>> GOOD AFTERNOON EVERYONE.
WELCOME TO THE WEDNESDAY
AFTERNOON LECTURE SERIES. I AM
-- CHIEF OF THE FUNCTIONAL--
SECTION IN THE NAIAB
LABORATORY, PLEASED TO
INTRODUCE OUR GUEST SPEAKER,
WHO IS CONDUCTING SOME OF THE
MOST EXCITING WORK TODAY ON
HIGH MASS SPECTROMETRY, AND
SOME CHROMATIN REGULATION FIRST
I HAVE FEW ANNOUNCEMENTS.
THE FIRST CONCERNS CONTINUING
MEDICAL EDUCATION CREDITS. THE
CME CODE FOR TODAY IS 50129. I
REPEAT THAT, 50129.
ALSO FOR THOSE OF YOU WATCHING
REMOTELY, YOU CAN SUBMIT
QUESTIONS AT ANY TIME, BY
CLICKING ON THE BUTTON JUST
BELOW YOUR VIDEOCAST WINDOW
THAT SAYS "SEND LIVE FEEDBACK."
FOR THOSE PRESENT HERE THERE
ARE TWO MICS IN THE AISLES THAT
YOU WILL BE ABLE TO USE AT THE
END OF THE LECTURE.
PLEASE NOTE ALSO THAT TODAY'S
LECTURE IS THE FINAL IN A
SERIES. WE WILL TAKE A
TWO-MONTH BREAK AND BEGIN A NEW
SEASON ON SEPTEMBER 11, WITH A
LECTURE BY DR. EVE MARDER
(SOUNDS LIKE) A BRANDEIS
UNIVERSITY.
OUR SPEAKER TODAY IS DR.
BENJAMIN GARCIA. THE RAYMOND H.
WALKOFF DISTINCT PROFESSOR OF
DIALOGICAL CHEMISTRY, PROFESSOR
OF BIOCHEMISTRY AND MOLECULAR
BIOPHYSICS AND HEAD OF THE
DEPARTMENT OF BIOCHEMISTRY AND
MOLECULAR BIOPHYSICS AT
WASHINGTON UNIVERSITY SCHOOL OF
MEDICINE.
DR. GARCIA RECEIVED HIS PHD IN
ANALYTICAL CHEMISTRY FROM THE
UNIVERSITY OF VIRGINIA,
CHARLOTTESVILLE. HE THEN SPENT
HIS POSTDOCTORAL YEAR AT THE
UNIVERSITY OF ILLINOIS,
URBANA-CHAMPAIGN, AND UNDER THE
MENTORSHIP OF NEIL-- FROM 2008
- 2012 HE WORKED AT PRINCETON
UNIVERSITY WHERE HE WAS AT THE
PROFESSOR. AND HE JOINED THE
UNIVERSITY OF PENNSYLVANIA
SCHOOL OF MEDICINE IS DIRECTOR
OF QUANTITATIVE PROTEOMICS. AND
IN 2019 HE BECAME THE--
PRESIDENTIAL PROFESSOR AND IN
2021 MOVED TO THE WASHINGTON
UNIVERSITY SCHOOL OF MEDICINE
IN ST. LOUIS WHERE HE ATTAINED
HIS CURRENT TITLE AS
DISTINGUISHED PROFESSOR.
HE IS VERY ACTIVE IN THE
PROTEOMICS COMMUNITY. HE IS
PART OF THE NOMINATING
COMMUNITY FOR THE HUMAN
PROTEOMIC ORGANIZATION. HEAD OF
THE DIVERSITY, EQUITY AND
INCLUSION COMMITTEE FOR THE
AMERICAN CHEMICAL SOCIETY AND
THE MENTOR FOR THE ASB,
NIH-FUNDED PROGRAM CALLED
MAXIMIZE OPPORTUNITY FOR
SCIENTIFIC ACADEMIC STUDY.
HE IS ALSO ASSOCIATE EDITOR OF
AMERICAN CHEMISTRY, AND THE
EDITORIAL BOARD MEMBER OF
NUMEROUS JOURNALS IN OUR FIELD
AND HAS RECEIVED SEVERAL HONORS
ALONG THE WAY AS WELL AS YOU
MIGHT IMAGINE, HE WAS NAMED A
HIGHLY CITED RESEARCHER IN
2022. HE HAS RECEIVED THE HUMAN
PROTEOMIC ORGANIZATION
DISCOVERY IN PROTEOMIC SCIENCE
AWARD. AND WAS ELECTED A FELLOW
OF THE ROYAL SOCIETY OF
CHEMISTRY.
BENJAMIN'S PRIMARY RESEARCH
FOCUS HAS BEEN TO CATEGORIZE
THE ROLE OF POST-TRANSLATIONAL
MODIFICATIONS OR PTMS ON
PROTEINS INVOLVED IN HUMAN
BIOLOGY, ESPECIALLY INTERESTED
IN UNDERSTANDING HOW PROTEINS
AND NUCLEIC ACID IS MEDIATE
CANONICAL FUNCTIONS. AND THAT
HE HAS BROUGHT EXPECTATION MANY
AREAS INCLUDING CHEMISTRY.
PARTICULARLY IN THE AREA OF MAS
SPECTROMETRY BASED CHEMISTRY,
AND HIS JOKE IS TO BRING MASS
SPECTROMETRY TO THE MASSES. AN
ANALYSIS OF HISTONE PTNS AND
CHROMATIN EXTRACTORS.
HE ALSO EXEMPLIFIES THAT BY HIS
RELENTLESS ADVOCACY DURING THE
YEARLY -- WHERE SEVERAL VERY,
VERY GOOD METHODS COMPETE TO
BECOME MIDDLE OF THE YEAR. AND
ALL OF THE YEARS AT LEAST
SEVERAL YEARS IN A ROW MASS
SPECTROMETRY HAS ONE. EVEN
AGAINST CRISPR. NOT A JOKE. IT
IS A SERIOUS COMPETITION.
IN MORE RECENT YEARS HE HAS
UPPING HIS GAME WITH
TRANSLATIONAL RESEARCH AND
DEEPER CONNECTION TO HUMAN
DISEASE.
THE TITLE OF BEN'S TODAY IS
QUANTITATIVE PROTEOMICS FOR
UNDERSTANDING EPIGENETICS
MECHANISMS. PLEASE HELP ME
WELCOME DR. BENJAMIN GARCIA.
>> [APPLAUSE]
>> DR. GARCIA: THANK YOU
ALEXANDRA, FOR A GREAT
INTRODUCTION AND BEING A
FANTASTIC HOST. IT HAS BEEN A
WONDERFUL DAY, MEETING MANY
INCREDIBLE SCIENTISTS AND
TRAINEES HERE. I HAVE REALLY
ENJOYED IT SO FAR.
AND SO WHAT I AM HOPING TO DO
TODAY IS GIVE EVERYONE A LITTLE
BIT OF KIND OF A TASTE OF SOME
OF THE TECHNOLOGY, AND
APPROACHES THAT WE HAVE BEEN
DEVELOPING IN THE LAST COUPLE
OF YEARS AND HOW WE ARE
APPLYING THEM, ONE, TWO
PROBLEMS IN CANCER EPIGENETICS
AND THINK WE HAVE PUBLISHED AND
A NEWER STORY THAT IS EMERGING
AS WELL.
I GUESS I WILL PROBABLY USE ...
LET ME SEE.
IF I USE THIS, WILL PEOPLE
ONLINE BE ABLE TO SEE THAT? OR
NO.
>> (OFF MIC)
>> OKAY GREAT. YEAH. RIGHT
HERE? YOU CAN SEE IT?
>> (OFF MIC)
>> DR. GARCIA: AND SO YEAH,
MAYBE FIRST BEFORE I GET INTO
THE TECHNOLOGY AND APPLICATIONS
OF WANT TO GIVE A COUPLE OF
SLIDES ON THE BACKGROUND. AND
I KNOW NOT EVERYONE WORKS ON
CHROMATIN AND EPIGENETIC
MECHANISMS. THAT IS OKAY.
NOBODY IS PERFECT.
BUT TO GIVE A LITTLE BIT OF
BACKGROUND WE HAVE METER
MOUNTED DNA THAT HAVE TO BE
COMPACTED INTO SMALL AREA IN
THE NUCLEUS, AND THE WAY THAT
NATURE DOES THIS IS BY TAKING
THE DNA AND WINDING IT ON A
BALL IS CALLED HISTONES,
SIMILAR TO WANTING A STRING OF
SPOOL, SO YOU HAVE TO COPIES OF
EACH OF THE CORE HISTONES AND
ABOUT 146 PAIRS MAKE UP THE
NUCLEUS. ESSENTIALLY FOLDED
INTO CHROMATIN FIBER IN OUR
CHROMOSOMES.
