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Welcome to the Clinical
Center Grand Rounds,

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a weekly series of educational
lectures for physicians and

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health care professionals
broadcast from the Clinical

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Center at the National
Institutes of Health in

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Bethesda, MD.

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The NIH Clinical Center is the
world's largest hospital totally

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dedicated to investigational
research and leads the global

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effort in training today's
investigators and discovering

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tomorrow's cures.

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Learn more by visiting us online
at http://clinicalcenter.nih.gov

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I AM DELIGHTED TO INTRODUCE OUR

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SPEAKER TODAY, Dr. JASON A.

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WATTS.

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A NEPHROLOGIST, SCIENTIST AND

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STADTMAN INVESTIGATOR IN THE EP

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AGAIN ET TICKS AND STEM CELL

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LABRADOR AT THE HEALTH SCIENCES

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NIEHS LOCATED IN NORTH CAROLINA.

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HE IS ALSO AN ADJUNCT ASSISTANT

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PROFESSOR OF MEDICINE AT DUKE

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UNIVERSITY AND A STAFF

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NEPHROLOGIST AT THE DURHAM

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VETERANS AFFAIR HOSPITAL.

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Dr. WATTS ANTICIPATED BOTH AN

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MD AND A PH.D FROM THE PEARLMAN

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SCHOOL OF UNIVERSITY AT THE IN

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PENNSYLVANIA WHERE HE STUDIES

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FOX A.

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HE COMPLETED RECEIPT DEPOSIT SEE

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IN INTERNAL MEDICINE AT DUKE

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UNIVERSITY AND A FELLOWSHIP IN

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NEPHROLOGY AT THE UNIVERSITY OF

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MICHIGAN WHERE HE PURSUED

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FURTHER RESEARCH ON THE

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REGULATION OF RNA, PAUSING.

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IN 2020 HE MOVED TO NIEHS AS A

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STADTMAN INVESTIGATOR AND LEADS

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THE TRANSCRIPTIONAL RESPONSES IN

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DISEASE GROUP WITH A SECONDARY

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APPOINTMENT IN THE GENOME I AM

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TEG RIGHT TEE AND STRUCTURAL

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BIOLOGY LABRADOR.

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HIS GROUP STUDIES HOW NUCLEIC AK

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SID STRUCTURES AND CHROMATIN

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ENVIRONMENTS INFLUENCE GENE

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TRANSCRIPTION.

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Dr. WATTS RECENTLY RECEIVED AN

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NIH DIRECTOR'S AWARD FOR HIS

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WORK ON THE NIH COVID VACCINE

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AND BOOSTER CLINIC TEAM AND HE

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WAS A RECIPIENT OF THE AMERICAN

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SOCIETY OF NEPHROLOGY MEDICAL

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FACULTY DEVELOPMENT AWARD IN

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2019.

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HE SERVED AS A MEMBER OF THE

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AMERICAN SOCIETY OF NEPHROLOGY

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AND THE AMERICAN SOCIETY OF

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HUMAN GENETICS AND IS BOARD

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CERTIFIED IN INTERNAL MEDICINE

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AND NEPHROLOGY.

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PLEASE JOIN ME IN WELCOMING OUR

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SPEAKER, Dr. JASON WATTS, FOR

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HIS PRESENTATION ENTITLED, SEE

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REQUEST DEPENT AROUND IS A

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DETERMINANT OF HUMAN GENE

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EXPRESSION.

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WELCOME,.

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THANK YOU SO MUCH FOR THE KIND

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INTRODUCTION AND I'M PLEASED TO

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BE HERE.

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SO, AS Dr. CHUNG MENTIONED,

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I'M A BASIC SCIENTIST AND A

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NEPHROLOGIST AND TODAY I WANT TO

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STUDY SEQUENCE IN THE

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TRANSMISSION AND HOW IT AFFECTS

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GENE EXPRESSION.

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I DON'T HAVE ANY DISCLOSURES.

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AND MY OBJECTIVES TODAY ARE TO

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INTRODUCE PROMOTER PROXIMAL' RNA

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PAUSING IN IT'S ROLL IN

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TRANSCRIPTION REGULATION AND THE

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ROLE OF SEQUENCE AND REGULATING

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PAUSEING AND I WANT TO TALK

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ABOUT A NEW ROLE OF PAUSE

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REGULATION THAT INVOLVES FOLDING

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OF NUCLEIC ACIDS AND THIS IS ONE

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WE FIND THAT REGULATES NON

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CODING RNAs.

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I'LL TALK ABOUT TRANSCRIPTION

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AND MOLECULAR BUYEL GEE BUT I

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WANT TO FRAME THE WORK THAT

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WE'RE DOING IN THE CONTEXT OF A

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CLINICAL CASE.

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SO THIS IS A PATIENT WHO WAS

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SEEN UP THE ROAD FROM ME AT

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DUKE.

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SO IT'S A 73-YEAR-OLD WOMAN WHO

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HAS INSTAGE KIDNEY DISEASE AND

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RECEIVED A DECEASED KIDNEY

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DONOR.

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SHE PRESENTED TO AN OUTSIDE

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HOSPITAL WITH SEVERAL DAYS OF

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NAUSEA AND VOMITING.

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IN HER LABS IN THE EMERGENCY

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ROOM SHOW SHE HAD HYPO ATMIA

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WITH 121 CAN NORMAL RENAL

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FUNCTIONS AND GIVEN THE HISTORY

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IN LABS, IT WAS THOUGHT SHE

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LIKELY HAD HYPO BOW LEM I CAN

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AND STARTED ON NORMAL CHALEE AN

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IN THE EMERGENCY ROOM AND

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ADMITTED FOR FURTHER MANAGEMENT.

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ON HER FIRST HOSPITAL DAY, SHE

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SUFFERED A CARDIAC ARREST.

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SHE SPENT TWO WEEKS IN THE

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INTENSIVE CARE UNIT INTUBATED

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AND SA DADEED.

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SHE HAD A BRAIN MRI THE DAY

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AFTER THE CARDIAC ARREST AND

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SHOWED CHRONIC H HE IS.

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HER HYPO ATREMIA WAS CORRECTED

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AND SHE WAS EXCAVATED AND IT WAS

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NEAR BASELINE BUT SHE HAD

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NEUROLOGICAL DEFICITS AND SHE

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HAD DIFFICULTY MANAGING SEE CRES

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AND DUE TO THOSE SYSTEMS SHE WAS

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TRANSFERRED FOR DUKE FOR FURTHER

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MANAGEMENT.

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AFTER HOSPITAL TRANSFER BECAUSE

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HER SYMPTOMS WEREN'T IMPROVING

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SHE HAD A SECOND MRI AND SHOULD

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WAS ABOUT FOUR WEEKS FROM HER

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INITIAL PRESENTATION AND AS

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INDICATED HERE, WITH THIS RED

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ARROW, THERE WERE ABNORMALITIES

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ON THE MRI SO THERE WERE LESIONS

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IDENTIFIED IN THE INTERIOR

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POPPED IN THE MADEL A ON FLARE

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AND T2 WEIGHTED IMAGES.

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THESE FINDINGS ARE COMPATIBLE

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WITH SYNDROME WITH THE INSERIOUS

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PAUSE AND THE CERVIC OWE

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MEDULLARY JUNCTION.

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IT'S UP WITH OF THE THINGS IN

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NEPHROLOGY FEAR THE MOST WHEN WE

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TAKE CARE OF PATIENTS TAZ CAN

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RESULT FROM TWO RAPID CORRECTION

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OF THAT LOW SODIUM AND LEADING

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TO NEUROLOGICAL INJURIES.

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SO IF WE LOOK AT THE SODIUM

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INTERPRETED FOTREND FOR THIS PAN

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SEE THAT AT PRESENTATION HER

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SODIUM WAS 121 AND IT DROPPED TO

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117 AND OVER HER COURSE IN THE

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INTENSIVE CARE UNIT IT WAS

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CORRECTED BUT THEY ACTUALLY

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THERE WAS A PERIOD WHERE SHE WAS

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HYPER AND SHE HAD OVER

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CORRECTION.

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AND AT SOME POINT DURING THIS

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PERIOD, THIS IS LIKELY WHEN SHE

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SUFFERED THIS DEMYELINATION SO

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IN I THINK A QUESTION IS HOW DO

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CELLS DEFEND AGAINST THIS

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OSMONIC STRESS FROM HAVING A

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CHANGE IN THIS SODIUM.

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IF WE HAVE A HYPER TONIC

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CONDITION OUTSIDE OF THE CELLS,

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WATER LEAVES CELLS EX THEY'RE

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GOING TO SHRINK.

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SO THIS IS TO DEFEND AGAINST

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THIS LOSS OF VOLUME, THERE'S AN

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INCREASE IN THE UPTAKE OF

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ORGANIC OSMOL SUCH AS CHLORINE.

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IN ADDITION TO CHANGES AT

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MEMBRANE THAT ALLOW THESE TO BE

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BROUGHT BACK INTO CELLS, THERE'S

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A CHANGE AT THE TRANSCRIPTION

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LEVEL IN GENES WHERE THEY GET UP

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REGULATED SO IT CONVERTS GLUCOSE

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TO SCOTIA TOLL AND THIS ALLOWS

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FOR WATER TO MOVE BACK INTO

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CELLS AND IT CAN DEFEND AGAINST

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THE VOLUME LOSS IN THIS HOW DO

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CELLS RESPONDED TO PHYSICAL

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LOGIC STRESS SUCH AS HYPER TONIC

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STRESS.

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SO WHAT I WANT TO TALK ABOUT

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TODAY, IN THE FIRST PART IS

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DISCUSSING OR TALKING ABOUT THE

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ROLE OF SEQUENCE IN REGULATING

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PRO MOTOR PROXIMAL PAUSING AND

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THIS IS A STORY ABOUT CODING

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AGAINST.

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GENES.

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AND THE SECOND PART LOOKING AT

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FOLDING OF RNA AND DNA IN A

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STRUCTURE CALLED AN R-LOOP AND

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HOW THAT PAUSES RNA IN THE BODY

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OF NON CODING GENES AND AT END

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I'M GOING TO COME BACK TO HOW

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LEARNING ABOUT PAUSING

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REGULATIONS MIGHT OFFER NEW

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INSIGHTS INTO WHAT GOES ON WITH

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OSMONIC DELINEATION.

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WHAT IT REFERS TO IS THIS

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ADDITIONAL LAYER OF

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TRANSCRIPTION REGULATION THAT

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HAPPENS IN OUR CELLS.

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SO AFTER THE PRELIMINARY GETS

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RECRUITED TO GENE REMOTERS, IT

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CAN BE AGAIN TO TRANSCRIBE AND

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PAUSE NEAR THE PROMOTERS.

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AND IT NEEDS ADDITIONAL CUES TO

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BE RELEASE INTERESTED THIS PAUSE

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TO THEM MOVE INTO PRODUCTIVE

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ELONGATION AND SYNTHESIZE RNA TO

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INCREASE GENE REGULATION.

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THIS MODE OF REGULATION IS FOUND

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IN ANIMALS AND IS NOT FOUND IN

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BACTERIA OR YEAST.

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SO, IN OUR CELLS IT'S MOST

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COMMON IN STRESS RESPONSE OVER

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DYNAMICALLY REGULATED GENES, THE

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KINDS OF GEEZ NECESSARY FOR

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MAINTAINING HOMEOSTASIS OR

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DEFENDING AGAINST STRESSES.

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SO WHAT HAPPENS IS THAT AFTER

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THE PRELIMINARY LANDS ON THE

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PROMOTER AND SI SYNTHESIZES THEA

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IT ENCOUNTERS TWO PAUSING

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COMPLEXES.

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AND THE ADDITIONAL SIGNAL IS A

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PHOSPHORYLATION EVENT THAT

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ALLOWS THE PAUSING COMPLEX TO BE

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RELEASED AND DSIF CHANGES FOR

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BEING A PAUSING FACTOR TO BEING

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ELONGATION FACTOR AND IT TRAVELS

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ALONG WITH THE RNA

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PRELIMINARIES.

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WE'VE BEEN LOOKING AT GENES THAT

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HAVE THE PAUSING COMPLEXES NELF

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AND DSIF AND ONE QUESTION ABOUT

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THIS MODE OF REGULATION IS WHY

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THIS OCCURS IN OUR CELLS AND NOT

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OTHER ORGANISMS.

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ONE THOUGHT IS THAT HAVING THE

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PRELIMINARIES ON THE PROMOTERS,

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ALLOWS FOR MORE UNIFORM GENE

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INDUCTION BETWEEN DIFFERENT CELL

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TYPES.

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SO I WOULD MAKE THE METAPHOR

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THAT THESE BMX BIKE LISTS

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REPRESENT PRELIMINARY LOADED ON

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THE PROMOTER AND THE PAUSED AT

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THE STARTING BLOCK SO WHEN THE

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STARTING PISTOL GOES OFF THEY

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GET RELEASED FROM THE PAUSE AND

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THE BAY CAN GO INTO THE RACE.

260
00:10:22,320 --> 00:10:24,480
AND SO WORK THAT WAS DONE SHOWED

261
00:10:24,480 --> 00:10:29,040
THAT DURING DEVELOPMENT, GENES

262
00:10:29,040 --> 00:10:32,320
THAT HAVE PAUSE ARE INDUCED MORE

263
00:10:32,320 --> 00:10:34,320
UNIFORMLY THAN GENES WITHOUT

264
00:10:34,320 --> 00:10:35,600
PAUSE PRELIMINARY ARY SO THERE'S

265
00:10:35,600 --> 00:10:38,560
A COMPONENT OF A RAPID START ON

266
00:10:38,560 --> 00:10:40,360
THE PROMOTER BUT EVEN MORE

267
00:10:40,360 --> 00:10:42,280
IMPORTANTLY THERE'S A MORE

268
00:10:42,280 --> 00:10:43,800
UNIFORM START OF HOW GENES CAN

269
00:10:43,800 --> 00:10:46,600
RESPOND TO A PARTICULAR STRESS.

270
00:10:46,600 --> 00:10:52,040
SO WHEN WE WERE LOOKING AT DSI

271
00:10:52,040 --> 00:10:55,440
AND FINDING IN GENES NELF THIS

272
00:10:55,440 --> 00:10:58,080
GENE WHICH I DESCRIBED AS BEING

273
00:10:58,080 --> 00:10:59,960
IMPORTANT FOR RESPONDING TO OS

274
00:10:59,960 --> 00:11:01,840
MOT TICK STRESS THESE PAUSING

275
00:11:01,840 --> 00:11:03,080
COMPLEXES ARE LOCATED AT THE

276
00:11:03,080 --> 00:11:03,560
PROMOTERS.

277
00:11:03,560 --> 00:11:06,160
SO THIS IS THE PATTERN THAT WE

278
00:11:06,160 --> 00:11:09,040
EXPECT AND IF THERE'S PROMOTER

279
00:11:09,040 --> 00:11:10,480
PROXIMAL PAUSING SO A QUESTION

280
00:11:10,480 --> 00:11:13,200
WE HAVE IS WHETHER THE PAUSING

281
00:11:13,200 --> 00:11:15,160
REGULATION PLAYS A ROLE IN

282
00:11:15,160 --> 00:11:17,480
RESPONDING TO HYPER CONNICK CRES

283
00:11:17,480 --> 00:11:20,800
SOSTRESSED SO WE WANTED TO MEASE

284
00:11:20,800 --> 00:11:24,600
WHERE THE PRELIMINARY IS

285
00:11:24,600 --> 00:11:25,440
LOCATED.

