGE IN THE
BIOLOGICAL RHYTHM SARAH PALIN A
CHANGE IN THE FREE-RUNNING
PERIOD, BEHAVIORAL ACTIVITIES,
THAT IS THE INHERENT CIRCADIAN
CLOCK OF THESE MICE.
AND THE REASON THAT WE EVEN
THOUGHT TO LOOK AT THAT IS
BECAUSE A COUPLE OF YEARS
BEFORE, SCHINDLER AND HIS
COLLEAGUES IN JANIVA
IMPLICATED -- I DON'T KNOW HOW
YOU CAN SEE THIS.
THIS IS TAKEN FROM A ARTICLE,
THAT THIS IS ONE OF TWO NEGATIVE
LIMBS IN THE CIRCADIAN CLOCK
INVOLVING A POSITIVE LIMB LED BY
BMOL1 FORMING A HETERODIMER WITH
CLOCK, ANDç REGULATING A POSITIVE
WAY THE GENE EXPRESSION OF MANY
GENES WE THINK OF AS BEING
REGULATED IN A CIRCADIAN MANNER
AND EVEN IN THE TISSUE.
AND ESPECIALLY IN THE NUCLEUS
WITH THE HYPOTHALAMUS.
BUT ONE OF THE GENES THAT THEY
REGULATE IS REVERB AND THAT
BEING A REPRESSOR BINDS TO THE
BMOL PROMOTOR SOMETHING WE
DEMONSTRATED IN MY LAB.
AND IT IS PERFECT FOR THIS
BECAUSE IT'S A NUCLEAR RECEPTOR
WE HAVE BEEN STUDYING FOR QUITE
SOMETIME AND IT LACKS A C
TERMINUS THAT AGAIN NUCLEAR
RECEPTOR WILL KNOW IS CRITICAL
FOR ACTIVATION OF TRANSCRIPTION
BY LIGAND.
IT BINDS NCOR AT HDAC3 AND LATER
IT IS SHOWN THAT NOT ONLY WAS
NCOR REQUIRED, BUT THAT THE
INTERACTION WAS REQUIRED
IN-VITRO AND TURNED OUT TO BE
THE CASE IN-VIVO AS WELL.
AND I WANT TO - EYE DON'T HAVE
TIME TO GO INTO IT BUT THIS
WHOLE PROCESS IS STABILIZED
FUNCTIONING AS A LIGAND.
THIS HAS SOME IMPLICATIONS WHICH
I AM HAPPY TO TALK ABOUT FOR
METABOLIC POINT OF VIEW BUT
BECAUSE OF TIME I'M NOT GOING TO
EMPHASIZE THAT HERE TODAY.
I WANTED TO EMPHASIZE THAT THE
CIRCADIAN PHENOTYPE OF THESE
MICE WAS MORE OR LESS EXPLAINED
BY THE DEFECT IN REVERB BECAUSE
IT COULD NOT INTERACT WITH
HDAC3.
THE CHANGES IN METABOLISM WE
NOTICED, THEY WERE PROTECTED
FROM INSULIN RESISTENCE AND
HYPERGLYSEMIA AND HIGH FAT DIET.
THAT WAS HARD TORE EXPLAIN.
WE ORIGINALLY FOCUSED ON ADIPOSE
TISSUE BECAUSE OUR LAB IS
FOCUSEFOCUSED ON THAT.
WHEN WE DID MICROARRAY STUDIES,
WE FOUND VERY LITTLE CHANGE IN
GENE EXPRESSION IN THE ADIPOSE
TISSUE WHEREAS THE LIVER SAW
SOME MAJOR CHANGE THAT IS ARE
AGAIN SUMMARIZED HERE.
I'M NOT GOING TO GO THROUGH THEM
IN DETAIL BUT WHAT I WANT TO DO
IS NOW FOCUS ON HOW IS THIS
OCCUR SOMETHING WHAT IS HDAC3
DOING WITH LIVER TO EFFECT
HEPATIC METABOLISM?
THERE ARE A LOT OF POTENTIAL
TARGETS.
THE REVERSE BECAUSE OF THIS, AND
IT HAS OTHER TARGETS, INCLUDING
INVOLVED IN GLUCOSE AND LIPID
METABOLISM, BUT ALSO ALL THE
OTHER NUCLEAR RECEPTORS AND
PPARs AND LXR AND THYROID
HORMONE RECEPTOR BUT ANY NUCLEAR
RECEPTOR THAT CAN INTERACT WITH
NCOR OR SMART AND HDAC3 IS A
POTENTIAL HERE AND THERE ARE
ALSO A BUNCH OF OTHER
TRANSCRIPTION FACTORS THAT HAVE
BEEN IMPLICATED AS POTENTIAL
INTERACTORS.
SO YOU CAN THINK OF THIS AS A
PROBLEM OF SYSTEMS BIOLOGY
BECAUSE YOU YOU HAVE THE SYSTEM
OF THIS COMPLEX THAT CAN
INTERACT WITH MANY DIFFERENT
TRANSCRIPTION FACTORS.
WE HAVE A PHENOTYPE PRESCRIBING
TO THE COMPLEX AND WE WANT TO
DECONSO LAWSUIT IT AND
UNDERSTAND THE INDIVIDUAL
COMPONENTS AND INTERACTORS IN
THE HOPE OF UNDERSTANDING FOR
EXAMPLE, WE THINK THE CIRCADIAN
CHANGES LAUNCH THE REVERB BUT
HOPEFULLY WE CAN FIND OTHER
CHANGES AND THEREFORE IMPART
MORE SPECIFICITY AND UNDERSTAND
THE PROCESS BETTER.
SO TO GO ABOUT THIS, WHAT WE
DECIDED TO DO, AND THIS IS
LARGELY THE WORK OF THE
LABORATORY, A GRADUATE STUDENT,
IS TO DETERMINE THE SISTRUM OF
HDAC IN THE LIVER.
AGAIN, ALL THE BINDING SITES OF
HDAC3 IN THE LIVER.
AND THIS TECHNOLOGY I THINK IS
HUGELY WELL-KNOWN HERE.
THE WORK OF MANY HISTONE
MODIFYING ENZYMES, GORDON WHO IS
PIONEERED NOT ONLY THE NUCLEAR
RECEPTORS, CHIP ASSAYS WITH ALSO
HYPERSENSITIVITY ASSAYS AND SO
ON, WHICH USE SIMILAR
TECHNOLOGIES.
FOR THOSE WHO HAVE NOT SEEN THIS
BEFORE, IN THE BEAUTY OF THIS,
AS YOU LOOK AT THE ENDOGENOUS
MOLECULES AND THEIR ENDOGENOUS
SETTING WITH THE NATURAL GENOME
OF THE TISSUE OR CELLS THAT
YOU'RE INTERESTED IN, HERE WE
ARE USING THE MOUSE LIVER.