IT WAS THOUGHT THAT THESE
HISTONE PROTEINS WERE VERY
STRUCTURAL COMPONENTS OF
CHROMATIN. BUT OVER THE LAST
2+ DECADES IT HAS BEEN FOUND
THAT THESE HISTONES HAVE A HUGE
ARRAY OF POST-TRANSLATIONAL
MODIFICATIONS. OUR DYNAMIC
PROTEINS THAT HELP REGULATE
GENE EXPRESSION PATTERNS.
AND SO, KIND OF FAST FORWARD
NOW BY MANY METHODS INCLUDING
MASS SPECTROMETRY. SEVERAL
POST-TRANSLATIONAL
MODIFICATIONS OF THESE SYSTEMS
HAVE BEEN DESCRIBED. YOU CAN
SEE A WHOLE HOST OF MANY
DIFFERENT CHEMICAL
MODIFICATIONS ACROSS MANY
CITES. ESSENTIALLY THE ENTIRE
PROTEIN OF THESE HISTONES.
HOWEVER, DEFINITELY I WOULD SAY
THE MAJORITY OF MODIFICATIONS
ARE FOUND ON THE INTERNAL
REGIONS OF THE PROTEINS. AND
MORE HI STOKE GEOMETRIC
MODIFICATIONS.
YOU CAN SEE HERE THERE SOME
SITES THAT HAVE MULTIPLE
MODIFICATIONS 10 OR 11
DIFFERENT TYPES OF
MODIFICATIONS ON THE SAME
RESIDUE.
AND ONE THAT KEEPS AN
ANALYTICAL CHEMIST LIKE MYSELF
VERY BUSY.
AND THESE GENE REGULATE GENE
EXPRESSION IN THESE
MODIFICATIONS, SHOWING IN
CARTOON FORM SOME OF THE MORE
ABUNDANT MODIFICATIONS LIKE
ACETYLATION. AND THESE
MODIFICATIONS HAVE BY
BIOLOGISTS ONE AT A TIME HAVE
BEEN LINKED TO VERY IMPORTANT
PROCESSES ESPECIALLY TURNING
GENES ON OR OFF. AND ALSO IN
THE DNA DAMAGE EVENTS AND
THINGS LIKE THAT AND THE
HISTORY ON ACETYLATION IN
GENERAL IS LINKED TO GENE
ACTIVATION. HISTONE ACETYLATION
IS A LITTLE BIT MORE COMPLEX.
THERE'S DIFFERENT DEGREES OF
METHYLATION. IT CAN ALSO BE
LINKED TO BOTH GENE ACTIVATION
OR GENE SILENCING DEPENDING ON
WHETHER MODIFICATIONS FOUND. IN
SO FOR EXAMPLE THIS WILL HERE
IS FOUND ON ACTIVE GENES AND
THE GENOME, PROMOTERS OF ACTIVE
GENE AND THIS ON THE SAME
HISTONE PROPERTY JUST A FEW
AMINO ACID DOWN FROM THIS OTHER
MODIFICATION ... LOST THE
POINTER. NOT SURE IF I HAVE
THAT POINTER ANYMORE. THERE IT
IS.
AND SO, THIS MODIFICATION, SAME
METHYLATION ON THE SAME
PROTEIN, A FEW AMINO ACIDS
DOWN, THIS IS ACTUALLY BEEN
FOUND IN THE HEADER CHROMATIC
REGIONS OF THE GENOME. AND
BECAUSE OF THIS RELATIONSHIP
THAT A LOT OF THEORIES AND
HYPOTHESES HAVE BEEN PUT FORTH
TO TRY TO EXPEND HOW THESE
MODIFICATIONS MAY REGULATE GENE
EXPRESSION. ONE OF THIS IS THE
HISTONE CODE OR PTM CODING
HYPOTHESIS THAT STATES WHICH
THESE MODIFICATIONS KIND OF--
WELL IF YOU THINK ABOUT IT IF
THERE IS A CODE THAT HAS TO BE
A WAY TO INTERPRET THIS CODE.
IT IS THOUGHT THAT THESE
MODIFICATIONS ACT AS A CODE BY
BRINGING PROTEINS THAT THEN
READ OR INTERPRET THE CODE TO
SPECIFIC PARTS OF THE GENOME.
AND SO IT IS NOT THAT THESE
MODIFICATIONS ACT AS A PLATFORM
FOR RECRUITING THESE PROTEINS
TO ACTIVATE GENES; AND IN
SUPPORT SEVERAL PROTEINS WITH
SPECIALIZED DOMAINS HAVE BEEN
FOUND TO BIND SPECIFIC SPOTS ON
THESE HISTONES AND THE GENOME.
AND THERE'S A LOT OF INTEREST
RIGHT NOW IN KIND OF
UNDERSTANDING-- ESPECIALLY IN
HUMAN DISEASES-- HOW THIS
EPIGENETIC MECHANISMS CAN GO
AWRY. NOW WITH A DECADES'
WORTH OR MORE OF LARGE-SCALE
GENOMIC EFFECTS, SEVERAL
EFFECTS THAT MODIFY HISTONES OR
PROTEINS THAT INTERACT WITH
HISTONES OR EVEN THE HISTONES
THEMSELVES BEEN FOUND MUTATED
IN VARIOUS CANCERS. SEVERAL
COMPANIES INCLUDING VERY LARGE
COMPANIES HAVE EPIGENETIC
ONCOLOGY DIVISIONS. AND THERE
ARE SMALLER COMPANIES THAT ONLY
FOCUS ON THIS WITH EPIGENETIC
INHIBITORS MAKING THEIR WAY
THROUGH CLINICAL TRIALS AND FDA
APPROVALS.
AND SO THERE'S A LOT OF
INTEREST IN UNDERSTANDING IF
YOU KNOW, WHAT YOU ARE SEEING
IN TERMS OF TRANSCRIPTIONAL
CHANGES INVOLVED EPIGENETIC
MECHANISMS. IN HUMAN DISEASE
AND BIOLOGY.
BUT I ALWAYS CANNOT TELL PEOPLE
THAT YOU REALLY KNOW YOU ARE
WORKING IN A GREY FIELD WHEN IT
MAKES IT INTO BLOCKBUSTER
MOVIES. AND SO HERE'S A FILM
STILL OF ONE OF THE SPIDER FILM
MOVIES FROM A LONG TIME AGO. SO
YOU SEE ALL THE SIGNS ON THE
BOARD EVERYTHING YOU NEED TO
KNOW TO GET SUPERHUMAN POWER
YOU MAY RECOGNIZE PHRASES OR
SCHEMATICS AND THINGS LIKE THAT
AND IF YOU LOOK RIGHT AT THE
BOTTOM HERE THERE IS SOMETHING
REALLY FAMILIAR. AND
NUCLEOSOME. WITH HISTONE HS3 K
27 METHYLATION ON IT SO WHOEVER
KNEW THEY HAD TO PUT THE MOST
IMPORTANT SIGNS ON THE BOARD IN
THIS MOVIE KNEW THEY HAD TO
INCLUDE EPIGENETIC MECHANISMS.
AND SO THERE'S A LOT OF
DIFFERENT WAYS TO LOOK AT
HISTONE MODIFICATIONS. THE
MOST COMMON APPROACHES USING
SITE-SPECIFIC ANTIBODIES WHICH
IF YOU USE ANTIBODIES CAN BE
BOTH A BLESSING AND A CURSE. IF
YOU HAVE A GOOD ANTIBODY YOU
GET ONE SINGLE MODIFICATION
DOES NOT EXIST IN YEAST, AND
THIS IS WHAT YOU WILL SEE. YOU
CAN SEE THE CROSS-REACTIVITY
WITH ANOTHER PROTEIN THAT HAS A
SIMILAR EPITOPE. AND YOU WILL
SEE A COUPLE OF OTHER BANDS
HERE. AND THIS RECOGNIZES THE
COMMON -- NOT RECOGNIZING THE
MODIFICATION AND SO THIS IS WHY
BIOLOGISTS ONLY SHOW THE LITTLE
SLIVER OF THE WESTERN BLOCK,
NOT THE WHOLE THING BECAUSE
THEN YOU WILL SEE ALL THE OTHER
PROTEINS THAT IT IS NOT
REACTING WITH YOU HAVE TO BE
CAREFUL.
ESPECIALLY WITH SUCH
MODIFICATION-DENSE PROTEINS
SUCH AS SYSTEMS TO REALLY
UNDERSTAND WHAT EPISODE -- AND
NOT GET WILL THAT IT IS ANOTHER
HISTONE OR SOMETHING ELSE.
