>> HI.

WELCOME TO THE WALS AFTERNOON

TALKS.

TODAY, WE HAVE DR. STEVE

GOVERNOR FROM COLUMBIA

UNIVERSITY.

HE IS AN INVITEEE OF THE

VIROLOGY SCHOOLS OF NIH AND HE

WILL TALK TODAY ABOUT TWO

TOPICS, THE SILENCING OF

RETROVIRUSES AND THE FACTORS

INVOLVED AND ALSO SOMETHING

INTERESTING AND INTRIGUING, CLAN

RETROVIRUSES.

SO FOR THOSE OF YOU WHO FOLLOWED

STEVE, YOU KNOW HE'S A PROFESSOR

AT COLUMBIA UNIVERSITY SINCE

1990.

HE IS ALSO A HOWARD HUGHES

INVESTIGATOR SINCE 1993.

AND HE WAS ENACTED TO THE

ACADEMY SCIENCES IN 2006.

HE IS INTERESTED IN A VARIETY OF

TOPICS.

HE IS A RENAISSANCE SCIENTIST.

HE IS INTERESTED MAINLY IN DNA

TECHNOLOGY BUT OVER HIS CAREER,

HE TACKLED MANY OTHER TOPICS.

HIS MAIN INTEREST HAS BEEN THE

WHOLE CELL FACTORS INVOLVED IN

STOCKING RETROVIRUSES.

HE TRAINED UNDER VERY, VERY

PROMINENT AND SUCCESSFUL

SCIENTIST, BOTH NOBEL LAUREATE

WINNERS, PAUL BERG AT STANFORD

DURING HIS GRAD SCHOOL YEARS AND

POSTDOC UNDER DAVID BALTIMORE AT

MIT.

IN ADDITION TO BEING A

SUCCESSFUL SCIENTIST AND

FANTASTIC PROFESSOR AT COLUMBIA

UNIVERSITY, TO GIVE YOU I A

NUMBER, HE PUBLISHED MORE THAN

300 PUBLICATIONS, 50 OF THEM

WERE IN THE JOURNALS OF CELL,

SCIENCE, NATURE.

BUT IN ADDITION TO THAT, I

WANTED TO TELL YOU TODAY ABOUT

ANOTHER ASPECT OF STEVE'S CAREER

IS STEVE IS A MENTOR.

STEVE MENTORED SO MANY PEOPLE,

MORE THAN 90% OF THEM ARE

ACTUALLY PIs AND STARTED THEIR

OWN LABS AND THREE OF THEM ARE

HERE AT NIH AND FOR THE SAKE OF

FULL DISCLOSURE, I'M ONE OF

THEM, ONE OF THE LUCKY ONES.

WHAT I WANTED TO SAY IS VERY

IMPORTANT.

YOU'LL HEAR FANTASTIC AND

ELEGANT SCIENCE TODAY AND

BEAUTIFUL SCIENCE THAT STEVE

ALSO FOSTERED AND ENCOURAGED US

TO DO OF HIS MEN TEES AND WE

WANT TO THANK HIM FOR HIS

MENTORSHIP.

TODAY YOU WILL HEAR ABOUT

ANOTHER TWO STORIES OF HIS

ELEGANT AND BEAUTIFUL SCIENCE,

AND AS I MENTIONED, PROTEINS OF

SILENCE RETROVIRUSES AND CLAN

RETROVIRUSES AND WE ARE THRILLED

YOU ACCEPTED THE INVITATION AND

WE LOOK FORWARD TO YOUR TALK.

STEVE?

[ APPLAUSE ]

>> THANK YOU VERY MUCH FOR THAT

NICE INTRODUCTION.

IT'S A TREMENDOUS PLEASURE TO BE

HERE.

I FEEL LIKE IT'S COMING HOME, IN

SOME WAYS.

I HAVE BEEN HERE SO MANY TIMES

AND HAVE SO MANY FRIENDS THEY

KNOW HERE.

SO IS IT A PLEASURE.

I'M GOING TO TRY TO TALK ABOUT,

BASICALLY, TWO SEEMINGLY AND IN

FACT, REALLY DIFFERENT TALKS.

THAT IS REPRESENTATIVE OF THE

WAY WE THINK AND CERTAINLY OF

THE KINDS OF DIFFERENT SCIENCES

WE DO IN MY LAB AT COLUMBIA.

SO I WILL BE TALKING FIRST ABOUT

SOME OF THE WORK THAT REALLY

REPRESENTS, I GUESS, MY DAY JOB,

WHICH IS WORKING ON THE MOUSE

AND LEUKEMIA VIRUSES, AND A

PARTICULAR INTEREST OF OURS IN

THAT AREA LATELY HAS BEEN A VERY

OLD, LON STANDING PROBLEM OF HOW

RETROVIRAL GENOMES ARE SILENCED

IN EMBYRONIC STEM CELLS.

SO I'LL TALK ABOUT THAT.

IT REPRESENTS A SIGNIFICANT

FOCUS OF THE LAB AND CERTAINLY

AN ONGOING AREA.

BUT THEN I HOPE LI SQUEEZE IN

TIME AT THE END FOR SOMETHING

THAT IS MORE OF A FUN PROJECT

FOR US ON THE SIDE.

AND IT IS MAYBE REPRESENTATIVE

OF OUR TENDENCY TO PICK UP ODD

BALL TOPICS AND EXPLORE THEM AS

MUCH AS WE CAN.

AND THERE IS SOME THREAD BETWEEN

THE TWO.

ALL RIGHT.

SO, WITH THAT, LET ME DIG RIGHT

IN.

TO SAY THAT THIS IMAGE HERE IS

AN IMAGE THAT I THINK MAYBE IS

IN THE LAB.

THIS REPRESENTS LEUKEMIA VIRUS

BUDDING FROM THE SURFACE OF

INFECTED PRODUCER CELL.