AND IT IS THAT YOU CHOP UP THE
DNA IN ONE WAY OR THE OTHER, AND
PRECIPITATE IT WITH A SPECIFIC
ANTICIPATE BODY AND MANY
LABORATORIES INTERROGATE VERY
SPECIFIC REGIONS OF THE GENOME
BY SOMETHING THAT CAN BE CALLED
CHROMATIN IP, CHIP, PCR WHERE
STEADY QUANTITATIVELY ORmy
QUALITATIVELY, USING SPECIFIC
PRIMERS, YOU INTERROGATE THE
GENOME.
YOU FIGURE OUT IF YOUR FACTOR
WAS THERE OR NOT.
WELL, THE ADVENT OF DEEP
SEQUENCING, WE CAN NOW DO CHIP
SEEK AND IN THIS CASE, INSTEAD
OF DOING A BIASED ANALYSIS AND
LOOKING AT THE SITES YOU'RE
INTERESTEDIN, YOU CAN LOOK AT
GENOME-WIDE AND THAT IS WHY THIS
IS CALLED SIS STROMICS.
AND THIS REQUIRES CHEAP DEEP
SEQUENCING WHICH IS NOW
POSSIBLE, ABOUT AS -- NOT AS
CHEEK AS WE LIKE BUT OUTSTANDING
BIOINFORMATICS.
NOW, I DEBATED INCLUDING THIS
BECAUSE IT IS SUCH A GENERAL
AUDIENCES AND IT'S A DETAIL.
BUT I THINK IT'S REALLY
IMPORTANT TO POINT OUT HOW MUCH
TO REALLY GET GENOME-WIDE DATA,
THAT'S GREAT.
BUT IF IT'S BAD DATA, BAD DATA
IS BAD DATA.
AND IT'S VERY IMPORTANT TO BE
CERTAIN, ESPECIALLY BECAUSE
THERE IS SO MUCH TIME AND EFFORT
SPENT IN ANALYZING THESE DATA.
AND DIFFERENT WAYS TO BEEN THIS,
ONE OF THE WAYS TO SAY THAT
WHATEVER THE SIGNAL YOU'RE
SEEING YOU WANT TO ASCRIBE
SHOULDN'T BE THERE IF YOU'RE
FACTOR IS NOT THERE.
WHAT WE TRY TO DO IS AS MUCH AS
POSSIBLE.
THIS IS AN EXAMPLE HERE TO
CONFIRM THE SPECIFICITY OF THE
CHIP ANTIBODY.
THIS IS THE WORK OF -- AS WE
HAVE MADE MICE THAT HAVE THE
HDAC3 ALLELE ON BOTH ALLELES.
WE CAN INFECT OR INJECT THESE
MICE WITH ADD NO ASSOCIATED
VIRUS.
CRE GOES INTO THE LIVER AND THAT
TAKES OUT HDAC3 IN THE LIVER.
WE DID LOOK AT THE CHIP
EXPERIMENT BIOCHIP PCR.
HERE IS SITES THAT WE FOUND BY
CHIP-SEQ WERE DUE TO HDAC3.
YOU CAN SEE THE SIGNAL GOES
AWAY.
THESE ARE JUST CONTROLLED UP.
THIS HAS FORMATTED WRONG.
THIS IS INSULIN, NOT EXPRESSED
IN THE LIVER, NO BINDING.
YOU CAN SEE THE SIGNAL GOES AWAY
AND THAT REASSURES US THAT WHEN
I TELL YOU THERE IS A SIGNAL, IT
REALLY IS BECAUSE OF HDAC3.
OKAY.
SO WHAT DID DANNY FIND?
SHE FIRST EXPERIMENTS AND THE
WAY THE CHIP-SEQ WORKS IS YOU
GET A BUNCH OF DNA, 36-40 BASE
PAIRS AND HERE YOU CAN SEE OR
BASES.
AND HERE YOU CAN SEE SHE GOT
ABOUT 28 MILLION LEADS.
AND THEN IT USED TO BE
DECONVOLUTED INTO PEAKS.
AND HOW YOU CALL YOUR PEAKS, IT
DEPENDS ON HOW YOU WANT TO DO
YOUR BIOIN FORMATTICS AND HOW
MANY FALSE POSITIVES AND FALSE
NEGATIVES YOU WANT TO ACCEPT.
HERE WE WANT ONE READ PER
MILLION AND IT HAS TO BE 9 TIMES
OR MORE GATOR THAN THAT OF THE
SURROUNDING DNA.
AND WITH THAT, WE SEE ABOUT
15,000 PEAKS OF BINDING OF
HDAC3.
BUT ONE THING THAT WE NOW ALWAYS
ASK, AND I WOULD SUGGEST THE
PEOPLE IN THE AUDIENCE KNOW IS
WHEN DID YOU DO THE EXPERIMENTS?
DAN DID THIS EXPERIMENT AT 5:00
P.M., WHICH IS ALSO CALLED DT10.
BUT DT0 IS WHEN THE LIGHTS GO ON
AND YOUR MOUSE FACILITATES.
SO THIS IS WHEN THE MICE ARE
GENERALLY NOT ACTIVE AND NOT
EATING.
AND WE PICTURE THIS FOR A
REASON.
THAT'S BECAUSE WE KNEW THAT
REVERB, NUCLEAR RECEPTOR IS
RECRUITING THIS HDAC3 AND IT HAS
A REALLY DYNAMIC CIRCADIAN
RHYTHM AND IT'S ALMOST GONE AT
5 A.M. OR CT22.
AND SO, ORIGINALLY, CONSIDERED
IN THESE EXPERIMENTS, THE NOTION
WAS, AS I IMPLIED EARLIER, THAT
WE WOULD KNOW THAT OR IMAGINE
AND WE TRY TO FIGURE OUT WHICH
OF THE SITES OF HDAC3 BINDING AT
THIS TIME POINT WERE DUE TO
REVERB AND BY LOOKING AT 5 A.M.
WHEN THERE IS LOW REVERB AROUND,
WE COULD INFER SOME OF THE SITES
WE SEE HERE BUT NOT HERE WERE
DUE TO OTHER FACTORS AND BY
DOING SIMILAR TYPES OF ANALYSIS,
WE COULD START ASCRIBING PEAKS
TO MATCH UPS BETWEEN THE FACTOR
AND THE COREPRESSOR COMPLEX.
AS I MENTIONED, OTHER
TRANSCRIPTION FACTORS POSSIBLE
AS WELL.