THE OTHER ISSUE WITH ANTIBODIES
AND HISTONE MODIFICATIONS IS I
SHOWED YOU HOW MANY DIFFERENT
MODIFICATIONS THERE ARE ON THIS
HISTONES. AND SO YOU CAN
IMAGINE THAT SOMEBODY'S
MODIFICATIONS MIGHT ACTUALLY
BLOCK THE ANTIBODY FROM
RECOGNIZING THE INTENDED SITE.
AND HERE IS AN EXAMPLE OF THIS.
JUST THE PEPTIDE-- ANTIBODY
AGAINST SERIES 10
PHOSPHORYLATION, THESE ARE --
VERY GOOD RECOGNIZING
PHOSPHORYLATION BY ITSELF BUT
STRUGGLES ACTUALLY MODIFIED
PEPTIDE WITH EITHER THE METHIL
OR SUBTLE GROUP AND DOES EXIST
IN VIVO BUT ABUNDANTLY. MUCH
MORE EASIER DETECTED WITH MASS
SPECTROMETRY THAT YOU CAN
ANTIBODIES. AND YOU CAN DO THIS
WITH --.
AND TO UNDERSTAND DIFFERENT
ASPECTS OF PROTEIN PTN
MODIFICATIONS AS WELL AND SO
TRYING TO CREATE VERY
QUANTITATIVE HIGH THROUGH
METHODS TO ANALYZE ADMINISTER
MODIFICATIONS AT A TIME.
TRYING TO UNDERSTAND WHICH
COMBINATIONS OR MODIFICATION
OCCUR TOGETHER BECAUSE OUR DATA
SHOWS THAT ONE MODIFICATION ON
THE HISTONE AT THE TIME JUST
DOES NOT EXIST. IS ALWAYS IN
COMBINATION WITH SOMETHING
ELSE. BUT IF YOU'RE ONLY USING
ANTIBODIES YOU ARE ONLY
RECOGNIZING THAT ONE
MODIFICATION.
WE HAVE DEVELOPED A LOT OF
METABOLIC LABELING APPROACHES,
COMBINING THAT WITH PROTEOMICS
TO BE ABLE TO UNDERSTAND THE
HISTONE DYNAMICS AND GET UP THE
KINETICS OF THESE MEDICATION
BECAUSE THEY'RE NOT STATIC THEY
ARE CONSTANTLY BEING PUT ON AND
OFTEN COMBINING GENOMICS WITH
THE DOWNSTREAM PROTEOMICS
READOUT SO HOPEFULLY ONE DAY WE
CAN GET TO THE POINT WHERE WE
ARE PULLING OUT OUR FAVORITE
GENE, AND IDENTIFYING ALL THE
PROTEINS AND HISTONE
MODIFICATIONS THAT ARE ALL THAT
GENE, YOU KNOW IN BOTH KIND OF
THE PHYSIOLOGICAL AND DISEASE
STATE.
SO, FOR THIS FIRST PART OF THE
TALK I WAS JUST GOING TO GIVE
AN UPDATE ON SOME NEWER
APPROACHES AND NEW TECHNOLOGY
THAT THE LAB HAS BEEN KIND OF
EVALUATING OVER THE LAST YEAR
+. WE ARE ALWAYS KIND OF ON
THE LOOKOUT FOR NEW TECHNOLOGY
THAT GIVES US SOME ADVANTAGES,
AND ALLOWS US TO DO THINGS THAT
WE WERE NOT ABLE TO DO BEFORE.
AND ONE OF THE CRUTCHES OF
QUANTITATIVE PROTEOMICS, YOU
KNOW, EXCEPT FOR IN THE LAST
YEAR OR SO, WAS THAT IT IS NOT
VERY HIGH THROUGHPUT. IT TAKES
A LONG TIME RUNNING AN HOUR OR
TWO HOUR GRADIENTS ON THESE
INSTRUMENTS. AND OUR GOLD
STANDARD IS RUN ON AN
INSTRUMENT TO DETECT HISTONE
MODIFICATIONS. IT WOULD TAKE 60
TO 90 MINUTES SO NOT A HIGH
THROUGHPUT APPROACH.
BUT NOW YOU HAVE A LOT OF
INSTRUMENTS THAT ARE COMING
OUT, BASED ON DIFFERENT MASS
ANALYZERS, AND ORBIT TRAPS
WHICH ARE HIGH-RESOLUTION
INSTRUMENTS THAT ARE MUCH
FASTER SCANNING INSTRUMENT SO
WE WERE INTERESTED TO EVALUATE
A FEW OF THESE. ONE WE ENDED UP
REALLY LIKING, NOT JUST FOR THE
HIGH THROUGHPUT ABILITIES WHICH
I WILL SHOW YOU IN A SECOND BUT
ALSO BECAUSE THE SYSTEM IS
EQUIPPED WITH A DIFFERENT
FRAGMENTATION MODE. ELECTRON
INACTIVATED DISASSOCIATION
WHICH ALLOWS US TO DO SOMETHING
THAT WE CANNOT DO IN TERMS OF
FRAGMENTATION SHOW SHOW YOU HOW
WE ARE DOING THAT IN DIFFERENT
WAYS AS WELL.
TO READ ABOUT THE TIME WE GOT
THIS INSTRUMENT, YOU KNOW, OUR
FRIENDS AT SIAX (SOUNDS LIKE)--
I WANT TO DISCLOSE I DON'T HAVE
A RESEARCH AGREEMENT OR
ANYTHING LIKE THAT IN PLACE. WE
ARE HAPPY CUSTOMERS. BUT OUR
FRIENDS AT SIAX PUBLISHES
PAPERS SAYING THAT ESSENTIALLY
IT COULD QUANTIFY ABOUT 1000
PROTEINS PER MINUTE ON THIS
INSTRUMENT.
KIND OF HARD TO READ THIS BUT
THIS IS A FIVE-MINUTE GRADIENT.
YOU CAN KIND OF SEE THAT SO
YOU ARE TALKING REALLY FAST
QUANTITATIVE PROTEOMICS. ALMOST
TOO GOOD TO BE TRUE. AND
HONESTLY, WHEN STUFF LIKE THIS
COMES OUT, ARE USUALLY KIND OF
SAY, I DON'T KNOW IF I WANT TO
BELIEVE THIS PROPAGANDA. I NEED
TO DO IT KIND OF FOR MYSELF. SO
I GOT ALL OF THE PARAMETERS
FROM PEOPLE AT SIAX, AND WAS
ABLE TO REPRODUCE. I HAD THIS
POSTDOC IN MY GOOGLE WAS ABLE
TO REPRODUCE THIS.
THIS IS OUR DATA HERE AGAIN
FIVE-MINUTE GRADIENTS. AND
ESSENTIALLY WE GET ANYWHERE
FROM 4500 TO 5000 PROTEINS IN
FIVE MINUTES DETECTED WITH 3 TO
4 DIFFERENT POINTS ACROSS THE
PEAK. AND SO THIS IS ACTUALLY
A REALLY, REALLY FAST
INSTRUMENT.
AND NOW THIS IS KIND OF OUR QC.
BEFORE WE WOULD RUN EITHER A
WHOLE PROTEOMIC AND KIND OF
RUNNER TO OUR GREATER MAYBE A
FEW SYNTHETIC STANDARDS AND RUN
LIKE HALF HOUR, AND NOW WE RUN
FIVE MINUTES QC BEFORE EVERY
BATCH OF SAMPLES. WE DO NOT GET
4500 TO 5000 PROTEINS IN FIVE
MINUTES, THEN THE INSTRUMENT IS
TO BE CLEANER SOMETHING NEEDS
TO BE ADJUSTED. SO WE WERE
QUITE HAPPY WITH THAT.
AND AS I MENTIONED, OUR
GRADIENT FOR NORMAL NANO LC MS
ANALYSIS HISTONE SYSTEM
MODIFICATIONS IS ABOUT 55
MINUTE GRADIENT. WE CREATED
SOME REALLY QUICK RATINGS TO
SEE IF WE COULD GET VERY
SIMILAR DATA FROM JUST THESE
MUCH FASTER HIGH THROUGHPUT
RUNS.
THE BIG DIFFERENCE HERE IS THAT
WE ARE USING MICRO FLOW LCS,
FOR THOSE IN THE PROTEOMIC
FIELD 5 TO 10 5L PER MINUTE,
FLOW RATE INSTEAD OF 200 NM
FLOWRATES, WE WANTED TO
EVALUATE THESE SO WE SET UP A
WHOLE DATA INDEPENDENT
ACQUISITION, TO GET THROUGH
SOME OF THESE TRANSITION MODES
TO OPTIMIZE THE WINDS WE WERE
ASSESSING IN THE MASS
SPECTROMETRY A LITTLE BIT MORE
DYNAMICALLY.