SO, THE STORY BEGINS WITH THE

VERY, VERY LONG-STANDING

REELINGS THAT THE MOUSE LEUKEMIA

VIRUSES THAT WE NORMALLY THINK

OF REPLICATING WELL AND DIVIDING

CELL, IS IN FACT, PROFOUNDLY

BLOCKED FROM REPLICATION IN

EMBYRONIC STEM CELLS.

SO THAT TYPE OF SILENCING

INCLUDES THE TWO TRADITIONAL

REAL EMBYRONIC STEM CELLS BUT A

VARIETY OF DIFFERENTIATED CELL

CELLS.

IN THOSE CELLS, IT'S ANYONE BONE

FOR A LONG, LONG TIME, THAT A

PARTICULAR STAGE IN THE LIFE

PSYCHE SELL PROFOUNDLY BLOCKED.

SO WHEN YOU TRY TO INFECT THOSE

CELLS, IN FACT, THE EARLIER

ADVANCE OF THE LIFE CYCLE COULD

BE PERFECTLY WELL.

VIRUS ENTERS THE CELL AND IF THE

CELLS ARE DIVIDING, THAT IS A

CRITICAL REQUIREMENT FOR

INFECTION BY THE MOUSE LEUKEMIA

VIRUSES.

TRANSCRIPTION WILL OCCUR AND

EVERYTHING OCCURS NORMALLY.

AT THAT POINT, THE LIFE OF THE

VIRUS STOPS.

THE DNA IS NOT TRANSCRIBED, AND

SO IN THAT SETTING, THE VIRUS IS

FROZEN POTENTIAL NETHAT STATE.

THAT FACTOR IS REALLY

APPRECIATED AS A PROBLEM VERY

EARLY ON WHEN PEOPLE STARTED TO

TRY TO DO GENE THERAPY BECAUSE

YOU WOULD WANT TO DELIVER GENE

ON A RETROVIRAL VECTOR INTO A

VARIETY OF PRIMITIVE CELLS,

HEMATOPOIETIC STEM CELLS OR TRUE

STEM CELLS AND THOSE EFFORTS

WERE IMMEDIATELY A FAILURE

BECAUSE THE GENES WERE

NOT -- PART OF THE BLOCK IS

ATTRIBUTABLE TO THE FACT THAT

THE PROMOTOR CONTAINED WITHIN

THE VIRAL LTR IS IN GENERAL,

POORLY ADAPTED FOR HIGH-LEVEL

EXPRESSION IN EMBYRONIC STEM

CELLS.

WE THINK SOME OF THE POSITIVE

TRANSCRIPTION FACTORS REQUIRED

FOR EXPRESSION OF THE VIRAL DNA

ARE AT LOW LEVELS.

BUT MORE PROFOUNDLY THERE IS AN

EXTREMELY POTENT BLOCK THAT IS

TARGETED TO A VERY SPECIFIC

ELEMENT IN THE RETROVIRAL DNA.

A SITE WE'LL TALK ABOUT CALLED

THE PRIMER BINDING SITE.

SO EMBYRONIC STEM CELLS

RECOGNIZE THE SITE AND USE THAT

SOMEHOW TO BLOCK TRANSCRIPTION.

AND SO THE PRIMER BINDING SITE,

WE NEED TO EXPLAIN.

SO THIS PBS ELEMENT IS A VERY

FAMOUS ASPECT OF THE RETRO VIRE

LIFE CYCLE.

IT'S A VERY SHORT, 18-19

NUCLEOTIDE ELEMENT FOUND IN ALL

RETROVIRUSES.

AND IN DEED, IN MANY OF THE

RETRO TRANSPOSONS IN OUR GENOME.

AND IT IS NOTED AS BEING

COMPLEMENTARY TO A CELLULAR

TRNA'S 3 PRIME END.

SO ITS MAIN FUNCTION THAT WE

THINK ABOUT IT HAVING IS IN

REGARD TO THE REVERSE

TRANSCRIPTION OF THE VIRAL

GENOME.

THIS IS AN ELEMENT WHOSE MAIN

GOAL IN LIFE IS TO ACT AT THE

RNA STAGE.

AND WHAT WE KNOW, OF COURSE

ABOUT IT, IS A CELLULAR TRNA

ALWAYS ONE PARTICULAR TRNA OF

THE CELL OF MANY, HAVE BEEN

CHOSEN BY A GIVEN VIRUS, AND

THAT TRNA YIELDS TO THE VIRAL

GENOME DURING THE ASSEMBLY AND

PRODUCTION OF VIRUS, AND THAT

PLAYS A CRITICAL ROLE AS THE

PRIMER FOR THE INITIATION OF DNA

SYNTHESIS IN THE NEXT ROUND WHEN

THAT VIRUS IS ENTERED TO THE

NEXT CELL.

SO DIFFERENT RETROVIRUSES

EVOLVED TO UTILIZE DIFFERENT

TRNAs AND IN PARTICULAR, OUR

FAVORITE LEUKEMIA VIRUS HAPPENS

TO BE PROLINE TRNA.

SO THERE IS ALWAYS, IN EACH

RETROVIRUS, A DIFFERENT PBS THAT

IS EVOLVED TO MATCH A GIVEN TRNA

AND SO THAT VIRUS HAS EVOLVED TO

USE THAT PARTICULAR TRNA AS A

PRIMER FOR DNA SYNTHESIS.

THAT ROUGHLY IS THE LINEAR

STRUCTURE WE THINK OF.

THIS RNA IS LONG, 8 OR 10 BASES

LONG AND THIS TRNA IS A

PARTICULAR PBS SITE NEAR THE 5

PRIME END OF THE GENOME.

SO WHEN THIS VIRUS DELIVERS THE

GENOME, THE FIRST THING THAT

HAPPENS IS THAT RIVERS

TRANSCRIPTASE EXTENDS THE 3

PRIME END OF THE TRN TOO.

MAKE A DNA MOLECULE AND THAT DNA

THEN TRANSLOCATES AND GOES

THROUGH A VARIETY OF OTHER

EVENTS TO FINALLY GIVE US THE

DNA GENOME.