286
00:11:25,440 --> 00:11:31,600
THE ASSAY IS THE PRO SEEK O WE

287
00:11:31,600 --> 00:11:33,240
TAKE CULTURAL CELLS AND PERFORM

288
00:11:33,240 --> 00:11:36,200
A RUN ON REACTION WITH BIO TIN A

289
00:11:36,200 --> 00:11:41,840
LATED NUCLEOTIDES SO THEY BE

290
00:11:41,840 --> 00:11:44,720
INCORPORATED BY THE DETECT THESE

291
00:11:44,720 --> 00:11:47,960
NEWLY ADDED MODIFIED NUCLEOTIDES

292
00:11:47,960 --> 00:11:55,600
AND PULL THOSE OUT OF CELLS.

293
00:11:55,600 --> 00:11:57,040
WE CAN USE SEQUENCING TO MAP

294
00:11:57,040 --> 00:11:59,280
THOSE BACK ON THE GENOME SO THE

295
00:11:59,280 --> 00:12:02,040
THREE PRIME END OF THE RNA, THE

296
00:12:02,040 --> 00:12:03,760
END OF THE RNA THAT HAS THIS

297
00:12:03,760 --> 00:12:05,800
MODIFIED NUCLEOTIDE IS A PROXY

298
00:12:05,800 --> 00:12:07,240
WHERE IT WAS LAST

299
00:12:07,240 --> 00:12:08,680
TRANSCRIPTIONALLY IMPACTED.

300
00:12:08,680 --> 00:12:12,280
SO WE CAN INFER WHERE THE PAUSE

301
00:12:12,280 --> 00:12:14,200
AS BEING LOCATIONS WHERE THERE'S

302
00:12:14,200 --> 00:12:18,440
ANNA CUMULUS OF THESE READS FROM

303
00:12:18,440 --> 00:12:19,960
PRO-SEQ SO IF IT'S NEAR THE

304
00:12:19,960 --> 00:12:24,800
PROMOTER IT'S THE PATTERN TA FIX

305
00:12:24,800 --> 00:12:25,520
THIS PAUSE PRELIMINARY.

306
00:12:25,520 --> 00:12:28,240
WHEN WE DO THIS ASSAY ON A CAP

307
00:12:28,240 --> 00:12:30,760
SER CELL LINE AND LOOK AT WHERE

308
00:12:30,760 --> 00:12:32,440
THE PRELIMINARY IS LOCATED ON

309
00:12:32,440 --> 00:12:35,480
THESE WITH WHAT WE SAW WITH NELF

310
00:12:35,480 --> 00:12:38,360
AND DFIS WE CAN SEE THE TYPE OF

311
00:12:38,360 --> 00:12:40,280
PROMOTER EVIDENT BY THE TIGHT OF

312
00:12:40,280 --> 00:12:42,520
THESE MARKS AND DETECT THERE'S

313
00:12:42,520 --> 00:12:44,320
SOME PRELIMINARY IN THE GENE

314
00:12:44,320 --> 00:12:46,320
BODY WHEN THESE CELLS ARE GROWN

315
00:12:46,320 --> 00:12:48,200
IN NORMAL CONDITIONS OR ISO

316
00:12:48,200 --> 00:12:50,080
TONIC CONDITIONS.

317
00:12:50,080 --> 00:12:52,200
SO THIS IS WHERE THE PRELIMINARY

318
00:12:52,200 --> 00:12:53,040
IS IS PAUSE.

319
00:12:53,040 --> 00:12:54,560
IN WHAT WE WOULD EXPECT IF

320
00:12:54,560 --> 00:12:56,120
THERE'S PAUSE RELEASED, THE GENE

321
00:12:56,120 --> 00:12:57,880
IS BEING INDUCED WE WOULD SEE A

322
00:12:57,880 --> 00:12:59,520
SHIFT WHERE THERE'S GOING TO BE

323
00:12:59,520 --> 00:13:00,800
FEWER PRELIMINARY ACCUMULATED AT

324
00:13:00,800 --> 00:13:03,640
THE PROMOTER AND WE EXPECT TO

325
00:13:03,640 --> 00:13:05,680
SEE MORE THAT GET RELEASED TO

326
00:13:05,680 --> 00:13:08,240
TRANSCRIBE IN THE GENE BODY.

327
00:13:08,240 --> 00:13:10,160
SO WE SHIFTED OURSELVES FROM EYE

328
00:13:10,160 --> 00:13:12,040
OWE TONIC CONDITIONS NOW TO

329
00:13:12,040 --> 00:13:13,840
HYPER TONIC CONDITIONS.

330
00:13:13,840 --> 00:13:15,000
THE OS MOT TICK STRESS

331
00:13:15,000 --> 00:13:16,280
CONDITIONS IN DEED WE CAN DETECT

332
00:13:16,280 --> 00:13:18,080
THERE'S AN INCREASE IN THE

333
00:13:18,080 --> 00:13:19,480
AMOUNT OF PRELIMINARIES IN THE

334
00:13:19,480 --> 00:13:21,880
GENE BODY IN THESE TWO

335
00:13:21,880 --> 00:13:23,040
REPLICATES AND WHAT I THINK

336
00:13:23,040 --> 00:13:24,680
STANDS OUT IS THAT THERE'S STILL

337
00:13:24,680 --> 00:13:29,240
A POPULATION OF REMAIN PAUSED IN

338
00:13:29,240 --> 00:13:31,240
THE PROMOTER SO EVEN THOUGH THE

339
00:13:31,240 --> 00:13:32,920
GENE HAS BEEN INDUCED THERE'S

340
00:13:32,920 --> 00:13:34,440
STILL SOME THAT ARE GETTING

341
00:13:34,440 --> 00:13:36,800
STUCK ON LIKE A SPEED BUMP WHERE

342
00:13:36,800 --> 00:13:40,360
SOMETHING THERE IS PAUSING IT TO

343
00:13:40,360 --> 00:13:41,120
ACCUMULATE.

344
00:13:41,120 --> 00:13:42,400
SO WE CONSIDERED THAT THERE MAY

345
00:13:42,400 --> 00:13:44,880
BE AN ADDITIONAL LAYER OF

346
00:13:44,880 --> 00:13:45,840
REGULATION BEYOND THESE PAUSING

347
00:13:45,840 --> 00:13:46,240
COMPLEXES.

348
00:13:46,240 --> 00:13:47,960
AND IT COULD BE THAT THERE'S AN

349
00:13:47,960 --> 00:13:51,120
UNDERLYING CONTRIBUTION OF THE

350
00:13:51,120 --> 00:13:52,520
NUCLEIC ACID SEQUENCE AND

351
00:13:52,520 --> 00:13:55,640
SOMETHING IN THE DNA OR THE RNA

352
00:13:55,640 --> 00:13:56,960
THAT CON TRIBUTES TO PAUSING

353
00:13:56,960 --> 00:13:57,480
REGULATION.

354
00:13:57,480 --> 00:14:01,680
AND THAT MAY PERSIST EVEN AFTER

355
00:14:01,680 --> 00:14:04,600
A RESPONDING TO THE STRESS.

356
00:14:04,600 --> 00:14:06,320
SO TO ADDRESS THIS QUESTION,

357
00:14:06,320 --> 00:14:08,200
WHAT WE DECIDED TO DO WAS

358
00:14:08,200 --> 00:14:10,800
PROFILE THE PATTERN OF PAUSING

359
00:14:10,800 --> 00:14:12,840
IN NORMAL HUMAN CELLS.

360
00:14:12,840 --> 00:14:16,640
AND AGAIN AS A NEFF R NEPHROLOGI

361
00:14:16,640 --> 00:14:19,080
WOULD HAVE LOVE TO DO THIS ON

362
00:14:19,080 --> 00:14:19,680
PRIMARY RENAL CELL TYPES BUT

363
00:14:19,680 --> 00:14:21,800
IT'S' YEAR TO GET A HOLD OF

364
00:14:21,800 --> 00:14:24,720
PRIMARY SKIN FIBER BLAST SO WE

365
00:14:24,720 --> 00:14:27,560
PROFILE TRANSCRIPTION IN FIBER

366
00:14:27,560 --> 00:14:32,080
BLASTS FROM BUY ONS SEE FROM

367
00:14:32,080 --> 00:14:33,040
OTHER PIES AND THE PAT YOUR

368
00:14:33,040 --> 00:14:34,560
WORSHIP OF PRELIMINARIES PAUSING

369
00:14:34,560 --> 00:14:40,960
AND MEASURED GENE EXPRESSION.

370
00:14:40,960 --> 00:14:43,920
I'M SNOWING A GENE WITH ROLLS IN

371
00:14:43,920 --> 00:14:45,440
PONDING STRESS SIMILAR TO SHOWED

372
00:14:45,440 --> 00:14:48,880
WHAT I AND WE CAN SEE THERE'S A

373
00:14:48,880 --> 00:14:50,440
ACCUMULATION AT THE PROMOTER

374
00:14:50,440 --> 00:14:52,280
CONSISTENT WITH PAUSING AND WE

375
00:14:52,280 --> 00:14:54,560
CAN DETECT SOME PRELIMINARY IN

376
00:14:54,560 --> 00:15:01,200
THE GENE BODY.

377
00:15:01,200 --> 00:15:03,320
WE PROFILED AND FIVEN DOSING

378
00:15:03,320 --> 00:15:04,160
CELLS FROM FIVE INDIVIDUALS AND

379
00:15:04,160 --> 00:15:07,720
WE CAN SEE As COULD THE FIVE CRE

380
00:15:07,720 --> 00:15:09,160
INDIVIDUALS IT ACCUMULATION IN

381
00:15:09,160 --> 00:15:10,920
THE PROMOTE YOU ARE AND ONE OF

382
00:15:10,920 --> 00:15:15,600
THE STRENGTH IS WE GET ONE GINO

383
00:15:15,600 --> 00:15:17,680
WIDE INFORMATION AND HIGH

384
00:15:17,680 --> 00:15:18,320
RESOLUTION INFORMATION.

385
00:15:18,320 --> 00:15:20,000
AND SO WE CAN ACTUALLY ZOOM IN

386
00:15:20,000 --> 00:15:22,080
AND SEE THAT THESE ARE NOT JUST

387
00:15:22,080 --> 00:15:24,200
ACCUMULATING IN THE PROMOTER,

388
00:15:24,200 --> 00:15:26,120
ACTUALLY ACCUMULATING AT THE

389
00:15:26,120 --> 00:15:28,040
SAME NUCLEOTIDE POSITION SO

390
00:15:28,040 --> 00:15:29,840
ACROSS THESE FIVE INDIVIDUALS IT

391
00:15:29,840 --> 00:15:32,080
SEEMS IT'S PAUSING PRECISELY TO

392
00:15:32,080 --> 00:15:35,800
SPECIFIC NUCLEOTIDE.

393
00:15:35,800 --> 00:15:37,960
IS IT JUST AN OUT LIER?

394
00:15:37,960 --> 00:15:40,200
WE CAN LOOK AT THOUSANDS OF

395
00:15:40,200 --> 00:15:42,160
GENES SO HERE I'M NOW DISPLAYING

396
00:15:42,160 --> 00:15:44,160
THE PROCEED DATA AS A HEAT MAP.

397
00:15:44,160 --> 00:15:46,040
EACH ROW ON THIS HEAT MAP IS A

398
00:15:46,040 --> 00:15:47,440
INDIVIDUAL GENE AND THE GENES

399
00:15:47,440 --> 00:15:49,720
ARE ARRANGED SO THE NUCLEOTIDE

400
00:15:49,720 --> 00:15:54,200
POSITION WITH THE MOST IS

401
00:15:54,200 --> 00:15:54,800
GETTING FARTHER FROM THE START

402
00:15:54,800 --> 00:15:56,320
SITE AS WE MOVE DOWN THE PLOT.

403
00:15:56,320 --> 00:15:57,680
THIS IS THE DATA FROM ONE

404
00:15:57,680 --> 00:15:58,880
INDIVIDUAL AND IF YOU LOOK AT

405
00:15:58,880 --> 00:16:00,440
THE DATA FROM ALL FIVE OF THE

406
00:16:00,440 --> 00:16:02,080
INDIVIDUALS THAT WE PROFILE,

407
00:16:02,080 --> 00:16:02,960
HOPEFULLY YOU WOULD AGREE WITH

408
00:16:02,960 --> 00:16:05,640
ME THE PATTERN OF WHERE THE

409
00:16:05,640 --> 00:16:07,880
PAUSE IS VERY SIMILAR ACROSS

410
00:16:07,880 --> 00:16:08,520
INDIVIDUALS.

411
00:16:08,520 --> 00:16:13,240
AND IT'S STATISTICALLY

412
00:16:13,240 --> 00:16:14,760
CORRELATED THAT THE CORRELATIONS

413
00:16:14,760 --> 00:16:17,280
AND THE VERY SMALL P VALUE.

414
00:16:17,280 --> 00:16:19,760
AND MORE THAN JUST THE HIGHLY

415
00:16:19,760 --> 00:16:21,760
CORRELATED LOCATIONS OF WHERE

416
00:16:21,760 --> 00:16:27,640
THE PAUSE WE CAN FIND OVER 1300

417
00:16:27,640 --> 00:16:29,640
GENES WHERE THE POL LL ACROSS

418
00:16:29,640 --> 00:16:32,560
ALL FIVE INDIVIDUALS SO PATTERNS

419
00:16:32,560 --> 00:16:34,200
SIMILAR FOR WHAT HE SHOWED.

420
00:16:34,200 --> 00:16:36,880
SO IT SEEMS ACROSS THOUSANDS OF

421
00:16:36,880 --> 00:16:40,320
GENES WHERE THE PAUSE VERY

422
00:16:40,320 --> 00:16:40,920
PRECISE.

423
00:16:40,920 --> 00:16:42,960
THIS IS ALL LOOKING AT WITHIN

424
00:16:42,960 --> 00:16:44,960
THE SAME CELL TYPE.

425
00:16:44,960 --> 00:16:47,680
SO WE WONDERED, DOES THIS

426
00:16:47,680 --> 00:16:49,560
PRECISION OF PAUSING ORGANIZE

427
00:16:49,560 --> 00:16:51,560
SIMILARLY OF WHERE THE PAUSES

428
00:16:51,560 --> 00:16:54,320
THAT EXTEND TO OTHER CELL TYPES.

429
00:16:54,320 --> 00:16:56,680
SO WE HAVE BEEN PROFILING FIBER

430
00:16:56,680 --> 00:16:58,800
BLAST FROM ADULT SKIN AND WE

431
00:16:58,800 --> 00:17:02,640
ALSO LOOKED AT NEONATAL FIBER

432
00:17:02,640 --> 00:17:08,640
BLASTS FROM FORE SKIN AND LOOKED

433
00:17:08,640 --> 00:17:12,840
AT TUBAL CELL LINE AND A TIME OR

434
00:17:12,840 --> 00:17:14,520
SEE PRESS OR AND IT'S IN THE

435
00:17:14,520 --> 00:17:15,560
PROMOTER AND ACROSS THESE

436
00:17:15,560 --> 00:17:17,920
DIFFERENT CELL CONTEXT, WE CAN

437
00:17:17,920 --> 00:17:20,080
SEE THAT THE POL LL IT'S AT THE

438
00:17:20,080 --> 00:17:21,720
SAME NUCLEOTIDE POSITION.

439
00:17:21,720 --> 00:17:25,040
WE TOOK THIS A STEP FURTHER AND

440
00:17:25,040 --> 00:17:28,000
LOOKED THIS IN DATA WE DIDN'T

441
00:17:28,000 --> 00:17:29,400
HAVE ANY PART GENERATING.

442
00:17:29,400 --> 00:17:31,560
THIS IS LOOKING IN A LEUKEMIA

443
00:17:31,560 --> 00:17:33,320
CELL LINE AND WE CAN SEE IT'S

444
00:17:33,320 --> 00:17:36,240
ACCUMULATING AT THE PROMOTER AND

445
00:17:36,240 --> 00:17:39,000
THE SAME NUCLEOTIDE POSITION SO

446
00:17:39,000 --> 00:17:40,800
ACROSS CELL TYPES, ACROSS LABS

447
00:17:40,800 --> 00:17:42,760
DOING THE ASSAY WE OTHER SEE

448
00:17:42,760 --> 00:17:45,520
THERE'S A HIGH DEGREE OF

449
00:17:45,520 --> 00:17:47,680
SIMILARLY WHERE IT'S PAUSING IN

450
00:17:47,680 --> 00:17:49,440
THESE GENE PROMOTERS.

