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I AM DELIGHTED TO INTRODUCE OUR
SPEAKER TODAY, Dr. JASON A.
WATTS.
A NEPHROLOGIST, SCIENTIST AND
STADTMAN INVESTIGATOR IN THE EP
AGAIN ET TICKS AND STEM CELL
LABRADOR AT THE HEALTH SCIENCES
NIEHS LOCATED IN NORTH CAROLINA.
HE IS ALSO AN ADJUNCT ASSISTANT
PROFESSOR OF MEDICINE AT DUKE
UNIVERSITY AND A STAFF
NEPHROLOGIST AT THE DURHAM
VETERANS AFFAIR HOSPITAL.
Dr. WATTS ANTICIPATED BOTH AN
MD AND A PH.D FROM THE PEARLMAN
SCHOOL OF UNIVERSITY AT THE IN
PENNSYLVANIA WHERE HE STUDIES
FOX A.
HE COMPLETED RECEIPT DEPOSIT SEE
IN INTERNAL MEDICINE AT DUKE
UNIVERSITY AND A FELLOWSHIP IN
NEPHROLOGY AT THE UNIVERSITY OF
MICHIGAN WHERE HE PURSUED
FURTHER RESEARCH ON THE
REGULATION OF RNA, PAUSING.
IN 2020 HE MOVED TO NIEHS AS A
STADTMAN INVESTIGATOR AND LEADS
THE TRANSCRIPTIONAL RESPONSES IN
DISEASE GROUP WITH A SECONDARY
APPOINTMENT IN THE GENOME I AM
TEG RIGHT TEE AND STRUCTURAL
BIOLOGY LABRADOR.
HIS GROUP STUDIES HOW NUCLEIC AK
SID STRUCTURES AND CHROMATIN
ENVIRONMENTS INFLUENCE GENE
TRANSCRIPTION.
Dr. WATTS RECENTLY RECEIVED AN
NIH DIRECTOR'S AWARD FOR HIS
WORK ON THE NIH COVID VACCINE
AND BOOSTER CLINIC TEAM AND HE
WAS A RECIPIENT OF THE AMERICAN
SOCIETY OF NEPHROLOGY MEDICAL
FACULTY DEVELOPMENT AWARD IN
2019.
HE SERVED AS A MEMBER OF THE
AMERICAN SOCIETY OF NEPHROLOGY
AND THE AMERICAN SOCIETY OF
HUMAN GENETICS AND IS BOARD
CERTIFIED IN INTERNAL MEDICINE
AND NEPHROLOGY.
PLEASE JOIN ME IN WELCOMING OUR
SPEAKER, Dr. JASON WATTS, FOR
HIS PRESENTATION ENTITLED, SEE
REQUEST DEPENT AROUND IS A
DETERMINANT OF HUMAN GENE
EXPRESSION.
WELCOME,.
THANK YOU SO MUCH FOR THE KIND
INTRODUCTION AND I'M PLEASED TO
BE HERE.
SO, AS Dr. CHUNG MENTIONED,
I'M A BASIC SCIENTIST AND A
NEPHROLOGIST AND TODAY I WANT TO
STUDY SEQUENCE IN THE
TRANSMISSION AND HOW IT AFFECTS
GENE EXPRESSION.
I DON'T HAVE ANY DISCLOSURES.
AND MY OBJECTIVES TODAY ARE TO
INTRODUCE PROMOTER PROXIMAL' RNA
PAUSING IN IT'S ROLL IN
TRANSCRIPTION REGULATION AND THE
ROLE OF SEQUENCE AND REGULATING
PAUSEING AND I WANT TO TALK
ABOUT A NEW ROLE OF PAUSE
REGULATION THAT INVOLVES FOLDING
OF NUCLEIC ACIDS AND THIS IS ONE
WE FIND THAT REGULATES NON
CODING RNAs.
I'LL TALK ABOUT TRANSCRIPTION
AND MOLECULAR BUYEL GEE BUT I
WANT TO FRAME THE WORK THAT
WE'RE DOING IN THE CONTEXT OF A
CLINICAL CASE.
SO THIS IS A PATIENT WHO WAS
SEEN UP THE ROAD FROM ME AT
DUKE.
SO IT'S A 73-YEAR-OLD WOMAN WHO
HAS INSTAGE KIDNEY DISEASE AND
RECEIVED A DECEASED KIDNEY
DONOR.
SHE PRESENTED TO AN OUTSIDE
HOSPITAL WITH SEVERAL DAYS OF
NAUSEA AND VOMITING.
IN HER LABS IN THE EMERGENCY
ROOM SHOW SHE HAD HYPO ATMIA
WITH 121 CAN NORMAL RENAL
FUNCTIONS AND GIVEN THE HISTORY
IN LABS, IT WAS THOUGHT SHE
LIKELY HAD HYPO BOW LEM I CAN
AND STARTED ON NORMAL CHALEE AN
IN THE EMERGENCY ROOM AND
ADMITTED FOR FURTHER MANAGEMENT.
ON HER FIRST HOSPITAL DAY, SHE
SUFFERED A CARDIAC ARREST.
SHE SPENT TWO WEEKS IN THE
INTENSIVE CARE UNIT INTUBATED
AND SA DADEED.
SHE HAD A BRAIN MRI THE DAY
AFTER THE CARDIAC ARREST AND
SHOWED CHRONIC H HE IS.
HER HYPO ATREMIA WAS CORRECTED
AND SHE WAS EXCAVATED AND IT WAS
NEAR BASELINE BUT SHE HAD
NEUROLOGICAL DEFICITS AND SHE
HAD DIFFICULTY MANAGING SEE CRES
AND DUE TO THOSE SYSTEMS SHE WAS
TRANSFERRED FOR DUKE FOR FURTHER
MANAGEMENT.
AFTER HOSPITAL TRANSFER BECAUSE
HER SYMPTOMS WEREN'T IMPROVING
SHE HAD A SECOND MRI AND SHOULD
WAS ABOUT FOUR WEEKS FROM HER
INITIAL PRESENTATION AND AS
INDICATED HERE, WITH THIS RED
ARROW, THERE WERE ABNORMALITIES
ON THE MRI SO THERE WERE LESIONS
IDENTIFIED IN THE INTERIOR
POPPED IN THE MADEL A ON FLARE
AND T2 WEIGHTED IMAGES.
THESE FINDINGS ARE COMPATIBLE
WITH SYNDROME WITH THE INSERIOUS
PAUSE AND THE CERVIC OWE
MEDULLARY JUNCTION.
IT'S UP WITH OF THE THINGS IN
NEPHROLOGY FEAR THE MOST WHEN WE
TAKE CARE OF PATIENTS TAZ CAN
RESULT FROM TWO RAPID CORRECTION
OF THAT LOW SODIUM AND LEADING
TO NEUROLOGICAL INJURIES.
SO IF WE LOOK AT THE SODIUM
INTERPRETED FOTREND FOR THIS PAN
SEE THAT AT PRESENTATION HER
SODIUM WAS 121 AND IT DROPPED TO
117 AND OVER HER COURSE IN THE
INTENSIVE CARE UNIT IT WAS
CORRECTED BUT THEY ACTUALLY
THERE WAS A PERIOD WHERE SHE WAS
HYPER AND SHE HAD OVER
CORRECTION.
AND AT SOME POINT DURING THIS
PERIOD, THIS IS LIKELY WHEN SHE
SUFFERED THIS DEMYELINATION SO
IN I THINK A QUESTION IS HOW DO
CELLS DEFEND AGAINST THIS
OSMONIC STRESS FROM HAVING A
CHANGE IN THIS SODIUM.
IF WE HAVE A HYPER TONIC
CONDITION OUTSIDE OF THE CELLS,
WATER LEAVES CELLS EX THEY'RE
GOING TO SHRINK.
SO THIS IS TO DEFEND AGAINST
THIS LOSS OF VOLUME, THERE'S AN
INCREASE IN THE UPTAKE OF
ORGANIC OSMOL SUCH AS CHLORINE.
IN ADDITION TO CHANGES AT
MEMBRANE THAT ALLOW THESE TO BE
BROUGHT BACK INTO CELLS, THERE'S
A CHANGE AT THE TRANSCRIPTION
LEVEL IN GENES WHERE THEY GET UP
REGULATED SO IT CONVERTS GLUCOSE
TO SCOTIA TOLL AND THIS ALLOWS
FOR WATER TO MOVE BACK INTO
CELLS AND IT CAN DEFEND AGAINST
THE VOLUME LOSS IN THIS HOW DO
CELLS RESPONDED TO PHYSICAL
LOGIC STRESS SUCH AS HYPER TONIC
STRESS.