SOCIETY PBS IS PLAYING

INCREDIBLY CRITICAL ROLE IN THE

LIFE OF THE VIRUS.

SO, IN FACT, OUR EMBYRONIC STEM

CELLS HAVE EVOLVED A SYSTEM THAT

RECOGNIZE THAT IS PBS AT A VERY

DIFFERENT TIME OF LIFE AT THE

DNA STAGE OF THE INTEGRATED

PROVIRUS.

SO THIS SILENCING OR RESTRICTION

OCCURS ONLY IN STEM CELLS THAT

WE KNOW OF.

WHEN YOU IN FACT HAVE A SILENT

PROVIRUS SITTING IN A PRIMITIVE

STEM CELL, AND IF YOU

DIFFERENTIATE THOSE CELLS, THEY

WILL LOSE THE RESTRICTION OR

PROFOUNDLY ATTENUATE THE

RESTRICTION AND AS A RULE, THE

PROVIRUS LIST BECOME INDUCED AND

EXPRESSION NOT INDUCED.

THE TRANSCRIPTION WILL BECOME

INDUCED.

WE PRESUME THERE RESTRICTION

SYSTEM EVOLVES TO PROTECT THE

GERMLINE FROM MUTAGENESIS.

NOTE THAT THIS BLOCK DOES NOT

PREVENT THE INSERTION OF THE DNA

TO THE CELL THAT WAS IMMEDIATELY

INFECTED, REVERSE TRANSCRIPTION

INTEGRATION STILL OCCURS, BUT

YOU DON'T HAVE AN ACTIVE VIREMIA

AND SPREAD FROM CELL TO CELL.

SO IT CERTAINLY WOULD LIMIT THE

INFECTION AND THE INTRODUCTION

OF DNA INTO THE GERMLINE.

PERHAPS AS WE'LL SAY LATER, A

MUCH MORE IMPORTANT ROLE HERE IS

TO KEEP SILENT ALL THE MANY

ENDOGENOUS RETROVIRUS DNAs WE

CARRY.

WE KNOW THE RESTRICTION IS

DEPENDENT ON THE ENTIRE PBS,

PRETTY MUCH, SO SINGLE-BASE PAIR

MUTATIONS ANYWHERE IN THE PBS

PRETTY MUCH, WILL KILL THE

RESTRICTION.

MANY YEARS AGO OTHERS LABS

WORKED WITH A SINGLE BASE PAIR

IN THE MIDDLE OF THE PB MALONEY

AND THAT RESTRICTED THE

PRESTRICTION.

IF YOU ALLOW THE VIRUS TO GO

THROUGH THE FORCE CYCLE OF

INFECTION, INTERESTINGLY, THAT

MUTATION IMMEDIATELY REVERTS

BACK TO THE WILDTYPE.

SO IF YOU WERE A CELL EVOLVING

RESTRICTION, THIS IS A PERFECT

SITE FOR YOU TO CHOOSE.

BECAUSE THE VIRUS REALLY CANNOT

MUTATE THIS PBS.

IT NEEDS THE SITE FOR ITS

EXISTENCE AND NEEDS TO BE ABLE

TO BIND TO THE TRNA TO THE SITE

AND THE PBS IS GENETICALLY

GENERATED BY REVERSE

TRANSCRIPTION USING THE TRNA

SEQUENCE ITSELF.

SO WHEN YOU HAVE FINISHED A

ROUND OF REVERSE TRANSCRIPTION,

EACH CYCLE CORRECTS ANY

MUTATIONS YOU MIGHT HAVE MADE

BECAUSE THE REVERSE

TRANSCRIPTION PROCESS DOESN'T

USE THE PBS OF THE VIRAL GENOME

TO MAKE THE DNA.

IT ACTUALLY FOR ONE OF THE

STRANDS, USES THE TRNA SEQUENCE

ITSELF.

SO VERY QUICKLY, THE VIRUS WILL

FORCEIBLY REVERT BACK IN A

MUTATION THAT MIGHT APPEAR IN

THE PBS BACK TO THE WILDTYPE

SEQUENCE.

SO, IT'S VERY HARD FOR THE VIRUS

TO ESCAPE THE RESTRICTION BY

MUTATION AND SO, THE RESTRICTION

THAT USES THAT SEQUENCE IS VERY,

VERY COLORFULLY ENGINEERED

PERFECTLY ATTUNE TO A CRITICAL

POINT ON THE VIRAL GENOME.

WHAT WAS KNOWN WHEN WHEN WE

STARTED IS THAT YES, THERE WAS

SOME ACTIVITY, SOME APPARENT

PROTEIN IN EMBYRONIC STEM CELLS

THAT RECOGNIZE THE PBS AND WE

COULD SEE THAT AS A MOBILITY

SHIFT ACTIVITY THAT WAS PRESENT

IN THE LINEUPAISE OF NUCLEI OF

EMBYRONIC STEM CELLS

SPECIFICALLY TARGETING STEM

CELLS THAT HAVE THE MLVPBS

ELEMENT.

ONE OF THE FEW WAYS A VIRUS CAN

ESCAPE THIS IS TO SWITCH THE

ENTIRE PBS TO MATCH A COM PLEALY

DIFFERENT TRNA AND RETROVIRUSES

EXIST AND THESE HAVE DONE THAT

OR MAKE USE OF A DIFFERENT TRNA.

ONE OF THE COMMON ONES THAT WE

WORK WITH IN THE LAB IS THE

VIRUS THAT USES GLUTAMINE RATHER

THAN PROLINE AS PRIMER, AND THAT

IS THE TRNA VIRUS CALLED THE

STEM CELL VIRUS.

WHY?

BECAUSE THAT VIRUS GROWS WELL ON

STEM CELLS.

GENE THERAPISTS WERE VERY HAPPY.

THEY COULD MAKE USE OF VIRUSES

LIKE THIS ONE THAT USED A

DIFFERENT TRNA BUT THE VIRUS CAN

JUST SWITCH QUICKLY.