SOME AREAS WE HAD MORE MODIFIED
SYSTEMS AND BIGGER WINDOWS
INSTEAD OF GOING THROUGH WITH
EQUAL WINDOWS LIKE WE DID IN
SYSTEMS WE COULD TAKE THE TIME
TO TAILOR AND OPTIMIZE THE
WINDOWS AND WE KNOW THE FOR THE
(CORRECTION) BINS WERE THE MOST
MODIFIED SYSTEMS COME OUT AND
WE LOOKED AT THE QUANTITATIVE
DATA WE WERE QUITE HAPPY WITH
IT SO YOU HAVE THE 55 AT A
NORMAL NANO C RUN AND THESE
CRAZY FAST FIVE MINUTE
GRADIENTS OR RUNS AND YOU CAN
SEE HERE PEAK SHAPE LOOKS
REALLY, REALLY STRONG.
WHEN YOU HAVE A PEPTIDE THAT
HAS MULTIPLE LYZENES AND HAVE A
MODIFICATION AS ISOLATION, YOU
CAN LOOK AT THE TRANSITION AND
YOU CAN TELL WHETHER
MODIFICATIONS ARE FOUND, UNTIL
WE GET ENOUGH POINTS ACROSS THE
PEAK TO BE ABLE TO
DIFFERENTIATE THESE ISOMERIC
UPSIDES. AND WE CAN DO THIS
REALLY WELL.
WE HAVE DEVELOPED A FEW
SOFTWARE TO BE ABLE TO HANDLE
THIS DATA AS WELL. IN TERMS OF
TOTAL NUMBER OF PEPTIDES
IDENTIFIED, OR THE CS PEPTIDES
THEY ARE VERY CONSISTENT ACROSS
ALL OF THESE DIFFERENT TYPES OF
RUNS, OUR GOLD STANDARD, NANO C
RUN, HOUR PLUS, AND THE TENANT
FIVE MINUTES. FAMILIES MIGHT BE
A LITTLE BIT QUICK SO WE LIKE
THE 10 MINUTE GRADIENT. BUT YOU
CAN SEE THE VERY SOLID ACROSS
ALL THE WAY IN TERMS OF THE
NUMBERS.
INCREDIBLY ROBUST INSTRUMENT AS
WELL. HERE YOU HAVE 100 REPEAT
INJECTIONS OF THE SAME SAMPLE
RIGHT ON TOP OF EACH OTHER.
AND YOU CAN SEE HERE RETENTION
TIME, THE NUMBER OF PEPTIDES IS
PRETTY CONSISTENT ACROSS LARGE
MAJORITY OF THESE RUNS.
AND SO NOW, WE CAN GET TO THE
POINT WHERE WE ARE ABLE TO WORK
WITH OUR CLINICAL COLLEAGUES.
WE HAVE MANY CLINICAL
COLLEAGUES AT PENN AND AT WASH
U, THAT CAME UP TO ME AND SAID
WE HAVE BEEN FOLLOWING THESE
PATIENTS FOR THE LAST FIVE
YEARS TAKING SAMPLES, BLOOD
SAMPLES EVERY MONTH. AND WE
HAVE THOUSANDS AND THOUSANDS OF
SAMPLES. WE WOULD LOVE TO
RUFFLE FOR HISTONE
MODIFICATIONS. I CAN'T RUN THAT
MANY SAMPLES. IT WOULD TAKE TOO
MUCH INSTRUMENT TIME.
BUT NOW WITH THIS 10 MINUTE
GRADIENT MAY BE ABOUT 17
MINUTES TOTAL RUN, WE CAN GET
TO THE POINT WHERE WE ARE
RUNNING HUNDREDS OR THOUSANDS
OF SAMPLES IN A WEEK. AND WE
CAN TAKE ON SOME OF THESE VERY
LARGE-SCALE CLINICAL PROJECTS.
OR THESE POPULATION TYPE OF
STUDIES AS WELL.
AND MENTIONED THIS ELECTRON
ACTIVATED DISASSOCIATION. THIS
IS VERY DIFFERENT. SOMETHING
THAT NOT ALL INSTRUMENTS HAVE
THE ABILITY TO USE ELECTRONS TO
FRAGMENT PEPTIDES. MOST
INSTRUMENTS USE COLLISIONS
WITHIN THE INNER GAS.
AND SO HERE YOU CAN SEE IF YOU
INCREASE THE ENERGY, YOU GET
THE FRAGMENTATION. IT GIVES YOU
VERY COMPLETE COVERAGE IN
BETWEEN ALMOST EVERY AMINO
ACID. YOU CAN TELL EXACTLY
WHERE THE MODIFICATION IS.
WHAT WE FOUND THAT WE ACTUALLY
WERE ABLE TO USE THIS IN A
DIFFERENT ROUTE TO HELP SOLVE A
REALLY DIFFICULT PROBLEM.
AND SO I HAD A COLLABORATOR AT
YALE, MATT SIMON, WHO SAID
ABOUT OTHER METHODS HE HAD
EVIDENCE THAT A LYSINE RESIDUE
WOULD HAVE AN ACETO GROUP AND
MONO GROUP TOGETHER. I NEVER
HEARD OF TWO MEDICATIONS ON THE
SAME LYSINE AT THE SAME TIME SO
HE WANTED TO KNOW WE COULD GET
MORE DIRECT EVIDENCE. AND THIS
IS THE SAME MASS AS ANOTHER
MODIFICATION CALL PROPIANO. WE
CANNOT TELL THEM APART IS BY
MASS WHEN WE FRAGMENT THESE
PEPTIDE TOGETHER FROM THE
SAMPLES WE SAW THERE WERE TWO
SLIGHTLY OVERLAPPING PEAKS. WE
FRAGMENTING THEM TOGETHER IT
BASICALLY GAVE US COMPLETE
COVERAGE. AND SO SINCE THESE
ARE THE EXACT SAME OSIBERIC
(SOUNDS LIKE) MATH WE CANNOT
ALL DIFFERENT BUT THE NICE
THING ABOUT THIS ASSOCIATION IS
THAT IT IS VERY TUNABLE. YOU
CAN CRANK UP THE ENERGY AND DO
THINGS THAT YOU CANNOT DO
ANOTHER INSTRUMENTS THAT HAVE
FRAGMENTATION LOADS WE CRANKED
UP THE ENERGY TO THE POINT WE
WERE BREAKING OFF THE
SIDECHAINS OF THE AMINO ACIDS.
AND WE BROKE OFF THE SIDECHAINS
OF THIS LYSINE THAT HAD THE
ACETYLMETHOL, WE SAW THAT THIS
WAS AN ACETYLMETHOL, AND THIS
WAS THE PROPIANIC GROUP AND WE
SAW THIS MEDICATION TOGETHER AT
ANY PROTEIN THAT IS INTERESTED
YOU AT THE SAME TIME.
AND SO NOW I WANT TO GET INTO
TWO PROJECT TO SHOW YOU HOW WE
ARE USING THE TECHNOLOGY. THIS
ONE WE HAVE GONE A LITTLE BIT
FARTHER AND PUBLISHED. WE HAVE
BEEN INTERESTED IN SOFT TISSUE
SARCOMA FOR A LONG TIME. AND
THERE'S A WHOLE HOST OF
MUTATIONS TO MANY DIFFERENT
TYPES OF SOFT TISSUE SARCOMAS,
THAT EITHER AFFECT CHROMATIN
REMODELING PROTEINS, WHICH IS
THE POLICONE OR HAVE THESE
INTERESTING FUSION PROTEINS, A
MODIFIED ENZYME USED IN
TRANSCRIPTION FACTORS OR
SOMETHING LIKE THIS. NOT REALLY
CLEAR WITH THE MOLECULAR
MECHANISMS FOR HOW THIS LEADS
TO THE PROGRESSION OF THE
CANCER.