SO WHAT I WANT TO TALK ABOUT
TODAY, IN THE FIRST PART IS
DISCUSSING OR TALKING ABOUT THE
ROLE OF SEQUENCE IN REGULATING
PRO MOTOR PROXIMAL PAUSING AND
THIS IS A STORY ABOUT CODING
AGAINST.
GENES.
AND THE SECOND PART LOOKING AT
FOLDING OF RNA AND DNA IN A
STRUCTURE CALLED AN R-LOOP AND
HOW THAT PAUSES RNA IN THE BODY
OF NON CODING GENES AND AT END
I'M GOING TO COME BACK TO HOW
LEARNING ABOUT PAUSING
REGULATIONS MIGHT OFFER NEW
INSIGHTS INTO WHAT GOES ON WITH
OSMONIC DELINEATION.
WHAT IT REFERS TO IS THIS
ADDITIONAL LAYER OF
TRANSCRIPTION REGULATION THAT
HAPPENS IN OUR CELLS.
SO AFTER THE PRELIMINARY GETS
RECRUITED TO GENE REMOTERS, IT
CAN BE AGAIN TO TRANSCRIBE AND
PAUSE NEAR THE PROMOTERS.
AND IT NEEDS ADDITIONAL CUES TO
BE RELEASE INTERESTED THIS PAUSE
TO THEM MOVE INTO PRODUCTIVE
ELONGATION AND SYNTHESIZE RNA TO
INCREASE GENE REGULATION.
THIS MODE OF REGULATION IS FOUND
IN ANIMALS AND IS NOT FOUND IN
BACTERIA OR YEAST.
SO, IN OUR CELLS IT'S MOST
COMMON IN STRESS RESPONSE OVER
DYNAMICALLY REGULATED GENES, THE
KINDS OF GEEZ NECESSARY FOR
MAINTAINING HOMEOSTASIS OR
DEFENDING AGAINST STRESSES.
SO WHAT HAPPENS IS THAT AFTER
THE PRELIMINARY LANDS ON THE
PROMOTER AND SI SYNTHESIZES THEA
IT ENCOUNTERS TWO PAUSING
COMPLEXES.
AND THE ADDITIONAL SIGNAL IS A
PHOSPHORYLATION EVENT THAT
ALLOWS THE PAUSING COMPLEX TO BE
RELEASED AND DSIF CHANGES FOR
BEING A PAUSING FACTOR TO BEING
ELONGATION FACTOR AND IT TRAVELS
ALONG WITH THE RNA
PRELIMINARIES.
WE'VE BEEN LOOKING AT GENES THAT
HAVE THE PAUSING COMPLEXES NELF
AND DSIF AND ONE QUESTION ABOUT
THIS MODE OF REGULATION IS WHY
THIS OCCURS IN OUR CELLS AND NOT
OTHER ORGANISMS.
ONE THOUGHT IS THAT HAVING THE
PRELIMINARIES ON THE PROMOTERS,
ALLOWS FOR MORE UNIFORM GENE
INDUCTION BETWEEN DIFFERENT CELL
TYPES.
SO I WOULD MAKE THE METAPHOR
THAT THESE BMX BIKE LISTS
REPRESENT PRELIMINARY LOADED ON
THE PROMOTER AND THE PAUSED AT
THE STARTING BLOCK SO WHEN THE
STARTING PISTOL GOES OFF THEY
GET RELEASED FROM THE PAUSE AND
THE BAY CAN GO INTO THE RACE.
AND SO WORK THAT WAS DONE SHOWED
THAT DURING DEVELOPMENT, GENES
THAT HAVE PAUSE ARE INDUCED MORE
UNIFORMLY THAN GENES WITHOUT
PAUSE PRELIMINARY ARY SO THERE'S
A COMPONENT OF A RAPID START ON
THE PROMOTER BUT EVEN MORE
IMPORTANTLY THERE'S A MORE
UNIFORM START OF HOW GENES CAN
RESPOND TO A PARTICULAR STRESS.
SO WHEN WE WERE LOOKING AT DSI
AND FINDING IN GENES NELF THIS
GENE WHICH I DESCRIBED AS BEING
IMPORTANT FOR RESPONDING TO OS
MOT TICK STRESS THESE PAUSING
COMPLEXES ARE LOCATED AT THE
PROMOTERS.
SO THIS IS THE PATTERN THAT WE
EXPECT AND IF THERE'S PROMOTER
PROXIMAL PAUSING SO A QUESTION
WE HAVE IS WHETHER THE PAUSING
REGULATION PLAYS A ROLE IN
RESPONDING TO HYPER CONNICK CRES
SOSTRESSED SO WE WANTED TO MEASE
WHERE THE PRELIMINARY IS
LOCATED.
THE ASSAY IS THE PRO SEEK O WE
TAKE CULTURAL CELLS AND PERFORM
A RUN ON REACTION WITH BIO TIN A
LATED NUCLEOTIDES SO THEY BE
INCORPORATED BY THE DETECT THESE
NEWLY ADDED MODIFIED NUCLEOTIDES
AND PULL THOSE OUT OF CELLS.
WE CAN USE SEQUENCING TO MAP
THOSE BACK ON THE GENOME SO THE
THREE PRIME END OF THE RNA, THE
END OF THE RNA THAT HAS THIS
MODIFIED NUCLEOTIDE IS A PROXY
WHERE IT WAS LAST
TRANSCRIPTIONALLY IMPACTED.
SO WE CAN INFER WHERE THE PAUSE
AS BEING LOCATIONS WHERE THERE'S
ANNA CUMULUS OF THESE READS FROM
PRO-SEQ SO IF IT'S NEAR THE
PROMOTER IT'S THE PATTERN TA FIX
THIS PAUSE PRELIMINARY.
WHEN WE DO THIS ASSAY ON A CAP
SER CELL LINE AND LOOK AT WHERE
THE PRELIMINARY IS LOCATED ON
THESE WITH WHAT WE SAW WITH NELF
AND DFIS WE CAN SEE THE TYPE OF
PROMOTER EVIDENT BY THE TIGHT OF
THESE MARKS AND DETECT THERE'S
SOME PRELIMINARY IN THE GENE
BODY WHEN THESE CELLS ARE GROWN
IN NORMAL CONDITIONS OR ISO
TONIC CONDITIONS.
SO THIS IS WHERE THE PRELIMINARY
IS IS PAUSE.
IN WHAT WE WOULD EXPECT IF
THERE'S PAUSE RELEASED, THE GENE
IS BEING INDUCED WE WOULD SEE A
SHIFT WHERE THERE'S GOING TO BE
FEWER PRELIMINARY ACCUMULATED AT
THE PROMOTER AND WE EXPECT TO
SEE MORE THAT GET RELEASED TO
TRANSCRIBE IN THE GENE BODY.
SO WE SHIFTED OURSELVES FROM EYE
OWE TONIC CONDITIONS NOW TO
HYPER TONIC CONDITIONS.
THE OS MOT TICK STRESS
CONDITIONS IN DEED WE CAN DETECT
THERE'S AN INCREASE IN THE
AMOUNT OF PRELIMINARIES IN THE
GENE BODY IN THESE TWO
REPLICATES AND WHAT I THINK
STANDS OUT IS THAT THERE'S STILL
A POPULATION OF REMAIN PAUSED IN
THE PROMOTER SO EVEN THOUGH THE
GENE HAS BEEN INDUCED THERE'S
STILL SOME THAT ARE GETTING
STUCK ON LIKE A SPEED BUMP WHERE
SOMETHING THERE IS PAUSING IT TO
ACCUMULATE.
SO WE CONSIDERED THAT THERE MAY
BE AN ADDITIONAL LAYER OF
REGULATION BEYOND THESE PAUSING
COMPLEXES.
AND IT COULD BE THAT THERE'S AN
UNDERLYING CONTRIBUTION OF THE
NUCLEIC ACID SEQUENCE AND
SOMETHING IN THE DNA OR THE RNA
THAT CON TRIBUTES TO PAUSING
REGULATION.
AND THAT MAY PERSIST EVEN AFTER
A RESPONDING TO THE STRESS.
