>> GOOD AFTERNOON, LADIES AND
GENTLEMEN.
TODAY I'LL TALK ONE OF MY
RESEARCH PROJECTS INCLUDING THE
CSB AND ACCELERATING CELLULAR
AGING.
EVERYONE GETS OLD.
WE DON'T WANT TO BUT IT'S
INEVITABLE UNLESS YOU CAN STOP
TIME.
SO SCIENTISTS WORK REALLY
HARD --
[NO AUDIO] EPIGENETICS ARE A RESEARCH
FIELD THAT STUDY
[NO AUDIO]
>> TO FORM NUCLEAR SCAFFOLD TO
GET INTO THE NUCLEAR.
SO IT IS WELL KNOWN THESE HAVE
SPECIFIC STRUCTURES WHICH
PROTRUDE INTO NUCLEARPLASM AND
PRONE TO VARIOUS TRANSLATIONAL
MODE FICTION SUCH AS
PHOSPHORYLATIONS AND ONE OF THE
OUTCOMES OF THE MODIFICATIONS IS
THAT IT ALTER THE ELECTROSTATIC
NATURE OF THE PROTEINS THERE BE
REGULATING THE INTERACTION FORCE
OF DNA MOLECULES.
BY THAT IT BECOMES LOOSE AND
CONDENSE AND REGULATES THE
TRANSCRIPTION STATUS OF THE GENE
INSIDE THESE REGIONS.
SO, WHEN THE CHROMATIN STRUCTURE
BECOMES LOOSE THEY CAN ACCESS
THE DNA REGIONS THERE BE
TRANSCRIPTION IS SWITCHED ON.
OTHERWISE IT CANNOT REACH THESE
DNA REGIONS.
AND THERE'S TRIMETHYLATION AND
THESE ARE TWO CATEGORIES.
THIS MODEL OF AGING WAS PROPOSED
MORE THAN TO -- 20 YEARS AGO AND
THE CHROMATIN STRUCTURE WAS
CAUSING ALTERED TRANSCRIPTION
PROFILE CAUSING THE AGING IN
PHENOTYPES.
AND SINCE THEN LOTS OF FOLLOW-UP
STUDIES HAVE IDENTIFIED THESE
CHROMATIN STRUCTURES GETTING
LOOSE AND THE TRIMETHYLATION IS
DECREASING BY SMALL LEVELS AND
CELLULAR LEVELS.
AND THE EXPRESSION WAS DECREASED
BY AGING.
IT'S BEEN INTERESTING THAT THESE
AGING SYNDROMES HAVE ALSO
REPORTED TO HAVING DECREASED
HEMATOCHROMATIN STRUCTURE AS
WELL AND WITH THE LOSS OR
ALTERED MODIFICATION RESULTS IN
LOSS OF TRANSCRIPTION PRECISION
WHICH IS CAUSING INSTABILITY
WHICH IS VERY TOXIC TO CELLS.
IT IS VERY WELL KNOWN IN THE
RUNNER SYNDROME CELLS THESE ARE
IMPORTED BUT IT WASN'T THE CASE
IN THE COKIN SYNDROME.
SO WHAT IS THE COKIN SYNDROME?
IT'S A RECESSSIVE DISEASE
ACCELERATED AGING DISORDER
CHARACTERIZED BY
NEURODEGENERATION, TROFISM AND
MOUSE MODELS HAVE DYSFUNCTION
WHICH IS CHARACTERIZED BY
INCREASED ENERGY TURNOVER OR
PHOSPHORYLATION AND ACCUMULATION
OF DAMAGED MITOCHONDRIA OR ATP
CONSUMPTION.
THESE TWO GENES HAVE DISTINCT
STRUCTURE BUT THE MUTATION HAS
VERY SIMILAR PHENOTYPES.
AND THE MAJORITY OF THE SYNDROME
IS ALSO KNOWN PATIENT SUFFERING
MORE AND THEY INDUCE DAMAGE
REPAIR AND SO IN 1936 THE
COCKAYNE SYNDROME WAS DISCOVERED
TO HAVE REGULATION AND MOST
RECENTLY IN 2017 CSB IS DIRECTLY
REGULATING THE TRANSCRIPTION OF
THE TRANSCRIPTION DEPRESSOR BY
DEGRADING THESE REPRESSORS ATF3
REPRESSORS.