BUT THE BIGGEST SURPRISE WE GOT
OUT OF ALL OF THIS IS THAT WHEN
DAN DID THIS ANALYSIS, AT
5 A.M., THEN THERE IS NO REVERB
AROUND BUT WE STILL EXPECTED TO
SEE ALL THE OTHER PEAKS
ASSOCIATED WITH ALL THE OTHER
SITES.
HE BASICALLY FOUND ALMOST NO
SPECIFIC BINDING OF HDAC3 TO THE
GENOME.
WHEN YOU LOOK AT IT IN TERMS OF
WHAT THOSE SITES WERE, AND THIS
IS A BIGGER CIRCLE THAN IT
SHOULD BE TO SCALE, SO WE CAN
ALL SEE IT.
THEY OVERLAP BUT IT'S 100 OUT OF
15,000.
NOW, THAT'S BIOINFORMATIC
ANALYSIS.
HOW CAN WE BE SURE?
FIRST OF ALL, IT'S NOT BECAUSE
HDAC3 IS GONE AT 5:00 A.M.
THERE IS PLENTY THERE.
SO WHAT HE DID WAS TO, THIS IS
TO PUT THE DATA IN THE FORM OF A
HEAT MAP, THIS IS ALL THE SITES
WHERE HDAC IS BOUND TO THE
GENOME AT THIS TIME OF DAY.
ALL THESE 15,000 SITES AND WHAT
YOU'RE SEEING IS WHEN IT'S A
COLOR OF RED OR WHITE, THERE IS
BINDING.
SO THIS IS NOT SURPRISING BUT
THIS IS WHAT CONTRIBUTED TO THIS
BLUE CIRCLE AND THE QUESTION IS,
IF YOU LOOK AT THESE SAME SITES
AT 5 A.M., YOU CAN SEE THEY ARE
EITHER GONE OR REALLY ATTENUATED
QUITE A BIT.
IF YOU LOOK AT IT IN A DIFFERENT
WAY AND YOU SAY, OKAY, I JUST
WANT TO KNOW WHAT THE AVERAGE
SIGNAL WAS AT THE CENTER OF
THESE, OF COURSE IT WAS VERY
HIGH AT 5:00 P.M. BUT AT 5 A.M.
IT WAS ALMOST -- IT WAS MUCH
ATTENUATED.
NOW, I THINK BECAUSE OF TIME,
I'M GOING TO SKIP THIS.
BUT THIS RHYTHM OF HDAC BINDING
TO GENOME IS MAINTAINED IN
CONSTANT DARKNESS AND ALSO CAN
BE INVERTED IF THE MICE ARE
PROVIDED WITH FOOD DURING THE
TIME WHEN THEY WOULD NORMALLY BE
INACTIVE.
SO I'M NOT GOING TO TAKE YOU
THROUGH THAT BUT THAT'S THE
CONCLUSION OF THESE TWO SLIDES.
YOU KNOW, IT WAS SURPRISING TO
US TO SEE SUCH A GREAT
ATTENUATION OF THE SIGNAL AT
5 A.M., THE MOST OBVIOUS
EXPLANATION IS REVERB WAS MORE
IMPORTANT THAN WE IMAGINED IT
WOULD BE FOR BRINGING HDAC GENES
IN THE LIVER.
TO ADDRESS THIS, WE DID THE
FOLLOWING.
FIRST WE LOOKED
BIOINFORMATICALLY UNDER THOSE
15,000 PEAKS AND WE SOUGHT SITE,
THE NUCLEAR RECEPTOR HALF SITE.
SECONDLY, WE PERFORMED CHIP SEEK
ANALYSIS FOR REVERB AND WHAT WE
FOUND WAS A REMARKABLE OVERLAP
SUCH THAT ABOUT 90% OF THE HDAC2
BINDING SITES OVERLAPPED RIGHT
ON TOP AND I'LL SHOW IF YOU A
MINUTE AN EXAMPLE OF THIS, ON
TOP OF THE REVERB BINDING SITES.
AND FINALLY, USING REVERB
KNOCKOUT MICE WE ATTAINED FROM
STOCKHOLM AND THEN BREAD TO 10
GENERATIONS ON BLACK 6 MICE TO
BE COMPARABLE TO THE OTHER
EXPERIMENTS, WHAT WE FIND WAS
SUMMARIZED IN THIS BLUE BOX HERE
AND FOR ALL SORTS OF GENES, IS
THAT WE LOSE THE LARGELY LOSE
THE RHYTHM THAT WE SEE BETWEEN
5:00 P.M. AND 5:00 A.M.
SUGGESTING THAT INDEED IT IS
REVERB THAT IS RECRUITING HDAC3
TO THE MAJORITY OF THESE SITES.
WHAT ABOUT NCOR?
THE AS I SET THIS UP FOR YOU, IT
WASIN BETWEEN SO IT TOOK US A
WHILE BUT WE FINALLY GOT AN
ANTIBODY THAT COULD GIVE US A
CHIP THAT WE BELIEVE FOR NCO R
AND WHAT LOGAN EVERETT, A
POSTDOC IN THE LAB HAS DONE
HERE, PERFORMED AN ANALYSIS
WHERE WHERE WE ARE LOOKING ON
THE Y AXEIS AND X AXIS A SIGNAL
OF NCOR.
WHAT IS APPARENT IS THAT THIS IS
REALLY VERY CLOSE TO THE UNITY
LINE SUGGESTING THAT THE BINDING
OF NCOR AND HDAC3 ARE REALLY
HIGHLY CORRELATED.
AND THE SITES LOOKING AT HERE
ARE ALL THE THE REVERB BINDING
SITES.
AND WHAT LOGAN DID WAS TO
COLOR-CODE REVERB BINDING BY A
STRENGTH OF BINDING GIVING A
THIRD DIMENSION AND SO THESE ARE
THE LOWEST SITES.
THESE ARE THE HIGHEST IN GREEN
AND THEN YELLOW AND RED ARE LESS
WELL SEPARATE AND WE CAN STILL
SEE, I THINK PRETTY CLEAR
SEPARATION THERE.
AND WHAT THIS TELLS US IS THERE
IS A CORRELATION BETWEEN THE
BINDING STRENGTH OF
REVERB -- NCOR AND THE HDAC3 BUT
ALSO OF REVERB.
I KNOW YOU HAVE REALLY
OUTSTANDING MODELERS HERE.
WE DO ALSO.
WE ARE ABLE TO MOD THEY'LL DATA.
FIRST I WANT TO SHOW YOU THAT
THESE POINTS ARE THE WAY SHOULD
BE TAKEN OUT BECAUSE THIS IS
WHERE THE INSPUT GREATER THAN
THE SIGNAL.
HERE IS THE SOPHISTICATED
MODELING ANALYSIS WE PERFORMED
WHICH I THINK SPEAKS VOLUMES.