SO TO ADDRESS THIS QUESTION,
WHAT WE DECIDED TO DO WAS
PROFILE THE PATTERN OF PAUSING
IN NORMAL HUMAN CELLS.
AND AGAIN AS A NEFF R NEPHROLOGI
WOULD HAVE LOVE TO DO THIS ON
PRIMARY RENAL CELL TYPES BUT
IT'S' YEAR TO GET A HOLD OF
PRIMARY SKIN FIBER BLAST SO WE
PROFILE TRANSCRIPTION IN FIBER
BLASTS FROM BUY ONS SEE FROM
OTHER PIES AND THE PAT YOUR
WORSHIP OF PRELIMINARIES PAUSING
AND MEASURED GENE EXPRESSION.
I'M SNOWING A GENE WITH ROLLS IN
PONDING STRESS SIMILAR TO SHOWED
WHAT I AND WE CAN SEE THERE'S A
ACCUMULATION AT THE PROMOTER
CONSISTENT WITH PAUSING AND WE
CAN DETECT SOME PRELIMINARY IN
THE GENE BODY.
WE PROFILED AND FIVEN DOSING
CELLS FROM FIVE INDIVIDUALS AND
WE CAN SEE As COULD THE FIVE CRE
INDIVIDUALS IT ACCUMULATION IN
THE PROMOTE YOU ARE AND ONE OF
THE STRENGTH IS WE GET ONE GINO
WIDE INFORMATION AND HIGH
RESOLUTION INFORMATION.
AND SO WE CAN ACTUALLY ZOOM IN
AND SEE THAT THESE ARE NOT JUST
ACCUMULATING IN THE PROMOTER,
ACTUALLY ACCUMULATING AT THE
SAME NUCLEOTIDE POSITION SO
ACROSS THESE FIVE INDIVIDUALS IT
SEEMS IT'S PAUSING PRECISELY TO
SPECIFIC NUCLEOTIDE.
IS IT JUST AN OUT LIER?
WE CAN LOOK AT THOUSANDS OF
GENES SO HERE I'M NOW DISPLAYING
THE PROCEED DATA AS A HEAT MAP.
EACH ROW ON THIS HEAT MAP IS A
INDIVIDUAL GENE AND THE GENES
ARE ARRANGED SO THE NUCLEOTIDE
POSITION WITH THE MOST IS
GETTING FARTHER FROM THE START
SITE AS WE MOVE DOWN THE PLOT.
THIS IS THE DATA FROM ONE
INDIVIDUAL AND IF YOU LOOK AT
THE DATA FROM ALL FIVE OF THE
INDIVIDUALS THAT WE PROFILE,
HOPEFULLY YOU WOULD AGREE WITH
ME THE PATTERN OF WHERE THE
PAUSE IS VERY SIMILAR ACROSS
INDIVIDUALS.
AND IT'S STATISTICALLY
CORRELATED THAT THE CORRELATIONS
AND THE VERY SMALL P VALUE.
AND MORE THAN JUST THE HIGHLY
CORRELATED LOCATIONS OF WHERE
THE PAUSE WE CAN FIND OVER 1300
GENES WHERE THE POL LL ACROSS
ALL FIVE INDIVIDUALS SO PATTERNS
SIMILAR FOR WHAT HE SHOWED.
SO IT SEEMS ACROSS THOUSANDS OF
GENES WHERE THE PAUSE VERY
PRECISE.
THIS IS ALL LOOKING AT WITHIN
THE SAME CELL TYPE.
SO WE WONDERED, DOES THIS
PRECISION OF PAUSING ORGANIZE
SIMILARLY OF WHERE THE PAUSES
THAT EXTEND TO OTHER CELL TYPES.
SO WE HAVE BEEN PROFILING FIBER
BLAST FROM ADULT SKIN AND WE
ALSO LOOKED AT NEONATAL FIBER
BLASTS FROM FORE SKIN AND LOOKED
AT TUBAL CELL LINE AND A TIME OR
SEE PRESS OR AND IT'S IN THE
PROMOTER AND ACROSS THESE
DIFFERENT CELL CONTEXT, WE CAN
SEE THAT THE POL LL IT'S AT THE
SAME NUCLEOTIDE POSITION.
WE TOOK THIS A STEP FURTHER AND
LOOKED THIS IN DATA WE DIDN'T
HAVE ANY PART GENERATING.
THIS IS LOOKING IN A LEUKEMIA
CELL LINE AND WE CAN SEE IT'S
ACCUMULATING AT THE PROMOTER AND
THE SAME NUCLEOTIDE POSITION SO
ACROSS CELL TYPES, ACROSS LABS
DOING THE ASSAY WE OTHER SEE
THERE'S A HIGH DEGREE OF
SIMILARLY WHERE IT'S PAUSING IN
THESE GENE PROMOTERS.
SO WE WANTED TO ASK, YOU KNOW,
ARE THERE SEQUENCE ELEMENTS THAT
CORRELATE WITH WHERE IT PAUSES.
SO ONE OF THE FIRST THINGS WE
LOOKED AT IN JUST THE UNDER
LYING GC CO CONTENT WE LOOKED AT
THESE 1300 CITES ACROSS
INDIVIDUALS AND FIBER BLASTS,
COMPARED TO LOCATIONS WITHOUT
POL LL PAUSE AND THEY'RE FAVORED
WITH REGIONS WITH HIGH GC
CONTENT AND WITHIN THE REGIONS
OF HIGH GC CONTENT THERE'S
SKEWING WITH THE Gs AND Cs
ARE LOCATED AND PAUSES TEND TO
OFFER AFTER SYNTHESIZING THE G
RICH RNA.
THIS IS WHAT THIS GC SKEW REFERS
TO AND THEN IF WE TAKE ADVANTAGE
OF THAT HIGH RESOLUTION FROM THE
ASSAY WE CAN ZOOM IN AND LOOK AT
NUCLEOTIDE LEVEL WHERE THE PAUSE
IS AND WE CAN SEE 66% OF THE
SITES WHERE WE OBSERVED THE
PAUSING IT INCURS AT A SITE AND
FREQUENTLY THE NEXT BASIS
APPEARING SO EITHER A G OR AN A
AND THEY'RE IN THE CONTEXT OF
WHAT WE REFER TO THINK ABOUT AS
A PAUSE MOTIVE AND I WON'T TAKE
YOU THROUGH THE ADDITIONAL
STATISTICAL ANALYSIS BUT EACH OF
THESE SEQUENCE ELEMENTS ARE
STATISTICALLY ASSOCIATED WITH
POL LL PAUSING SO WE HAVE A
ASSOCIATION BETWEEN THE UNDER
LINE SEQUENCES AND WHERE THE POL
LL PAUSES WE WANTED TO TEST HOW
MUCH OF A CONTRIBUTION IS THERE
OF THESE SEQUENCE ELEMENTS.
AND WE JUST DECIDED TO FOCUS ON
THE CONTRIBUTION OF PAUSING AT A
CYTISINICLINE AS CHANGING A
SINGLE BASE WOULD BE EASIER TO
DO RATHER THAN CHANGING THE GC
CONTENT NEAR PROMOTERS.
SO WHAT WE DID WAS TO AGAIN TAKE
ADVANTAGE OF THIS ASSAY WE GET
INFORMATION ABOUT WHERE THE POL
LL IS PAUSING NUCLEOTIDE
RESOLUTION SO WE LOOKED FOR
PAUSE SITES WHERE THE INDIVIDUAL
WAS HETERO SIGH GUS WHETHER THEY
HAVE A G OR C AND PAUSE SITE AND
ASK IF THERE'S A DIFFERENT IN
THE EXTENT OF PAUSING WHETHER
IT'S A C OR A G OR ANOTHER
NUCLEOTIDE AT THAT PAUSE SITE.
TO TAKE AN EXAMPLE OF THAT, THIS
IS A GENE 6 AND IT'S THE
PROMOTER AND WITHIN THE PROMOTER
REGION THERE'S A SITE WHERE THIS
INDIVIDUAL IS A C OR A G AND NOW
I'M SHOWING THE SEQUENCING READS
AND WHAT WE SEE AT THE G ALLELE
IN GEORGE IS THE IT'S ABLE TO
READ THROUGH THIS G AND THERE'S
NO PAUSES THAT ARE HERE.
BUT IF WE LOOK AT READS OF THE
TRANSCRIPTS THAT COME FROM THE C
BARRING ALLELE, WE CAN IDENTIFY
THESE TRUNCATED READS WHICH IS
WHERE THE POLYMERASES PAUSES
WHEN THERE'S A C BUT NOT A G
WHEN THE REST OF THE SURROUNDING
SEQUENCES IS IS THE SAME.