AND THERE IS EVEN HISTONES GET
MUTATED THEMSELVES. YOU CAN
IMAGINE IF YOU HAD LYSINE
RESIDUE AND NOW YOU HAVE A
ADDITION TO SOMETHING OF YOU
WIPEOUT MUTILATION. AND SO
THERE OF INTERVIEWS THAT OCCUR
IN ONE OF THE THING WE ARE
INTO THAT IS A MALIGNANT
PERIPHERAL NERVE SHEATH TUMOR
MPNST, MOST PATIENTS HAVE AN
UNDERLYING MUTATION TO THE
FIBROMA GENE AND THEN WE GET
INVITATION TO CANCER AND OLDER
PATIENTS CAN SPORADICALLY GET
THOSE SECOND MUTATIONS AND
PROGRESSES TO CANCER. IT IS A
RARE TUMOR, MAY BE SEVERAL
HUNDRED A YEAR THAT ARE
DESCRIBED. BUT IT IS VERY
AGGRESSIVE. LESS THAN 50%
FIVE-YEAR SURVIVAL RATE.
THERE'S NOT A LOT OF OPTIONS TO
TREAT. BASICALLY JUST SURGERY
AND RADIATION. AND ABOUT HALF
OF THE PATIENTS HAVE A RELAPSE.
THERE'S A LOT KNOWN ABOUT THE
MUTATIONS THAT LEAD TO MPNST.
BUT ABOUT 80% OF PATIENTS HAVE
A MUTATION TO ONE OR TWO
COMPONENTS OF THE POLYCHROME
REPRESSIVE COMPLEX, AND THIS IS
ACCOMPLISHED IN CREATE
TRI-METHYLATION AT HISTONE H3.
AND THIS IS A PATHOLOGIST
DIFFERENTIATE DIFFERENT MPNSTS
BUT STAINING. AND SO IT HAS A
DIFFERENT MUTATION AND YOU CAN
SEE THE BROWN STANDING OF THE
METHYLATION. AND YOU CAN SEE
THE LOSS OF THAT METHYLATION.
AND, THE LOSS IS PROBABLY ALSO
THE ONE THAT YOU KNOW LEADS TO
POOR SURVIVAL. AND SO YOU CAN
SEE BECKY FROM PATIENTS WITH
THE LOSS MUTATION VERSUS A
DIFFERENT MUTATION. IT RETAINS
P53 OR SOME OTHER TYPE OF
MUTATION.
HOWEVER, LOOKING AT HISTONE
H3K27 ME3 IS PROBABLY THE
MARKET FOR THIS PROBABLY NOT
THE BEST THING TO DO BECAUSE
K27 ME3 IS PROBABLY
OVEREXPRESSED A LOT OF
DIFFERENT CANCERS, ESPECIALLY
CANCERS THAT ARE VERY DIFFICULT
TO DISTINGUISH VERSUS MPNST,
RELEASE FROM THE PATHOLOGY
STANDPOINT.
AND SO STARTING THIS PROJECT WE
HAD A COUPLE OF QUESTIONS. ONE,
WE CAN UNDERSTAND THE FIRST
INITIAL LOSS. WE HAVE MUTATION
AND THE POLYCOM LOSING K27 ME3,
HOW DOES THIS FURTHER
REPROGRAMMED THE EPIGENOME?
WANTED TO ANSWER THIS WITH THE
PROFILING. AND WE ARE
EXPECTING BIG CHANGES HERE IN
TERMS OF THE EIPGENOMES. AND SO
HOW DOES THIS AFFECT
TRANSCRIPTION? ESPECIALLY
PROTEIN TRANSCRIPTION? AND SO
WE HAVE TWO GREAT POSTDOCS.
JOHN STARTED THE PROJECT AND
JOANNA IS CURRENTLY WORKING THE
PROJECT.
THE FIRST THING WE HAD TO DO IS
CONVINCE HERSELF TO GET HIGH
QUALITY PROTEOMIC DATA FROM
PARAFFIN- EMBEDDED SAMPLES, AND
A LOT OF TIMES MORE THE CELLS
ARE FROZEN. BUT PARAFFIN
EMBEDDED IN THESE WAX CHUNKS SO
WE HAD TO WORK OUT A LOT OF
METHODOLOGY TO GET HIGH QUALITY
HISTONES AND PROTEOMES OUT OF
THE SAMPLES. BUT IT DID TAKE A
FAIR AMOUNT OF OPTIMIZATION.
THIS IS GONNA TRIVIAL TO REALLY
RETRIEVING YOU KNOW PROTEIN
SAMPLES FROM THESE FFP BLOCK TO
THE BEEN SITTING HERE FOR 10
PLUS YEARS.
AND SO JUMPING INTO THE DATA,
IF WE TOOK FFP SAMPLES FROM
PATIENTS JUST OF THE NEURAL
FIBROMA MUTATION, ONES THAT HAD
POLICONE LOSS OR RETAIN YOU SEE
SOMETHING VERY DIFFERENT ABOUT
WANT TO HAVE THE POLY CONE
LOSS, LOOKING AT ABOUT 150
MODIFICATIONS, CLUSTER TOGETHER
IN THIS PRINCIPAL COMPONENT
ANALYSIS AND YOU CAN SEE A
LITTLE BIT BETTER IN THE
VOLCANO PLOT. SO THESE ARE ALL
THE HISTONES MARKS UPREGULATED.
IN THE PATIENTS WITH POLYCLONAL
LOSS ARE DOWN RELATED,
ESPECIALLY FROM THE
NEUROFIBROMATOSIS POLY CONE
RETAINED, AND THE SAMPLES AND
USING SEE THE LOSS OF THE K27
DIE. AND OTHER THINGS ARE UP
AND DOWN REGULATED. AND WE HAVE
SEEN THAT IN THEIR OTHER
HISTONE MARKS SUCH AS THE
K4H420 METHYLATION THAT IS DOWN
REGULATED. AND THEN YOU HAVE
ALL OF THESE METHYLATION AND
ACETYLATION THAT ARE
UPREGULATED AS WELL.
IN BLUE IS THE POLYCHROME
RETAIN. IN RED IS POLYCHROME
LAWSON WHEN YOU LOSE YOU HAVE
HIGHER LEVELS OF H4 ACETILATION
ISOFORMS.
IT IS ONLY PARTIALLY TRUE. WHAT
WE ENDED UP FINDING FROM THE
DATA WAS THAT THERE'S PEPTIDES
THAT HAVE K27 MONO DIE AND TRI
DIE. IT WILL LOOK AT THE TOTAL
LEVELS OF K36 METHYL, THEY ARE
FOUND OFTEN WITH K27 MONODINE
TRY, K27. IN THE DISEASE STATE
YOU LOSE THE K27 METHYLATION.
AND SO BASICALLY YOU JUST HAVE
K36 ALONE WITH THE UNMODIFIED
K27.
>> IT IS LOSING THE K 27 WE
SEEK A 36 INCREASING.
WHAT THE DATA IS TELLING US
WERE GOING FROM A SILENCING
MARK OF K 27 BOTH BY ITSELF IT
MAY BE WITH SOME ACTIVE
HISTONE MARKS THAT THERE IT
ITSELF IS INTERESTING BUT IN
THE DISEASE STATE YOU LOSE A
LOT OF KEY 27 THERE IS GLOBAL
DOMINANT WITH HISTONE MARKS
FOUND WE DID SEE THAT K 27
DIMETHYL AND TRIMETHYL ARE
BOTH DOWN REGULATED.
THAT IS INTERESTING ITSELF AS
WELL BECAUSE AND OTHER CANCERS
I TOLD YOU K 27 AND TRIMETHYL
BUT AND OTHERS DIMETHYL IS NOT.
THESE TWO ARE VERY SPECIFIC
MARKERS.
AND JOHN IS A RESIDENT
PATHOLOGIST HAS GONE BACK TO
THE CLINICS AND USING THESE
TWO MARKS TO DIFFERENTIATE.
SO WE KNOW WHAT HAPPENS TO THE
APPLE GENOME IT GOES FOR CLOSE
DATE TO OPEN ACTIVE BASED ON
THE HISTONE MARKS.
WHAT ABOUT THE PROTEINS OR
GENE EXPRESSION?
ANOTHER VAULT A NO PLOT AND
YOU CAN SEE A LOT OF THINGS
ARE UPREGULATED AND WHEN WE DO
THE ANALYSIS ALL PROTEINS ARE
INVOLVED TO KEEP AN OPEN
ACTIVE CHROMATIN WITH
REMODELERS AS WELL AS PROTEINS
INVOLVED IS TRANSCRIPTION.
THIS MATCHES REALLY WELL WITH
HISTONE DATA GOING FROM A
CLOSED CHROMATIN TO MORE OPEN
AND ACTIVE.
AND IT SUPPORTS THAT WE HAVE
PROTEINS THAT MAINTAIN
TRANSCRIPTION.
BUT AS YOU CAN SEE A LOT OF
PROTEINS ARE DOWN REGULATED.