IT HAS TO MAKE MANY, MANY

MODIFICATIONS TO ADAPT USE A

DIFFERENT TRNA BECAUSE THERE ARE

OTHER POINTS OF CONTACT ON THE

GENOME BESIDES THE PBS.

SO, WE ALL KNEW THAT THERE WAS

SOME FACTOR IN EMBYRONIC STEM

CELLS RECOGNIZED IN THE PBS AND

THEN SOMEHOW SILENCING THE

NEARBY INTEGRATED PROVIRUS FROM

AND PREVENTING IT FROM BEING

TRANSCRIBED.

WE KNEW THE SILENCING HAD A

RANGE WHICH WAS QUITE LARGE,

HENCE THE KB.

ONE COULD MANIPULATE THE PBS IN

VARIOUS RECORDED CONSTRUCTS AND

IN A RANGE.

IT ACTED UP DREAM DOWNSTREAM

INDEPENDENT OF ORIENTATION AND

IT WOULD SILENCE PROMOTORS THAT

WERE WITHIN MANY OF THE PBS.

IF YOU MUTATED IT BY HAND, YOU

COMPLETELY LOST THE RESTRICTION,

SO SOMETHING WAS RECOGNIZING

APPARENTLY ALL 18 NUCLEOTIDES OF

THE PBS IN DOING THE SILENCING.

WE ARE VERY TALENTED POSTDOCS

AND THEY CAME TO THE LAB AND SET

TOUT IDENTIFY AND CHARACTERIZE

THIS ACTIVITY.

SO, THE ACTIVITY WAS MANIFEST

HERE AS THE GEL SHIFT ACTIVITY

AND IT WAS VERY WELL BEHAVED.

SO IT WOULD BIND AND SHIFT,

LABEL DNAs AND GO TO THE SITE.

IT WOULD BE RELATIVELY SOLUBLE

AND IT WAS A VERY LARGE HIGH,

MOLECULAR WADE COMPLEX.

THE GEL SHIFTED DNA USUALLY BUT

IT WAS RELATIVELY STABLE AND

WELL BEHAVED.

IT WOULD FRACTIONATE IN MANY

WAYS.

YOU COULD PUT IT OVER COLUMNS

AND SO IT -- WE COULD START OUT

TRYING TO PURIFY IT.

AND WORKED OUT A PURIFICATION

SCHEME SHOWN HERE.

WE'LL WALK YOU THROUGH IT.

AT THE END OF MANY OF THESE

STEPS IN PURIFICATION, HE HAS

SOMETHING THAT WAS BY NO MEANS

PURE.

BUT WERE CERTAINLY VERY PURIFIED

COMPARED TO A CERTAIN NUCLEAR

LINEUPAISE AND STILL HAD THE

INITIAL WELL BEHAVED SHIFT

ACTIVITY.

SO WE SENT THAT OFF TO THE MASS

SPECK PEOPLE AND THE MAJOR

PROTEIN THEY IDENTIFIED FOR US

WAS THIS PROTEIN, TRIM 28.

AND THAT WAS VERY SATISFYING

BECAUSE TRIM 28 IS A MEMBER OF A

VERY WELL STUDIED FAMILY NOW,

THE TRIM FAMILY.

THEY ARE THE TRIPARTIED MOTIF

FAMILY.

IT HAS A RING FINGER, B BOX,

ANDY COILED COIL AND THEY ARE

KNOWN TO BE KEY ANTIVIRAL

PROTEINS.

MOST OF THE MEMBERS OF THIS

FAMILY HAVE VARIOUS ACTIVITIES

INVOLVED WITH SILENCING VARIOUS

VIRUSES.

THIS ONE IN PARTICULAR WAS

WELL-KNOWN AND STUDIED AND HAS

OTHER NAMES.

AND IT WAS WELL-KNOWN IN VERY

DIFFERENT SETTINGS TO BE A

REPRESSOR, TO BE A COREPRESSOR.

IT ACTS ON DOZENS, MAYBE

HUNDREDS OF GENES, AND IT IS

THOUGHT TO ACT WITH OR KNOWN TO

ACT BY BINDING TO THE CRAB BOX

DOMAINS OF ZINC

FINGER-CONTAINING PROTEINS, THAT

WERE THEY THEMSELVES DNA-BINDING

PROTEINS.

SO TRIM 28 ITSELF,ATIVE 1 IS NOT

A DNA BINDING PROTEIN, BUT

TETHERED TO DNA BY THESE ZINC

FINGER GUYS AND SOMEHOW HAD

SILENCING ACTIVITY IN MANY

DIFFERENT SETTINGS.

PROBABLY MOST STUDIED BY FRANK

RUSH AND MANY OTHER LABS WHO

STUDIED THESE ACTIVITIES IN A

VARIETY OF CELLS, NOT INCLUDING

EMBYRONIC STEM CELLS IN

PARTICULAR.

IT IS KNOWN WHEN IT IS

TOGETHERRERED TO DN TOO.

INTERACT WITH A WHOLE HOST OF

PROTEINS -- TETHERED  --

AND WE ARE CONTINUING TO TRY TO

EXPAND THE LIST OF PROTEINS THAT

INTERACT WITH IT.

THEY ARE INVOLVED IN THIS

REPRESSION ACTIVITY.

THIGH INCLUDE CHROMATIN

MODIFIERS LIKE THE NURD COMPLEX,

CALLED HP1, AND ESET, THE

HISTONE H3K9 METHYL TRANSFERASE.

SO I THINK IT'S APPROPRIATE TO

THINK OF TRIM 28 AS A BRIDGING

MOLECULE THAT BRIDGES BETWEEN

VARIOUS DNA BINDING PROTEINS AND

THE MACHINERY THAT IS REQUIRED

TO MEDIATE THE SILENCING.

AND THOSE INCLUDE GUY THAT IS

WILL PLAY WITH CHROMATIN, MODIFY

HISTONES IN THE CHROMATIN AND

EVENTUALLY ALSO DNA METHYL

TRANSFERASE WILL SILENCE

EVENTUALLY THE DNA AS WELL.