SO WE CAN DETECT A ALLELE LIKE
EFFECT OF THIS C AT THE PAUSE
SITE.
WE DID THAT COMPARISON LOOKING
AT C VERSUS G, C VERSUS T, C
VERSUS A AND ALL THOSE CASES WE
FOUND THERE WAS SIGNIFICANTLY
MORE PAUSES THAT OCCURRED AT THE
C BEARING ALLELE.
SO WE ASKED IF WE CAN DETECT
ANNA LEGAL DIFFERENCE IN THE
EXTENT OF PAUSING BECAUSE DOES
THIS LEAD TO A DIFFERENCE IN
GENE EXPRESSION LEVELS.
SO WE TOOK ADVANTAGE OF
INFORMATION FROM THE GENOTYPE
TISSUE EXPRESSION BASE WHICH HAS
INFORMATION ABOUT UNDER LYING
GENOTYPE AS WELL AS GENE
EXPRESSION FROM HUNDREDS OF
INDIVIDUALS.
AND WE CAN FIND EXAMPLES WHERE
THERE WERE POLY MORPHISMS AT
PAUSE SITES WE IDENTIFIED THAT
WERE PRESENT IN THE GTEX
DATABASE.
A GENE THAT HAD THISSEN CODES A
WHAT WE CAN SEE IS INDIVIDUALS
WHO ARE HOMO SIGH GUS FOR A C AT
THE PAUSE SITE HAVE LOWER
EXPRESSION AS COMPARED TO
INDIVIDUALS WITH A T AT THE
PAUSE SITE.
O WE THINK THERE'S MORE PAUSING
ON AND GREATER LEAD TO GO FEW
MAKING IT INTO PRODUCTI PRODUCTE
ELONGATION AND ONE TISSUE TYPE
WHERE YOU CAN LOOK AT EXPRESSION
OF Z21 ACROSS MULTIPLE TISSUE
AND WHAT WE FOUND ACROSS THE
TISSUE WHERE HEAD INFORMATION
FROM GTX IS ALL CASES
INDIVIDUALS WHO WERE HOMO SIGH
GUS FOR A C HAVE LOWER
EXPRESSION OF THIS.
>>Gina: COMPARED TO INDIVIDUALS
WITH A T.
SO IT SEEMS THAT THIS PAUSING
CAN THEN LEAD TO DIFFERENCE THIS
IS EXPRESSION THAT PAUSING ON
THE C MORE MUCH RESULTS IN FEWER
POLYMERASES IN DIFFERENT
ELONGATION.
WE WANT TO TEST THAT
EXPERIMENTALLY.
SO THE APPROACH WAS TO USE A
REPORTER ASSAY.
SO WE CLONED THE PROMOTERS OF
GENES THAT HAVE PAUSE
PRELIMINARY OR PAUSE
CYTISINICLINE AND THEN USED CITE
DIRECTED IMMUNE' GENESIS TO
CHANGE THAT C TO A T AND ASK IS
THERE A CHANGE IN REPORTER
ACTIVITY.
AND SO WE DID THAT WITH THREE
GENES AND I SHOWED YOU DATA FOR
BL CAP EARLIER THAT THERE'S THE
ACCUMULATES AT A C AND MY O1E IS
A MIO SIN WHICH ENCODES A
PROTEIN THAT MUTATED CAN LEAD TO
KIDNEY DISEASE AND ALL THREE OF
THESE PROMOTERS MAKING THE
SINGLE NUCLEOTIDE CHANGE FROM A
CYTISINICLINE AT THE PAUSE SITE
OR A C TO A T IS IS SUFFICIENT
TO THE SIGNIFICANT INCREASE IN
REPORTER ACTIVITY.
SO WE CAN SEE THERE'S ALLELE I
CAN PAUSING IN THE GTX DATA AND
IN EXPRESSION AND HERE
EXPERIMENTALLY WE CAN TEST AND
SHOW THAT MAKING THAT SINGLE
CHANGE IS SUFFICIENT TO ALTER
REPORTER GENE ACTIVITY.
SO WHAT WE WANT TODAY DO IS TO
KNOW WHETHER THESE CHANGES IN
THE EXTENT OF PAUSING MUCH FOR
THE CHANGES AND EXPRESSION THAT
RESULT FROM DIFFERENCES IN
PAUSING, ARE THOSE THAT LARGE
ENOUGH TO CONTRIBUTE TO
DIFFERENCES THAT WE MIGHT SEE IN
THE CLINIC.
SO WE GOT A CLUE THAT THIS
PAUSING OR ALLELEIC PAUSING CAN
EFFECT DISEASE LOOKING AT
REDUCKING SO I INTRODUCED THIS
IN ITS ROLE OF CONVERTING
GLUCOSE TO SORBITOL WHEN
DEFENDING AGAINST STRESS.
FOR PATIENTS THAT HAVE DIABETES
AND THERE'S A HIGHER ABUNDANCE
OF GLUCOSE, THE INCREASE IN
GLUCOSE LEADS TO MORE OR GREATER
GENERATION OF SORBITOL.
IT'S THOUGHT THE INAPPROPRIATE
ACCUMULATION OF SORBITOL INSIDE
CELLS CAN CONTRIBUTE TO THE
DEVELOPMENT OF DIABETIC
COMPLICATIONS SO CATARACT,
DIABETES EYE AND NERVE DISEASE
AND KIDNEY DISEASE THROUGH PART
ARE RESULT FROM INAPPROPRIATE
GENERATIONS OF SORBITOL SO
THERE'S SEVERAL PAPERS THAT HAVE
IDENTIFIED A POLY MORPHISM IN
ALL THOSE WHICH COP FIRST
INCREASE RISK OF DIABETIC
COMPLICATIONS SO THIS IS DATA
COMING FROM A PAPER IN 2019
LOOKING AT DIABETIC RECEIPT
INOPATHY AND THEY OBSERVED
COMPARING INDIVIDUALS WHO ARE
HOMO SIGH GUS FOR A C AT THIS
NIP AND INDIVIDUALS THAT ARE
HOMO SIGH GUS FOR A T HAD A THEY
DIDN'T HAVE AN GUY OF WHAT WAS
CONTRIBUTE TO GO THAT RISK.
SO WE CONSIDERED THAT THE
DIFFERENCE IN THIS RISK COULD BE
RELATE TODAY DIFFERENT EVERY DIL
PAUSING AT THIS AND WE USED THE
APPROACH THAT I SHOWED A SLIDE
OR TWO ASK WE CLONED THE
PROMOTER EITHER WITH THE C OR
WITH THE T AND THEN TESTED THE
ACTIVITIES IN A ASSAY.
SO WE DID THIS IN THREE
DIFFERENT CELL LINES AND WE GOT
THE SAME THREE RESULTS IN ALL
THREE CELL LINES THAT SINGLE
NUCLEOTIDE DIFFERENCE BETWEEN C
AND T RESULTED IN A SIGNIFICANT
DIFFERENCE IN REPORTER GENE
ACTIVITIES.
SO THE SAME PATTERN AND THESE
CELLS ARE HETERO SIGH OWE GUS OR
POLY MORE PICK FOR THIS AND SO
IN THE CHROMATIN ENVIRONMENT AND
THE NATIVE CONTEXT WE CAN ASK IF
THERE'S A DIFFERENCE IN ALLELEIC
EXPRESSION WITH THE GENE BEING
IN THE SAME CELLS AND THE EXACT
SAME CAN B.
SO WE MEASURE THE ALLELE I CAN
EXPRESSION SO WHAT WE SAW -- SOE
SAME PATTERN THAT WE'VE SEEN
RIGHT AT PAUSING MORE MUCH ON
AND WE HAVE LOWER EXPRESSION AND
LESS ON THE T WE GET GREATER
EXPRESSION.
ALL THOSE INDUCING IN RESPONSE
TO HYPER TONIC STRESS SO WE
CULTURE THESE CELLS IN TYPIER
TONIC CONDITIONS AND INDUCED IN
BOTH CASES BUT WE SEE A MUCH
GREATER INCREASE IN ALL THOSE
EXPRESSION OF THE T BEARING
ALLELE AS COMPARED TO THE C
BEARING ALLELE.