THEY ARE INVOLVED IN IMMUNE
RESPONSE SO THAT COMPLEX
PROCESSING INTERFERON
SIGNALING THAT POTENTIALLY
COULD LEAD TO A PATIENT BEING
MORE IMMUNOCOMPROMISED.
JUST LOOK AT CLASS ONE AND
CLASS TWO PROTEINS YOU CAN SEE
A HIGHER LEVEL OF THE PROTEIN
GROUPS.
YOU CAN SEE THIS JUST BY BIRDS
EYE STAINING FOR BOTH OF THE
HLA MOLECULES.
HIGHER STATING GOES TO LOWER,
SO THE DATA TELLS US THE
HISTONE MARKS THERE IS MORE
ACTIVE OPEN HISTONE MARKS.
AND A SUBSET ALSO THAT IS
UPREGULATED AS WELL MOSTLY
INVOLVED WITH KEEPING THE OPEN
ACTIVE CHROMATIN THAN WITH THE
ANTIGEN PATHWAY IS DOWN
REGULATED.
'S AND NOW THE QUESTION ARISES
YOU LOSE A REPRESSOR WITH A
SILENCING PROTEIN.
I UNDERSTAND HOW YOU CAN LEAD
TO ACTIVATION AND TARGETS, BUT
HOW DO YOU GET MORE
OPPRESSION?
SO JOHN WENT BACK TO THE DATA
AND FOUND THE PROTEIN WAS ALSO
UPREGULATED.
AND IN FACT THESE PATIENTS
HAVE HYPER METHYLATION AND
HIGHER LEVELS OF DNA
METHYLATION SO WE HAVE
DEVELOPED METHODS TO ANNALEE
ANALYZED A CLAY CASTLE AND
MODIFICATIONS WE SEE
METHYLATION'S EVEN THEN DNA
DAMAGE MARKERS THAT ARE ALL
UPREGULATED IN THESE PATIENTS
AS WELL.
IF YOU LOOK AT WHERE IN THE
GENOME HYPER METHYLATION IS
FOUND RED IS LOW AND BLUE IS
HIGH.
LOW METHYLATION TOO HIGH.
YOU CAN SEE HIGHER METHYLATION
IN THE PATIENT'S AND ALL WITH
THE PROMOTERS OF THINGS WE SAW
DOWN REGULATED AND THEN
INTERFERE ON THE PATHWAY AND
ALL THE IMMUNE RESPONSE
PATHWAYS.
SO THE PICTURE IS NOW NOW WITH
LOSSES OPEN ACTIVE CHROMATIN
IT IS EXPECTED BUT IMMUNE
RESPONSES DOWN REGULATED THEN
YOU HAVE DNA METHYLATION
ANOTHER SILENCING MARK AND
PROMOTERS OF THE GENES.
SO WHAT HAPPENS IF WE
MANIPULATE THE PATHWAYS AND
RESTORE ACTIVITY?
OR IF WE KNOCKED DOWN OTHER
ENZYMES OF THE HISTONE MARKS
THAT ARE REGULATED OR DNA
METHYLATION?
SO I TOLD YOU BEFORE MAYBE IN
THE NORMAL STATE YOU HAVE K 27
A SILENCING MARK AND IN THE
DISEASE STATE YOU LOSE THE K
27 METHYLATION SO YOU HAVE K
36 OR OTHER MARKS FOUND ALONE.
WHAT HAPPENS IF WE RESTORE THE
MUTATED SUBUNITS AND TRY TO IF
WE REMOVE THE K 36 AND IF WE
CAN GET TO THE STATE.
AND THEN WE CAN HAVE THE
ENTIRE COMPLEX TOGETHER THAT
IS WHAT WE COULDN'T DO WITH
THE MUTATION WE CAN'T PUT OUT
THE ENTIRE COMPLEX AND THEN
THE METHYLATION LEVELS COME
BACK.
WE CAN KNOCKED DOWN AND ASK T.
WE KNOCKED DOWN THAT NSD WE
HAVE CREATED THAT'S LOOK AT
THE PROFILES OR THE NSD
KNOCKDOWN IS BASICALLY THE
SAME GENES WHAT WE SAW BEFORE
BEING AFFECTED SUCH AS THE
INTERFERON GENES AND OTHER
TRANSCRIPTIONAL BUT BASICALLY
THIS SAYS THESE DIFFERENT
HISTONE MARKS ARE FOUND ON THE
SAME GENOME.
HOW SUSCEPTIBLE ARE THESE
CELLS TO THE KNOCKDOWN OR
REMOVAL OF K 36 METHYLATION?
THOSE THAT HAVE A DIFFERENT
MUTATION THE CELLS ARE NOT
SENSITIVE.
BUT IN THOSE THAT LOSE YOU CAN
SEE HOW SENSITIVE THEY ARE TO
LOSE NSD WE CAN DO A TRICK OF
REMOVING ACROSS THE GENOME IT
IS THE SAME STORY.
THE CELLS HAVE A DIFFERENT
MUTATION THOSE THAT HAVE THE
LOST MUTATION DO CARE THEY
LOSE K 36.
IT'S A DIFFERENT WAY.
THIS COULD BE A THERAPEUTIC
VULNERABILITY IN THE FUTURE.
WITH THE DNA METHYLATION
INHIBITOR YOU WILL INCREASE OR
KILL ANY CELLS THAT HAVE DNA
METHYLATION AND INHIBITION IF
YOU INCREASE THE CONCENTRATION
YOU CAN SEE HOW MUCH MORE
SENSITIVE EVEN THE CELLS THAT
CAN KILL THEM BUT I TELL
SENSITIVE THEY ARE TO THE
METHYLATION THEN YOU HAVE
EXPRESSION OF THOSE GENES
WHICH CONFIRMS WHICH WE
THOUGHT WAS THE MECHANISM WHEN
IT COMES INTO SILENCE THE
IMMUNE RESPONSE AND THEN YOU
GET UP REGULATION OF THOSE
THAT WERE DOWN REGULATED
BEFORE.
BUT THE PROBLEM IS TO RESTORE
PROTEIN ACTIVITY WITH A
PATIENT IS DIFFICULT AND THERE
IS NO NSD INHIBITOR THERE ARE
NOT VIABLE OPTIONS.
BUT THERE IS A LOT OF OTHER
HISTONE MARKS THAT ARE
EXPRESSED ESPECIALLY WITH
ACETYLATION.
AND WITH HISTONE THEY SHARE A
MECHANISM OF ACTION.
WE THOUGHT WE COULD PUSH THE
CELLS TO A HYPER ACETYLATED
STATE TO REDUCE TOXICITY.
WE CAN DO THIS SO WE CAN DO
THIS WITH THE AGE THREE AND
EACH FOR ACETYLATION THOSE
LEVELS ARE READY HIRE AT A
BASELINE LEVEL AND WHEN WE ADD
THE INHIBITOR THE CELLS REALLY
GO HIGH.
SO WE DO FEEL WE ARE GETTING
TO THIS POINT.
GETTING TO THE KILL CURVES IN
TERMS OF THE INCREASING
CONCENTRATION THESE THREE IN
RED HAVE THE MUTATION AND ARE
MUCH MORE SENSITIVE THAN THEN
WITH THE INHIBITOR WE CAN HAVE
A EXPRESSION OF ALL OF THE HLA
MOLECULES THEY HAD REPRESSED
IN THE DISEASE STATE.
AND YOU CAN SEE THAT SHIFTS
THE ABILITY.
MORE WITH THE A STACK
INHIBITOR BUT USING A
COMBINATION AND A SINGLE DOSE
OF DNA METHYLATION AND YOU CAN
SEE THE TWO TOGETHER ARE MORE
IMPORTANT THING AND KILLING
THE CELLS THAN JUST THE AGE
DECK INHIBITOR ALONE.
WITH THE QUANTITATIVE POINT OF
VIEW WE LEARNED A LOT ABOUT
THE SYSTEM WITH EPIGENETIC
REPROGRAMMING AND HOW THIS
COULD LEAD TO PROGRESSION OF
CANCER.
WE PUBLISH THE INITIAL PAPER
YEARS AGO.
QUICKLY AND WANT TO SHARE A
NEW PROJECT IT IS IN PROCESS.
NOT A COMPLETE STORY BUT WE
ALWAYS HAD A QUESTION IF YOU
HAD A HIGHLY MODIFIED HISTONE
AND YOU WANT TO REMOVE THE
MODIFICATIONS HOW DO YOU DO
THIS EFFICIENTLY?
AND THEN TO REMOVE THE
MODIFICATION THAT SEEMS LIKE A
LOT OF WORK.