451
00:17:49,440 --> 00:17:51,120
SO WE WANTED TO ASK, YOU KNOW,

452
00:17:51,120 --> 00:17:55,000
ARE THERE SEQUENCE ELEMENTS THAT

453
00:17:55,000 --> 00:17:57,520
CORRELATE WITH WHERE IT PAUSES.

454
00:17:57,520 --> 00:17:59,120
SO ONE OF THE FIRST THINGS WE

455
00:17:59,120 --> 00:18:01,680
LOOKED AT IN JUST THE UNDER

456
00:18:01,680 --> 00:18:08,000
LYING GC CO CONTENT WE LOOKED AT

457
00:18:08,000 --> 00:18:08,480
THESE 1300 CITES ACROSS

458
00:18:08,480 --> 00:18:10,240
INDIVIDUALS AND FIBER BLASTS,

459
00:18:10,240 --> 00:18:14,560
COMPARED TO LOCATIONS WITHOUT

460
00:18:14,560 --> 00:18:16,000
POL LL PAUSE AND THEY'RE FAVORED

461
00:18:16,000 --> 00:18:18,120
WITH REGIONS WITH HIGH GC

462
00:18:18,120 --> 00:18:19,280
CONTENT AND WITHIN THE REGIONS

463
00:18:19,280 --> 00:18:21,400
OF HIGH GC CONTENT THERE'S

464
00:18:21,400 --> 00:18:23,640
SKEWING WITH THE Gs AND Cs

465
00:18:23,640 --> 00:18:25,560
ARE LOCATED AND PAUSES TEND TO

466
00:18:25,560 --> 00:18:30,000
OFFER AFTER SYNTHESIZING THE G

467
00:18:30,000 --> 00:18:30,280
RICH RNA.

468
00:18:30,280 --> 00:18:32,600
THIS IS WHAT THIS GC SKEW REFERS

469
00:18:32,600 --> 00:18:33,800
TO AND THEN IF WE TAKE ADVANTAGE

470
00:18:33,800 --> 00:18:38,080
OF THAT HIGH RESOLUTION FROM THE

471
00:18:38,080 --> 00:18:40,240
ASSAY WE CAN ZOOM IN AND LOOK AT

472
00:18:40,240 --> 00:18:42,160
NUCLEOTIDE LEVEL WHERE THE PAUSE

473
00:18:42,160 --> 00:18:44,480
IS AND WE CAN SEE 66% OF THE

474
00:18:44,480 --> 00:18:46,760
SITES WHERE WE OBSERVED THE

475
00:18:46,760 --> 00:18:49,120
PAUSING IT INCURS AT A SITE AND

476
00:18:49,120 --> 00:18:51,080
FREQUENTLY THE NEXT BASIS

477
00:18:51,080 --> 00:18:53,640
APPEARING SO EITHER A G OR AN A

478
00:18:53,640 --> 00:18:54,920
AND THEY'RE IN THE CONTEXT OF

479
00:18:54,920 --> 00:18:57,760
WHAT WE REFER TO THINK ABOUT AS

480
00:18:57,760 --> 00:19:00,040
A PAUSE MOTIVE AND I WON'T TAKE

481
00:19:00,040 --> 00:19:02,240
YOU THROUGH THE ADDITIONAL

482
00:19:02,240 --> 00:19:03,400
STATISTICAL ANALYSIS BUT EACH OF

483
00:19:03,400 --> 00:19:06,760
THESE SEQUENCE ELEMENTS ARE

484
00:19:06,760 --> 00:19:10,600
STATISTICALLY ASSOCIATED WITH

485
00:19:10,600 --> 00:19:11,680
POL LL PAUSING SO WE HAVE A

486
00:19:11,680 --> 00:19:13,040
ASSOCIATION BETWEEN THE UNDER

487
00:19:13,040 --> 00:19:14,280
LINE SEQUENCES AND WHERE THE POL

488
00:19:14,280 --> 00:19:16,800
LL PAUSES WE WANTED TO TEST HOW

489
00:19:16,800 --> 00:19:18,200
MUCH OF A CONTRIBUTION IS THERE

490
00:19:18,200 --> 00:19:20,040
OF THESE SEQUENCE ELEMENTS.

491
00:19:20,040 --> 00:19:22,280
AND WE JUST DECIDED TO FOCUS ON

492
00:19:22,280 --> 00:19:25,600
THE CONTRIBUTION OF PAUSING AT A

493
00:19:25,600 --> 00:19:26,920
CYTISINICLINE AS CHANGING A

494
00:19:26,920 --> 00:19:28,320
SINGLE BASE WOULD BE EASIER TO

495
00:19:28,320 --> 00:19:30,240
DO RATHER THAN CHANGING THE GC

496
00:19:30,240 --> 00:19:32,160
CONTENT NEAR PROMOTERS.

497
00:19:32,160 --> 00:19:35,960
SO WHAT WE DID WAS TO AGAIN TAKE

498
00:19:35,960 --> 00:19:37,160
ADVANTAGE OF THIS ASSAY WE GET

499
00:19:37,160 --> 00:19:38,800
INFORMATION ABOUT WHERE THE POL

500
00:19:38,800 --> 00:19:41,560
LL IS PAUSING NUCLEOTIDE

501
00:19:41,560 --> 00:19:43,600
RESOLUTION SO WE LOOKED FOR

502
00:19:43,600 --> 00:19:46,520
PAUSE SITES WHERE THE INDIVIDUAL

503
00:19:46,520 --> 00:19:49,880
WAS HETERO SIGH GUS WHETHER THEY

504
00:19:49,880 --> 00:19:52,480
HAVE A G OR C AND PAUSE SITE AND

505
00:19:52,480 --> 00:19:53,760
ASK IF THERE'S A DIFFERENT IN

506
00:19:53,760 --> 00:19:55,760
THE EXTENT OF PAUSING WHETHER

507
00:19:55,760 --> 00:19:58,200
IT'S A C OR A G OR ANOTHER

508
00:19:58,200 --> 00:20:04,000
NUCLEOTIDE AT THAT PAUSE SITE.

509
00:20:04,000 --> 00:20:07,760
TO TAKE AN EXAMPLE OF THAT, THIS

510
00:20:07,760 --> 00:20:09,560
IS A GENE 6 AND IT'S THE

511
00:20:09,560 --> 00:20:12,040
PROMOTER AND WITHIN THE PROMOTER

512
00:20:12,040 --> 00:20:14,840
REGION THERE'S A SITE WHERE THIS

513
00:20:14,840 --> 00:20:17,800
INDIVIDUAL IS A C OR A G AND NOW

514
00:20:17,800 --> 00:20:21,080
I'M SHOWING THE SEQUENCING READS

515
00:20:21,080 --> 00:20:23,520
AND WHAT WE SEE AT THE G ALLELE

516
00:20:23,520 --> 00:20:26,360
IN GEORGE IS THE IT'S ABLE TO

517
00:20:26,360 --> 00:20:27,880
READ THROUGH THIS G AND THERE'S

518
00:20:27,880 --> 00:20:29,680
NO PAUSES THAT ARE HERE.

519
00:20:29,680 --> 00:20:33,640
BUT IF WE LOOK AT READS OF THE

520
00:20:33,640 --> 00:20:36,720
TRANSCRIPTS THAT COME FROM THE C

521
00:20:36,720 --> 00:20:38,160
BARRING ALLELE, WE CAN IDENTIFY

522
00:20:38,160 --> 00:20:42,680
THESE TRUNCATED READS WHICH IS

523
00:20:42,680 --> 00:20:45,520
WHERE THE POLYMERASES PAUSES

524
00:20:45,520 --> 00:20:47,560
WHEN THERE'S A C BUT NOT A G

525
00:20:47,560 --> 00:20:49,760
WHEN THE REST OF THE SURROUNDING

526
00:20:49,760 --> 00:20:51,200
SEQUENCES IS IS THE SAME.

527
00:20:51,200 --> 00:20:54,160
SO WE CAN DETECT A ALLELE LIKE

528
00:20:54,160 --> 00:20:56,280
EFFECT OF THIS C AT THE PAUSE

529
00:20:56,280 --> 00:20:56,760
SITE.

530
00:20:56,760 --> 00:20:58,280
WE DID THAT COMPARISON LOOKING

531
00:20:58,280 --> 00:21:02,080
AT C VERSUS G, C VERSUS T, C

532
00:21:02,080 --> 00:21:03,640
VERSUS A AND ALL THOSE CASES WE

533
00:21:03,640 --> 00:21:05,360
FOUND THERE WAS SIGNIFICANTLY

534
00:21:05,360 --> 00:21:10,160
MORE PAUSES THAT OCCURRED AT THE

535
00:21:10,160 --> 00:21:10,800
C BEARING ALLELE.

536
00:21:10,800 --> 00:21:13,600
SO WE ASKED IF WE CAN DETECT

537
00:21:13,600 --> 00:21:14,560
ANNA LEGAL DIFFERENCE IN THE

538
00:21:14,560 --> 00:21:16,160
EXTENT OF PAUSING BECAUSE DOES

539
00:21:16,160 --> 00:21:19,000
THIS LEAD TO A DIFFERENCE IN

540
00:21:19,000 --> 00:21:20,920
GENE EXPRESSION LEVELS.

541
00:21:20,920 --> 00:21:23,840
SO WE TOOK ADVANTAGE OF

542
00:21:23,840 --> 00:21:26,360
INFORMATION FROM THE GENOTYPE

543
00:21:26,360 --> 00:21:27,880
TISSUE EXPRESSION BASE WHICH HAS

544
00:21:27,880 --> 00:21:30,040
INFORMATION ABOUT UNDER LYING

545
00:21:30,040 --> 00:21:30,880
GENOTYPE AS WELL AS GENE

546
00:21:30,880 --> 00:21:32,560
EXPRESSION FROM HUNDREDS OF

547
00:21:32,560 --> 00:21:32,920
INDIVIDUALS.

548
00:21:32,920 --> 00:21:34,520
AND WE CAN FIND EXAMPLES WHERE

549
00:21:34,520 --> 00:21:39,000
THERE WERE POLY MORPHISMS AT

550
00:21:39,000 --> 00:21:40,960
PAUSE SITES WE IDENTIFIED THAT

551
00:21:40,960 --> 00:21:46,000
WERE PRESENT IN THE GTEX

552
00:21:46,000 --> 00:21:46,360
DATABASE.

553
00:21:46,360 --> 00:21:50,040
A GENE THAT HAD THISSEN CODES A

554
00:21:50,040 --> 00:21:51,760
WHAT WE CAN SEE IS INDIVIDUALS

555
00:21:51,760 --> 00:21:55,880
WHO ARE HOMO SIGH GUS FOR A C AT

556
00:21:55,880 --> 00:21:57,520
THE PAUSE SITE HAVE LOWER

557
00:21:57,520 --> 00:21:58,480
EXPRESSION AS COMPARED TO

558
00:21:58,480 --> 00:21:59,920
INDIVIDUALS WITH A T AT THE

559
00:21:59,920 --> 00:22:01,240
PAUSE SITE.

560
00:22:01,240 --> 00:22:06,160
O WE THINK THERE'S MORE PAUSING

561
00:22:06,160 --> 00:22:09,080
ON AND GREATER LEAD TO GO FEW

562
00:22:09,080 --> 00:22:17,400
MAKING IT INTO PRODUCTI PRODUCTE

563
00:22:17,400 --> 00:22:18,640
ELONGATION AND ONE TISSUE TYPE

564
00:22:18,640 --> 00:22:22,280
WHERE YOU CAN LOOK AT EXPRESSION

565
00:22:22,280 --> 00:22:25,160
OF Z21 ACROSS MULTIPLE TISSUE

566
00:22:25,160 --> 00:22:28,000
AND WHAT WE FOUND ACROSS THE

567
00:22:28,000 --> 00:22:29,440
TISSUE WHERE HEAD INFORMATION

568
00:22:29,440 --> 00:22:32,320
FROM GTX IS ALL CASES

569
00:22:32,320 --> 00:22:34,360
INDIVIDUALS WHO WERE HOMO SIGH

570
00:22:34,360 --> 00:22:36,360
GUS FOR A C HAVE LOWER

571
00:22:36,360 --> 00:22:38,520
EXPRESSION OF THIS.

572
00:22:38,520 --> 00:22:39,280
>>Gina: COMPARED TO
INDIVIDUALS

573
00:22:39,280 --> 00:22:39,960
WITH A T.

574
00:22:39,960 --> 00:22:42,480
SO IT SEEMS THAT THIS PAUSING

575
00:22:42,480 --> 00:22:44,920
CAN THEN LEAD TO DIFFERENCE THIS

576
00:22:44,920 --> 00:22:46,720
IS EXPRESSION THAT PAUSING ON

577
00:22:46,720 --> 00:22:52,080
THE C MORE MUCH RESULTS IN FEWER

578
00:22:52,080 --> 00:22:54,360
POLYMERASES IN DIFFERENT

579
00:22:54,360 --> 00:22:54,840
ELONGATION.

580
00:22:54,840 --> 00:22:58,080
WE WANT TO TEST THAT

581
00:22:58,080 --> 00:22:58,800
EXPERIMENTALLY.

582
00:22:58,800 --> 00:23:00,680
SO THE APPROACH WAS TO USE A

583
00:23:00,680 --> 00:23:01,240
REPORTER ASSAY.

584
00:23:01,240 --> 00:23:04,080
SO WE CLONED THE PROMOTERS OF

585
00:23:04,080 --> 00:23:05,880
GENES THAT HAVE PAUSE

586
00:23:05,880 --> 00:23:07,200
PRELIMINARY OR PAUSE

587
00:23:07,200 --> 00:23:10,640
CYTISINICLINE AND THEN USED CITE

588
00:23:10,640 --> 00:23:12,280
DIRECTED IMMUNE' GENESIS TO

589
00:23:12,280 --> 00:23:14,280
CHANGE THAT C TO A T AND ASK IS

590
00:23:14,280 --> 00:23:17,200
THERE A CHANGE IN REPORTER

591
00:23:17,200 --> 00:23:17,680
ACTIVITY.

592
00:23:17,680 --> 00:23:20,120
AND SO WE DID THAT WITH THREE

593
00:23:20,120 --> 00:23:23,440
GENES AND I SHOWED YOU DATA FOR

594
00:23:23,440 --> 00:23:28,320
BL CAP EARLIER THAT THERE'S THE

595
00:23:28,320 --> 00:23:32,920
ACCUMULATES AT A C AND MY O1E IS

596
00:23:32,920 --> 00:23:36,760
A MIO SIN WHICH ENCODES A

597
00:23:36,760 --> 00:23:38,840
PROTEIN THAT MUTATED CAN LEAD TO

598
00:23:38,840 --> 00:23:40,840
KIDNEY DISEASE AND ALL THREE OF

599
00:23:40,840 --> 00:23:43,000
THESE PROMOTERS MAKING THE

600
00:23:43,000 --> 00:23:45,120
SINGLE NUCLEOTIDE CHANGE FROM A

601
00:23:45,120 --> 00:23:46,800
CYTISINICLINE AT THE PAUSE SITE

602
00:23:46,800 --> 00:23:49,280
OR A C TO A T IS IS SUFFICIENT

603
00:23:49,280 --> 00:23:51,680
TO THE SIGNIFICANT INCREASE IN

604
00:23:51,680 --> 00:23:52,840
REPORTER ACTIVITY.