SO WE THINK THIS IS WHAT COULD
EXPLAIN THIS INCREASED RISK OF
DEVELOPING DIABETIC
COMPLICATIONS IS LESS POLY EMERY
THAT CAN LEAD TO GREATER
EXPRESSION OF ALL THOSE REDUCK
TASE AND INCREASE IN THE
GENERATION OF SORBITOL SO THIS
IS A HINT OF DIFFERENCES IN THE
EXTENT OF PAUSING COULD EXPLAIN
SOME DIFFERENCES IN GENE
EXPRESSION BETWEEN INDIVIDUALS.
SO IT SUMMARIZES THIS BEGINNING
PART OF THE TALK AND WHAT I'M
INTRODUCED IS THAT THE IT PAUSES
NEAR PROMOTERS AND THERE'S A
COMPONENT OR CONTRIBUTION OF
PAUSING REGULATION MEDIATED BY
THESE PAUSING COMMENT PLEXES
NELT AND DFIS AND THERE'S A ROLE
OR A CONTRIBUTION OF THE
UNDERLYING SEQUENCE IN COP
CONTRIBUTING TO THESE PAUSES.
WE THINK IT'S A SEQUENCE
MEDIATED OR SIS REGULATORY
ELEMENTS WITH THESE PROTEIN OR
TRANSACTING FACTORS AND WE THINK
THIS IS WHAT IT COUNTS FOR THE
PRECISE PAUSING THAT WE SEE AT
MANY OF THESE GENES WHERE POL LL
IS ACCUMULATING AT THE SAME
NUCLEOTIDE POSITION ACROSS CELL
TYPES AND ACROSS DIFFERENT
INDIVIDUALS.
SO I THINK OF THIS KIND OF
SEQUENCE DEPENDENT PAUSING AND
THAT'S THINK A GENETIC COMPONENT
OF PAUSING AND THIS LEADS TO A
HIGH DEGREE OF SIMILARLY WE SEE
ACROSS CELL TYPES OF
INDIVIDUALS.
SO NOW, WHAT I WANT TO CHANGE TO
IS A DIFFERENT MODE OF SIS
REGULATION AND THIS IS ONE THAT
IS NOT DEPENDENT ON THE PRIMARY
SEQUENCE OF THE DNA AND THE RNA
BUT MORE ABOUT THE SHAPE OF THE
NUCLEIC ACIDS AND THIS IS AN
EPIGENETIC ROLE OF PAUSING.
AND ONE THAT WE THINK CAN BE
MORE DYNAMIC.
SO, BEFORE KIND OF -- I'LL COME
BACK TO HOW WE SEE THIS
ASSOCIATION BETWEEN THE
STRUCTURE OF NUCLEIC ACIDS AND
PAUSING AND I'LL TAKE A DETOUR
AND SHARE SOME RESULTS ABOUT OUR
LOOPS AND COME BACK TO HOW IT'S
REL VENT IN PLUM POL LL PAUSE.
WORK DONE IF VIVIAN CHUNG'S
LAUGH WHEN I WAS THERE AS A
POSTDOC FOCUSED ON THE ROLE OR
BIOLOGY TO R-LOOP FORM WHEN WE
HAVE THE PRELIMINARY
TRANSCRIBING AND IT CAN DNA AND
CREATE AN RNA-DNA HYBRID.
THIS LEAVES THE NON TEMPLATES
BRAND SINGLE STRAND BETWEEN THE
THREE STRANDED STRUCTURES.
SO APP OBSERVATION PUBLISHED IN
A PAPER IN 2020 IS THAT BASIC
REPAIR ENZYME SOMETHING CALLED
THE DNA GLY CLOSE LAIDS IS ABLE
TO REMOVE THE BASE FROM THE RNA
IN AN RNA-DNA HYBRID SO THE
CONTEXT OF AN R-LOOP.
SO TO SHOW THIS LITTLE
DIFFERENTLY, SO THIS IS A WITH
THE SUGAR AND THE BASE AND THE
MPG IS ABLE TO CLEVE THIS BOND
AND WE'RE LEFT WITH THIS SUGAR
IN THE RNA.
AND WE CAN SHOW THAT THESE BASIC
RNAs WHILE RARE, ARE VERY
PREVALENT IN CELLS AND AT THE
TIME LED TO A QUESTION, WHAT'S
THE BIOLOGICAL FUNCTION OF THESE
BASIC RNAs.
SO WITH SOME ADDITIONAL
BIOCHEMICAL WORK, WHAT WE
REALIZED IS THAT WE CAN HAVE
THESE R-LOOPS THAT FORM.
IT CAN BE RECOGNIZED BY A WRITER
COMPLEX THE MET CAL 314 COMPLEX
THAT CAN MODIFY METHYLATED TO
CREATE AN M6A MODIFICATION AND
THESE MODIFIED ARE RECOGNIZED BY
MPG AND REMOVED TO LEAVE THE
SAME BASIC RNA.
SO IT SHOWS MORE IN THE
STRUCTURES AND NOT JUST THESE
CARTOONS, SO HERE IS THE BASE
AND THE 314 COMPLEX ADDS THIS
GROUP AND THIS MODIFIED
NUCLEOTIDE IS RECOGNIZED BY MPG
AND IT IT IS ABLE TO REMOVE THE
SPACE.
SO THEN WE UNDER OR TO HAVE AN
IDEA OF WHAT THESE BASIC RNAs
ARE DOING IN CELLS WE ASKED
WHERE ARE THEY LOCATED IN CELLS?
SO WE DID PULL DOWNS TO ASK
WHERE MPG IS FOUND IN THE GENOME
AND WE LOOKED WHERE THESE MIGHT
OCCUR IN R-LOOPS.
SO WE DID THIS ANNAL LOOKING AT
THE CO OCCURRENCE OF THESE
R-LOOPS AND MPG AND WE
IDENTIFIED NEARLY 2,000 NON
CODING RNA GENES WHICH HAVE
THESE MODIFIED R-LOOPS.
SO, THEN WE LOOKED AT THE
PATTERN OF TRANSCRIPTION AND WE
SAW THE PLOT SO WE HAVE AN
IMMUNO PARTICIPATION THAT PULLS
DOWN R-LOOPS AND IT'S IN THE
GREEN TRACE AND WE USE PROCEED
DATA TO PROFILE WHERE THE IT'S
LOCATED AND WE SEE THIS
ACCUMULATION OF THE PATTERN VERY
SIMILAR TO WHAT SEE APPROXIMATE
NEAR THE PROMOTERS WHERE IT'S
PAUSING THAT THIS ACCUMULATION
OCCURS UPSTREAM OF THESE
MODIFIED R-LOOPS.
SO IT SEEMS IT'S TRAPS
DESCRIBING ALONG AND IT RUNS
INTO THESE MODIFIED R-LOOPS AND
IT PAUSES.
SO NOW WE HAVE THIS IDEA THAT
THE MODIFICATION OR THIS
CREATION OF A BASIC R-LOOPS
SITES CAN CONFER SOME REGULATION
OF TRANSCRIPTION OR CAUSE THE
POL LL TO PAUSE SO WE WANTED TO
ASK NOW, WHAT IS THE ROLE OF THE
CONTRIBUTION OF THESE MODIFIED R
LOOPS IN REGULATING PAUSING AND
HOW THIS MIGHT EFFECT GENE
EXPRESSION.
THE DATA I'M GOING TO SHOW IS
FOR A NON CODING RNA THAT WE
DISCOVERED THAT'S UPSTREAM OF
APOE AND WE TORNADO IT ANSWER OR
FOR NO CODING RNA AND THIS NO
CODING RNA HAS ONE OF THESE
R-LOOPS MODIFIED TO HAVE BASIC
SCIENCE AND I'LL SHOW YOU HOW WE
HAVE THIS PATTERN WHERE THE POL
LL PAUSES AND UPSTREAM OF THESE
MODIFIED R-LOOPS IN THIS NON
CODING RNA.
AND THE REASON WE FOCUSED ON
THIS NON CODING RNA IS BECAUSE
IT'S NEAR SUCH AN IMPORTANT
DISEASE ASSOCIATED GENE AND IT
IT ENCODES PROTEIN E AND WHICH
IS AN IMPORTANT RISK FACTOR FOR
ALZHEIMER'S DISEASE.
SO, I'M SURE THIS AUDIENCE WELL
KNOWS THAT IT'S A CARRIER
PROTEINS FOR LIPIDS AND
CHOLESTEROL ASK THIS PROTEIN IS
POLY MORPHIC AND CHANGES THAT
CARRY THE ALLELE AOPE4 HAVE A
RISK FACTOR FOR LATE ON SET
ALZHEIMER'S.