SO THIS WAS A VERY ATTRACTIVE

PROTEIN FOR US TO BE WORKING

THAT THE LEVEL.

SO FIRST WE WANTED TO DON FIRM

THAT YES, THIS WAS REALLY -- WE

WANTED TO CONFIRM -- THIS WAS A

COMPONENT OF THE COMPLEX THAT WE

WERE ASS IING UP TO THIS POINT

AS THE GEL SHIFT ACTIVITY.

AS AING.

SO LOOKING BACK THROUGH ALL THE

FRACTIONATIONS, HE COULD SHOW

VERY QUICKLY THIS PROTEIN'S

PRESENCE MEASURED BY WESTERN

BLOT, TRACKED PERFECTLY WITH THE

DNA BINDING ACTIVITY AND WE'LL

WALK YOU THROUGH THAT.

HERE IS THE LAST COLUMN.

HERE IS THE FRACTIONING.

HERE IS THE GEL SHIFT ACTIVITY

AND IN DEED HERE IS THE PROTEIN

TRACKING WITH THE GEL SHIFT

ACTIVITY.

SO IT WAS CLEARLY PART OF THE

PURIFIED COMPLEX.

AND IT WAS INDEED PRESENT ON THE

GEL SHIFT BAND ITSELF.

AND HE COULD SHOW THAT BECAUSE

HERE IS THE NICE SHIFT WORKING

AS USUAL.

AND IF WE WOULD ADD ANTIBODIES

TO TRIM 28, WE GOT A VERY NICE

SUPER SHIFT.

SO THE PROTEIN WAS THERE.

IT WAS BEING RECOGNIZED BY THE

ANTIBODY AND NICELY SUPER

SHIFTED TO A VERY DISCRETE

HIGHER MOBILITY OR LOWER

MOBILITY BAN SO IT WAS CLEARLY

THERE.

AND THAT BAND BEHAVED AS IT

SHOULD IN EVERY WAY.

IT WOULD COMPETE WITH COLD DNA

JUST AS THE AUTHENTIC BAND DID.

EVERYTHING ABOUT THIS WAS

CLEARLY INDICATIVE OF THE

PROTEIN BEING PRESENT IN THE

SHIFT COMPLEX ITSELF.

IT HAD TO BE EXPRESSED IN THE

CELLS THAT WERE MEDIATING THE

SHIFT, FORMING THE COMPLEX,

BECAUSE IF YOU DID RNAI MEDIATE

THE KNOCK DOWN OF THIS GENE IN

THE EC CELL, WE COULD SHOW WE

COULD ELIMINATE THE PROTEIN

DETECTED BY WESTERN BLOT AS

SHOWN HERE, AND WHEN WE DID THAT

WITH APPROPRIATE RNAIs,

CONCORDANTLY WITH THE DECREASE

IN PROTEIN LEVEL, WE GOT

DECREASES IN THE GEL SHIFT

ACTIVITY SHOWN HERE.

THIS WAS TRACKING THAT WAS VERY

GOOD.

THIS WAS NOT DUE TO SOME

OFF-TARGET ACTIVITY OF THE RNAI

BECAUSE WE COULD RESTORE THE GEL

SHIFT BY REEXPRESSING RNAI

RESISTANT VERSION WITH A TAG

WHICH WE DID HERE.

THE TAG ADDS EXTRA MOLECULAR

WEIGHTS TO THE GEL SHIFT AS IT

GETS BIGGER.

THE PROTEIN GETS BIGGER.

AND THIS WAS DELIVERED TO THE

CELLS WITHIN ADNO-VIRUS VECTOR.

SO BY ALL THE CRITERIA.

NOW THIS SHIFTED REPLACED AND

THE BAND HAD A TAG SO WE COULD

SUPER SHIFT WITH ANTIBODIES TO

MIX WHICH WE COULD NOT DO

NORMALLY.

SO BY ALL THE POSSIBLE CRITERIA,

WE COULD THINK OF, THE GENE HAD

TO BE EXPRESSED BY THE CELLS TO

CREATE THE COMPLEX THAT WOULD BE

UNDERGOING THE GEL SHIFT.

IN THIS -- AND THIS WAS ACTIVE

INDEED IN THE CELLS.

THIS WAS REQUIRED NOT JUST FOR

THE GEL SHIFT BUT REQUIRED FOR

THE SILENCING ACTIVITY.

SO FOR THESE EXPERIMENTS, WHAT

WE WOULD DO TYPICALLY IS MEASURE

THE EFFICIENCY OF CONVECTION, IF

YOU WILL, THE EFFICIENCY OF

TRANSFER OF A REPORTER GENE INTO

CELLS USING RETROVIRAL VECT

AUTHORITIES MAKE USE OF EITHER

THE WILDTYPE PRO PBS CONSTRUCT,

WHICH WOULD BE SILENCED IN THE

ESL, AND ONE THAT MADE USE OF A

MUTANT BB.

THAT WOULD NOT BE SILENCED SO

THESE GENOMES WOULD BE IDENTICAL

THROUGHOUT WITH THE EXCEPTION OF

A SINGLE BASE PAIR CHANGE IN THE

PBS.

WE WOULD AVOIDING CORRECTION

ISSUE MOST OFTEN BY TRANSFECTING

THE DNAs BUT TWO OTHER TRICKS

WE COULD THEFT THESE.

BY MEASURING THE RATIO

EXPRESSION WAS TO REPORTERS, WE

COULD ASK THE EXTENT OF THE

SILENCING AND THE EFFICIENCY OF

THE SILENCING AND IN PARTICULAR,

CELL TYPE.

IF WE COMPARE THESE FOR THE

DELIVERY SAY OF GP18 RESISTENCE,

THESE ARE EQUALLY EFFECTIVE.

THE RATIO WOULD BE 1.

IF WE ENT INTO EMBYRONIC STEM

CELLS OR EMBYRONIC CARCINOMA

CELLS WHICH FLARE-UP JUST AS

WELL, THE RATIO WOULD BE IN THE

REALM OF 100.