AND THIS HAS SO MUCH
PHOSPHORYLATION THE REASON IS
WHEN THERE IS DAMAGE, PART ONE
IS RECRUITING TO THE DNA DAMAGE
AND LOOKING AT THE
PHOSPHORYLATION BRANCHES AND
IT'S KNOWN TO REPLACE --
DISPLACE THEM FROM DAMAGED
CHROMATIN.
BUT WHEN THERE'S NO FUNCTIONAL
CSB SUCH AS COCKAYNE SYNDROME
CELLS IT EXISTS IN THE DAMAGED
DNA WHICH CONSUMES SO MUCH OF
CELLULAR CHANGE ASSOCIATED WITH
PHENOTYPES.
AND DESPITE THE IMPORTANCE THE
FUTURE OF THESE ARE SO UNSTABLE
AND IT'S HARD TO DETECT BY THE
METHOD USING COMMERCIALLY
AVAILABLE ANTIBODIES AND HARD TO
DO SOMETHING LIKE CHP SEQUENCING
BUT TECHNOLOGY WAS DEVELOPED AND
THEY HAVE IDENTIFIED OKAY DATIVE
DAMAGE INDUCED AND THEY'RE
MOSTLY ENRICHED IN DENSE
CHROMATIN REGIONSES.
SO BASED ON THIS -- REGIONS.
SO BASED ON THIS FACT, SOME
QUESTIONS AROSE IN MY MIND.
IS HEMATOCHROMATIN STRUCTURE
DESTROYED IN THE CELLS AND IF
YES DO THEY CORRELATE WITH THE
CELLS AND WHAT IS THE CAUSE OF
HEMATOCHROMATIN DISRUPTION IN
THE CELLS AND WHICH DNA HAVE THE
MOST IN THE CELLS.
SO I STARTED AN EXPERIMENT GREW
THE CELLS THAT ARE CSB MUTATED
AND THESE ARE FROM CSA TO CSB
PATIENT FIBROBLASTS.
AND THERE ARE TWO COMPLEMENTARY
CONTROL CELLS COMPENSATED BY THE
WILD TYPE COUNTERPARTS.
I ALWAYS NOTICE THE PATIENT
CELLS HAD ENLARGED NUCLEUS.
YOU CAN SEE THE SIZE HAS BEEN
INCREASED IN CSB OR CSA PATIENT
CELLS BUT THE INTENSITY WHICH
REPRESENTS THE DENSITY OF
NUCLEAR DNA THE MUTATED CELLS
HAVE SIGNIFICANTLY LESS DNA
DENSITY COMPARED TO THE CONTROL
CELLS.
I SUSPECT THEY'RE UNDERGOING
SOME KIND OF CHROMATIN
ALTERATIONS AND SUBJECT USING
DIFFERENT HISTOMARKS.
YOU CAN SEE THE CSB YOU --
MUTATED CELLS HAVE
TRIMETHYLATION AS A MARKER FOR
CHROMATIN.
AND TO TEST THE POSSIBILITY THE
DECREASE OF TRIMETHYLATION IS A
DIRECT CONSEQUENCE OF CSB LOST,
I GENERATED INDUCIBLE KNOCK-DOWN
CELLS OF CSA AND CSB INTO
COMMONLY USED LAB SLIDES.
YOU CAN SEE INDUCED KNOCK-DOWN
OF CSB RESULTED IN REDUCED THE
TRIMETHYLATION MARK IN THE
CELLS.
I SO -- IT LEADS TO DECOMBINING
AND THIS IS IMPORTANT IN PATIENT
CELLS AND A TESTED HOW THE PARTS
ACCUMULATE ON THE CELLS BY
REDUCING OKAY DATIVE STRESS AND
WHEN I LOOKED AT THE CSB CELLS,
YOU CAN SEE THAT THE CSB CELLS
ARE REACTING MUCH MORE SENSITIVE
COMPARED TO THE CONTROL CELLS
UNLIKE CSA CELLS AND USING
ANOTHER DAMAGING AGENT I
CONFIRMED THAT THE CSB CELLS ARE
MUCH MORE SENSITIVE TO OXIDATIVE
ADVANTAGE.