IN ORDER TO OVER SIMPLIFY A
LARGE BODY OF LITERATURE, APOE
MADE IN ASTROCYTES AND IF YOU
HAVE THE NORMAL ALLELE THE APOE3
ALLELE THERE'S NORMAL LIPID
TRANSPORT TO THE NEURONS AND
THEY REMAIN HEALTHY.
IF YOU HAVE THE APOE34, IT CAN
LEAD TO LIPID TRANSPORT AND
CONTRIBUTE TO THE NEURO
DEGENERATION WE SEE IN THE MULTI
RISK DISEASE.
WE THOUGHT IF THERE'S A NOVEL
NON CODING RNA UPSTREAM THAT
COULD CONTRIBUTE TO APOE
REGULATION THIS WOULD BE AN
IMPORTANT NON CODING RNA TO SIDE
AND PART TWO OF WHAT WE THINK IS
GOING ON WITH THIS NON CODING
RNA AND LET ME SHOW YOU THE
DATA.
>>THIS NON CODING RNA IS TRANCE
DESCRIBED IN FIBER BLASTS AND
THIS IS DATA WHERE WE'RE SHOWING
ENRICHMENT TO THE PRESENCE OF
THIS NON CODING RNA.
WHAT I WOULD POINT OUT, IF
APPROXIMATE TTHERE'SA CHANGE INF
TRANSCRIPTION UPSTREAM WHERE
THIS R LINK IS FOUND SO THIS RNA
HAS THE PATTERN THAT WE OBSERVED
ON AVERAGE WHERE THERE'S THE
PAUSE UPSTREAM OF WHERE THESE
ARE END INDICATED.
WE DID ADDITIONAL WORK TO LOOK
USING CITE SPECIFIC ASSAYS WHERE
YOU CAN USE PCR AND YOU CAN DO
RNA IMMUNO PRECIPITATION AND
SHOW MPG BINDS TO THIS RNA AND
DETECT THERE ARE A BASIC RNA IN
THIS NON CODING OF TRAPS
DESCRIPTION UPSTREAM OF APOE.
OUR MODEL IS YOU CAN FORM A
R-LOOP ASK IT'S RECOGNIZED BY
M6A LEAVING THIS A BASIC SITE SO
WE ASKED IF WE PERTURBED THE
GENERATION OF THESE MODIFIED
R-LOOPS WOULD IT LEAD TO A
CHANGE IN ANSWER TRANSCRIPTION
OR A CHANGE IN APOE EXPRESSION?
SO WE LOOKED IN OR COP PAIRED
APOE EXPRESSION IN PATIENTS WHO
HAVE A MUTATION WITH A DIFFERENT
GENE THAT RESULTS IN FEWER
R-LOOPS.
AND SO IF WE COMPARE THE APOE
EXPRESSION IN CONTROLS TO
INDIVIDUALS WHO HAVE FEWER
R-LOOPS, WE SEE THAT THE
PATIENTS WITH FEWER R-LOOPS SHOW
HIGHER EXPRESSION OF APOE.
NEXT WE KNOCKED DOWN METAL 3 TO
PREVENT FORMATION OF N6A AND WE
SAW THAT LOSS OF METTL3 LEADS TO
AN INCREASE IN APOE EXPRESSION.
AND THEN WE KNOCKED CELLS OF MPG
SO THEY WON'T AND DON'T FORM AS
A BASIC SITE AND AGAIN WE SEE
THE SAME PATTERN THAT WE
KNOCKDOWN MPG WE CAN DETECT AND
INCREASE IN THE EXPRESSION OF
APOE AND I'M SHOWING THE
SEQUENCING DATA AND YOU CAN SEE
BY THREE OR SEVEN DAYS OF MPG
KNOCK DOWN THERE'S AN INCREASE
IN APOE EXPRESSION.
SO, WHETHER WE DECREASE THE
ABUNDANCE OF THESE MODIFIED BY
DECREASING THE ABUNDANCE OF
R-LOOPS AND M6A MODIFICATION OR
PREVENTING THE FORMATION OF
SITES AND R-LOOPS ALL THREE OF
THESE LEAD TO AN INCREASE OF THE
EXPRESSION OF APOE.
SO WE HAVE AN IDEA THAT THE RACE
IS MARCHING ALONG IN THIS NON
CODING RNA AND IT RUNS INTO THIS
MODIFIED R LOOP AND PAUSES AND
IT SEEMS LIKE WE REMOVE THE
BARRIERS, THIS R LOOP BY
PERTURBING R-LOOPS AND ALLOWS
FOR FULL TRANSCRIPTION OF AND
THAT CONTRIBUTE TO ACTIVATION OF
APOE.
AND SO TO TEST WHETHER SIP THAT
SIS OF A FULL LENGTH ANSWER OF A
FULL LENGTH NON CODING RNA
WHETHER IT CORRELATES CAN DRIVE
APOE EXPRESSION, WE LOOKED AT
TRANSCRIPTION AND CELLS WHICH
EXPRESS APOE AND THEN COMPARED
IT TO TRANSCRIPTION IN CELLS
WHICH DO NOT EXPRESS APOE SO
HERE I'M SHOWING RNA SEQUENCING
DATA FROM A EXPRESS APOE AND
WE'RE DETECTING SEQUENCING READS
AT THE FIVE PRIME END OF APOE
AND COMPARED THAT TO CELLS,
THESE ARE WHITE CELLS, FROM THE
SAME INDIVIDUAL, WHERE APOE IS
NOT EXPRESSED.
AND WHEN WE LOOK AT
TRANSCRIPTION OF ANSWER, WHAT WE
SEE IS THE CELL TYPE WHERE APOE
IS ACTIVE WE CAN DETECT FULL
LENGTH OR FULL TRANSCRIPTION
WHERE WE ONLY DETECT TRUNCATED
OF PAUSED IN THIS CELLS WHERE
APOE IS SILENT.
WE ALSO LOOKED AT PUBLICLY
AVAILABLE DATA FROM THE ENCODE
PROJECT WHERE THEY USED A BETTER
CAPTURE TRANSCRIPTS AND HERE
WE'RE LOOKING AT A LIVER CELL
LINE OR TRANSFORM D CELLS AND
THE PATTERN IS THE SAME.
THE LIVER CELL TYPE EXPRESSES
APOE BUT THE B CELLS IS IS
SILENT AND IN LIVER CELLS WE CAN
DETECT THE EXPRESSION OF A FULL
LENGTH ANSWER AND WHEREAS IN B
CELLS WE DON'T DETECT FULL
LENGTH ANSWERS.
SO WHAT WE -- THIS SUPPORTS THIS
MODEL THAT THE R-LOOP OR THE
FORMATION OF THIS MODIFIED
R-LOOP IS CAUSING THEM TO PAUSE
ON THIS NON CODING RNA AND
REGULATING WHETHER YOU GET A
FULL EXPRESSION OF FULL LENGTH
ANSWER OR WHETHER YOU GET THIS
SHORT AROUND RNA.
WHAT WE THINK HAPPENED IS THERE
IS IS INITIAL TRANSCRIPTION OF
THIS NON CODING RNA AND
FORMATION OF AN R-LOOP, THIS
R-LOOP CAN BE MODIFIED BY THE ME
IT TTL TO CREATE M6A AND THAT
M6A IS RECOGNIZED BY MPG AND
WHERE CLEVE AND WE THINK THE
R-LOOP CONTRIBUTES TO STABLATION
THAT RESULTS IN SUBSEQUENT
PRELIMINARY PAUSE WHAT IS THEY
RUN INTO THIS STRUCTURE.
IF USING EXPERIMENTAL, IF WE
PREVENT THE FORMATION OF THESE
MODIFIED R-LOOPS, WE CAN SEE
THAT THERE'S SYNTHESIS OF A FULL
LENGTH ANSWER AND THAT CAN LEAD
TO ACTIVATION OF APOE.
WE THINK THAT THIS MODE OF
TRANSCRIPTRANSCRIPTION AND THINT
REGULATION OF APOE EXPRESSION IN
PARTICULAR IN THE CONDITION TEXT
OF ALZHEIMER'S DISEASE.