605
00:23:52,840 --> 00:23:54,920
SO WE CAN SEE THERE'S ALLELE I

606
00:23:54,920 --> 00:24:01,600
CAN PAUSING IN THE GTX DATA AND

607
00:24:01,600 --> 00:24:04,360
IN EXPRESSION AND HERE

608
00:24:04,360 --> 00:24:05,280
EXPERIMENTALLY WE CAN TEST AND

609
00:24:05,280 --> 00:24:07,480
SHOW THAT MAKING THAT SINGLE

610
00:24:07,480 --> 00:24:08,880
CHANGE IS SUFFICIENT TO ALTER

611
00:24:08,880 --> 00:24:11,000
REPORTER GENE ACTIVITY.

612
00:24:11,000 --> 00:24:13,440
SO WHAT WE WANT TODAY DO IS TO

613
00:24:13,440 --> 00:24:14,360
KNOW WHETHER THESE CHANGES IN

614
00:24:14,360 --> 00:24:17,360
THE EXTENT OF PAUSING MUCH FOR

615
00:24:17,360 --> 00:24:18,800
THE CHANGES AND EXPRESSION THAT

616
00:24:18,800 --> 00:24:20,080
RESULT FROM DIFFERENCES IN

617
00:24:20,080 --> 00:24:22,640
PAUSING, ARE THOSE THAT LARGE

618
00:24:22,640 --> 00:24:24,480
ENOUGH TO CONTRIBUTE TO

619
00:24:24,480 --> 00:24:25,680
DIFFERENCES THAT WE MIGHT SEE IN

620
00:24:25,680 --> 00:24:26,960
THE CLINIC.

621
00:24:26,960 --> 00:24:29,920
SO WE GOT A CLUE THAT THIS

622
00:24:29,920 --> 00:24:34,040
PAUSING OR ALLELEIC PAUSING CAN

623
00:24:34,040 --> 00:24:38,280
EFFECT DISEASE LOOKING AT

624
00:24:38,280 --> 00:24:39,520
REDUCKING SO I INTRODUCED THIS

625
00:24:39,520 --> 00:24:43,240
IN ITS ROLE OF CONVERTING

626
00:24:43,240 --> 00:24:48,240
GLUCOSE TO SORBITOL WHEN

627
00:24:48,240 --> 00:24:48,920
DEFENDING AGAINST STRESS.

628
00:24:48,920 --> 00:24:50,720
FOR PATIENTS THAT HAVE DIABETES

629
00:24:50,720 --> 00:24:52,600
AND THERE'S A HIGHER ABUNDANCE

630
00:24:52,600 --> 00:24:55,080
OF GLUCOSE, THE INCREASE IN

631
00:24:55,080 --> 00:24:57,320
GLUCOSE LEADS TO MORE OR GREATER

632
00:24:57,320 --> 00:24:59,520
GENERATION OF SORBITOL.

633
00:24:59,520 --> 00:25:01,480
IT'S THOUGHT THE INAPPROPRIATE

634
00:25:01,480 --> 00:25:04,040
ACCUMULATION OF SORBITOL INSIDE

635
00:25:04,040 --> 00:25:07,000
CELLS CAN CONTRIBUTE TO THE

636
00:25:07,000 --> 00:25:08,760
DEVELOPMENT OF DIABETIC

637
00:25:08,760 --> 00:25:11,640
COMPLICATIONS SO CATARACT,

638
00:25:11,640 --> 00:25:12,440
DIABETES EYE AND NERVE DISEASE

639
00:25:12,440 --> 00:25:16,000
AND KIDNEY DISEASE THROUGH PART

640
00:25:16,000 --> 00:25:17,800
ARE RESULT FROM INAPPROPRIATE

641
00:25:17,800 --> 00:25:20,520
GENERATIONS OF SORBITOL SO

642
00:25:20,520 --> 00:25:22,040
THERE'S SEVERAL PAPERS THAT HAVE

643
00:25:22,040 --> 00:25:25,680
IDENTIFIED A POLY MORPHISM IN

644
00:25:25,680 --> 00:25:28,480
ALL THOSE WHICH COP FIRST

645
00:25:28,480 --> 00:25:30,320
INCREASE RISK OF DIABETIC

646
00:25:30,320 --> 00:25:31,760
COMPLICATIONS SO THIS IS DATA

647
00:25:31,760 --> 00:25:33,520
COMING FROM A PAPER IN 2019

648
00:25:33,520 --> 00:25:38,240
LOOKING AT DIABETIC RECEIPT

649
00:25:38,240 --> 00:25:40,240
INOPATHY AND THEY OBSERVED

650
00:25:40,240 --> 00:25:42,960
COMPARING INDIVIDUALS WHO ARE

651
00:25:42,960 --> 00:25:46,000
HOMO SIGH GUS FOR A C AT THIS

652
00:25:46,000 --> 00:25:48,040
NIP AND INDIVIDUALS THAT ARE

653
00:25:48,040 --> 00:25:58,600
HOMO SIGH GUS FOR A T HAD A THEY

654
00:26:02,720 --> 00:26:05,880
DIDN'T HAVE AN GUY OF WHAT WAS

655
00:26:05,880 --> 00:26:16,360
CONTRIBUTE TO GO THAT RISK.

656
00:26:21,280 --> 00:26:23,280
SO WE CONSIDERED THAT THE

657
00:26:23,280 --> 00:26:27,160
DIFFERENCE IN THIS RISK COULD BE

658
00:26:27,160 --> 00:26:32,120
RELATE TODAY DIFFERENT EVERY DIL

659
00:26:32,120 --> 00:26:34,280
PAUSING AT THIS AND WE USED THE

660
00:26:34,280 --> 00:26:35,680
APPROACH THAT I SHOWED A SLIDE

661
00:26:35,680 --> 00:26:38,480
OR TWO ASK WE CLONED THE

662
00:26:38,480 --> 00:26:40,360
PROMOTER EITHER WITH THE C OR

663
00:26:40,360 --> 00:26:44,600
WITH THE T AND THEN TESTED THE

664
00:26:44,600 --> 00:26:47,200
ACTIVITIES IN A ASSAY.

665
00:26:47,200 --> 00:26:48,640
SO WE DID THIS IN THREE

666
00:26:48,640 --> 00:26:50,120
DIFFERENT CELL LINES AND WE GOT

667
00:26:50,120 --> 00:26:53,040
THE SAME THREE RESULTS IN ALL

668
00:26:53,040 --> 00:26:55,680
THREE CELL LINES THAT SINGLE

669
00:26:55,680 --> 00:26:56,400
NUCLEOTIDE DIFFERENCE BETWEEN C

670
00:26:56,400 --> 00:26:58,840
AND T RESULTED IN A SIGNIFICANT

671
00:26:58,840 --> 00:27:00,800
DIFFERENCE IN REPORTER GENE

672
00:27:00,800 --> 00:27:01,240
ACTIVITIES.

673
00:27:01,240 --> 00:27:09,720
SO THE SAME PATTERN AND THESE

674
00:27:09,720 --> 00:27:14,560
CELLS ARE HETERO SIGH OWE GUS OR

675
00:27:14,560 --> 00:27:18,080
POLY MORE PICK FOR THIS AND SO

676
00:27:18,080 --> 00:27:19,640
IN THE CHROMATIN ENVIRONMENT AND

677
00:27:19,640 --> 00:27:22,480
THE NATIVE CONTEXT WE CAN ASK IF

678
00:27:22,480 --> 00:27:24,720
THERE'S A DIFFERENCE IN ALLELEIC

679
00:27:24,720 --> 00:27:25,800
EXPRESSION WITH THE GENE BEING

680
00:27:25,800 --> 00:27:31,600
IN THE SAME CELLS AND THE EXACT

681
00:27:31,600 --> 00:27:34,680
SAME CAN B.

682
00:27:34,680 --> 00:27:37,040
SO WE MEASURE THE ALLELE I CAN

683
00:27:37,040 --> 00:27:47,640
EXPRESSION SO WHAT WE SAW -- SOE

684
00:27:48,760 --> 00:27:50,640
SAME PATTERN THAT WE'VE SEEN

685
00:27:50,640 --> 00:27:54,320
RIGHT AT PAUSING MORE MUCH ON

686
00:27:54,320 --> 00:27:54,960
AND WE HAVE LOWER EXPRESSION AND

687
00:27:54,960 --> 00:27:57,360
LESS ON THE T WE GET GREATER

688
00:27:57,360 --> 00:27:58,120
EXPRESSION.

689
00:27:58,120 --> 00:27:59,720
ALL THOSE INDUCING IN RESPONSE

690
00:27:59,720 --> 00:28:04,240
TO HYPER TONIC STRESS SO WE

691
00:28:04,240 --> 00:28:06,360
CULTURE THESE CELLS IN TYPIER

692
00:28:06,360 --> 00:28:07,880
TONIC CONDITIONS AND INDUCED IN

693
00:28:07,880 --> 00:28:10,040
BOTH CASES BUT WE SEE A MUCH

694
00:28:10,040 --> 00:28:13,520
GREATER INCREASE IN ALL THOSE

695
00:28:13,520 --> 00:28:15,320
EXPRESSION OF THE T BEARING

696
00:28:15,320 --> 00:28:17,000
ALLELE AS COMPARED TO THE C

697
00:28:17,000 --> 00:28:17,680
BEARING ALLELE.

698
00:28:17,680 --> 00:28:20,120
SO WE THINK THIS IS WHAT COULD

699
00:28:20,120 --> 00:28:22,200
EXPLAIN THIS INCREASED RISK OF

700
00:28:22,200 --> 00:28:26,320
DEVELOPING DIABETIC

701
00:28:26,320 --> 00:28:29,960
COMPLICATIONS IS LESS POLY EMERY

702
00:28:29,960 --> 00:28:32,400
THAT CAN LEAD TO GREATER

703
00:28:32,400 --> 00:28:34,920
EXPRESSION OF ALL THOSE REDUCK

704
00:28:34,920 --> 00:28:36,320
TASE AND INCREASE IN THE

705
00:28:36,320 --> 00:28:39,040
GENERATION OF SORBITOL SO THIS

706
00:28:39,040 --> 00:28:40,120
IS A HINT OF DIFFERENCES IN THE

707
00:28:40,120 --> 00:28:41,560
EXTENT OF PAUSING COULD EXPLAIN

708
00:28:41,560 --> 00:28:43,560
SOME DIFFERENCES IN GENE

709
00:28:43,560 --> 00:28:45,680
EXPRESSION BETWEEN INDIVIDUALS.

710
00:28:45,680 --> 00:28:48,160
SO IT SUMMARIZES THIS BEGINNING

711
00:28:48,160 --> 00:28:49,960
PART OF THE TALK AND WHAT I'M

712
00:28:49,960 --> 00:28:52,520
INTRODUCED IS THAT THE IT PAUSES

713
00:28:52,520 --> 00:28:55,920
NEAR PROMOTERS AND THERE'S A

714
00:28:55,920 --> 00:28:58,280
COMPONENT OR CONTRIBUTION OF

715
00:28:58,280 --> 00:29:00,560
PAUSING REGULATION MEDIATED BY

716
00:29:00,560 --> 00:29:03,720
THESE PAUSING COMMENT PLEXES

717
00:29:03,720 --> 00:29:06,920
NELT AND DFIS AND THERE'S A ROLE

718
00:29:06,920 --> 00:29:08,560
OR A CONTRIBUTION OF THE

719
00:29:08,560 --> 00:29:10,920
UNDERLYING SEQUENCE IN COP

720
00:29:10,920 --> 00:29:13,920
CONTRIBUTING TO THESE PAUSES.

721
00:29:13,920 --> 00:29:16,760
WE THINK IT'S A SEQUENCE

722
00:29:16,760 --> 00:29:18,760
MEDIATED OR SIS REGULATORY

723
00:29:18,760 --> 00:29:20,720
ELEMENTS WITH THESE PROTEIN OR

724
00:29:20,720 --> 00:29:22,000
TRANSACTING FACTORS AND WE THINK

725
00:29:22,000 --> 00:29:24,200
THIS IS WHAT IT COUNTS FOR THE

726
00:29:24,200 --> 00:29:25,960
PRECISE PAUSING THAT WE SEE AT

727
00:29:25,960 --> 00:29:29,320
MANY OF THESE GENES WHERE POL LL

728
00:29:29,320 --> 00:29:32,080
IS ACCUMULATING AT THE SAME

729
00:29:32,080 --> 00:29:33,960
NUCLEOTIDE POSITION ACROSS CELL

730
00:29:33,960 --> 00:29:37,720
TYPES AND ACROSS DIFFERENT

731
00:29:37,720 --> 00:29:38,200
INDIVIDUALS.

732
00:29:38,200 --> 00:29:42,600
SO I THINK OF THIS KIND OF

733
00:29:42,600 --> 00:29:44,640
SEQUENCE DEPENDENT PAUSING AND

734
00:29:44,640 --> 00:29:46,040
THAT'S THINK A GENETIC COMPONENT

735
00:29:46,040 --> 00:29:53,040
OF PAUSING AND THIS LEADS TO A

736
00:29:53,040 --> 00:29:55,640
HIGH DEGREE OF SIMILARLY WE SEE

737
00:29:55,640 --> 00:29:57,080
ACROSS CELL TYPES OF

738
00:29:57,080 --> 00:29:57,720
INDIVIDUALS.

739
00:29:57,720 --> 00:30:00,200
SO NOW, WHAT I WANT TO CHANGE TO

740
00:30:00,200 --> 00:30:02,520
IS A DIFFERENT MODE OF SIS

741
00:30:02,520 --> 00:30:03,840
REGULATION AND THIS IS ONE THAT

742
00:30:03,840 --> 00:30:09,560
IS NOT DEPENDENT ON THE PRIMARY

743
00:30:09,560 --> 00:30:11,440
SEQUENCE OF THE DNA AND THE RNA

744
00:30:11,440 --> 00:30:16,560
BUT MORE ABOUT THE SHAPE OF THE

745
00:30:16,560 --> 00:30:19,560
NUCLEIC ACIDS AND THIS IS AN

746
00:30:19,560 --> 00:30:21,360
EPIGENETIC ROLE OF PAUSING.

747
00:30:21,360 --> 00:30:23,240
AND ONE THAT WE THINK CAN BE

748
00:30:23,240 --> 00:30:25,120
MORE DYNAMIC.

749
00:30:25,120 --> 00:30:29,080
SO, BEFORE KIND OF -- I'LL COME

750
00:30:29,080 --> 00:30:31,720
BACK TO HOW WE SEE THIS

751
00:30:31,720 --> 00:30:33,000
ASSOCIATION BETWEEN THE

752
00:30:33,000 --> 00:30:35,560
STRUCTURE OF NUCLEIC ACIDS AND

753
00:30:35,560 --> 00:30:38,160
PAUSING AND I'LL TAKE A DETOUR

754
00:30:38,160 --> 00:30:43,560
AND SHARE SOME RESULTS ABOUT OUR

755
00:30:43,560 --> 00:30:48,480
LOOPS AND COME BACK TO HOW IT'S

756
00:30:48,480 --> 00:30:49,800
REL VENT IN PLUM POL LL PAUSE.

757
00:30:49,800 --> 00:30:51,840
WORK DONE IF VIVIAN CHUNG'S

758
00:30:51,840 --> 00:30:53,400
LAUGH WHEN I WAS THERE AS A

759
00:30:53,400 --> 00:30:58,320
POSTDOC FOCUSED ON THE ROLE OR

760
00:30:58,320 --> 00:31:00,440
BIOLOGY TO R-LOOP FORM WHEN WE

761
00:31:00,440 --> 00:31:02,400
HAVE THE PRELIMINARY

762
00:31:02,400 --> 00:31:07,960
TRANSCRIBING AND IT CAN DNA AND

763
00:31:07,960 --> 00:31:09,640
CREATE AN RNA-DNA HYBRID.

