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