BUT WHAT I'VE SHOWN SO FAR HAVE
BEEN YOU KNOW, DYNAMICALLY
CHANGING THE EXTENT OF R-LOOP
FORMATION USING EXPERIMENTAL
MANIPULATIONS SO THE QUESTION WE
HAD IS WHETHER CELLS DYNAMICALLY
REGULATE THE FORMATION OF THESE
R-LOOPS OR DYNAMICALLY REGULATE
THE SYNTHESIS OF A FULL-LENGTH
ANSWER TO CHANGE APOE REGULATION
IN RESPONSE TO STRESS.
SO I THINK AS A NEPHROLOGIST I
HAVE A KIDNEY BIAS TOWARDS
THINGS AND WE POINT OUT THAT
APOE IS ALSO IMPLICATED IN SOME
FORMS OF CHRONIC KIDNEY DISEASE.
THERE ARE MOUSE MODELS WHERE
APOE HAS BEEN KNOCKED OUT AND
THOSE MICE DEVELOPED KIDNEY
DISEASE.
THERE ARE REPORTS IN THE
LITERATURE OF MUTATIONS AT APOE
THAT CAN LEAD TO GLOMREULOPATHY
OR INJURY TO THE GLOMERULOUS AND
YOU CAN SEE A CORRELATION WHERE
LOWER KIDNEY FUNCTION IS
ASSOCIATED WITH LOWER APOE
EXPRESSION.
THESE RESULTS IN CONTEXT WITH
THE DATA FOR MOUSE MODELS
SUGGESTED THAT APOE COULD PLAY A
PROTECTIVE ROLE IN THE KIDNEY.
SO WE WANTED TO ASK WHETHER THE
EXPRESSION OF ANSWER MIGHT
CHANGE IN RESPONSE TO STRESS TO
LEAD TO INDUCTION OF APOE.
AND OSMONIC STRESS IS ONE WE
LOOKED AT IN THE LAB SO WE TOOK
CULTURE CELLS AND THEN SUBJECTED
THEM TO OSMOTIC STRESS AND ASKED
TO WHAT HAPPENS TO EXPRESSION OF
ANSWER AND SURE ENOUGH, PUTTING
CELLS IN STRESS CONDITIONS
RESULTED IN A SIGNIFICANT
INCREASE IN THE EXPRESSION OF
FULL LENGTH ANSWERS AND THIS
INCREASE AND EXPRESSION OF FULL
LENGTH ANSWER CORRELATED WITH AN
INCREASE IN APOE EXPRESSION AT
THE RNA LEVEL AND WE CAN ALSO
DETECT AN INCREASE IN APOE
EXPRESSION AT PROTEIN LEVEL.
SO THIS IS EVIDENCE THAT THERE
COULD BE DYNAMIC REGULATION OF
THESE R-LOOP MEDIATED PAUSE TO
ALLOW GENE EXPRESSION TO RESPOND
TO STRESS.
SO ON THE ONE HAND, THIS IS A
VERY SATISFYING RESULT.
BUT ON THE OTHER HAND, IT WAS A
LITTLE BIT CONFUSING AS APOE WAS
NOT KNOWN TO BE AN OS MONDAY I
CAN STRESS RESPONSIVE GENE.
AND WE CAN DETECT APOE IN THE
MEDIA AFTER THIS OS MOT TICK
STRESS SO HERE WE CAN SEE THAT
THERE'S AN INCREASE I IN ITS GRN
THESE COMPANIES.
SO WHAT WE DID WAS TAKE THE
CONDITION MEDIA THAT HAS HIGHER
LEVELS OF APOE EXPRESSION OR
MEDIA FROM CELLS GROWN IN ISO
TONIC CONDITIONS AND BRING THEM
UP THE SAME.
MAKE THEM EQUALLY HYPER TONIC
AND WE PUT THIS CONDITIONED
MEDIA OR THE NON CONDITIONED
MEDIA AND ASKED WHETHER THERE
WAS A DIFFERENCE IN A POP TOE
SIS AND HOW CELLS WERE ABLE TO
RESPOND TO A OSMOTIC STRESS.
THE CELLS IN MEDIA WITH HIGHER
LEVELS OF APOE EXPRESSION IS
HYPER TONIC CONDITION MEDIA,
THEY HAD LOWER LEVELS OF
APOPTOSIS COMPARED TO CELLS IN
HYPER TONIC MEDIA.
WE REPEATED THIS WITH ADDING
HIGHER CONCENTRATION OF SALT
INCREASING THE EXTENT OF THE
OSMOTIC STRESS AND WE SAW THE
SAME PATTERN WHERE THE CELLS IN
THE MEDIA WITH HIGHER LEVELS OF
APO E AND THEY EXPERIENCED LESS
APOPTOSIS SO THEY WERE BETTER
ABLE TO RESPOND TO THIS STRESS.
SO THIS MIGHT SUGGEST THAT
INCREASING APOE EXPRESSION COULD
BE A PROTECTIVE AGAINST
APOPTOSIS AND FACED WITH OSMOTIC
STRESS AND THIS IDEA OF APOE
PLAYING A CYTO PROTECTIVE ROLE
IS CONSISTENT WITH DATA LOOKING
AT ITS ROLE IN THE CENTRAL
NERVOUS SYSTEM WHERE IT'S THE
THOUGHT TO PLAY OR COP TRIBUTE
TO CYTO PROTECTION IN THE BRAIN
AS WELL.
AND THAT'S WHERE I THINK I HAVE
AN AH-HA MOMENT AND THE CELL
TYPE IS IS DYSFUNCTIONAL AND I
HAVE TO GO BACK TO A CASE I
STARTED WITH.
IT'S ASTROCYTES THAT NEED TO
RESPOND TO THIS OSMOTIC STRESS
AND IT'S ASTROCYTES WHOSE
DYSFUNCTION CON TRIBUTES TO THE
DEVELOPMENT OF ALZHEIMER'S
DISEASE.
WE WERE ABLE TO LOOK IN
PUBLISHED DATA OF EXPRESSION IN
ASTROCYTES AND WE CAN SEE THAT
THIS NON CODING RNA IS EXPRESSED
TO HAVE IN ASTROCYTES AND THAT
THE EXPRESSION OF ANSWER OF FULL
LENGTH ANSWER CORRELATES WITH
THE EXPRESSION OF APOE AND SO WE
THINK THAT THIS TRANSCRIPTION
REGULATORY MECHANISM WERE
MODIFIED R-LOOPS CAN PAUSE
TRANSCRIPTION IN THIS NON CODING
RNA TO REGULATE APOE WE THINK
THIS MECHANISM CONTRIBUTES TO
THE REGULATION OF APOE
EXPRESSION IN ASTROCYTES SO TO
COME BACK TO THE CASE I BEGAN
THIS DISCUSSION WITH AND THIS
PATIENT WHO COMES TO THE
HOSPITAL WHO IS HYPO AND SPENT
TIME IN THE ICU ASK HAS OVER
CORRECTION OF HER HYPO ATREMIA
AND SURFS WITH OSMOTIC
DEMYELINATION, THIS IS COMMON IN
THE HOSPITAL AND NOT ALL
PATIENTS WHO ARE HYPOATREMIC
WILL HAVE DEHIGHATION AND I
THINK OF IDENTIFYING THE PAUSING
MECHANISMS THAT HAVE R-LOOPS AND
LED TO APOE COULD BE AN OS MOW
PROTECTED FACTOR AND PERHAPS
THIS IS A PREVIOUSLY
UNAPPRECIATED COMPONENT THAT CAN
CONTRIBUTE TO WHICH PATIENTS
EXPERIENCE OSMOTIC DEMYELINATION
VERSE WHIZ ARE PROTECTED AND
THOSE ABLE TO RESPOND AND HAVE
AN INCREASE IN APOE EXPRESSION
OR IT COULD BE THAT FOLKS THAT
CARRY THE APOE FOR RISK ALLELE,
THAT MAYBE THOSE PATIENTS ARE
MORE AT RISK FOR DEVELOPING
OSMOTIC DEMILE ENATION.
SO I HOPE I WAS ABLE TO SHOW
WITH THESE TWO STORIES,
ILLUSTRATING THE CONTRIBUTION OF
UNDERLYING SEQUENCE AND THE
REGULATION OF PAUSING AND
REGULATION OF GENE EXPRESSION IN
THE FIRST STORY LOOKING AT THIS
COMPONENT OF THE PRIMARY
SEQUENCE AND GENETIC PAUSING
WHICH SEEMS TO CONFER PRECISION
OR SIMILARITIES OF WHERE THE
PAUSES ACROSS CELL TYPES AND
INDIVIDUALS AND THE STORY I JUST
TALKED ABOUT IS EPIGENETIC
REGULATION OF PAUSE TAG DEPENDS
ON THE FORMATION OR DYNAMIC
FORMATION OF THESE MODIFIED
R-LOOP STRUCTURES.