THIS WAS GOOD SILENCING.

IF WE LOOKED AT THE KNOCK DOWN

WITH THE SCRAMBLED RNAI, THE

RESTRICTION REMAINS BUT IF WE

SUCCESSFUL KNOCK DOWN TRIM 28

USING A TARGETED RNAI, WE LOST

VIRTUALLY ALL OF THE SPECIFIC

SILENCING AND THE RATIO CAME

BACK CLOSE TO FULL.

SO, INDEED, TRIM 28 HAD TO BE

EXPRESSED FOR THE SHIFT BUT ALSO

HAD TO BE EXPRESSED FOR THE

SILENCING.

SO WE KNEW AT THIS POINT TRIM 28

WAS THE CRITICAL COMPONENT OF

THE SILENCING COMPLEX.

IT WAS PRESENT.

IT WAS REQUIRED.

BUT CERTAINLY IT WAS NOT THE

WHOLE STORY BECAUSE AS I SAID,

TRIM 28 IS NEEDED BUT IT'S NOT

SUFFICIENT.

IT'S NOT THE ES CELL.

AND IT'S QUITE ABUNDANT AND IN

ALL CELLS.

IT'S NOT WORKING IN

DIFFERENTIATED CELLS.

IT'S NOT AN ES-CELL SPECIFIC

THING MAKING THEM RESTRICTED.

AND OF COURSE IT'S NOT A

DNA-BINDING PROTEIN ITSELF.

SO IT WAS SOMETHING MORE

IMPORTANT GOING ON AND WE REALLY

WANTED THEN TO KNOW WHAT WAS THE

DNA BINDING COMPONENT OF THE

COMPLEX SEEING ITSELF THE PBS

AND PRESUMABLY WHAT WOULD BE

THEN THE ES-CELL SPECIFIC ASPECT

OF THE -- THAT REQUIRES FURTHER

PURIFICATION.

WE DIDN'T SEE ANYTHING OBVIOUS

IN THE MAS SPECK RESULTS WITHOUT

THAT FURTHER PURIFICATION THAT

ACCOUNTED FOR THE ACTIVITY AND

NOW WE COULD AS A -- ADD A TRIM

28 AFFINITY STEP WHICH IS A HUGE

PURIFICATION STEP.

SO HAVING BEEN PULLED OUT

ANYTHING IN THIS MOST HIGHLY

PURIFIED FRACTION THAT IS ALSO

HAD TRIM 28 AND THEN SENT THAT

OFF TO MASS SPECK, WE WERE ABLE

TO FIND A SMALLER NUMBER OF

PROTEINS THAT WERE CANDIDATES

AND THE KEY ONE TURNED OUT TO BE

THIS GUY.

ZINC FINGER PROTEIN 809.

THAT IS THE CRITICAL COMPONENT

AND I'LL SHOW YOU SOME OF THE

TESTS WE USED TO CONFIRM THAT

809 IS THE KEY.

SO, ZFP809 WAS A PREVIOUSLY

UNKNOWN MEMBER OF A VERY, VERY

LARGE FAMILY.

THEY ARE NOW PROBABLY AROUND 400

ZINC FINGER PROTEINS IN THE

GENOME.

SO THIS IS ONE OF THE LARGEST

FAMILIES, MAYBE OUTSIDE OF THE

OLFACTORY RECEPTOR FAMILY.

THIS GUY CONTAINS A KRAB DOMAIN,

THIS IS TO WHICH 28 BINDS AND

THIS HAS 7 ZINC FINGERS.

THESE ARE OF THE VERY

TRADITIONAL TYPE.

SO THESE ZINC FINGERS BROADLY

RECOGNIZE ABOUT THREE BASE PARIS

OF DNA.

SO THESE 7 WOULD BE IN PRINCIPAL

RESPONSIBLE FOR RECOGNIZING

ABOUT 21 OR SO BASE PAIRS.

A REASONABLE STRETCH OF DNA.

WE WOULD CERTAINLY PREDICT THAT

NOW WE KNOW THIS IS A DNA

BINDING PROTEIN.

ONE OF THE THINGS WE FOUND OUT

VERY EARLY ON IS THE C TERMINAL

50 AMINO ACIDS MAKES THE PROTEIN

UNBELIEVABLY TOXIC.

INTOL RATED IN DIFFERENTIATED

CELLS AND EVEN SOMEWHAT TOXIC IN

EMBYRONIC CELLS AS WELL.

SO THIS IS NOT A PROTEIN THAT IS

EASILY EXPRESSED.

WE HAVE NO IDEA WHY IT'S TOXIC.

WHEN WE TRY TO TRANSFECT

EXPRESSION CONSTRUCTS INTO MOST

CELLS, THEY QUICKLY DIE.

IF YOU REMOVE THE LAST 15 AMINO

ACIDS, THE PROTEIN LOST MOST OF

ITS TOXICITY, NOT ALL TESTIFY

BUT IT WAS WORKABLE AND IT

RETAINED THE DNA BINDING

ACTIVITIES AND THE SILENCING

ACTIVITIES WE NEEDED.

SO IT RE9s THIS DAY AN UNKNOWN

ISSUE OF HOW IT IS AND WHY IT IS

THIS PROTEIN IS SO TOXIC IN

NORMAL CELLS AND EVEN TOX NICK

EMBYRONIC STEM CELL.

PERHAPS THAT TOXITY EXPLAINS WHY

THIS PROTEIN IS NOT JUST

EXPRESSED ALL THE TIME TO

PROTECT CELLS FROM RETROVIRUSES.

SO, ONE OF THE THINGS WE

IMMEDIATELY COULD SHOW IS

RECOMBINANT ZFP809 MADE IN

E.COLI, ALL BY ITSELF, WITH NO

OTHER MAMMALIAN PROTEIN PRESENT

WAS CAPABLE OF RECOGNIZING THE

COALINE PBS.

SO, HERE IS THAT EXPRESSION.