SO, WHAT I HAVE SHOWN YOU SO FAR
IS THAT UNEXPECTEDLY HDAC3
REALLY HAD A DRAMATICALLY
DIFFERENT RECRUITMENT TO THE
GENOME AT 5:00 P.M. WHERE WE SAW
15,000 SITES AND AT 5 A.M. WHERE
WE BARELY SAW IT ON THE GENOME
AT ALL.
NOW WE WANT TO KNOW WHAT IT IS
DOING.
TAKING IT FROM INTEGRATED
PHYSIOLOGY.
WE WANT TO UNDERSTAND THE
PHYSIOLOGICAL FUNCTION OF THIS.
TO GET THIS, WE WANTED TO KNOW A
MORE BIOCHEMICAL QUESTION.
IS IT CAUSING HISTONE
DEACETYLATION?
AND THAT IS AN IMPORTANT POINT
BECAUSE THERE HAS BEEN QUESTION
ABOUT THIS.
CASEY AND COLLEAGUES HERE AT THE
NIH HAVE SHOWN VERY ELEGANTLY
THAT HISTONE DEACETYLASES
COUNTER INTUITIVELY ARE
ASSOCIATED WITH ACTIVELY
EXPRESSED GENES.
AND WE SEE THE SAME THING HERE.
THERE ARE ASSOCIATED WITH GENES
THAT ARE ON THE CELL BUT WHAT WE
THINK WE HAVE UNCOVERED IS THAT
THEY HAVE TO BE ON BECAUSE
OTHERWISE THEY COULDN'T BE
MODULATED.
GENES THAT ARE OFF CONTINUOUSLY
DON'T NEED TO BE MODULATED.
AND WHEN YOU CAN SEE THIS IS
PLAYING A ROLE IS BY LOOKING
DEEPLY INTO DEEP SEQUENCING AND
HISTONE ACETYLATION.
SO WHAT DAN DID WAS TO PERFORM
CHIP-SEQ FOR HISTONE H3 LYSINE 9
AND I DON'T THINK YOU CAN SEE IT
TOO WELL.
YOU WILL HAVE TO TAKE MY WORD
FOR THIS.
BUT AT CT22 WHEN THERE IS AT
5 A.M. VERY LITTLE REVERB OF
HDAC3 THERE AND THERE IS MORE
RED HERE AT THESE SITES.
AND THERE IS LESS RED HERE.
SO THERE IS LESS HISTONE
ACETYLATION AT THE TIME OF HDAC3
IS PRESENT, WHICH FITS THIS
NOTION THAT HDAC3 IS CAUSING
HISTONE DEACETYLATION.
BUT IT'S JUST A CORRELATION.
IT COULD BE THE OTHER WAY
AROUND.
IT COULD BE THAT HAVING LESS
HISTONE ACETYLATION RECRUITS
HDAC3.
THANKFULLY, ALTHOUGH CAUSE AND
EFFECT CAN BE TOUGH IN THIS
PARTICULAR TYPE OF ANALYSIS,
HERE WE HAVE TO OUR USE, THE
MICE IN WHICH WE TAKE OUT HDAC
IN THE LIVER.
WHEN HE DOES THIS AND PROVIDES
THE SAMPLES AFTER A WEEK, SHE
THEN LOOKS -- AND THIS IS TO
TAKE OUT THE HDAC3 AND LOOKS AT
HIS PHONE ACETYLATION IN THE
KNOCK OUT AT 5:00 P.M. AND NOW
IT LOOKS LIKE 5 A.M., NOT 5:00
P.M.
AND I THINK I'LL SHOW YOU THIS.
IT IS MORE GENETICALLY SHOWN
HERE.
IT'S COMPLICATED.
YOU HAVE TO LOOK AT THE
DIFFERENCES OF THESE CURVING.
HERE IS 5:00 P.M. AND 5:00 A.M.
YOU CAN SEE THAT AT 5:00 P.M.,
THE HDAC3 KNOCKOUT IS MORE
HISTONE ACETYLATION THAN AT 5:00
A.M.
AND THIS WILL JUST TELL US THAT
SUMMARIZED HERE IN CARTOON FORM,
THAT AT 5:00 P.M., WHEN REVERB
IS BRINGING NCOR AND HDAC3 TO
THESE GENES, THERE IS LESS
ACETYLATION THAN AT 5 A.M. WHEN
THEY ARE NOT THERE.
THE NOTION WOULD BE THAT THIS IS
HAVING AN AFFECT ON
TRANSCRIPTION.
THE MEDIATOR OF MOST OF YOU
CARRY OUT GENE EXPRESSION IS
POLYMERASE TWO.
AND I THINK YOU CAN SEE HERE
THAT THESE GENES THAT ARE BOUND
BY HDAC3 AT 5:00 P.M., THERE IS
MUCH LESS RNA POLYMERASE.
AND WHEN HDAC3 AND REVERB ARE
KNOCKED OUT, THERE IS MORE
POLYMERASE AND THAT'S SUMMARIZED
HERE.
AND THEN WE WENT BACK TO THE
BIAS IN WHICH WE KNOCKED OUT
HDAC3 IN THE LIVER AND LOOKED AT
GENE EXPRESSION BY MICROARRAY
ANALYSIS OR TRANSCRIPTOMICS AND
LOOKED AT THE GENES THAT WERE
EITHER ALL THE GENES IN THE
MICROARRAY.
THE GENES THAT WERE DOWN
REGULATED, OR THE GENES UP
REGULATE AND YOU HAD CAN SEE
IT'S ALL IN THE GENES THAT ARE
UP REGULATED THAT HAVE DRAMATIC
ENRICHMENTS FOR BINDING SITES
FOR HDAC3.
SO, THAT FILLS IN THIS PIECE OF
THE PUZZEL AND TAKEN TOGETHER,
THE CONCLUSION IS PROFOUND IN A
WAY THAT IT IS THE RECRUITMENT
OF HDAC3 THAT IS ON A DAILY
BASIS CAUSING REMODELING OF THE
EPIGENOME AND CHANGES IN GENE
EXPRESSION.
SO JUST TO SHOW YOU WHAT THIS
LOOKS LIKE ON ONE TRACK, THIS IS
OUR FAVORITE GENE.
THIS IS REVERB BINDING SO THE
HEIGHT OF THE PEAKS ARE HERE,
PROBABLY MULTIPLE BINDING SITES.
BUT THE HEIGHT OF THE PEAK HERE
IS PROPORTIONATE TO THE BINDING
STRENGTH OR THE NUMBER OF
SIGNALS WE GET.
AND YOU CAN SEE, IT IS THERE AT
CD10 NOT AT 5 A.M., NOT A BIG
SURPRISE BECAUSE THERE IS HARDLY
ANY REVERB IN THE CELL AT 5:00
A.M.