AND SO TO CONCLUDE, I HOPE I'VE
SHOWN YOU THE RNA PRELIMINARY
PAUSING IS A IMPORTANT AND
CRITICAL ASPECT OF TRANSCRIPTION
REGULATION THAT PAUSING NEAR THE
PROMOTER IS IS REGULATE BID A
COMBINATION OF THESE PROTEIN
PAUSING COMPLEXES AND THE
UNDERLYING SEQUENCE AND WE HAVE
IDENTIFIED THIS NEW REGULATORY
MECHANISM WHERE A SUBSET OF NON
CODING RNA AND IT ALLOWED US TO
IDENTIFY APOE HAS AN OSMOTIC
STRESS RESPONSE PROTEIN.
I'D LIKE TO THANK THE FOLKS AT
MY LAD.
I SHOWED DATA AND AS WELL AS
SOME DATA FROM MA AND WINNIE AND
DISCUSSING REDUCK TASE AND WE
GET SUPPORT FROM DON DELL KIRK
AND RODRIGUEZ DID THE BY OWE
CHEMISTRY LOOKING AT THE BETWEEN
M6A AND THIS WORK WAS STARTED
WHEN I WAS A POSTDOC AND IT HAS
CONTINUED AS A COLLABORATION NOW
THAT I HAVE ESTABLISHED MY OWN
GROUP AND THE CLINICAL CASE THAT
I PRESENTED WAS GENEROUSLY
SHARED BY ONE O AND IN ADDITION
FROM SUPPORT WE HAVE SUPPORT
FROM THE CAREER DEVELOPMENT A
CARD FROM ASN.
I APPRECIATE YOUR ATTENTION AND
I WILL BE HAPPY TO ANSWER ANY
QUESTIONS NOW OR E-MAIL.
>>THANK YOU O MUC SO MUCH, Dr.
WATTS.
IT WAS A GREAT TALK.
I LOVE THE WAY YOU DID THE FULL
TRANSLATIONAL SEQUENCE OF
TALKING ABOUT THE CASE AND GOING
BACK TO THE LAB AND BACK AND
FOURTH.
I DON'T SEE QUESTIONS IN THE
CHAT BUT I HAVE A QUESTION FOR
YOU.
SO YOU DID TALK ABOUT THE
GENETICS SEQUENCE, WHAT'S THERE
IN THE GENOME AND HOW IT EFFECTS
PAUSING AND YOU TALKED ABOUT ALL
THESE OTHER FACTORS THAT COULD
ACTUALLY ADDITIONALLY
DYNAMICALLY EFFECT THAT SYSTEM.
SO, I MEAN, HOW DO YOU DECIDE --
SO IS THERE A PATH FORWARD TO
SOME TYPE OF AN INTERVENTION?
THE QUESTION IS, WHAT'S
MODIFIABLE?
SO THE GENE SEQUENCES ARE THERE
RIGHT, BUT WHAT DO YOU SEE AS
THE POTENTIAL APPLICATION OF
THIS TYPE OF MECHANISTIC STUDY
TOWARDS SOME KIND OF CLINICAL
INTERVENTION?
>>Jason Watts: THAT'S A GOOD
QUESTION.
TO THE FIRST PART WE'RE FOCUSING
ON PRIMARY SEQUENCE AND PAUSING
AT THE SITE OZONE IS SIMILAR
ACROSS INDIVIDUALS INTERESTED IN
PURSUING THAT AND IN LOOKING AT
HOW POLY MORPHISMS COULD EXPLAIN
DIFFERENCES IN INDIVIDUALS IN
TERMS OF HOW GENES ARE EXPRESSED
AND WE'RE VERY EXCITED ABOUT
THIS NEW OBSERVATION ABOUT,
R-LOOP MEDIATED PAUSING IN NON
CODING RNAs IN THAT PROVIDES A
POTENTIAL TARGET TO DEVELOP
THEIR PIECE AND SO IF WE'RE
TALKING ABOUT APOE FOR EXAMPLE,
IF YOU ARE A CARRIER OF THE E-4
RISK ALLELE, IT MAY BE
ADVANTAGEOUS IF YOU HAVE LOWER
EXPRESSION OF THAT RISK A LEGAL.
SO WE THINK THAT TARGETING THIS
NON CODING RNA, COULD LEAD TO
RESULT IN DECREASED EXPRESSION
OF THE RISK ALLELE AND THAT
COULD BE PROTECTIVE IN TERMS OF
PREVENTING THE ONSET OF DEMENTIA
OR SLOWING THE PROGRESSION OF
DEMENTIA SO WANTING TO BETTER
UNDERSTAND HOW THIS NON CODE RNA
IS IS REGULATED AND HOW TURNING
IT ON LEADS TO AN INCREASE IN
APOE EXPRESSION IS AN ACTIVE
AREA OF WORK IN THE LAB RIGHT
NOW.
>>IT'S ALSO SUPER INTERESTING
THAT YOU MOVE FROM THE KIDNEY
AND TO THE BRAIN.
THESE ARE UBIQUITOUS MECHANISMS
SO HOW DO YOU THEN, AS A
NEPHROLOGIST, START THINKING
ABOUT THESE OTHER AREAS OF
APPLICATION?
DO YOU COLLABORATE WITH OTHER
LABS?
DO YOU TALK TO PEOPLE IN OTHER
FIELDS?
>>Jason Watts: SO, AT LEAST FOR
THE PARTICULARLY MAYBE ENAMORED
OR INTERESTED IN THIS LINK WITH
OSMOTIC DEMILE ENATION WHICH I
FULLY ACKNOWLEDGE THAT FAIRLY
RARE COMPLICATION BUT LOOKING
FOR COLLABORATORS AND VERY
INTERESTED IN WEATHER APOE
EXPRESSION IS DIFFERENT OR MIGHT
BE A RISK FACTOR FOR THOSE WHO
ARE GOING TO DEVELOP OSMOTIC
DEMILE ENATION.
I ALLUDED TO DOING THE
EXPERIMENT WHERE WE SHOW THAT
THERE'S MORE OR LESS APOPTOSIS
WHEN THERE'S HIGHER APOE
EXPRESSION AND IT WAS DONE IN
CELLS SO WE HAVE A COUPLE
EXAMPLES OF CHRONIC KIDNEY
DISEASES WHERE IT SEEMS THAT
THERE'S DIFFERENTIAL EXPRESSION
OF APOE AND I THINK MORE
INTERESTINGLY DIFFERENTIAL
EXPRESSION OF ANSWER THIS NON
CODING RNA THAT REGULATES APOE
SO ANOTHER PROJECT FOR ANOTHER
PERSON IN LAB IS LOOKING AT HOW
THIS REGULATORY MECHANISM IS
GERMANE TO KIDNEY BIOLOGY AND IN
PARTICULAR, INTO TUBAL BIOLOGY.
>>I DON'T SEE ANY OTHER
QUESTIONS RIGHT NOW FROM THE
VIDEO CAST WEBSITE.
DO YOU HAVE ANY CLOSING
COMMENTS, Dr. WATTS?
WOULD YOU LIKE TO MAKE TO THE
AUDIENCE BEFORE WE END?
>>I JUST APPRECIATE THOSE FOLKS
WHO ATTENDED.
AND YOUR QUESTIONS, Dr. CHUNG,
GAVE ME AN OPPORTUNITY TO ALLUDE
TO ON GOING WORK EX WE'LL LOOK
IN PARTICULAR AT THIS
RELATIONSHIP BETWEEN ANSWER AND
APOE IN KIDNEY DISEASE.
>>THAT'S GREAT.
ALL RIGHT.
SO I WANT TO THANK EVERYONE FOR
THEIR ATTENDANCE AND THEIR
ATTENTION.
JUST TO REMIND YOU THE CME CODE
TODAY IS 4454 AND WE WANT TO
THANK Dr. WATTS FOR HIS
BRILLIANT TALK AND BEST OF LUCK
TO YOU.
>>Jason Watts: THANK YOU, VERY
MUCH.
TAKE CARE, EVERYONE.
>>THANK YOU SO MUCH.