BUT WE ALWAYS THOUGHT IF YOU
WANT TO REMOVE THOSE
MODIFICATIONS THEN CLICK THIS
AND THEN THE HISTONE WOULD BE
EXCHANGED.
THERE COULD BE A LOT OF
CONSEQUENCES HERE WITH
MODIFICATIONS AND OTHER
PROTEINS THAT ARE ATTRACTED
BUT THEN YOU CAN REMOVE THESE
INTERACTIONS.
AND IN FACT HISTONE
CARDIOLOGISTS HAS BEEN SHOWN
IN MANY CONTEXT TO BE INVOLVED
IN A FEW DIFFERENT PROSTHESES
ESPECIALLY CANCER
PROGRESSION.
PROTEASE IS THAT HAS BEEN
IDENTIFIED AS A HISTONE AND
THEN WITH THOSE SPECIFIC SITES
ON THE PROTEIN AND ALSO THERE
HAS BEEN AND AS A WAY TO
REMOVE TALES AS METHYLATION.
THERE IS A A FEW PAPERS HERE
AND THERE IS INTERESTING WE
SHOULD SPENT A LOT OF TIME ON
NUCLEUS BUT THERE ARE PAPERS
THAT SHOW TRANSCRIPTION
FACTORS AND THIS AFFECTS
DIFFERENTIATION AS WELL.
SEE YOU CAN SEE HERE IT
AFFECTS THE MOUSE COLOR AND
HAIR SO ARE THERE ANY OTHER
SUBSTRATES IN THE NUCLEUS?
FOCUSING ON HISTONES BUT WE
HAVE DATA ON OTHERS AS WELL.
WHAT WE ACTUALLY FOUND LOOKING
BACK THROUGH SOME DATA WE
DIDN'T CHOP UP WITH A PROTEASE
BUT BACK DIRECTLY WE FOUND A
LOT OF SITES THAT THIS IS
HISTONE H2 A AND THEN WE
INCUBATED THAT AND SAW THERE
WERE SOME SITES THAT WERE
HIGHLY PREFERRED WHICH MATCHED
THE IN VIVO DATA.
SO LOOKING AT ANY PROCESS THAT
HAD A DIFFERENTIATION OF THE
HISTONE FRAGMENT EVENTS AND
GOING TO MOUSE EMBRYONIC STEM
CELLS THEY COULD BE MANY
DIFFERENT LINEAGES AND THE
EMBRYONIC DIFFERENTIATION SO
WE WENT TO VISIT THIS WITH THE
QUANTITATIVE POINT OF VIEW TO
MAKE EMBRYONIC BODIES AND THE
NEURON CELLS AND WHAT WE FOUND
IS IT IS HARD TO SEE THAT
THERE IS A LIGHTER FRAGMENT
THAT IS LIGHTING UP HERE AND
TO LOOK AT THE FRAGMENTS WE
CAN SEE THAT DURING A FOUR-DAY
DIFFERENTIATION WE HAVE
CLEAVAGE SITES ESPECIALLY ON
DAY FOUR.
THERE IS CLEAVAGE AND
FRAGMENTS WE THOUGHT SO
BECAUSE THERE IS SIMILARITY IN
THE MAJOR SITES WITH H2 A BUT
INCREASING DIFFERENTIATION
EXPRESSION IS A MATURE FORM
YOU CAN SEE ON DAY FOR IT IS
PRETTY HIGH.
SO WE KNOCK IT DOWN WITH THE
EMBRYONIC STEM CELLS AND WE
CAN SEE SEVERAL SITES THAT GO
DOWN ESPECIALLY ALL 23 IT
DECREASES IN ABUNDANCE SOME
DON'T CHANGE BUT OTHER
PROTEASE COULD AFFECT THAT MAY
BE PROCESSING ARTIFACTS OF
WHAT WE ARE DOING?
SPENT SOME SHOW
DIFFERENTIATION ON OF
CLEAVAGE.
SO WHY WOULD YOU NEED THE
PROTEASE CRACKS IF YOU HAVE
MODIFICATIONS IT'S A GREAT WAY
TO REMOVE THOSE
MODIFICATIONS.
LOOK AT H2 A IT HAS THOSE THAT
CAN BE ACETYLATED AT A LOWER
LEVEL.
AND OVER DIFFERENTIATION YOU
CAN SEE THE ABUNDANCE GOES UP
FOR ALL OF THESE AND THEN IT
DIPS A DAY FOR SO WHAT HAPPENS
WHEN WE KNOCK IT DOWN HERE
DURING THE DIFFERENTIATION?
AND THE CONTROL DAY FOR IT
GOES DOWN BUT THEN THE LEVELS
DON'T GO DOWN ANYMORE AND THE
NUMBER FOUR YOU CAN SEE THE
LEVELS DROP AND THEN WHEN WE
KNOCK IT DOWN THE LEVELS DON'T
DROP ANYMORE.
THIS TOLD US THAT CAPS ON IS A
PROTEASE.
WE WANT TO SEE WHERE CANINE
ACETYLATION WAS BOTH IN THE
CONTROL AND DOWN SO HERE WHAT
YOU CAN SEE BETWEEN DAY TWO
AND DAY FOR IN THE CONTROL
THEY HAVE WHAT DECREASES A
LITTLE BIT YOU CAN SEE THIS
BUT WHEN YOU KNOCK DOWN IN
SOME CASES IT GOES UP IS WHAT
YOU WOULD EXPECT SO
ESSENTIALLY WHEN YOU COMPARE
DAY TO THROUGH DAY FOR THOSE
THAT GO DOWN DO NOT GO DOWN
ANYMORE AND THE EXPRESSION
LEVELS THE NUMBER OF PEAKS DAY
TO THROUGH DAY FOR YOU CAN SEE
THEM GOING DOWN BUT INCREASE
HERE THE ANALYSIS SHOWS ALL
THINGS INVOLVED IN CELLULAR
DIFFERENTIATION ESPECIALLY TO
THE NERVOUS SYSTEM NOT A LOT
IS KNOWN ABOUT ACETYLATED H2
WAY BUT THE WHOLE IDEA IT IS
PROBABLY THERE IS SOMETHING
RECOGNIZE THE MODIFICATION.
WE THOUGHT TO DO AN EXPERIMENT
WHAT COULD BE RECOGNIZE SO WE
SAW SYNTHETIC PEPTIDES EITHER
WITH OR WITHOUT THE
ACETYLATION AND THE PULL DOWNS
FROM EXTRACTS TO THE PRETTY
ONYX WHERE WE COMPARED H2 A
ALLOSTATIC, CANINE OR THE
COMBINATION TOGETHER.
AND FOR THOSE THAT MIGHT
RECOGNIZE THESE IT IS THE
COMPLEX SEVERAL DOMAINS AND
THEN THEY COULD BIND THE
ACETYLATED SITES.
IT DOESN'T PULL THEM AS STRONG
AS HE ACETYLATED.
BUT THIS IS ALL UNIT SPECIFIC
TO THE COMPLEX.
AND P BETH IS KNOWN TO PLAY A
ROLE AND THEN TO SEE
POTENTIALLY WITH H2A AND
EMBRYONIC STEM CELLS.
AND THEN TO BE TURNED ON A
STAPLE WANT TO DIFFERENTIATE
AND THEN IT COMES UP WITH THE
GENE REGULATION AND ALLOWS THE
CELLS TO DIFFERENTIATE INTO
DIFFERENT LINEAGES.
SO AS YOU CAN SEE HERE THESE
ARE THE PEAKS PR BM ONE WITH
H2 A CANINE.
SO YOU HAVE A LARGER NUMBER OF
PEAKS WITH A CANINE AS THE
PROMOTER.
IN TERMS OF THE EXPRESSION AND
THEN WITH THE SETTLE IN THE
SAME WITH THE PR BM EXPRESSION
IS NOT AS STRONG AS THOSE THAT
HAVE IT WITH THE H2A.
HE WENT TO DO EXPERIMENTS TO
SEE IF IT WOULD MANIPULATE TO
CLIP H2A.
WE EXPRESS FULLY INTO CELLS
AND THAT SHOWS IT IS MISSING A
LOT OF THE PROTEINS AND THERE
IS ACTUALLY PROTEINS THAT ARE
UPREGULATED SO THESE ARE
EXCHANGE FACTORS OR NUCLEOSOME
ASSEMBLY FACTORS OF THESE
COULD BE THE PROTEINS THAT
RECOGNIZE REMOVING THEM.
AND WITH THOSE NUCLEOSOME'S
WHEN YOU LOOK AT THE FULL ONE
WITH THE CLUB.