SO WE SIMPLY, THESE ARE JUST

BACTERIAL LINEUPAISE EXPRESSING

THE PROTEIN AND WE ARE ASKING

THOSE TO BIND TO DNAs IN THE

GEL SHIFT ASSAY MUCH LIKE THE

ONES WE WOULD RUN IN NUCLEAR

EXTRACTS AND WHAT WE FOUND IS

YES, IF YOU EXPRESS 809, YOU GET

VERY NICE PROLINE SPECIFIC GEL

SHIFTS AND IF YOU CHANGE ANY ONE

OF THESE BASE PAIRS, IF YOU

CHANGE THESE, YOU LOSE ALL OF

THAT SPECIFIC DNA BINDING

ACTIVITY.

TWO OTHER PROTEINS WE ALSO

PURIFIED IN THE COMPLEX, AND

THEREFORE WERE CANDIDATES, DID

NOT DO THAT.

ANOTHER ZINC FINGER PROTEIN

DIDN'T DO IT.

A VERY INTERESTING PROTEIN

CALLED L1TE1 WHICH IS RELATED TO

L1 ELEMENTS, DOESN'T DO IT.

BUT WE THEN IS GENUINELY INDEED

PART OF THE COMPLEX.

WE DON'T KNOW WHAT IT'S ROLE IS,

WE ARE WORKING ON IT.

809 WAS THE GUY THAT CLEARLY

RECOGNIZED THE PBSDNA.

AND THE SHIFT THAT IS GENERATED

WAS OF A MUCH HIGHER MOBILITY

AND THE GEL SHIFT WOULD HAVE

LEFT.

SO IT WAS NOT RE-CREATING THE

FULL COMPLEX.

WE CAN'T DO THAT OF COURSE.

BUT IF WE MIXED IT WITH

BACTERIAL LINEUP ACE EXPRESSING

TRIM 28, THE TWO OF THOSE

PROTEINS TOGETHER BOUND AND MADE

A MUCH LARGER COMPLEX.

THEY WERE ON THEIR OWN,

RE-CREATING A CLOSER-TO-REALITY

COMPLEX SHOWN ON THE DNA.

HERE IS 809 DOING THIS SMALL

SHIFT ALONE.

HERE IT'S NOT DOING IT ON A

MUTANT PBS BUT HERE IS THE

MIXTURE OF INDETERMINANT 809 AND

TRIM 28 FORMING A LARGER COMPLEX

TOGETHER, AND YOU CAN SEE THAT

THE COMPARABLE AMOUNTS, LOW

AMOUNTS OF 809, NOW WILL BIND

DNA IN THE PRESENCE OF TRIM 28

AT FAR LOWER CONCENTRATIONS.

SO THIS AMOUNT OF 809 IS IN THE

LOW END AND WITH TRIM 28 NOW,

THE AFFINITY HIGHER.

THE GEL SHIFT IS MUCH BETTER.

AND CLOSER TO THE NORMAL SIZE OF

THE GEL SHIFT MADE IN AN

AUTHENTIC MAMMALIAN CELL.

THIS IS A HIGH SHIFT AND IT'S

NOT VERY SENSITIVE TO THE SIZE

OF THE COMPLEX.

SO WE THINK THERE ARE ACTUALLY

MANY MORE PROTEINS MISSING IN OR

NOT PRESENT IN THIS BACTERIAL

RECOMBINANT FORM COMPLEX THAT

ARE PRESENT IN THE REAL ONES,

EVEN THOUGH WE DON'T SEE MUCH

FURTHER SHIFT.

IT RETAINS PLASTICITY WHERE A

SINGLE BASE PAIR KILLS THIS

BINDING.

THESE WERE TWO OF THE KEY

PLAYERS.

MAYBE MOST INTERESTING WAS THE

IN FACT IF WE SIMPLY EXPRESS 809

DE NOVO IN A DIFFERENTIATED

CELL, THAT WAS ALL IT TOOK FOR A

NORMAL CELL TO RE-CREATE THE GEL

SHIFT COMPLEX.

EVERYTHING ELSE THAT WAS NEEDED

WAS THERE.

SO WE COULD WORK WITH ENDOGENOUS

LEVELS OF TRIM 28.

IF IT WAS A NORMAL CELL, THAT

CELL WILL CREATE THE GEL BINDING

SHIFT, DNA BINDING COMPLEX VERY

NICELY.

SO, HERE WHAT WE HAVE DONE IS

SIMPLY TRANSEFFECT THESE CELLS,

IT WORKS IN A VARIETY OF CELLS,

WITH EXPRESSION CONSTRUCT FOR

809.

SO, CLONE 5 WITH THE CLONE THAT

EXPRESSED NICE LEVELS OF 809 IN

SPITE OF THE TOXICITY ISSUE.

WE ARE USING NOW THE CRITICISM

TERMINAL TRUNCATED VERSION TO

AVOID THE TOXICITY AND THAT CELL

WILL HAVE DNA BIND BINDING

ACTIVITY THAT SHIFTS PROLINE.

DOES NOT SHIFT B2.

IT IS SUPER SHIFTED WITH

ANTIBODIES AS IT SHOULD BE TO

TRIM 28.

SINCE REARE NOW EXPRESSING A

FLAG TAG VERSION OF 809, IT CAN

BE SHIFTED WITH FLAGS.

SO 809 AS WELL AS TRIM 28 ARE

PRESENT IN THE COMPLEX IN THE

WESTERN BLOT HERE JUST CONFIRM

ALL THE CONSTRUCTS.

SO CLONE 9 DOES NOT EXPRESS

ZFP809 DOESN'T RE-CREATE THE

SHIFT.

AND THIS TOPIC EXPRESSION OF 809

IN A DIFFERENTIATED CELL NOT

ONLY RE-CREATED THE GEL SHIFT

COMPLEX BUT ALSO CORRECTLY

RE-CREATED THE ANTIVIRAL

ACTIVITY, THE SILENCING

ACTIVITY.