BUT PLENTY OF NCOR.
NCOR HAS NOT CHANGED AND IT'S
RECRUITMENT IS SPECIFIC TO THE
REVERB SITE AND IT IS ALSO ONLY
AT 5:00 P.M.
AND HDAC3, WHICH I DID SHOW YOU
DOES NOT CHANGE IN ITS AMOUNT,
IS ALSO, THIS IS THAT DRAMATIC
RECRUITMENT I WAS TALKING ABOUT.
SO HERE IS THE BHAL GENE, THIS
IS HAPPENING AT 15,000 SITES IN
THE GENOME.
HERE IS WHAT THE HISTONE
ACETYLATION LOOKS LIKE.
IT'S COMPLICATED BECAUSE THERE
IS A CLEARING OF MORE
NUCLEOSOME-FREE REGION HERE.
THE SIDES HAVE MORE HISTONE
ACETYLATION THERE IS EVEN MORE
IN THE CENTER CONSISTENT WITH
THE NOTION WHEN HDAC3 IS THERE,
LIKE THERE IS LESS HISTONE
ACETYLATION.
AND THE PAUL 2 BEING CORRELATING
WITH THE LACK OF HISTONE
ACETYLATION IS ACTUALLY VERY
VALUABLE BECAUSE YOU MIGHT
THINK, DESPITE EVERYTHING I DID
TO TRY TO CONVINCE YOU THESE
WERE NOT FACT ULUAL DATA, WE ARE
JUST NOT ABLE TO DO CHIP VERY
WELL AT 5:00 A.M.
MAYBE SHE IS JUST NOT ON HER
GAME AT 5 A.M. LIKE 5:00 P.M.
BUT IT'S THE SAME SAMPLE AND
ANOTHER ANTIBODY AND YOU CAN SEE
THERE IS A SIGNAL HERE AND NOT
HERE.
SO THAT'S NOT IT.
IT'S REALLY A BIOLOGICAL RESULT.
SO FINALLY, AS I ALLUDED TO, WE
WANT TO KNOW THE FUNCTION.
WE WANT TO DECONVOLUTE THIS IN
TERMS OF WHAT FACTOR IS
RECRUITING THE COPREPRESSOR AND
THE ROLE OF HISTONE ACETYLATION
AND SO ON.
WHAT IS THE ROLE IN BIOLOGY.
AND I HAVE TO EVENTUALLY LINK IT
TO LIPID METABOLISM BECAUSE I
STARTED BY TELLING YOU WE WERE
GOING TO EXPLAIN THE PHENOMENON
OF INCREASED LYPOGENESIS DURING
THE NIGHT IN RODENTS.
THE WAY WE GOT AT THIS IS ALSO
BIOINFORMATIC APPROACH.
FIRST WE LOOKED TO THE GENES
DOWN BY BOTH HDAC3 AND REVERB AT
5:00 P.M. AND YOU CAN SEE THESE
ARE METABOLIC PROCESSES.
BUT WHEN YOU EVEN ADD IN ANOTHER
MUCH MORE STRINGENT AND
BIOLOGICALLY RELEVANT CRITERION,
THE GENE IS UP REGULATED A WEEK
AFTER WE KNOCK OUT HDAC3 IN THE
LIVER AS WELL AS HAVING THE
BINDING SITE, LIPID MEMETABOLISM
JUMPS TO THE TOP.
SO WE HAD A PREPARED BIND FOR
LIPID METABOLISM PHENOTYPE UP
AND THAT'S WHAT I'M GOING SHOW
YOU.
I'M LOOKING AT THIS IN A COUPLE
OF DIFFERENT WAYS.
THIS IS TAKEN USING A TRANSGENIC
APPROACH IN WHICH WE MATED CROSS
TO MICE OR HDAC3 WITH MICE THAT
EXPRESSED THE CLEAR RECOM NAS
OFF THE ALBUMIN, LIVER SPECIFIC
BUT BEGINS IN DEPARTMENT AND
CONTINUES TO ADULTHOOD.
WHEN YOU LOOK AT THESE MICE,
THIS IS WHAT THEY LOOK LIKE AND
REALLY PERCEPTIVE PEOPLE CAN SEE
A BULGE HERE.
THIS IS -- I DON'T KNOW HOW WELL
IT'S PROJECTING.
BUT THERE IS A LARGER LIVER AND
IT IS MORE PAIL.
SO WHEN YOU TAKE IT OUT, IT WAS
CLEAR THAT LOSS OF HDAC3 IN
LIVER CAUSES THE FATTY LIVER.
THIS IS IN 3-MONTH-OLD MICE FED
NORMAL CHOW.
VERY IMPORTANT.
BECAUSE THE DIET IS NORMALLY A
HUGE REGGATIVE FACTOR.
EVEN ON NORMAL CHOW, WE SEE
FATTY LIVER.
THIS COULD BE A DEVELOPMENTAL
PROCESS.
THAT'S WHERE THIS MODEL AGAIN
COME IN VERY HANDY.
BECAUSE WE CAN DO THIS TO THE
ADULTS.
WHICH WE DO THESE EXPERIMENTS
AND TAKES OUT HDAC3, LEAVES THE
MICE ON NORMAL CHOW AND LOOKS
TWO WEEKS LATER AFTER CONTROLS,
WE GOT A GFP VERSUS THE ONES
THAT HAD THE KNOCKOUT, YOU CAN
SEE IT REALLY IS A DRAMATIC
INCREASE IN THE STAINING
STAINING FOR NEUTRAL LIPID IN
THE LIVER.
THIS IS JUST A SECTION.
BUT IF YOU GRIND UP THE LIVER
AND LOOK AT TOTAL TRIGLYCERIDE,
THERE IS A 8-9 FOLD INCREASE.
A COUPLE OF MONTHS ON A HIGH
FIGHT DIET, THEY WOULD
INDIVIDUAL ABOUT A TWO FOLD
INCREASE.
THIS IS A MARKED INCREASE N
COLLABORATION WITH OUR STABLE
ISOTOPE CORE, WE WERE ABLE TO
DEMONSTRATE AN INCREASE IN
DE NOVO ACTIVE GENESIS IN THESE
LIVERS SUGGESTING THAT HDAC3 AND
REVERB IS CONTROLLING THIS AND
ACTUALLY WE THINK REVERB IS
IMPORTANT EVEN IN THIS ASPECT
BECAUSE THE KNOCKOUT HAS A FATTY
LIVER.
A LESSER DEGREE AND ONE OF THE
REASONS IS POTENTIALLY BECAUSE
THERE IS REVERB DATA
COMPENSATING IN THE LAB TESTING
THIS BY LOOKING AT THE BETA AND
DOUBLE KNOCKOUT BUT I DON'T HAVE
THOSE RESULTS YET.