AND I WANTED TO SEE
SYSTEMATICALLY USING REAL-TIME
PCR HOW THE TRIMETHYLATION HAS
BEEN ALTERED IN MUTATED CELLS.
THESE THREE REPRESENTATIVE
REGIONS.
THIS REPRESENTS THE HOUSEKEEPING
GENE.
THEY ALMOST DON'T HAVE
HETEROCHROMATIN STRUCTURE AND
THE REGION IS A DENSE REGION.
SO I CONFIRMED THAT THESE
REGIONS AS WELL AS OTHER REGIONS
IN GENERAL DNA ALL THE REGIONS
MOST OF ALL THE REGIONS HAD
DECREASED HETEROCHROMATIN IN THE
CELLS.
I TRIED TO DO THIS ASSESSMENT.
AND WITH THE METHOD WHEN THERE
IS DAMAGE I FIXED THEM WITH
HIGHER PERCENTAGE COMPARED TO
JUST CONVENTIONAL CHROMATIN AND
USED 4% INSTEAD OF 1%.
AND INSTEAD OF USING ANTIBODIES
IN THE OXIDATION AND WE GAVE
CFCR FOR SEQUENCING.
THESE ARE THE RESULTS OF WHICH
ARE INDUCED.
IN THE CSB MUTATED OR CONTROLLED
CELLS AND YOU CAN SEE THESE
HYDROGEN PEROXIDE TREATMENT
INDUCED PARLYLATION IN THESE
REGIONS.
AND I CONFIRMED THAT WITH
ANOTHER DNA THE CSB MUTATED
CELLS HAVE MORE ACCUMULATION BUT
NOT IN THE CHROMATINIZED REGION.
AND I LOOKED AT THE SEQUENCING
TO CONFIRM THE RESULT.
AND I COULDN'T FIND DISTRIBUTION
BETWEEN THE CELLS WHICH IS
DISAPPOINTING AND WHEN I DID A
DETAILED STUDY CALCULATING PEAK
ENRICHMENT NOT ENRICHED IN CSB
MUTATED CELLS I COULD FIND THAT
GSS TRANSCRIPTION FLANKING
REGIONS ARE MORE PAR SENSITIVE
THAN MUTATED CELLS.
AND AS YOU CAN SEE THE CSB
MUTATED CELLS ARE HAVING MUCH
LESS CHROMATIN ON THE START SIDE
THAN THE MUTATED CELLS.
AND TO EXCLUDE THE POSSIBILITY
THAT THESE TRANSCRIPTION SITES
AND PEAK REGIONS, I DID THE
CALCULATIONS AND IT TURNED OUT
MOST OF THESE HETEROCHROMATIN
PEAK REGIONS ARE FAR FROM
TRANSCRIPTION START SIDE
INCLUDING THE POSSIBILITY THESE
TWO REGIONS ARE OVERLAPPING.
AND I DID THE ALIGNMENT OF THE
SEQUENCING PIECE TO THE
TRANSCRIPTION START SITE OR THE
REGIONS AND I GOT CONSISTENT
RESULTS WITH REAL-TIME RESULTS.
THE CELLS HAVE MORE PARYLATION
AND THERE WAS MINIMAL CHANGES IN
HETEROCHROMATIN REGIONS.
AND AS WE TRY TO UNDERSTAND WHY
THE CHROMATIN WAS DECREASED IN
THESE CELLS, I SCREENED FOR THE
FACTORS THAT AFFECT
TRIMETHYLATION CSA AND CSB
CELLS.
AND I FOUND THIS THESE TWO
METHODS WERE SIGNIFICANTLY
DECREASED IN CSB MUTATED CELLS.
AND WE HAD PROTEIN LEVELS THAT
WITH DECREASED MUTATED CELLS.
AND I TRIED TO UNDERSTAND WHY
THESE TWO METHODS OF EXPRESSION
WAS DECREASED IN MUTATED CELLS
AND IN THE LITERATURE RESEARCH
AND CAME UP WITH THE PAPER IN
2017 AND THEY CLAIM THAT THE
PROTEOLYSIS AND THE OCCUPANCY
MEANS MUTATED CELLS ARE EASIER
IN THE TARGET GENES.
AND I CONFIRMED BY SEARCHING THE
DATABASE AND THE TRANSCRIPTION
FACTOR DATABASE AND FOUND IT WAS
THE TARGET.