764
00:31:09,640 --> 00:31:12,040
THIS LEAVES THE NON TEMPLATES

765
00:31:12,040 --> 00:31:13,920
BRAND SINGLE STRAND BETWEEN THE

766
00:31:13,920 --> 00:31:16,560
THREE STRANDED STRUCTURES.

767
00:31:16,560 --> 00:31:18,000
SO APP OBSERVATION PUBLISHED IN

768
00:31:18,000 --> 00:31:22,200
A PAPER IN 2020 IS THAT BASIC

769
00:31:22,200 --> 00:31:24,000
REPAIR ENZYME SOMETHING CALLED

770
00:31:24,000 --> 00:31:30,120
THE DNA GLY CLOSE LAIDS IS ABLE

771
00:31:30,120 --> 00:31:34,320
TO REMOVE THE BASE FROM THE RNA

772
00:31:34,320 --> 00:31:36,360
IN AN RNA-DNA HYBRID SO THE

773
00:31:36,360 --> 00:31:37,840
CONTEXT OF AN R-LOOP.

774
00:31:37,840 --> 00:31:41,560
SO TO SHOW THIS LITTLE

775
00:31:41,560 --> 00:31:43,520
DIFFERENTLY, SO THIS IS A WITH

776
00:31:43,520 --> 00:31:47,560
THE SUGAR AND THE BASE AND THE

777
00:31:47,560 --> 00:31:51,120
MPG IS ABLE TO CLEVE THIS BOND

778
00:31:51,120 --> 00:31:57,600
AND WE'RE LEFT WITH THIS SUGAR

779
00:31:57,600 --> 00:31:57,920
IN THE RNA.

780
00:31:57,920 --> 00:32:01,160
AND WE CAN SHOW THAT THESE BASIC

781
00:32:01,160 --> 00:32:03,440
RNAs WHILE RARE, ARE VERY

782
00:32:03,440 --> 00:32:05,040
PREVALENT IN CELLS AND AT THE

783
00:32:05,040 --> 00:32:06,480
TIME LED TO A QUESTION, WHAT'S

784
00:32:06,480 --> 00:32:10,720
THE BIOLOGICAL FUNCTION OF THESE

785
00:32:10,720 --> 00:32:11,840
BASIC RNAs.

786
00:32:11,840 --> 00:32:14,320
SO WITH SOME ADDITIONAL

787
00:32:14,320 --> 00:32:15,880
BIOCHEMICAL WORK, WHAT WE

788
00:32:15,880 --> 00:32:18,160
REALIZED IS THAT WE CAN HAVE

789
00:32:18,160 --> 00:32:20,160
THESE R-LOOPS THAT FORM.

790
00:32:20,160 --> 00:32:23,680
IT CAN BE RECOGNIZED BY A WRITER

791
00:32:23,680 --> 00:32:27,360
COMPLEX THE MET CAL 314 COMPLEX

792
00:32:27,360 --> 00:32:31,200
THAT CAN MODIFY METHYLATED TO

793
00:32:31,200 --> 00:32:33,960
CREATE AN M6A MODIFICATION AND

794
00:32:33,960 --> 00:32:37,240
THESE MODIFIED ARE RECOGNIZED BY

795
00:32:37,240 --> 00:32:41,120
MPG AND REMOVED TO LEAVE THE

796
00:32:41,120 --> 00:32:41,480
SAME BASIC RNA.

797
00:32:41,480 --> 00:32:42,680
SO IT SHOWS MORE IN THE

798
00:32:42,680 --> 00:32:45,720
STRUCTURES AND NOT JUST THESE

799
00:32:45,720 --> 00:32:48,760
CARTOONS, SO HERE IS THE BASE

800
00:32:48,760 --> 00:32:51,280
AND THE 314 COMPLEX ADDS THIS

801
00:32:51,280 --> 00:32:53,160
GROUP AND THIS MODIFIED

802
00:32:53,160 --> 00:32:54,640
NUCLEOTIDE IS RECOGNIZED BY MPG

803
00:32:54,640 --> 00:32:58,840
AND IT IT IS ABLE TO REMOVE THE

804
00:32:58,840 --> 00:32:59,960
SPACE.

805
00:32:59,960 --> 00:33:02,320
SO THEN WE UNDER OR TO HAVE AN

806
00:33:02,320 --> 00:33:05,160
IDEA OF WHAT THESE BASIC RNAs

807
00:33:05,160 --> 00:33:06,720
ARE DOING IN CELLS WE ASKED

808
00:33:06,720 --> 00:33:09,920
WHERE ARE THEY LOCATED IN CELLS?

809
00:33:09,920 --> 00:33:13,800
SO WE DID PULL DOWNS TO ASK

810
00:33:13,800 --> 00:33:20,320
WHERE MPG IS FOUND IN THE GENOME

811
00:33:20,320 --> 00:33:22,320
AND WE LOOKED WHERE THESE MIGHT

812
00:33:22,320 --> 00:33:23,600
OCCUR IN R-LOOPS.

813
00:33:23,600 --> 00:33:26,720
SO WE DID THIS ANNAL LOOKING AT

814
00:33:26,720 --> 00:33:29,760
THE CO OCCURRENCE OF THESE

815
00:33:29,760 --> 00:33:32,200
R-LOOPS AND MPG AND WE

816
00:33:32,200 --> 00:33:34,560
IDENTIFIED NEARLY 2,000 NON

817
00:33:34,560 --> 00:33:39,960
CODING RNA GENES WHICH HAVE

818
00:33:39,960 --> 00:33:43,360
THESE MODIFIED R-LOOPS.

819
00:33:43,360 --> 00:33:46,800
SO, THEN WE LOOKED AT THE

820
00:33:46,800 --> 00:33:52,400
PATTERN OF TRANSCRIPTION AND WE

821
00:33:52,400 --> 00:33:55,120
SAW THE PLOT SO WE HAVE AN

822
00:33:55,120 --> 00:33:57,040
IMMUNO PARTICIPATION THAT PULLS

823
00:33:57,040 --> 00:33:59,960
DOWN R-LOOPS AND IT'S IN THE

824
00:33:59,960 --> 00:34:00,720
GREEN TRACE AND WE USE PROCEED

825
00:34:00,720 --> 00:34:04,040
DATA TO PROFILE WHERE THE IT'S

826
00:34:04,040 --> 00:34:05,960
LOCATED AND WE SEE THIS

827
00:34:05,960 --> 00:34:07,360
ACCUMULATION OF THE PATTERN VERY

828
00:34:07,360 --> 00:34:10,320
SIMILAR TO WHAT SEE APPROXIMATE

829
00:34:10,320 --> 00:34:11,960
NEAR THE PROMOTERS WHERE IT'S

830
00:34:11,960 --> 00:34:13,960
PAUSING THAT THIS ACCUMULATION

831
00:34:13,960 --> 00:34:16,240
OCCURS UPSTREAM OF THESE

832
00:34:16,240 --> 00:34:16,840
MODIFIED R-LOOPS.

833
00:34:16,840 --> 00:34:19,680
SO IT SEEMS IT'S TRAPS

834
00:34:19,680 --> 00:34:22,280
DESCRIBING ALONG AND IT RUNS

835
00:34:22,280 --> 00:34:24,000
INTO THESE MODIFIED R-LOOPS AND

836
00:34:24,000 --> 00:34:25,080
IT PAUSES.

837
00:34:25,080 --> 00:34:28,560
SO NOW WE HAVE THIS IDEA THAT

838
00:34:28,560 --> 00:34:30,360
THE MODIFICATION OR THIS

839
00:34:30,360 --> 00:34:34,720
CREATION OF A BASIC R-LOOPS

840
00:34:34,720 --> 00:34:38,040
SITES CAN CONFER SOME REGULATION

841
00:34:38,040 --> 00:34:40,680
OF TRANSCRIPTION OR CAUSE THE

842
00:34:40,680 --> 00:34:42,400
POL LL TO PAUSE SO WE WANTED TO

843
00:34:42,400 --> 00:34:45,840
ASK NOW, WHAT IS THE ROLE OF THE

844
00:34:45,840 --> 00:34:49,200
CONTRIBUTION OF THESE MODIFIED R

845
00:34:49,200 --> 00:34:50,960
LOOPS IN REGULATING PAUSING AND

846
00:34:50,960 --> 00:34:57,600
HOW THIS MIGHT EFFECT GENE

847
00:34:57,600 --> 00:34:58,520
EXPRESSION.

848
00:34:58,520 --> 00:34:59,640
THE DATA I'M GOING TO SHOW IS

849
00:34:59,640 --> 00:35:02,760
FOR A NON CODING RNA THAT WE

850
00:35:02,760 --> 00:35:05,520
DISCOVERED THAT'S UPSTREAM OF

851
00:35:05,520 --> 00:35:10,480
APOE AND WE TORNADO IT ANSWER OR

852
00:35:10,480 --> 00:35:13,120
FOR NO CODING RNA AND THIS NO

853
00:35:13,120 --> 00:35:16,000
CODING RNA HAS ONE OF THESE

854
00:35:16,000 --> 00:35:17,320
R-LOOPS MODIFIED TO HAVE BASIC

855
00:35:17,320 --> 00:35:18,760
SCIENCE AND I'LL SHOW YOU HOW WE

856
00:35:18,760 --> 00:35:21,040
HAVE THIS PATTERN WHERE THE POL

857
00:35:21,040 --> 00:35:25,000
LL PAUSES AND UPSTREAM OF THESE

858
00:35:25,000 --> 00:35:27,280
MODIFIED R-LOOPS IN THIS NON

859
00:35:27,280 --> 00:35:27,720
CODING RNA.

860
00:35:27,720 --> 00:35:29,480
AND THE REASON WE FOCUSED ON

861
00:35:29,480 --> 00:35:31,760
THIS NON CODING RNA IS BECAUSE

862
00:35:31,760 --> 00:35:35,920
IT'S NEAR SUCH AN IMPORTANT

863
00:35:35,920 --> 00:35:39,720
DISEASE ASSOCIATED GENE AND IT

864
00:35:39,720 --> 00:35:41,600
IT ENCODES PROTEIN E AND WHICH

865
00:35:41,600 --> 00:35:44,880
IS AN IMPORTANT RISK FACTOR FOR

866
00:35:44,880 --> 00:35:47,800
ALZHEIMER'S DISEASE.

867
00:35:47,800 --> 00:35:49,480
SO, I'M SURE THIS AUDIENCE WELL

868
00:35:49,480 --> 00:35:51,720
KNOWS THAT IT'S A CARRIER

869
00:35:51,720 --> 00:35:56,080
PROTEINS FOR LIPIDS AND

870
00:35:56,080 --> 00:35:58,680
CHOLESTEROL ASK THIS PROTEIN IS

871
00:35:58,680 --> 00:36:01,280
POLY MORPHIC AND CHANGES THAT

872
00:36:01,280 --> 00:36:04,480
CARRY THE ALLELE AOPE4 HAVE A

873
00:36:04,480 --> 00:36:07,320
RISK FACTOR FOR LATE ON SET

874
00:36:07,320 --> 00:36:07,640
ALZHEIMER'S.

875
00:36:07,640 --> 00:36:09,680
IN ORDER TO OVER SIMPLIFY A

876
00:36:09,680 --> 00:36:12,680
LARGE BODY OF LITERATURE, APOE

877
00:36:12,680 --> 00:36:15,520
MADE IN ASTROCYTES AND IF YOU

878
00:36:15,520 --> 00:36:18,760
HAVE THE NORMAL ALLELE THE APOE3

879
00:36:18,760 --> 00:36:21,000
ALLELE THERE'S NORMAL LIPID

880
00:36:21,000 --> 00:36:23,240
TRANSPORT TO THE NEURONS AND

881
00:36:23,240 --> 00:36:24,200
THEY REMAIN HEALTHY.

882
00:36:24,200 --> 00:36:26,840
IF YOU HAVE THE APOE34, IT CAN

883
00:36:26,840 --> 00:36:28,560
LEAD TO LIPID TRANSPORT AND

884
00:36:28,560 --> 00:36:30,440
CONTRIBUTE TO THE NEURO

885
00:36:30,440 --> 00:36:32,360
DEGENERATION WE SEE IN THE MULTI

886
00:36:32,360 --> 00:36:33,520
RISK DISEASE.

887
00:36:33,520 --> 00:36:35,440
WE THOUGHT IF THERE'S A NOVEL

888
00:36:35,440 --> 00:36:39,520
NON CODING RNA UPSTREAM THAT

889
00:36:39,520 --> 00:36:41,520
COULD CONTRIBUTE TO APOE

890
00:36:41,520 --> 00:36:42,920
REGULATION THIS WOULD BE AN

891
00:36:42,920 --> 00:36:47,160
IMPORTANT NON CODING RNA TO SIDE

892
00:36:47,160 --> 00:36:48,640
AND PART TWO OF WHAT WE THINK IS

893
00:36:48,640 --> 00:36:53,000
GOING ON WITH THIS NON CODING

894
00:36:53,000 --> 00:36:56,600
RNA AND LET ME SHOW YOU THE

895
00:36:56,600 --> 00:37:06,680
DATA.

896
00:37:15,920 --> 00:37:18,720
>>THIS NON CODING RNA IS
TRANCE

897
00:37:18,720 --> 00:37:22,320
DESCRIBED IN FIBER BLASTS AND

898
00:37:22,320 --> 00:37:24,320
THIS IS DATA WHERE WE'RE SHOWING

899
00:37:24,320 --> 00:37:26,200
ENRICHMENT TO THE PRESENCE OF

900
00:37:26,200 --> 00:37:27,240
THIS NON CODING RNA.

901
00:37:27,240 --> 00:37:28,720
WHAT I WOULD POINT OUT, IF

902
00:37:28,720 --> 00:37:32,840
APPROXIMATE TTHERE'SA CHANGE INF

903
00:37:32,840 --> 00:37:34,080
TRANSCRIPTION UPSTREAM WHERE

904
00:37:34,080 --> 00:37:36,040
THIS R LINK IS FOUND SO THIS RNA

905
00:37:36,040 --> 00:37:41,280
HAS THE PATTERN THAT WE OBSERVED

906
00:37:41,280 --> 00:37:45,240
ON AVERAGE WHERE THERE'S THE

907
00:37:45,240 --> 00:37:48,280
PAUSE UPSTREAM OF WHERE THESE

908
00:37:48,280 --> 00:37:48,720
ARE END INDICATED.

909
00:37:48,720 --> 00:37:53,280
WE DID ADDITIONAL WORK TO LOOK

910
00:37:53,280 --> 00:37:57,200
USING CITE SPECIFIC ASSAYS WHERE

911
00:37:57,200 --> 00:38:00,600
YOU CAN USE PCR AND YOU CAN DO

912
00:38:00,600 --> 00:38:04,800
RNA IMMUNO PRECIPITATION AND

913
00:38:04,800 --> 00:38:06,920
SHOW MPG BINDS TO THIS RNA AND

914
00:38:06,920 --> 00:38:10,920
DETECT THERE ARE A BASIC RNA IN

915
00:38:10,920 --> 00:38:13,160
THIS NON CODING OF TRAPS

916
00:38:13,160 --> 00:38:16,120
DESCRIPTION UPSTREAM OF APOE.

917
00:38:16,120 --> 00:38:18,920
OUR MODEL IS YOU CAN FORM A

918
00:38:18,920 --> 00:38:25,120
R-LOOP ASK IT'S RECOGNIZED BY

919
00:38:25,120 --> 00:38:27,520
M6A LEAVING THIS A BASIC SITE SO

920
00:38:27,520 --> 00:38:31,400
WE ASKED IF WE PERTURBED THE

921
00:38:31,400 --> 00:38:33,200
GENERATION OF THESE MODIFIED

922
00:38:33,200 --> 00:38:34,600
R-LOOPS WOULD IT LEAD TO A

923
00:38:34,600 --> 00:38:36,840
CHANGE IN ANSWER TRANSCRIPTION

924
00:38:36,840 --> 00:38:40,000
OR A CHANGE IN APOE EXPRESSION?