SO WHAT I STARTED DOING WAS SHOW
YOU WE HAVE KNOWN FOR A LONG
TIME THERE IS A CIRCADIAN RHYTHM
OF LYPOGENESIS IN THE LIVER.
THIS IS A COMPUTER VERSIONY TOLD
YOU.
BUT I WANT TO EMPHASIZE THAT IN
THE NIGHT TIME PERIOD, WHEN THEY
ARE ACTIVE AND FEEDING, THEY
HAVE LOW LEVELS OF REVERB, AND
HDAC RECRUITMENT IS LOW AND THAT
IS CORRELATING WITH THE
LYPOGENESIS.
12 HOURS LATER, REVERB LEVEL IS
HIGH.
HDAC RECRUITMENT IS HIGH AND
RECRUITED TO THE GENOME AND
THERE IS REDUCTION IN HISTONE
ACETYLATION.
AND I THINK POTENTIALLY THE MOST
REMARKABLE THING ABOUT THIS,
OTHER THAN A VERY SPECIFIC TEAM
OF GENES GOING TO A -- PROTEINS
GOING TO SPECIFIC, LITTLE OF
SITES IN THE GENOME, IS THAT
THIS IS HAPPENING ON A DAILY
BASIS, DAY AND NIGHT AND DAY AND
NIGHT AND IN FACT, THERE IS A
PHYSIOLOGY CIRCADIAN PHYSIOLOGY
METABOLISM KEEPING LYPOGENESIS
LIKE THIS AND IT'S RELATED TO
OUR FEEDING AND FASTING AND SO
FORTH.
AND IT'S THE ROLE OF THE OR LOSS
OF REVERB THAT BUILDS UP THE
TRIGLYCERIDES IN THE LIVER ONLY
TO FALL DOWN AGAIN WHEN THE
REVERB COMES UP GOES TO THE
GENOME AND BRINGS NCOR AND THE
HDAC3 AND TURNS OFF GENE
EXPRESSION AS WELL AS HAVING THE
OXIDATION AS WELL.
AND IF WE JUST LOOK DAY AND
NIGHT, WE ONLY SEE -- TOTAL LIP
SID JUST LIKE THIS.
BUT IMPORTANCE IS REALLY BROUGHT
OUT IN THE EXPERIMENTS IN WHICH
WE KNOCK OUT HDAC3 AND YOU CAN'T
UNDO THE SYNTHESIS AND IT
CONTINUES UNABATED AND IT REALLY
BECOMES DRAMATICALLY INCREASED
AS I HAVE SHOWN YOU WHICH THIS
SORT OF PHYSIOLOGY BEING COUNTER
MANNEDDED BY THE LOSS OF HDAC IN
THE ARTIFICIAL SETTING, REALLY
SHOWS YOU HOW DEVASTATING THIS
COULD BE IN THE LONG RUN.
SO IN SUMMARY, THIS IS RECRUITED
TO TARGET CHANGE IN THE LIVER
REVERB AND THAT RESULTS IN THE
CIRCADIAN EPIGENOME.
HISTONE MODIFICATION CHANGES AND
THIS MODULATES THE LIPID
METABOLISM.
SO THE TAKE HOME MESSAGE IS
THIS, EVERY DAY, REVERSIBLE
CHANGES IN THE CISTROMES WHERE
HDAC3 IS BINDING AND OTHER
FACTORS ARE BINDING AND
EPIGENOMIC CHANGES ILLUSTRATED
HERE BY HISTONE ACETYLATION THAT
ARE CURRENT THROUGHOUT THE
GENOME, THERE IS NOT A SPECIFIC
CLUSTERING OF THESE 15,000
SITES.
ALTHOUGH IT IS HIGHLY SPECIFIC.
THERE ARE MILLIONS OF POTENTIAL
SITES.
I WOULD SUGGEST THAT IN ADDITION
TO THIS SPECIFIC INTEREST THAT
WE HAVE AND THE UNDERSTANDING OF
THIS AS A PHYSIOLOGIC MECHANISM
UNDERLYING CIRCADIAN METABOLISM,
TRYING TO UNDERSTAND THE
PATHOPHYSIOLOGY OF LIVER DISEASE
AND FATTY LIVER AND SO ON, ABOVE
THAT, THIS WORK SHOWS AS PROOF
OF CONCEPT THAT WE CAN INTEGRATE
THESE OHMICS, AND THE CORE BANDS
METABOLOMICS.
AND PHYSIOLOGIY AND
PATHOPHYSIOLOGY, INTEGRATIVE
PHYSIOLOGY AND ACTUALLY USE IT
TO EXPLORE AND UNDERSTAND THE
ASPECTS OF BIOLOGY THAT WE
DIDN'T KNOW BEFORE.
WE DIDN'T KNOW WHAT REGULATES
THE CIRCADIAN RHYTHM OF LI
ID -- LIPID MEMETABOLISM IN THE
LIVER AND THIS GAVE US THE CLUES 
CLUES.
I'D LIKE TO THANKS THE PEOPLE IN
MY GROUP.
THIS IS A FAIRLY RECENT SHOT OF
THE GROUP AND THE PERSON WHO DID
THE MAJORITY OF CHIP-SEQ WORK, A
TERRIFIC GRADUATE STUDENT IN THE
LABORATORY AND GREAT HELP FROM
ANNA WHO DID THE WORK ON REVERB
KNOCKOUT AND SHANNON, ALONG WITH
THERESA DEVELOPED THE HDAC3, AND
THERESA ALSO DEVELOPED AND DID
THE WORK ON THE NCORHDAC3
INTERACTION MUTATIONS.
BUT I JUST QUICKLY WENT THROUGH
THAT.
A LOT OF THE BASIC WORK WE HAVE
DONE ON THE CIRCADIAN BIOLOGY,
IS LAEI WHO HAS HER OWN
LABORATORY AS ASSISTANT
PROFESSOR AT UNIVERSITY OF
MICHIGAN.
I CAN'T EMPHASIZE ENOUGH HOW
IMPORTANT THE COLLABORATION OF
SHIRLEY WAS ON THE
BIOINFORMATICS.
AND I WANT TO THANK BOB AND
JONATHAN, THE DIRECTOR AND A
GREAT COLLEAGUE AND DIRECTOR OF
OUR FUNCTIONAL GENOMICS CORE OF
OUR PENN DIABETES CENTER, NIDDK
FUNDED AND A LOT OF THE
INFRASTRUCTURE FOR THE WORK IS
PROVIDED BY THE NUCLEAR
SIGNALING ATLAS AND ALL THE WORK
I DESCRIBED WAS FUNDED BY THE
NIDDK AND CELEBRATING ITS
60th ANNIVERSARY.