AND FROM THAT DATABASE I FOUND
THE TWO TRANSFERASES WERE BEING
OCCUPIED BY FBF3.
I ANALYZED THE SEQUENCING DATA
FOUND THE BINDING TO THE
PROMOTERS OF THE TWO GENES BUT
NOT IN THE CONTROL CELLS.
WE TRIED TO RECOVER THE
HETEROCHROMATIN MARK AND SEE HOW
IT WORKED IN DAMAGE INDUCED
OSCILLATION ACCUMULATION.
I GENERATED THE MUTATED CELLS
AND WHEN I INDUCED THE TWO I CAN
SEE IT COVERS THE
TRIMETHYLATION.
AND I FOUND IT IN THE INDUCED
CELLS AND I ALSO CONFIRMED THE
RESULTS BY REAL TIME PCR SHOWING
THE INDUCED EXPRESSION DECREASES
THE OSCILLATION ACCUMULATION.
SINCE PATIENTS HAVE
CHARACTERISTICS OF MITOCHONDRIAL
DYSFUNCTION, I WANTED TO SEE IF
THIS COULD RECOVER THE ABNORMAL
MITOCHONDRIAL PHENOTYPES IN THE
CELLS AND YOU CAN SEE IT HAS
MUTATED CELLS AND BY THAT YOU
CAN SEE DRAMATICALLY DECREASED
THE OSCILLATION AND WE TEST THE
LEVELS AND THE WHETHER WE USE
THIS OR NOT IT HAD NO EFFECT AND
HAS A RATE IN THE ALL THE
PROFILES BUT WHEN YOU INDUCED
THE SETDB1 RECEPTOR IT RECOVERS
TO ALMOST NORMAL LEVEL WITH THE
MITOCHONDRIAL PROFILES.
AND FINALLY, I WANTED TO TEST IF
THESE DECREASING PHENOTYPE IS
ALSO TRULY IN CHRONOLOGICAL
AGING MODEL.
I GOT THESE FIBROBLAST CELLS
FROM HEALTHY HUMAN CANDIDATES
EITHER FEMALE OR MALE.
AND STAINED IT FOR CSB OR SET
DB1 EXPRESSION LEVEL AND THE
LEVEL DECREASES BY AGING AND THE
SETDB1 EXPRESSION LEVEL
DECREASES BY AGING.
AND NOT ONLY IN HUMAN CELLS.
I TRIED TO TEST IF IT'S TRUE IN
MICE.
BRA
BRAIN-TISSUE EXTRACTION AND THE
HARD LEVELS ARE SIGNIFICANTLY
INCREASING IN THIS LEVELS AND
THE CSB LEVELS WAS DECREASED AND
THE EXPRESSION WAS DECREASED AND
AS EXPECTED, THE TRIMETHYLATION
WAS DECREASED.
SO, TO SUM UP, WHEN THERE IS
NORMAL CSB, CSB IS SUFFICIENT
AND THE LEVELS CAN INDUCE THE
TRIMETHYLATION WHICH LEADS TO
MITOCHONDRIAL HOMEOSTASIS BUT
WHEN THERE'S NO OR LESS
FUNCTIONAL CSB AN OLD ORGANISM
EXPRESSION LEVELS DECREASE AND
SETDB1 LEVEL DECREASED AND BY
THAT THERE'S LESS TRIMETHYLATION
LEVELS WHICH LEADS TO
MITOCHONDRIA HOMEOSTASIS.
NOW I CAN ANSWER THE QUESTIONS
THEY HAD BEFORE I DID THESE
EXPERIMENTS.
FIRST, IT'S DISRUPTED IN IN THE
CS CELLS.
YES, DEFICIENT CELLS HAVE LESS
TRIMETHYLATION LEVELS THAN
CONTROL CELLS WHILE CSA CELLS
ARE NOT AFFECTED.
SO DOES IT CORRELATE WITH THE
PATHOLOGY OF CELLS.
I FOUND THE TRIMETHYLATION
REDUCED THE STRESS AND INDUCED
PARYLATION AND LOOKED AT THE
DEFICIENT CELLS AND I FOUND THAT
THE CSB MUTATED CELLS ARE
EXPRESSING LESS AND BY LOOKING
AT THE PRODUCT THE DEFICIENT
CELLS ARE GREATLY INCREASED
ESPECIALLY ON THE TRANSCRIPTION
FLANKING REGIONS WHERE LEVELS
ARE DECREASED IN THE MUTANT
CELLS.