925
00:38:40,000 --> 00:38:44,680
SO WE LOOKED IN OR COP PAIRED

926
00:38:44,680 --> 00:38:45,880
APOE EXPRESSION IN PATIENTS WHO

927
00:38:45,880 --> 00:38:47,360
HAVE A MUTATION WITH A DIFFERENT

928
00:38:47,360 --> 00:38:49,280
GENE THAT RESULTS IN FEWER

929
00:38:49,280 --> 00:38:49,880
R-LOOPS.

930
00:38:49,880 --> 00:38:54,640
AND SO IF WE COMPARE THE APOE

931
00:38:54,640 --> 00:38:55,560
EXPRESSION IN CONTROLS TO

932
00:38:55,560 --> 00:38:57,480
INDIVIDUALS WHO HAVE FEWER

933
00:38:57,480 --> 00:38:59,000
R-LOOPS, WE SEE THAT THE

934
00:38:59,000 --> 00:39:01,760
PATIENTS WITH FEWER R-LOOPS SHOW

935
00:39:01,760 --> 00:39:06,080
HIGHER EXPRESSION OF APOE.

936
00:39:06,080 --> 00:39:08,680
NEXT WE KNOCKED DOWN METAL 3 TO

937
00:39:08,680 --> 00:39:12,200
PREVENT FORMATION OF N6A AND WE

938
00:39:12,200 --> 00:39:16,000
SAW THAT LOSS OF METTL3 LEADS TO

939
00:39:16,000 --> 00:39:18,960
AN INCREASE IN APOE EXPRESSION.

940
00:39:18,960 --> 00:39:22,560
AND THEN WE KNOCKED CELLS OF MPG

941
00:39:22,560 --> 00:39:25,520
SO THEY WON'T AND DON'T FORM AS

942
00:39:25,520 --> 00:39:27,000
A BASIC SITE AND AGAIN WE SEE

943
00:39:27,000 --> 00:39:29,320
THE SAME PATTERN THAT WE

944
00:39:29,320 --> 00:39:32,720
KNOCKDOWN MPG WE CAN DETECT AND

945
00:39:32,720 --> 00:39:36,200
INCREASE IN THE EXPRESSION OF

946
00:39:36,200 --> 00:39:37,520
APOE AND I'M SHOWING THE

947
00:39:37,520 --> 00:39:39,040
SEQUENCING DATA AND YOU CAN SEE

948
00:39:39,040 --> 00:39:41,520
BY THREE OR SEVEN DAYS OF MPG

949
00:39:41,520 --> 00:39:42,720
KNOCK DOWN THERE'S AN INCREASE

950
00:39:42,720 --> 00:39:45,200
IN APOE EXPRESSION.

951
00:39:45,200 --> 00:39:47,240
SO, WHETHER WE DECREASE THE

952
00:39:47,240 --> 00:39:50,360
ABUNDANCE OF THESE MODIFIED BY

953
00:39:50,360 --> 00:39:54,680
DECREASING THE ABUNDANCE OF

954
00:39:54,680 --> 00:39:57,280
R-LOOPS AND M6A MODIFICATION OR

955
00:39:57,280 --> 00:39:58,840
PREVENTING THE FORMATION OF

956
00:39:58,840 --> 00:40:01,960
SITES AND R-LOOPS ALL THREE OF

957
00:40:01,960 --> 00:40:04,680
THESE LEAD TO AN INCREASE OF THE

958
00:40:04,680 --> 00:40:06,080
EXPRESSION OF APOE.

959
00:40:06,080 --> 00:40:08,760
SO WE HAVE AN IDEA THAT THE RACE

960
00:40:08,760 --> 00:40:10,440
IS MARCHING ALONG IN THIS NON

961
00:40:10,440 --> 00:40:14,240
CODING RNA AND IT RUNS INTO THIS

962
00:40:14,240 --> 00:40:15,400
MODIFIED R LOOP AND PAUSES AND

963
00:40:15,400 --> 00:40:17,600
IT SEEMS LIKE WE REMOVE THE

964
00:40:17,600 --> 00:40:24,240
BARRIERS, THIS R LOOP BY

965
00:40:24,240 --> 00:40:26,480
PERTURBING R-LOOPS AND ALLOWS

966
00:40:26,480 --> 00:40:27,880
FOR FULL TRANSCRIPTION OF AND

967
00:40:27,880 --> 00:40:30,040
THAT CONTRIBUTE TO ACTIVATION OF

968
00:40:30,040 --> 00:40:30,640
APOE.

969
00:40:30,640 --> 00:40:33,760
AND SO TO TEST WHETHER SIP THAT

970
00:40:33,760 --> 00:40:36,400
SIS OF A FULL LENGTH ANSWER OF A

971
00:40:36,400 --> 00:40:39,040
FULL LENGTH NON CODING RNA

972
00:40:39,040 --> 00:40:41,240
WHETHER IT CORRELATES CAN DRIVE

973
00:40:41,240 --> 00:40:43,160
APOE EXPRESSION, WE LOOKED AT

974
00:40:43,160 --> 00:40:44,520
TRANSCRIPTION AND CELLS WHICH

975
00:40:44,520 --> 00:40:46,640
EXPRESS APOE AND THEN COMPARED

976
00:40:46,640 --> 00:40:48,920
IT TO TRANSCRIPTION IN CELLS

977
00:40:48,920 --> 00:40:51,400
WHICH DO NOT EXPRESS APOE SO

978
00:40:51,400 --> 00:40:55,400
HERE I'M SHOWING RNA SEQUENCING

979
00:40:55,400 --> 00:40:58,360
DATA FROM A EXPRESS APOE AND

980
00:40:58,360 --> 00:41:00,600
WE'RE DETECTING SEQUENCING READS

981
00:41:00,600 --> 00:41:02,760
AT THE FIVE PRIME END OF APOE

982
00:41:02,760 --> 00:41:05,920
AND COMPARED THAT TO CELLS,

983
00:41:05,920 --> 00:41:08,000
THESE ARE WHITE CELLS, FROM THE

984
00:41:08,000 --> 00:41:09,920
SAME INDIVIDUAL, WHERE APOE IS

985
00:41:09,920 --> 00:41:11,000
NOT EXPRESSED.

986
00:41:11,000 --> 00:41:12,480
AND WHEN WE LOOK AT

987
00:41:12,480 --> 00:41:13,840
TRANSCRIPTION OF ANSWER, WHAT WE

988
00:41:13,840 --> 00:41:16,360
SEE IS THE CELL TYPE WHERE APOE

989
00:41:16,360 --> 00:41:20,960
IS ACTIVE WE CAN DETECT FULL

990
00:41:20,960 --> 00:41:22,880
LENGTH OR FULL TRANSCRIPTION

991
00:41:22,880 --> 00:41:26,640
WHERE WE ONLY DETECT TRUNCATED

992
00:41:26,640 --> 00:41:29,520
OF PAUSED IN THIS CELLS WHERE

993
00:41:29,520 --> 00:41:31,080
APOE IS SILENT.

994
00:41:31,080 --> 00:41:33,320
WE ALSO LOOKED AT PUBLICLY

995
00:41:33,320 --> 00:41:35,360
AVAILABLE DATA FROM THE ENCODE

996
00:41:35,360 --> 00:41:41,720
PROJECT WHERE THEY USED A BETTER

997
00:41:41,720 --> 00:41:42,920
CAPTURE TRANSCRIPTS AND HERE

998
00:41:42,920 --> 00:41:46,960
WE'RE LOOKING AT A LIVER CELL

999
00:41:46,960 --> 00:41:50,560
LINE OR TRANSFORM D CELLS AND

1000
00:41:50,560 --> 00:41:52,720
THE PATTERN IS THE SAME.

1001
00:41:52,720 --> 00:41:55,440
THE LIVER CELL TYPE EXPRESSES

1002
00:41:55,440 --> 00:41:57,800
APOE BUT THE B CELLS IS IS

1003
00:41:57,800 --> 00:42:00,040
SILENT AND IN LIVER CELLS WE CAN

1004
00:42:00,040 --> 00:42:02,320
DETECT THE EXPRESSION OF A FULL

1005
00:42:02,320 --> 00:42:04,000
LENGTH ANSWER AND WHEREAS IN B

1006
00:42:04,000 --> 00:42:06,160
CELLS WE DON'T DETECT FULL

1007
00:42:06,160 --> 00:42:07,840
LENGTH ANSWERS.

1008
00:42:07,840 --> 00:42:11,400
SO WHAT WE -- THIS SUPPORTS THIS

1009
00:42:11,400 --> 00:42:13,280
MODEL THAT THE R-LOOP OR THE

1010
00:42:13,280 --> 00:42:15,160
FORMATION OF THIS MODIFIED

1011
00:42:15,160 --> 00:42:17,520
R-LOOP IS CAUSING THEM TO PAUSE

1012
00:42:17,520 --> 00:42:19,760
ON THIS NON CODING RNA AND

1013
00:42:19,760 --> 00:42:21,840
REGULATING WHETHER YOU GET A

1014
00:42:21,840 --> 00:42:23,280
FULL EXPRESSION OF FULL LENGTH

1015
00:42:23,280 --> 00:42:32,840
ANSWER OR WHETHER YOU GET THIS

1016
00:42:32,840 --> 00:42:33,400
SHORT AROUND RNA.

1017
00:42:33,400 --> 00:42:36,240
WHAT WE THINK HAPPENED IS THERE

1018
00:42:36,240 --> 00:42:37,840
IS IS INITIAL TRANSCRIPTION OF

1019
00:42:37,840 --> 00:42:39,840
THIS NON CODING RNA AND

1020
00:42:39,840 --> 00:42:42,400
FORMATION OF AN R-LOOP, THIS

1021
00:42:42,400 --> 00:42:45,720
R-LOOP CAN BE MODIFIED BY THE ME

1022
00:42:45,720 --> 00:42:50,440
IT TTL TO CREATE M6A AND THAT

1023
00:42:50,440 --> 00:42:58,680
M6A IS RECOGNIZED BY MPG AND

1024
00:42:58,680 --> 00:43:03,560
WHERE CLEVE AND WE THINK THE

1025
00:43:03,560 --> 00:43:06,920
R-LOOP CONTRIBUTES TO STABLATION

1026
00:43:06,920 --> 00:43:09,360
THAT RESULTS IN SUBSEQUENT

1027
00:43:09,360 --> 00:43:10,080
PRELIMINARY PAUSE WHAT IS THEY

1028
00:43:10,080 --> 00:43:12,240
RUN INTO THIS STRUCTURE.

1029
00:43:12,240 --> 00:43:15,680
IF USING EXPERIMENTAL, IF WE

1030
00:43:15,680 --> 00:43:17,800
PREVENT THE FORMATION OF THESE

1031
00:43:17,800 --> 00:43:20,240
MODIFIED R-LOOPS, WE CAN SEE

1032
00:43:20,240 --> 00:43:22,240
THAT THERE'S SYNTHESIS OF A FULL

1033
00:43:22,240 --> 00:43:24,160
LENGTH ANSWER AND THAT CAN LEAD

1034
00:43:24,160 --> 00:43:26,920
TO ACTIVATION OF APOE.

1035
00:43:26,920 --> 00:43:31,000
WE THINK THAT THIS MODE OF

1036
00:43:31,000 --> 00:43:35,800
TRANSCRIPTRANSCRIPTION AND THINT

1037
00:43:35,800 --> 00:43:37,720
REGULATION OF APOE EXPRESSION IN

1038
00:43:37,720 --> 00:43:39,960
PARTICULAR IN THE CONDITION TEXT

1039
00:43:39,960 --> 00:43:42,080
OF ALZHEIMER'S DISEASE.

1040
00:43:42,080 --> 00:43:44,280
BUT WHAT I'VE SHOWN SO FAR HAVE

1041
00:43:44,280 --> 00:43:46,280
BEEN YOU KNOW, DYNAMICALLY

1042
00:43:46,280 --> 00:43:48,480
CHANGING THE EXTENT OF R-LOOP

1043
00:43:48,480 --> 00:43:50,480
FORMATION USING EXPERIMENTAL

1044
00:43:50,480 --> 00:43:51,600
MANIPULATIONS SO THE QUESTION WE

1045
00:43:51,600 --> 00:43:53,920
HAD IS WHETHER CELLS DYNAMICALLY

1046
00:43:53,920 --> 00:43:57,760
REGULATE THE FORMATION OF THESE

1047
00:43:57,760 --> 00:43:59,800
R-LOOPS OR DYNAMICALLY REGULATE

1048
00:43:59,800 --> 00:44:02,520
THE SYNTHESIS OF A FULL-LENGTH

1049
00:44:02,520 --> 00:44:04,840
ANSWER TO CHANGE APOE REGULATION

1050
00:44:04,840 --> 00:44:06,520
IN RESPONSE TO STRESS.

1051
00:44:06,520 --> 00:44:10,040
SO I THINK AS A NEPHROLOGIST I

1052
00:44:10,040 --> 00:44:12,200
HAVE A KIDNEY BIAS TOWARDS

1053
00:44:12,200 --> 00:44:14,560
THINGS AND WE POINT OUT THAT

1054
00:44:14,560 --> 00:44:17,600
APOE IS ALSO IMPLICATED IN SOME

1055
00:44:17,600 --> 00:44:19,360
FORMS OF CHRONIC KIDNEY DISEASE.

1056
00:44:19,360 --> 00:44:21,560
THERE ARE MOUSE MODELS WHERE

1057
00:44:21,560 --> 00:44:23,920
APOE HAS BEEN KNOCKED OUT AND

1058
00:44:23,920 --> 00:44:26,040
THOSE MICE DEVELOPED KIDNEY

1059
00:44:26,040 --> 00:44:26,960
DISEASE.

1060
00:44:26,960 --> 00:44:28,280
THERE ARE REPORTS IN THE

1061
00:44:28,280 --> 00:44:31,120
LITERATURE OF MUTATIONS AT APOE

1062
00:44:31,120 --> 00:44:39,040
THAT CAN LEAD TO GLOMREULOPATHY

1063
00:44:39,040 --> 00:44:44,320
OR INJURY TO THE GLOMERULOUS AND

1064
00:44:44,320 --> 00:44:48,320
YOU CAN SEE A CORRELATION WHERE

1065
00:44:48,320 --> 00:44:49,960
LOWER KIDNEY FUNCTION IS

1066
00:44:49,960 --> 00:44:53,000
ASSOCIATED WITH LOWER APOE

1067
00:44:53,000 --> 00:44:53,400
EXPRESSION.

1068
00:44:53,400 --> 00:44:55,000
THESE RESULTS IN CONTEXT WITH

1069
00:44:55,000 --> 00:44:57,360
THE DATA FOR MOUSE MODELS

1070
00:44:57,360 --> 00:45:00,200
SUGGESTED THAT APOE COULD PLAY A

1071
00:45:00,200 --> 00:45:02,960
PROTECTIVE ROLE IN THE KIDNEY.

1072
00:45:02,960 --> 00:45:05,440
SO WE WANTED TO ASK WHETHER THE

1073
00:45:05,440 --> 00:45:06,760
EXPRESSION OF ANSWER MIGHT

1074
00:45:06,760 --> 00:45:10,120
CHANGE IN RESPONSE TO STRESS TO

1075
00:45:10,120 --> 00:45:14,200
LEAD TO INDUCTION OF APOE.