EVERY DAY THERE IS ANOTHER PARTY
FOR THE NIDDK HERE AND THERE
SHOULD BE BECAUSE IT'S DONE SO
MANY GREAT THINGS.
BUT FOR MY OWN WORK IN
PARTICULAR, NONE OF THIS WOULD
BE POSSIBLE WITHOUT THE GREAT
SUPPORT I RECEIVED FROM THE
NIDDK.
AND FINALLY, FOR THOSE OF YOU
WHO ARE TAKING A TRAIN TO
PHILADELPHIA OR BEYOND, TAKE A
LOOK TO YOUR LEFT AND YOU'LL SEE
OUR BRAND NEW BUILDING.
WE ARE ON THE 12th FLOOR.
FRANCIS COLLINS WAS VISITING US
YESTERDAY EVENING TO THE OPENING
CEREMONY FOR THE NEW BUILDING.
AND SO, ONE THING HE GOT TO SEE,
WHICH I'M GOING TO SHARE WITH
YOU IS THAT -- AND I DON'T THINK
OVERHEAD PERMITTED THIS.
BUT THEY HAD A CAKE IN THE SHAPE
OF THE BUILDING.
AND OUR INSTITUTE FOR DIABETES
AND OBESITY AND METABOLISM IS
HERE.
AND FINALLY, I WANT TO SHOW YOU,
WE JUST LITERALLY MOVED IN 10
DAYS AGO AND THE DAY WE MOVED IN
IT RAINED LIKE IT DID YESTERDAY.
AND WE LOOK OUT AT CENTER CITY
PHILADELPHIA AND HAVE A
FANTASTIC VIEW.
10 DAYS AGO, THIS IS THE VIEW
FROM OUR LABORATORIY AND FROM MY
OFFICE.
AND IT REMINDED ME OF HOW LUCKY
WE ARE TO BE ABLE TO DO SCIENCE
IN THE U.S.
HOW IMPORTANT THE NIH IS THE AND
HOW WE ARE ALL IN SPARED BY MANY
THINGS BUT INCLUDING WHAT I
TRIED TO START THIS WORK ON,
WHICH IS THE IMPORTANCE OF
UNDERSTANDING THE CAUSES OF
OBESITY, DIABETES AND GETTING TO
THE NEXT GENERATION OF
TREATMENTS AND SO I'LL END WITH
THIS FANTASTIC REPORT THAT WAS
LED BY NIDDK AND JUDY AND MANY
OTHERS.
I PLAYED A SMALL PART.
THERE IS A GREAT BLUEPRINT FOR
THE FUTURE.
SO, THANK YOU FOR THE HONOR OF
GIVING THIS LECTURE AND I WOULD
LOVE TO TAKE ANY QUESTIONS.
[APPLAUSE]
>> THAT WAS A LOVELY TALK.
WOULD YOUi] COMMENT ON HDAC3 IN
REGULATION GLYCOGENOSIS OR
REGULATION --
>> YES, MARK, THAT'S A GREAT
QUESTION.
GLUCOSE METABOLISM.
AND ONE THING WHICH WE HAVEN'T
PUBLISHED THAT WE JUST RECENTLY
HAVE THESE DATA SO I HOPE THIS
IS TRUE BUT I THINK IT IS.
WE REALLY, BECAUSE OF THE WORK
OF MANY OUTSTANDING
LABORATORIES, THIS WOULD BE A
GREAT MODEL OF FATTY LIVER
INDUCED INSULIN RESISTINS AND WE
EXPECTED TO SEE ABNORMALITIES OF
INSULIN RESIST EXPENSE IS
DIABETES AND SO ON.
INFACT, OUR METABOLIC
PHENOTYPING CORE DIRECTOR SHOW
THESE MICE HAVE PERFECTLY NORMAL
GLUCOSE AND NORMAL NEWICOSE
OUTPUT AND NOT INSULIN RESISTANT
IN THE LIVER.
SO IT'S SOME OTHER PATHWAY.
THIS IS A MODEL IN WHICH THEY
HAVE ASSOCIATED MAYBE THE
AUDIENCE SOME PEOPLE IN THE
WORLD WHO WOULD NOT LIKE THEM TO
BE ASSOCIATED YOU BUT THIS IS A
CASE WHERE THEY ARE.
>> YOU SHOWED THE GREAT DATA
WITH HDAC RECRUITMENT AND K9
ACETYLATION AND YOU HOW MUCH DO
OTHER CHROMATIN MARKS PLAY A
ROLE AND ESPECIALLY WHEN YOU DO
THE CRE INJECTION, HOW MUCH OF
THOSE OTHER MARKS A FACTOR?
>> THAT'S A GREAT QUESTION ABOUT
OTHER CHROMATIN MARKS.
WE HAVE NOT LOOKED GENOME-WIDE
AND WE ARE THINKING ABOUT IT.
WE ARE ACTUALLY DEBATING, GIVEN
THESE THINGS COST MONEY AND TAKE
TIME AND ANALYSIS, EXACTLY WHAT
THE NEXT THINGS TO DO.
ONE THING LARGELY, WOE MIGHT
PRIORITIZE ABOVE THAT LOOKING AT
HYPERSENSITIVITY AND THEN GO
BACK AND LOOK AT THE MARKS
ASSOCIATED WITH THAT.
>> HOW COULD WE TRANSLATE THIS
IN HUMAN PATIENTS IN WE COULD
LOOK AT THE ANTIBODY USING MRI
AND SO, IS THERE ANYTHING WE
COULD DO TO SIMULATE IT IN HUMAN
PATIENTS AND SEE THIS?
>> IT'S A GREAT QUESTION.
YES.
IT BOILS DOWN TO HOW DO WE THEN
TRANSLATES TO HUMANS?
AND IT'S VERY DIFFICULT TO KNOW
THAT.
EVEN IF THE QUESTION -- IT'S
CLEAR THAT HUMANS, ALTHOUGH I
BUILT THE PREMISE ON THE
NIGHT-SHIFT WORKERS, EVEN THAT
HAS NOT BEEN EXTREMELY WELL
STUDIED.
THERE THE PEOPLE HAVE DONE THAT
WORK HAVE LARGELY USED HEPATIC
ENZYMES.
SO YOU NOT ONLY HAVE FATTY LIVER
BUT YOU HAVE HEPATITIS.
THEY DO GO OFF IN NIGHT-SHIFT
WORKERS.
BUT THE STUDIES ARE POOR AND NOT
WELL CONTROLLED.
SOMEONE NEEDS TO DO THE STUDY
FATTY LIVER IN NIGHT SCHIFF
WORKERS AND THOSE BEING PLANNED.