AND FINALLY, I WOULD LIKE TO
THANK DR. GORE FOR GIVING ME A
GREAT OPPORTUNITY TO DO RESEARCH
IN HIS WONDERFUL LAB AND GIVING
ME THE OPPORTUNITY TO SPEAK IN
FRONT OF THESE SCIENTISTS TODAY.
I WOULD LIKE TO THANK OUR
RESEARCH AND PRESENTATION AND
WOULD LIKE TO SPECIALLY THANK
EDWARD KIM BY STUDENT.
HE'S SUCH A DEDICATED STUDENT
AND I WOULD LIKE TO THANK DR.
TYLER FOR THEIR DEDICATION IN MY
STUDY.
THANK YOU SO MUCH FOR YOUR
ATTENTION.
[APPLAUSE]
>> ALL RIGHT.
THANKS.
SO DO YOU WANT TO RUN IT OR
SHALL I DO IT?
>> DR. LEE, WONDERFUL TALK.
IF YOU CAN STAY THERE PEOPLE
WILL ASK QUESTIONS FROM
DIFFERENT SITES.
LET'S GO TO STONY BROOK, NEW
YORK.
>> HI, I HAVE A QUESTION THAT'S
KIND OF A GENERAL ONE AND MAYBE
TOO OBVIOUS BUT IF THE CHROMATIN
EFFECTS OF THE CSB DEFICIENCY
ACCOUNT FOR THE ACCELERATING
AGING PHENOTYPE WHAT IS GOING ON
WITH CSA, WHY ARE THE PHENOTYPES
SO CONVERGENT?
>> SO BOTH PHENOTYPES ARE KNOWN
TO HAVE SIMILAR PHENOTYPES BUT
THE SYMPTOMS ARE LESS SEVERE
THAN CSB MUTATED CELLS.
I THINK THE CHROMATIN
CONTRIBUTING THE DIFFERENCES IN -
CSA INDUCED SECURITIES.
>> COULD YOU REMIND US OF CSA
CELLS SHOWED THE SAME
MITOCHONDRIAL DEFECTS AS CSB?
>> YES, THEY DO BUT I DIDN'T
TEST IT IN MICE.
SO ONE POSSIBILITY IS WORKING
DIRECTLY ON MITOCHONDRIA AND ONE
WORKS ON THE CHROMATIN EFFECTS?
>> MAYBE.
>> OKAY.
VERY GOOD.
PLEASE MUTE IN STONEY BOOK.
LET'S GO TO PITTSBURGH.
>> CAN YOU HEAR ME OKAY?
>> YES.
>> A WHILE AGO THERE WAS CSB
INVOLVED WITH TRANSCRIPTION
REPAIR USING A BEAUTIFUL FISH
ASSAY AND WONDERING IF SOME OF
THE RESULTS YOU'RE SEEING FROM
HYDROGEN PEROXIDE CAN BE RELATED
TO THE TRANSCRIPTION REPAIR IN
CSB CELLS.
>> I HAVEN'T LOOKED INTO THAT
BUT AS YOU CAN SEE IN MY DATA,
THE ACCUMULATION WAS MORE
SEVERELY AT THE SITE THAN IN
HETEROCHROMATIC REGIONS WHICH
REPRESENTS THESE TRANSCRIPTION
IS MORE PRONE TO CSB
TRANSCRIPTION.
SO I THINK IT HAS TO DO WITH
THIS ACTIVITY.
>> THANK YOU VERY MUCH FOR AN
INTERESTING TALK.
>> THANK YOU.
>> OKAY.
THANK YOU.
PLEASE MUTE IN PITTSBURGH.
LET'S GO TO NIEHS IN RESEARCH,
TRIANGLE PARK.
>> I HAVE A QUESTION.
WHAT IS THE CONCENTRATION OF
PEROXIDE.
THE OTHER QUESTION IS ARE THERE
ANY TRANSCRIPTIONAL MARKERS?
>> PLEASE IDENTIFY YOURSELF AT
NIEHS.
>> NATALI VITORIMA.