AND WHEN YOU HAVE EXPERIMENTS
YOU CAN SEE HERE THAT THE
CONTROL KNOCKDOWN ARE THE
LEVELS OF THE PR BM AND THEN
YOU CAN SEE THESE ARE LEVELS
WITH THE HISTONE MARK AND IF
YOU JUST PULL OUT A TRACK YOU
CAN SEE HERE WHEN WE KNOCK
THIS OUT THE PEAK GETS A
LITTLE STRONGER FROM H2 WAY.
AND HERE AS WELL.
YOU CAN SEE PR BM.
SO IN THE KNOCKDOWN WE GET
STRONGER H2 WAY.
AND CLIPPING TO THE ABILITY TO
BE FOUND AT SPECIFIC GENES AND
THE GENOME THAT HAVE H2A.
LASTLY WITH THE FULL LINKAGE
TO A WE CAN SEE THE HISTONE
MARKS ARE QUITE DIFFERENT AND
THE CLIPPED H2 A IS FOUND WITH
MORE ACTIVE GENES.
WE KNOW IT'S GETTING CLIPPED.
WITH MORE ACETYLATED H2A.
AND THOSE THAT ARE MORE ACTIVE
MARKS.
AND THOSE THAT ARE MORE LINKED
TO GENE SILENCING.
THE CLIPPED H2 WAY YOU CAN
IMAGINE IT CANNOT SAY IN THE
NUCLEOSOME FOREVER SO IF YOU
DO A POST CHASE TYPE OF
EXPERIMENT AND LOOK AT THE
INCORPORATION OR THE CLIPPED
H2 WAY WE CAN SEE ONCE WE ADD
USING SOME PRO RODEO PSALM
INHIBITORS THESE DO NOT HAVE
IT BUT THEY ARE INHIBITING A
PROTEIN SYNTHESIS YOU CAN SEE
THE LEVELS STAY FOR SEVERAL
HOURS.
IS NOT AS STABLE FOR A
FULL-LENGTH THAT IS A WORK IN
PROGRESS WITH A DIFFERENT
MECHANISM THAT COULD BE
INVOLVED AND TO RECOGNIZE LOW
LEVEL ACETYLATION.
SHOWING THE TECHNOLOGY
ADVANCES.
TO FOLLOW THE EPIGENETIC
CHANGES THAT OCCUR BECAUSE OF
THESE MUTATIONS AND THE SOFT
TISSUES.
THAT GIVES A LOT OF TARGETS
THAT TRY TO GET DATA IN THE
MOUSE MODELS AND KEEP MARCHING
TOWARDS WITH THE THERAPEUTIC
INTERVENTION.
IT IS ON THE BASIC SCIENCE
SIDE BUT AND TO REGULATE
HISTONE MODIFICATIONS DUE TO A
MUCH MORE PERMANENT TYPE OF
WAY.
AND WAS STEM CELL
DIFFERENTIATION.
THANK YOU TO THE GROUP.
AND CONTRIBUTED TO THE
DIFFERENT PROJECTS AND ALSO
ONE OF THE PEOPLE WORKING ON
PROJECT AND THOSE WORKING ON
THE TECHNOLOGY ADVANCES AS
WELL AND I'LL BE HAPPY TO
ANSWER ANY QUESTIONS.
THANK YOU.
[APPLAUSE]
>> IT LOOKS LIKE WE HAVE SOME
QUESTIONS.
>> I HAVE A COUPLE OF
TECHNICAL QUESTIONS AND THEN
TO DESCRIBE THE QUANTIFICATION
OF THE POST- TRANSLATIONAL
MODIFICATION OF HISTONES IS
EXTREMELY INTERESTING.
AS YOU POINTED OUT IS ALSO
VERY CHALLENGING.
THE NUMBER OF MODIFICATIONS
AND DENSITY AND THE NUMBER OF
POSSIBLE COMBINATIONS.
I WONDER WHAT YOUR THOUGHTS
ARE ON THE RELATIVE MERITS OF
TOP-DOWN ANALYSIS VERSUS
BOTTOM-UP?
AND WHAT IS THE THINKING WITH
REGARD TO BOTTOM-UP ANALYSIS
AS TO WHAT PROTEASE TO USE?
THAT COULD BE PROBLEMATIC.
>> GOOD QUESTION.
IT REALLY GETS BACK TO
EVERYTHING I SHOWED YOU WAS
BOTTOM-UP.
SMALLER PEPTIDES.
BUT IT REALLY DEPENDS ON THE
QUESTIONS YOU ARE ASKING.
IF YOU ASK ABOUT COMBINATIONS
OF MODIFICATIONS AND WANT TO
LOOK AT A BIGGER STRETCH YOU
HAVE TO DO MIDDLE OR TOP DOWN.
THERE ARE CHALLENGES THERE
BOTTOM-UP IS MUCH MORE FASTER
AND QUANTITATIVE AND EASIER TO
DO TOP-DOWN BASICALLY THE
OPPOSITE.
WE HAVE DONE A LOT AND BOTH
WORLDS.
BUT TO ANSWER BIOLOGICAL
QUESTIONS RIGHT NOW MOST
PEOPLE STILL PREFER BOTTOM-UP.
YOU CAN GET OUT WHAT IS
HAPPENING IF YOU DO SEE
SOMETHING HINTING AT A
COMBINATION WE SAW A PEPTIDE K
27 K 36 METHYLATION CHANGING.
I DIDN'T SHOW YOU THE DATA BUT
WE WANT TO EXTEND THAT TO SEE
WHAT OTHER METHYLATION WE HAVE
DONE OTHER TOP-DOWN AND EXTEND
TO OTHER MODIFICATIONS SO IT
IS A LOCAL BINARY COMBINATIONS
WHICH IS DEPENDS ON THE
QUESTION.
BUT DEFINITELY GOING TO MIDDLE
AND TOP-DOWN YOU BETTER BE
PREPARED TO SPEND MORE EFFORT
ON THAT.
AND THE PROTEASE.
I DIDN'T GO OVER THE COMPLETE
PLATFORM OF WHAT WE DO.
BUT FOR BOTTOM-UP WE
CHEMICALLY DERIVATIZED THE
HISTONE TO BLOCK THE LYSINE.
IT WILL BE NATURALLY BLOCKED
SO WE CHEMICALLY MODIFY TO
BLOCK THE UNMODIFIED AND THEN
THEY ARE NICELY SPREAD OUT SO
WITH THIS APPROACH YOU CAN GET
A DEAR 90 PERCENT SEQUENCE
COVERAGE.
>> I MAY HAVE MISSED IT BUT IF
IT IS TARGETED TO ALL HISTONES
OR WHAT IS TARGETING IT?
>> A PREVIOUS PAPER THAT I
MENTIONED DOES SHOW IT CAN HIT
HISTONE AGE THREE BUT FROM OUR
OWN DATA IT SEEMS H2 WAY WE
SEE MORE FUNDED FRAGMENTS.
BUT WE DO SEE A LITTLE BIT
EVEN IN OUR SAMPLES AND LITTLE
BIT OF EACH TO BE BUT IT IS
PROMISCUOUS AND HITTING A
LITTLE BIT OF EVERYTHING.
>> IS TARGETED TO CERTAIN
PARTS OF THE GENOME?
>> I THINK SO.
IT LOOKS LIKE IT IS MOSTLY
AFFECTING PATTERNS THAT HAVE
THE ACETYLATED H2 WAY AND
THAT'S NOT EVERYWHERE IN THE
GENOME.
AND HOW IT IS TARGETED TO THAT
WE HAVE NO IDEA BUT IT SEEMS
TO BE TARGETED TO THE
ACETYLATED FORM THAT IS LESS
THAN HALF A PERCENT OF THE
ENTIRE H2 WAY MARK SO THERE IS
SOMETHING VERY SPECIFIC ABOUT
IT BEING TARGETED BUT WE HAVE
NO CLUE WHAT THAT IS WE'VE
TRIED TO DO SOME IP TO SEE
WHAT IS BINDING BUT NOTHING
THAT MAKES A LOT OF SENSE.
MAYBE WE NEED TO USE A
CROSS-LINK SO WE DON'T LOSE
WHATEVER IT IS INTERACTING
WITH.
THAT IS A GOOD QUESTION.
I DON'T KNOW HOW IT IS BEING
TARGETED.
>> I HAVE A QUESTION BUT IT'S
MORE GENERAL.
WE SEE A LOT FRAGMENTS.
THERE COULD BE A LOT OF
PROTEASE GETTING INTO THE
NUCLEUS AND SPECIFICALLY -- --
SPECIFICALLY CHROMATIN.
>> I THINK WE WILL CONCLUDE.
THANK YOU VERY MUCH.
[APPLAUSE]