TO MY KNOWLEDGE, THAT DATA HAS
NOT BEEN REPORTED
[INDISCERNIBLE]
>> AND THEN TO ASK WHETHER THIS
MECHANISM REGULATES IT, IT'S
VERY DIFFICULT.
I DON'T HAVE A NONINVASIVE WAY
TO DO THAT.
AND IT'S SOMETHING WE WOULD LOVE
TO DO AND PEOPLE HAVE
SUGGESTIONS.
BUT FOR NOW, I THINK WE HAVE TO
USE THE MOUSE AS A MODEL.
>> AS PART OF THE INFERENCE
BETWEEN 5 A.M. AND 5:00 P.M.,
HOW MUCH IS DUE TO THE DARKNESS,
DAY -- IF YOU RESTRICT FOOD
DURING NIGHT AND WHAT IS THE
PREDICTION IF WHAT WOULD THAT
BRING?
>> THAT'S A GREAT QUESTION.
DURING THE TIME IN THE NIGHT, IS
ALSO WHEN THEY ARE EATING.
I RUSHED BY IT BUT WHEN WE ONLY
FEED THEM THIS THE DAYTIME, WE
SHIFT THE RHYTHM.
SUCH THAT IT IS THE OPPOSITE OF
WHAT IT WAS WHEN THEY WERE
VOLUNTARILY TAKING 75% OF THEIR
FOOD IN THE DARK, COME IS WHAT
THEY DO WHEN THEY ARE LEFT TO
FREE RUN ON DARK CYCLES.
SO YOU COULD SAY, THAT SHOWS YOU
IT IS REALLY THE FOOD AND NOT
THE CLOCK.
BUT THE REASON IT'S NOT SO CLEAR
IS THAT WE HAVE SHOWN THE LIVER
CLOCK ITSELF ISEN TRAINED BY
FOOD IN PREFERENCE TO LIGHT.
SO WE ACTUALLY TAKE THAT -- IN
GENERAL, THE CIRCADIAN FIELDS
TAKE THAT AS A SIGN OF THE
CLOCK.
BUT THERE WERE STUDIES WE ARE
DOING NOW, FOR EXAMPLE, FASTING
IN MICE, THE PROBLEM THERE IS
THE MICE ARE NOT THE SAME AT THE
BEGINNING AS
THE -- METABOLICALLY AT THE
BEGINNING OR END OF THE FAST.
WE KNOW THERE IS NO
FOOD -- TAKING OUT FOOD AS A
VARIABLE.
SO JUST WAITING FOR THOSE
STUDIES WHICH GET IN FROM
ANOTHER POINT.
>> I WAS JUST WONDERING IN THE
CLINICAL TRIALS FROM THE
PATIENTS WHO RECEIVED HDAC
INHIBITORS, DO YOU SEE
ALTERATIONS IN LIPID PROFILES?
>> THAT'S A GREAT QUESTION.
ONE WE HAVE BEEN INTERESTED IN.
I CAN ONLY TELL YOU THAT IF YOU
DO A PUBMED SEARCH, ALONG THE
LINES OF YOUR QUESTION OF
WHETHER HDAC INHIBITORS GIVEN TO
CANCER PATIENTS AND OTHERS
CLINICALLY CAUSE HEPATIC
SEETOSIS, THE ANSWER IS, IT'S
CLEAR FROM PEARLIC ACID, THAT
HAPPENS TO BE GIVEN MORE TO
PATIENTS WITH NEUROPSYCHOLOGICAL
PROBLEMS THAN WITH CANCER, BUT
THEY DO GET FATTY LIVER.
OTHERWISE IT'S LESS CLEAR.
ONE THING I WANT TO ALSO
EMPHASIZE THAT LESS FROM THIS
WORK BUT FROM A SEPARATE PROJECT
WHICH I HAVEN'T PUBLISHED BUT I
DIDN'T PRESENT HERE.
BUT WE KNOCKED OUT HDAC3 IN
MACROPHAGES WHERE WE REALLY
CONTROL THINGS BETTER THAN WE
CAN IN THE LIVER.
AND WE HAVE AN INTERESTING
PHENOTYPE.
WE DO NOT SEE THE SAME THING
WITH HDAC INHIBITORS AND I THINK
WHAT THAT TELLS SUSTHAT HDAC
INHIBITORS HAVE OFF TARGET
EFFECTS AND THEY AFFECT OTHER
HDACs THAT MIGHT HAVE OPPOSITE
EFFECTS ON THESE PARAMETERS.
AND SO, ON THE ONE HAND, ONE OF
THE JUSTIFICATIONS FOR OUR WORK
HAS BEEN THIS HELPS US
UNDERSTAND BETTER WHAT WE ARE
DOING PATIENTS WHO GIVE HDAC
INHIBITORS BUT IT REALLY
UNDERSCORES HOW MUCH BETTER IT
WOULD BE IF WE SPECIFICALLY TOOK
OUT THE HDA CLAMP DOES WHAT WE
WANT TO DO AND NOT THE ONES THAT
ARE CAUSING THOSE EFFECTS.
>> HAVE YOU SEEN OR CHECKED
WHETHER THERE IS A DIFFERENCE IN
THE RATE OF FATTY ACIDS FROM AT
POSE TISSUE RELATED TO THE
KNOCKOUT MICE AND SO WHETHER THE
FATTY ACCUMULATION IN THE LIVER
IS PURELY DUE TO LYPOGENESIS IN
THE LIVER OR ALSO BECAUSE OF
INCREASED SUPPLY OF FREE FATTY
ACIDS?
>> IT IS A GOOD QUESTION.
IN THE CASE OF THE NCOR-HDAC3
MUTATION I GLOSSED OVER BUT
WHICH IS IN EVERY CELL IN THE
BODY.
WE LOOKED AT THAT AND THERE WAS
NO DIFFERENCE IN THESE MICE
BECAUSE IT WAS LIVER-SPECIFIC.
WE DIDN'T FOCUS ON A PRIMARY
AFFECT ON AT POSE TISSUE.
WE DID NOTICE ALTERATIONS -- NO
CHANGE IN FAT MASS.
BUT WE DID NOTICE ALTERATIONS IN
LIPID OXIDATION AS THEY WOULD
HAVE TO BE TO GET THAT LEVEL OF
ACCUMULATION.
BUT WE ASSUMED AT THE MOMENT
THOSE ARE AUTONOMOUS IN THE
LIVER.
BUT WE HAVEN'T FORMERLY RULED
OUT SOME SECONDARY EFFECT
[APPLAUSE]
>> I THINK DR. LAZAR HAS GIVEN
US A LOT TO THINK ABOUT.
PLEASE JOIN US NEXT DOOR AT THE
LIBRARY WHERE YOU CAN MEET HIM
AND HAVE REFRESHMENTS.
THANKS AGAIN.