1076
00:45:14,200 --> 00:45:16,640
AND OSMONIC STRESS IS ONE WE

1077
00:45:16,640 --> 00:45:19,440
LOOKED AT IN THE LAB SO WE TOOK

1078
00:45:19,440 --> 00:45:22,160
CULTURE CELLS AND THEN SUBJECTED

1079
00:45:22,160 --> 00:45:24,440
THEM TO OSMOTIC STRESS AND ASKED

1080
00:45:24,440 --> 00:45:26,280
TO WHAT HAPPENS TO EXPRESSION OF

1081
00:45:26,280 --> 00:45:29,080
ANSWER AND SURE ENOUGH, PUTTING

1082
00:45:29,080 --> 00:45:31,040
CELLS IN STRESS CONDITIONS

1083
00:45:31,040 --> 00:45:32,880
RESULTED IN A SIGNIFICANT

1084
00:45:32,880 --> 00:45:35,240
INCREASE IN THE EXPRESSION OF

1085
00:45:35,240 --> 00:45:37,240
FULL LENGTH ANSWERS AND THIS

1086
00:45:37,240 --> 00:45:39,160
INCREASE AND EXPRESSION OF FULL

1087
00:45:39,160 --> 00:45:41,640
LENGTH ANSWER CORRELATED WITH AN

1088
00:45:41,640 --> 00:45:45,280
INCREASE IN APOE EXPRESSION AT

1089
00:45:45,280 --> 00:45:46,240
THE RNA LEVEL AND WE CAN ALSO

1090
00:45:46,240 --> 00:45:48,800
DETECT AN INCREASE IN APOE

1091
00:45:48,800 --> 00:45:50,120
EXPRESSION AT PROTEIN LEVEL.

1092
00:45:50,120 --> 00:45:51,600
SO THIS IS EVIDENCE THAT THERE

1093
00:45:51,600 --> 00:45:54,280
COULD BE DYNAMIC REGULATION OF

1094
00:45:54,280 --> 00:45:56,920
THESE R-LOOP MEDIATED PAUSE TO

1095
00:45:56,920 --> 00:45:59,680
ALLOW GENE EXPRESSION TO RESPOND

1096
00:45:59,680 --> 00:46:00,360
TO STRESS.

1097
00:46:00,360 --> 00:46:03,080
SO ON THE ONE HAND, THIS IS A

1098
00:46:03,080 --> 00:46:03,640
VERY SATISFYING RESULT.

1099
00:46:03,640 --> 00:46:05,040
BUT ON THE OTHER HAND, IT WAS A

1100
00:46:05,040 --> 00:46:07,960
LITTLE BIT CONFUSING AS APOE WAS

1101
00:46:07,960 --> 00:46:11,280
NOT KNOWN TO BE AN OS MONDAY I

1102
00:46:11,280 --> 00:46:21,760
CAN STRESS RESPONSIVE GENE.

1103
00:46:25,320 --> 00:46:28,320
AND WE CAN DETECT APOE IN THE

1104
00:46:28,320 --> 00:46:30,640
MEDIA AFTER THIS OS MOT TICK

1105
00:46:30,640 --> 00:46:31,840
STRESS SO HERE WE CAN SEE THAT

1106
00:46:31,840 --> 00:46:41,360
THERE'S AN INCREASE I IN ITS GRN

1107
00:46:41,360 --> 00:46:41,720
THESE COMPANIES.

1108
00:46:41,720 --> 00:46:43,520
SO WHAT WE DID WAS TAKE THE

1109
00:46:43,520 --> 00:46:45,400
CONDITION MEDIA THAT HAS HIGHER

1110
00:46:45,400 --> 00:46:47,440
LEVELS OF APOE EXPRESSION OR

1111
00:46:47,440 --> 00:46:50,200
MEDIA FROM CELLS GROWN IN ISO

1112
00:46:50,200 --> 00:46:51,440
TONIC CONDITIONS AND BRING THEM

1113
00:46:51,440 --> 00:46:53,080
UP THE SAME.

1114
00:46:53,080 --> 00:46:55,000
MAKE THEM EQUALLY HYPER TONIC

1115
00:46:55,000 --> 00:46:56,800
AND WE PUT THIS CONDITIONED

1116
00:46:56,800 --> 00:46:59,640
MEDIA OR THE NON CONDITIONED

1117
00:46:59,640 --> 00:47:00,840
MEDIA AND ASKED WHETHER THERE

1118
00:47:00,840 --> 00:47:03,560
WAS A DIFFERENCE IN A POP TOE

1119
00:47:03,560 --> 00:47:05,600
SIS AND HOW CELLS WERE ABLE TO

1120
00:47:05,600 --> 00:47:10,360
RESPOND TO A OSMOTIC STRESS.

1121
00:47:10,360 --> 00:47:12,240
THE CELLS IN MEDIA WITH HIGHER

1122
00:47:12,240 --> 00:47:15,320
LEVELS OF APOE EXPRESSION IS

1123
00:47:15,320 --> 00:47:16,720
HYPER TONIC CONDITION MEDIA,

1124
00:47:16,720 --> 00:47:20,760
THEY HAD LOWER LEVELS OF

1125
00:47:20,760 --> 00:47:23,280
APOPTOSIS COMPARED TO CELLS IN

1126
00:47:23,280 --> 00:47:24,560
HYPER TONIC MEDIA.

1127
00:47:24,560 --> 00:47:26,560
WE REPEATED THIS WITH ADDING

1128
00:47:26,560 --> 00:47:28,640
HIGHER CONCENTRATION OF SALT

1129
00:47:28,640 --> 00:47:30,640
INCREASING THE EXTENT OF THE

1130
00:47:30,640 --> 00:47:31,720
OSMOTIC STRESS AND WE SAW THE

1131
00:47:31,720 --> 00:47:33,120
SAME PATTERN WHERE THE CELLS IN

1132
00:47:33,120 --> 00:47:37,480
THE MEDIA WITH HIGHER LEVELS OF

1133
00:47:37,480 --> 00:47:43,600
APO E AND THEY EXPERIENCED LESS

1134
00:47:43,600 --> 00:47:44,560
APOPTOSIS SO THEY WERE BETTER

1135
00:47:44,560 --> 00:47:47,400
ABLE TO RESPOND TO THIS STRESS.

1136
00:47:47,400 --> 00:47:49,400
SO THIS MIGHT SUGGEST THAT

1137
00:47:49,400 --> 00:47:51,520
INCREASING APOE EXPRESSION COULD

1138
00:47:51,520 --> 00:47:55,600
BE A PROTECTIVE AGAINST

1139
00:47:55,600 --> 00:47:57,840
APOPTOSIS AND FACED WITH OSMOTIC

1140
00:47:57,840 --> 00:48:03,200
STRESS AND THIS IDEA OF APOE

1141
00:48:03,200 --> 00:48:04,880
PLAYING A CYTO PROTECTIVE ROLE

1142
00:48:04,880 --> 00:48:06,280
IS CONSISTENT WITH DATA LOOKING

1143
00:48:06,280 --> 00:48:08,200
AT ITS ROLE IN THE CENTRAL

1144
00:48:08,200 --> 00:48:10,560
NERVOUS SYSTEM WHERE IT'S THE

1145
00:48:10,560 --> 00:48:12,360
THOUGHT TO PLAY OR COP TRIBUTE

1146
00:48:12,360 --> 00:48:14,440
TO CYTO PROTECTION IN THE BRAIN

1147
00:48:14,440 --> 00:48:17,160
AS WELL.

1148
00:48:17,160 --> 00:48:22,080
AND THAT'S WHERE I THINK I HAVE

1149
00:48:22,080 --> 00:48:24,720
AN AH-HA MOMENT AND THE CELL

1150
00:48:24,720 --> 00:48:26,400
TYPE IS IS DYSFUNCTIONAL AND I

1151
00:48:26,400 --> 00:48:29,760
HAVE TO GO BACK TO A CASE I

1152
00:48:29,760 --> 00:48:30,480
STARTED WITH.

1153
00:48:30,480 --> 00:48:32,360
IT'S ASTROCYTES THAT NEED TO

1154
00:48:32,360 --> 00:48:34,240
RESPOND TO THIS OSMOTIC STRESS

1155
00:48:34,240 --> 00:48:37,440
AND IT'S ASTROCYTES WHOSE

1156
00:48:37,440 --> 00:48:38,840
DYSFUNCTION CON TRIBUTES TO THE

1157
00:48:38,840 --> 00:48:42,440
DEVELOPMENT OF ALZHEIMER'S

1158
00:48:42,440 --> 00:48:48,960
DISEASE.

1159
00:48:48,960 --> 00:48:50,640
WE WERE ABLE TO LOOK IN

1160
00:48:50,640 --> 00:48:52,120
PUBLISHED DATA OF EXPRESSION IN

1161
00:48:52,120 --> 00:48:53,080
ASTROCYTES AND WE CAN SEE THAT

1162
00:48:53,080 --> 00:48:55,920
THIS NON CODING RNA IS EXPRESSED

1163
00:48:55,920 --> 00:48:58,240
TO HAVE IN ASTROCYTES AND THAT

1164
00:48:58,240 --> 00:49:00,240
THE EXPRESSION OF ANSWER OF FULL

1165
00:49:00,240 --> 00:49:01,560
LENGTH ANSWER CORRELATES WITH

1166
00:49:01,560 --> 00:49:03,800
THE EXPRESSION OF APOE AND SO WE

1167
00:49:03,800 --> 00:49:06,200
THINK THAT THIS TRANSCRIPTION

1168
00:49:06,200 --> 00:49:08,600
REGULATORY MECHANISM WERE

1169
00:49:08,600 --> 00:49:10,120
MODIFIED R-LOOPS CAN PAUSE

1170
00:49:10,120 --> 00:49:13,920
TRANSCRIPTION IN THIS NON CODING

1171
00:49:13,920 --> 00:49:16,840
RNA TO REGULATE APOE WE THINK

1172
00:49:16,840 --> 00:49:18,280
THIS MECHANISM CONTRIBUTES TO

1173
00:49:18,280 --> 00:49:22,400
THE REGULATION OF APOE

1174
00:49:22,400 --> 00:49:24,360
EXPRESSION IN ASTROCYTES SO TO

1175
00:49:24,360 --> 00:49:25,840
COME BACK TO THE CASE I BEGAN

1176
00:49:25,840 --> 00:49:26,960
THIS DISCUSSION WITH AND THIS

1177
00:49:26,960 --> 00:49:27,960
PATIENT WHO COMES TO THE

1178
00:49:27,960 --> 00:49:31,360
HOSPITAL WHO IS HYPO AND SPENT

1179
00:49:31,360 --> 00:49:33,160
TIME IN THE ICU ASK HAS OVER

1180
00:49:33,160 --> 00:49:39,880
CORRECTION OF HER HYPO ATREMIA

1181
00:49:39,880 --> 00:49:44,320
AND SURFS WITH OSMOTIC

1182
00:49:44,320 --> 00:49:45,200
DEMYELINATION, THIS IS COMMON IN

1183
00:49:45,200 --> 00:49:46,680
THE HOSPITAL AND NOT ALL

1184
00:49:46,680 --> 00:49:51,840
PATIENTS WHO ARE HYPOATREMIC

1185
00:49:51,840 --> 00:49:57,320
WILL HAVE DEHIGHATION AND I

1186
00:49:57,320 --> 00:50:01,320
THINK OF IDENTIFYING THE PAUSING

1187
00:50:01,320 --> 00:50:03,400
MECHANISMS THAT HAVE R-LOOPS AND

1188
00:50:03,400 --> 00:50:07,360
LED TO APOE COULD BE AN OS MOW

1189
00:50:07,360 --> 00:50:09,240
PROTECTED FACTOR AND PERHAPS

1190
00:50:09,240 --> 00:50:11,280
THIS IS A PREVIOUSLY

1191
00:50:11,280 --> 00:50:14,200
UNAPPRECIATED COMPONENT THAT CAN

1192
00:50:14,200 --> 00:50:16,800
CONTRIBUTE TO WHICH PATIENTS

1193
00:50:16,800 --> 00:50:19,840
EXPERIENCE OSMOTIC DEMYELINATION

1194
00:50:19,840 --> 00:50:22,320
VERSE WHIZ ARE PROTECTED AND

1195
00:50:22,320 --> 00:50:23,480
THOSE ABLE TO RESPOND AND HAVE

1196
00:50:23,480 --> 00:50:25,640
AN INCREASE IN APOE EXPRESSION

1197
00:50:25,640 --> 00:50:27,760
OR IT COULD BE THAT FOLKS THAT

1198
00:50:27,760 --> 00:50:31,160
CARRY THE APOE FOR RISK ALLELE,

1199
00:50:31,160 --> 00:50:32,240
THAT MAYBE THOSE PATIENTS ARE

1200
00:50:32,240 --> 00:50:36,520
MORE AT RISK FOR DEVELOPING

1201
00:50:36,520 --> 00:50:37,720
OSMOTIC DEMILE ENATION.

1202
00:50:37,720 --> 00:50:39,640
SO I HOPE I WAS ABLE TO SHOW

1203
00:50:39,640 --> 00:50:42,440
WITH THESE TWO STORIES,

1204
00:50:42,440 --> 00:50:43,880
ILLUSTRATING THE CONTRIBUTION OF

1205
00:50:43,880 --> 00:50:46,120
UNDERLYING SEQUENCE AND THE

1206
00:50:46,120 --> 00:50:47,400
REGULATION OF PAUSING AND

1207
00:50:47,400 --> 00:50:48,800
REGULATION OF GENE EXPRESSION IN

1208
00:50:48,800 --> 00:50:50,720
THE FIRST STORY LOOKING AT THIS

1209
00:50:50,720 --> 00:50:52,720
COMPONENT OF THE PRIMARY

1210
00:50:52,720 --> 00:50:55,120
SEQUENCE AND GENETIC PAUSING

1211
00:50:55,120 --> 00:50:57,600
WHICH SEEMS TO CONFER PRECISION

1212
00:50:57,600 --> 00:50:59,720
OR SIMILARITIES OF WHERE THE

1213
00:50:59,720 --> 00:51:01,280
PAUSES ACROSS CELL TYPES AND

1214
00:51:01,280 --> 00:51:03,760
INDIVIDUALS AND THE STORY I JUST

1215
00:51:03,760 --> 00:51:06,360
TALKED ABOUT IS EPIGENETIC

1216
00:51:06,360 --> 00:51:07,320
REGULATION OF PAUSE TAG DEPENDS

1217
00:51:07,320 --> 00:51:10,600
ON THE FORMATION OR DYNAMIC

1218
00:51:10,600 --> 00:51:13,080
FORMATION OF THESE MODIFIED

1219
00:51:13,080 --> 00:51:13,760
R-LOOP STRUCTURES.

1220
00:51:13,760 --> 00:51:16,320
AND SO TO CONCLUDE, I HOPE I'VE

1221
00:51:16,320 --> 00:51:18,160
SHOWN YOU THE RNA PRELIMINARY

1222
00:51:18,160 --> 00:51:20,080
PAUSING IS A IMPORTANT AND

1223
00:51:20,080 --> 00:51:22,600
CRITICAL ASPECT OF TRANSCRIPTION

1224
00:51:22,600 --> 00:51:23,880
REGULATION THAT PAUSING NEAR THE

1225
00:51:23,880 --> 00:51:25,920
PROMOTER IS IS REGULATE BID A

1226
00:51:25,920 --> 00:51:28,840
COMBINATION OF THESE PROTEIN

1227
00:51:28,840 --> 00:51:31,200
PAUSING COMPLEXES AND THE

1228
00:51:31,200 --> 00:51:33,080
UNDERLYING SEQUENCE AND WE HAVE

1229
00:51:33,080 --> 00:51:37,480
IDENTIFIED THIS NEW REGULATORY

1230
00:51:37,480 --> 00:51:42,400
MECHANISM WHERE A SUBSET OF NON

1231
00:51:42,400 --> 00:51:44,040
CODING RNA AND IT ALLOWED US TO

1232
00:51:44,040 --> 00:51:49,400
IDENTIFY APOE HAS AN OSMOTIC

1233
00:51:49,400 --> 00:51:51,320
STRESS RESPONSE PROTEIN.