>> I COULDN'T HEAR YOU VERY
CLEARLY.
I'M NOT SURE I'M UNDERSTANDING.
I USED THE HYDROGEN PEROXIDE FOR
30 MINUTES.
AND IT SEEMED IN MORE THAN 30
MINUTES THE CELLS START TO DIE.
I WANTED TO SEE ACUTE
ACCUMULATION ON THE CELLS BY
THAT CONCENTRATION.
TO ANSWER THE SECOND QUESTION, I
SEARCHED FOR THE CONTROVERSIES
AND METHYLASES AND I SAW
DISTINCT GENE EXPRESSION BUT I
CANNOT TELL YOU AT THE MOMENT.
>> WHAT IS THE LEVEL OF CELL
DEATHS WHEN YOU USE THE 2.5
PEROXIDE IN YOUR EXPERIMENTS?
TO EXCLUDE THE POSSIBILITY OF
SECONDARY EFFECT?
>> THANK YOU.
PLEASE MUTE AT NIEHS AND LET'S
GO TO LEXINGTON, KENTUCKY.
UNIVERSITY OF KENTUCKY, DAVID.
>> WE'RE HERE BUT WE DON'T HAVE
ANY QUESTIONS.
I JUST WANT TO THANK DR. LEE FOR
AN INTERESTING TALK.
>> PONG LET'S GO TO
>> OKAY, LET'S GO TO UNIVERSITY
OF GAINESVILLE.
>> CAN YOU COMMENT ON THE
POSSIBILITY THAT IT'S EITHER AN
INCREASE IN PARYLATION OR IF
IT'S A DECREASE IN REMOVAL OF
THE PAR AND THE OTHER QUESTION
IS CAN YOU EXPLAIN AGAIN THE
REDUCTION AND INCREASED PAR.
?
THANK YOU.
>> IT IS WELL KNOWN THAT FORMING
CHROMATIN BY MINIMIZING DNA
EXPOSURE TO THE ENVIRONMENT IS
VERY GOOD FOR DNA PROTECTION.
SO I THINK MORE HETEROCHROMATIN
FORMATION IS CAUSING A MORE
BENEFICIAL EFFECT FROM
PROTECTING DNA FROM GETTING
DAMAGED IN THE FIRST PLACE.
AND WHAT'S THE SECOND QUESTION?
>> IS IT A COUPLELATION --
ACCUMULATION OF PAR OR
PARYLATION BEING DECREASED?
>> IT MAKES MUCH MORE PAR
ACCUMULATION.
SO YOU STARTED TO MENTION THAT
CSB COULD NOT LOCALIZE TO THE
CELL SITES.
IS THERE A SEQUENCE FOR THE
GENE?
CAN YOU EXPLAIN?
>> SO WHENEVER THERE IS DNA
DAMAGE OR REGULATED DAMAGE, CSB
CAN BE THERE TO REPAIR THE
DAMAGED DNA.
BACK IN 2017 IT WAS IDENTIFIED
CSB HAS CONSENSUS BINDING
SEQUENCE THAT IS VERY SIMILAR TO
INTERACTING SEQUENCE.
>> HAVE YOU LOOK AT INHIBITERS?
>> I HAVEN'T.
I'M SORRY.
>> THAT'S A QUICK ANSWER.
WE CAN GO AROUND IF PEOPLE HAVE
ADDITIONAL QUESTIONS.
JUST UNMUTE AND JUMP IN.
>> I HAVE A QUESTION FROM THE
UNIVERSITY OF FLORIDA.
SO YOU SHOW THAT THE EXPRESSION
IS INTACT TO THE ATF IN THE
CELLS AND RATHER YOU HAVE LESS
TRIMETHYL RESIDUES OF THESE AND
MORE DNA.
DO YOU KNOW IF THERE'S ALSO A
REEFFECT IN THE RECRUIT --
DEFECT OF THE RECRUITMENT IN THE
CELL OR PURELY AN EXPRESSION
DEFECT?
>> I HAVEN'T TESTED FOR SETDB1
RECRUITMENT TO THESE DNA BUT IN
MY SPONSOR, THERE'S A DRE CRASE
IN SETDB1 AND I THINK THE
EXPRESSION LEVEL DECREASES.
>> THANK YOU VERY MUCH.