1234
00:51:51,320 --> 00:51:53,400
I'D LIKE TO THANK THE FOLKS AT

1235
00:51:53,400 --> 00:51:53,720
MY LAD.

1236
00:51:53,720 --> 00:51:55,000
I SHOWED DATA AND AS WELL AS

1237
00:51:55,000 --> 00:51:58,680
SOME DATA FROM MA AND WINNIE AND

1238
00:51:58,680 --> 00:51:59,400
DISCUSSING REDUCK TASE AND WE

1239
00:51:59,400 --> 00:52:01,760
GET SUPPORT FROM DON DELL KIRK

1240
00:52:01,760 --> 00:52:04,440
AND RODRIGUEZ DID THE BY OWE

1241
00:52:04,440 --> 00:52:08,760
CHEMISTRY LOOKING AT THE BETWEEN

1242
00:52:08,760 --> 00:52:11,400
M6A AND THIS WORK WAS STARTED 

1243
00:52:11,400 --> 00:52:13,800
WHEN I WAS A POSTDOC AND IT HAS

1244
00:52:13,800 --> 00:52:15,320
CONTINUED AS A COLLABORATION NOW

1245
00:52:15,320 --> 00:52:16,720
THAT I HAVE ESTABLISHED MY OWN

1246
00:52:16,720 --> 00:52:18,360
GROUP AND THE CLINICAL CASE THAT

1247
00:52:18,360 --> 00:52:21,800
I PRESENTED WAS GENEROUSLY

1248
00:52:21,800 --> 00:52:28,800
SHARED BY ONE O AND IN ADDITION

1249
00:52:28,800 --> 00:52:29,840
FROM SUPPORT WE HAVE SUPPORT

1250
00:52:29,840 --> 00:52:31,480
FROM THE CAREER DEVELOPMENT A

1251
00:52:31,480 --> 00:52:34,040
CARD FROM ASN.

1252
00:52:34,040 --> 00:52:35,120
I APPRECIATE YOUR ATTENTION AND

1253
00:52:35,120 --> 00:52:37,040
I WILL BE HAPPY TO ANSWER ANY

1254
00:52:37,040 --> 00:52:42,480
QUESTIONS NOW OR E-MAIL.

1255
00:52:42,480 --> 00:52:47,280
>>THANK YOU O MUC SO
MUCH, Dr.

1256
00:52:47,280 --> 00:52:47,600
WATTS.

1257
00:52:47,600 --> 00:52:48,640
IT WAS A GREAT TALK.

1258
00:52:48,640 --> 00:52:50,920
I LOVE THE WAY YOU DID THE FULL

1259
00:52:50,920 --> 00:52:53,400
TRANSLATIONAL SEQUENCE OF

1260
00:52:53,400 --> 00:52:54,880
TALKING ABOUT THE CASE AND GOING

1261
00:52:54,880 --> 00:52:57,120
BACK TO THE LAB AND BACK AND

1262
00:52:57,120 --> 00:52:58,080
FOURTH.

1263
00:52:58,080 --> 00:52:59,000
I DON'T SEE QUESTIONS IN THE

1264
00:52:59,000 --> 00:53:00,080
CHAT BUT I HAVE A QUESTION FOR

1265
00:53:00,080 --> 00:53:00,520
YOU.

1266
00:53:00,520 --> 00:53:05,200
SO YOU DID TALK ABOUT THE

1267
00:53:05,200 --> 00:53:06,360
GENETICS SEQUENCE, WHAT'S THERE

1268
00:53:06,360 --> 00:53:08,920
IN THE GENOME AND HOW IT EFFECTS

1269
00:53:08,920 --> 00:53:10,640
PAUSING AND YOU TALKED ABOUT ALL

1270
00:53:10,640 --> 00:53:13,680
THESE OTHER FACTORS THAT COULD

1271
00:53:13,680 --> 00:53:17,080
ACTUALLY ADDITIONALLY

1272
00:53:17,080 --> 00:53:18,720
DYNAMICALLY EFFECT THAT SYSTEM.

1273
00:53:18,720 --> 00:53:22,160
SO, I MEAN, HOW DO YOU DECIDE --

1274
00:53:22,160 --> 00:53:23,840
SO IS THERE A PATH FORWARD TO

1275
00:53:23,840 --> 00:53:26,920
SOME TYPE OF AN INTERVENTION?

1276
00:53:26,920 --> 00:53:28,440
THE QUESTION IS, WHAT'S

1277
00:53:28,440 --> 00:53:29,200
MODIFIABLE?

1278
00:53:29,200 --> 00:53:30,960
SO THE GENE SEQUENCES ARE THERE

1279
00:53:30,960 --> 00:53:32,800
RIGHT, BUT WHAT DO YOU SEE AS

1280
00:53:32,800 --> 00:53:34,280
THE POTENTIAL APPLICATION OF

1281
00:53:34,280 --> 00:53:36,960
THIS TYPE OF MECHANISTIC STUDY

1282
00:53:36,960 --> 00:53:41,400
TOWARDS SOME KIND OF CLINICAL

1283
00:53:41,400 --> 00:53:43,920
INTERVENTION?

1284
00:53:43,920 --> 00:53:44,560
>>Jason Watts: THAT'S A
GOOD

1285
00:53:44,560 --> 00:53:44,920
QUESTION.

1286
00:53:44,920 --> 00:53:46,880
TO THE FIRST PART WE'RE FOCUSING

1287
00:53:46,880 --> 00:53:50,080
ON PRIMARY SEQUENCE AND PAUSING

1288
00:53:50,080 --> 00:53:52,160
AT THE SITE OZONE IS SIMILAR

1289
00:53:52,160 --> 00:53:53,880
ACROSS INDIVIDUALS INTERESTED IN

1290
00:53:53,880 --> 00:53:56,040
PURSUING THAT AND IN LOOKING AT

1291
00:53:56,040 --> 00:53:58,960
HOW POLY MORPHISMS COULD EXPLAIN

1292
00:53:58,960 --> 00:53:59,920
DIFFERENCES IN INDIVIDUALS IN

1293
00:53:59,920 --> 00:54:02,600
TERMS OF HOW GENES ARE EXPRESSED

1294
00:54:02,600 --> 00:54:04,160
AND WE'RE VERY EXCITED ABOUT

1295
00:54:04,160 --> 00:54:07,280
THIS NEW OBSERVATION ABOUT,

1296
00:54:07,280 --> 00:54:08,600
R-LOOP MEDIATED PAUSING IN NON

1297
00:54:08,600 --> 00:54:12,240
CODING RNAs IN THAT PROVIDES A

1298
00:54:12,240 --> 00:54:13,840
POTENTIAL TARGET TO DEVELOP

1299
00:54:13,840 --> 00:54:15,360
THEIR PIECE AND SO IF WE'RE

1300
00:54:15,360 --> 00:54:17,040
TALKING ABOUT APOE FOR EXAMPLE,

1301
00:54:17,040 --> 00:54:20,000
IF YOU ARE A CARRIER OF THE E-4

1302
00:54:20,000 --> 00:54:22,920
RISK ALLELE, IT MAY BE

1303
00:54:22,920 --> 00:54:25,800
ADVANTAGEOUS IF YOU HAVE LOWER

1304
00:54:25,800 --> 00:54:26,960
EXPRESSION OF THAT RISK A LEGAL.

1305
00:54:26,960 --> 00:54:29,000
SO WE THINK THAT TARGETING THIS

1306
00:54:29,000 --> 00:54:31,920
NON CODING RNA, COULD LEAD TO

1307
00:54:31,920 --> 00:54:34,160
RESULT IN DECREASED EXPRESSION

1308
00:54:34,160 --> 00:54:38,120
OF THE RISK ALLELE AND THAT

1309
00:54:38,120 --> 00:54:40,000
COULD BE PROTECTIVE IN TERMS OF

1310
00:54:40,000 --> 00:54:42,320
PREVENTING THE ONSET OF DEMENTIA

1311
00:54:42,320 --> 00:54:44,160
OR SLOWING THE PROGRESSION OF

1312
00:54:44,160 --> 00:54:45,600
DEMENTIA SO WANTING TO BETTER

1313
00:54:45,600 --> 00:54:48,680
UNDERSTAND HOW THIS NON CODE RNA

1314
00:54:48,680 --> 00:54:50,760
IS IS REGULATED AND HOW TURNING

1315
00:54:50,760 --> 00:54:53,240
IT ON LEADS TO AN INCREASE IN

1316
00:54:53,240 --> 00:54:54,440
APOE EXPRESSION IS AN ACTIVE

1317
00:54:54,440 --> 00:54:57,000
AREA OF WORK IN THE LAB RIGHT

1318
00:54:57,000 --> 00:54:57,400
NOW.

1319
00:54:57,400 --> 00:55:03,880
>>IT'S ALSO SUPER
INTERESTING

1320
00:55:03,880 --> 00:55:05,080
THAT YOU MOVE FROM THE KIDNEY

1321
00:55:05,080 --> 00:55:07,080
AND TO THE BRAIN.

1322
00:55:07,080 --> 00:55:09,600
THESE ARE UBIQUITOUS MECHANISMS

1323
00:55:09,600 --> 00:55:13,360
SO HOW DO YOU THEN, AS A

1324
00:55:13,360 --> 00:55:14,080
NEPHROLOGIST, START THINKING

1325
00:55:14,080 --> 00:55:16,200
ABOUT THESE OTHER AREAS OF

1326
00:55:16,200 --> 00:55:16,520
APPLICATION?

1327
00:55:16,520 --> 00:55:18,000
DO YOU COLLABORATE WITH OTHER

1328
00:55:18,000 --> 00:55:18,200
LABS?

1329
00:55:18,200 --> 00:55:21,080
DO YOU TALK TO PEOPLE IN OTHER

1330
00:55:21,080 --> 00:55:22,120
FIELDS?

1331
00:55:22,120 --> 00:55:25,400
>>Jason Watts: SO, AT
LEAST FOR

1332
00:55:25,400 --> 00:55:27,760
THE PARTICULARLY MAYBE ENAMORED

1333
00:55:27,760 --> 00:55:30,360
OR INTERESTED IN THIS LINK WITH

1334
00:55:30,360 --> 00:55:31,760
OSMOTIC DEMILE ENATION WHICH I

1335
00:55:31,760 --> 00:55:34,040
FULLY ACKNOWLEDGE THAT FAIRLY

1336
00:55:34,040 --> 00:55:37,120
RARE COMPLICATION BUT LOOKING

1337
00:55:37,120 --> 00:55:38,640
FOR COLLABORATORS AND VERY

1338
00:55:38,640 --> 00:55:40,520
INTERESTED IN WEATHER APOE

1339
00:55:40,520 --> 00:55:41,960
EXPRESSION IS DIFFERENT OR MIGHT

1340
00:55:41,960 --> 00:55:43,960
BE A RISK FACTOR FOR THOSE WHO

1341
00:55:43,960 --> 00:55:48,240
ARE GOING TO DEVELOP OSMOTIC

1342
00:55:48,240 --> 00:55:48,960
DEMILE ENATION.

1343
00:55:48,960 --> 00:55:52,680
I ALLUDED TO DOING THE

1344
00:55:52,680 --> 00:55:54,520
EXPERIMENT WHERE WE SHOW THAT

1345
00:55:54,520 --> 00:55:57,640
THERE'S MORE OR LESS APOPTOSIS

1346
00:55:57,640 --> 00:55:59,720
WHEN THERE'S HIGHER APOE

1347
00:55:59,720 --> 00:56:01,200
EXPRESSION AND IT WAS DONE IN

1348
00:56:01,200 --> 00:56:04,920
CELLS SO WE HAVE A COUPLE

1349
00:56:04,920 --> 00:56:06,720
EXAMPLES OF CHRONIC KIDNEY

1350
00:56:06,720 --> 00:56:08,680
DISEASES WHERE IT SEEMS THAT

1351
00:56:08,680 --> 00:56:10,320
THERE'S DIFFERENTIAL EXPRESSION

1352
00:56:10,320 --> 00:56:14,080
OF APOE AND I THINK MORE

1353
00:56:14,080 --> 00:56:15,120
INTERESTINGLY DIFFERENTIAL

1354
00:56:15,120 --> 00:56:16,320
EXPRESSION OF ANSWER THIS NON

1355
00:56:16,320 --> 00:56:19,480
CODING RNA THAT REGULATES APOE

1356
00:56:19,480 --> 00:56:20,560
SO ANOTHER PROJECT FOR ANOTHER

1357
00:56:20,560 --> 00:56:22,320
PERSON IN LAB IS LOOKING AT HOW

1358
00:56:22,320 --> 00:56:26,200
THIS REGULATORY MECHANISM IS

1359
00:56:26,200 --> 00:56:31,080
GERMANE TO KIDNEY BIOLOGY AND IN

1360
00:56:31,080 --> 00:56:36,840
PARTICULAR, INTO TUBAL BIOLOGY.

1361
00:56:36,840 --> 00:56:38,920
>>I DON'T SEE ANY OTHER

1362
00:56:38,920 --> 00:56:40,720
QUESTIONS RIGHT NOW FROM THE

1363
00:56:40,720 --> 00:56:43,960
VIDEO CAST WEBSITE.

1364
00:56:43,960 --> 00:56:45,920
DO YOU HAVE ANY CLOSING

1365
00:56:45,920 --> 00:56:46,760
COMMENTS, Dr. WATTS?

1366
00:56:46,760 --> 00:56:48,760
WOULD YOU LIKE TO MAKE TO THE

1367
00:56:48,760 --> 00:56:50,920
AUDIENCE BEFORE WE END?

1368
00:56:50,920 --> 00:56:52,800
>>I JUST APPRECIATE THOSE
FOLKS

1369
00:56:52,800 --> 00:56:54,120
WHO ATTENDED.

1370
00:56:54,120 --> 00:56:55,440
AND YOUR QUESTIONS, Dr. CHUNG,

1371
00:56:55,440 --> 00:56:57,760
GAVE ME AN OPPORTUNITY TO ALLUDE

1372
00:56:57,760 --> 00:57:01,360
TO ON GOING WORK EX WE'LL LOOK

1373
00:57:01,360 --> 00:57:02,080
IN PARTICULAR AT THIS

1374
00:57:02,080 --> 00:57:03,760
RELATIONSHIP BETWEEN ANSWER AND

1375
00:57:03,760 --> 00:57:06,040
APOE IN KIDNEY DISEASE.

1376
00:57:06,040 --> 00:57:08,560
>>THAT'S GREAT.

1377
00:57:08,560 --> 00:57:08,920
ALL RIGHT.

1378
00:57:08,920 --> 00:57:10,800
SO I WANT TO THANK EVERYONE FOR

1379
00:57:10,800 --> 00:57:12,920
THEIR ATTENDANCE AND THEIR

1380
00:57:12,920 --> 00:57:13,440
ATTENTION.

1381
00:57:13,440 --> 00:57:16,400
JUST TO REMIND YOU THE CME CODE

1382
00:57:16,400 --> 00:57:18,960
TODAY IS 4454 AND WE WANT TO

1383
00:57:18,960 --> 00:57:20,920
THANK Dr. WATTS FOR HIS

1384
00:57:20,920 --> 00:57:22,560
BRILLIANT TALK AND BEST OF LUCK

1385
00:57:22,560 --> 00:57:22,960
TO YOU.

1386
00:57:22,960 --> 00:57:23,720
>>Jason Watts: THANK YOU,
VERY

1387
00:57:23,720 --> 00:57:24,320
MUCH.

1388
00:57:24,320 --> 00:57:25,000
TAKE CARE, EVERYONE.

1389
00:57:25,000 --> 00:57:36,240
>>THANK YOU SO MUCH.