BUT HERE WE TAKE A WHOLE PANEL

OF THESE CLONES THAT HAVE BEEN

TRANSFECTED SO TO EXPRESS 809.

THE SUCCESSFUL ONES ARE

INDICATED HERE BY THE WESTERN

BLOT AND FOR 809, THOSE CLONES

THAT DON'T HAVE IT, DON'T.

AND THEN WE SIMPLY TESTED THOSE

THROUGH THAT SAME PAIR OF

RETROVIRAL GENOMES, PROLINE OR

NOT.

AND WE MEASURE A RATIO OF THEIR

EXPRESSION AND MEASURE THE

SILENCING AND SO EACH OF THE

CLONES THAT DON'T EXPRESS 809

HAVE RATIOS OF EXPRESSION

ESSENTIALLY AT 1.

THEY DON'T CARE WHERE THE VIRUS

USES PROLINE OR NOT BUT EVERY

TIME YOU EXPRESS 809, YOU GET

VERY NICE PROLINE-SPECIFIC

SILENCING CLOSE TO THE REALM.

SO THE MERE EXPRESSION EVER 809

WAS SUFFICIENT FOR THE CELL TO

UTILIZE ALL THE REST OF THE

MACHINERY THAT IT ALREADY HAD.

TO CREATE THE SILENCING.

AND THAT WAS NOT ONLY MANIFESTED

SILENCING A REPORTER THAT WAS

EMBEDDED IN THE RETROVIRAL

GENOME.

THE REPORTER COULD BE UNDER

RESISTENCE OR LUCIFERASE BUT IT

COULD ALSO BE REPRESSED

AUTHENTIC VIRUS REPLICATION.

FOR THESE EXPERIMENTS, WE ARE

SIMPLY INFECTING CELLS ON DAY

ZERO WITH VIRUS THAT IS USE

EITHER PROLINE OR THE STEM CELL,

GLUTAMINE PBS, AND THESE VIRUSES

ARE OTHERWISE VIRTUALLY

IDENTICAL.

AND THOSE TWO VIRUSES REPLICATE

EQUALLY WELL ON ORDINARY CELLS,

DIFFERENTIATED CELLS.

IT'S THE CELL THAT DON'T EXPRESS

809 THAT REPLICATE PRETTY MUCH

EQUALLY WELL F THEY NOW HAVE

BEEN ENGINEERED TO EXPRESS 809,

THE PROLINE VIRUSES IS

COMPLETELY SILENT TO PREVENT

FROM REPLICATING WHERE THE

GLUTAMINE REPLICATES AND SPREADS

VERY WELL.

WE USED THE MEASUREMENT OF THE

REVERSE TRANSCRIPTASE ACTIVITY

PRESENT IN THE MEDIUM AS OUR

READ OUT OF VIRAL SPREADS IN

EXPERIMENTS.

SO THIS WAS THE ONLY GUY.

THE GUY YOU NEEDED TO EXPRESS TO

MAKE CELLS BECOME

EMBYRONIC-LIKE, AT LEAST IN

REGARDS TO THEIR SILENCING OF

RETROVIRAL GENOME.

SO WE HAVE DONE A LOT OF WORK

LATELY TO TRY TO LOOK AT A BIT

MORE ABOUT HOW THE COMPLEX

WORKS.

WE KNOW THAT GENUINELY SITTING

ON THE DNA OF THE SILENCED

RETROVIRUSES.

THIS WAS SORT OF OUR ENTREE INTO

THAT.

THESE ARE ESSENTIALLY CHIP

EXPERIMENTS WHERE WE ARE

CROSSLINKING THE COMPLEX ON DNA,

FRAGMENTING,

IMMUNOPRECIPITATING, UNLINKING

AND DOING PC.

TO ASK WHAT WE HAVE RECOVERED IN

THE CHROMATIN IP AND WHAT WE

FIND IS THAT YES, 809 IS VERY

NICELY CROSSLINKING TO THE

PRIMERS THAT ATTACK THE PB IS.

THERE IS THAT CROSSLINK.

TRIM 28, OF COURSE IT'S MUCH

MORE DISTANT FROM THE DNA SORE

THE CROSSLINKING IS DIFFICULT

BUT IT'S CLEARLIY THERE AND NOT

THERE IN THE CASE WHERE IS THE

VIRUS THAT USE GLUTAMINE.

WE ARE SURE THAT THIS WHOLE

COMPLEX IS SITOTH DNA IN VIVO IN

CELLS  --

SITTING ON THE DNA -- WE ASKED,

IS THIS RIGHT?

ANY TRUE EMBYRONIC STEM CELL

SPECIFIC GUY.

WE WERE PLANNING TO EMBARK ON A

LONG PROGRAM OF LOOKING AT THE

PROMOTOR OF THE 809 GENE ASK HOW

IS IT THAT IT WAS EXPRESSED SO

WELL IN EMBYRONIC STEM CELLS AND

NOT IN OTHER CELLS AND WE WERE

SURPRISED TO FIND THAT THIS IS

NOT THE CASE.

THAT IN FACT, 809 MESS GER RNA

IS PRESENT IN ALL CELLS.

IT IS EXPRESSED WELL AS MEASURED

BY RTPCR IN EMBYRONIC STEM CELLS

BUT ALSO EXPRESSED IN

FIBROBLASTS MANY OTHER

DIFFERENTIATED CELLS AT

COMPARABLE LEVELS FOUND IN ES

LIFE CELLS.

WE FOUND THAT THE PROTEIN IS

VERY SPECIFIC TO ES-CELLS.

AS WE CAN TELL WHAT WE HAVE,

IT'S NOT EXPRESSED IN

DIFFERENTIATED CELLS EVEN THOUGH

THE MESSAGE IS.

SO AT THIS POINT, ALL WE KNOW IS

THAT THERE MUST BE SOME POST

TRANSCRIPTIONAL REGULATION GOING

ON IN THE VAST MAJORITY OF OUR

CELLS THAT PREVENTS SOMETHING OR

THAT PREVENTS THE CORRECT

FORMATION OF THE PROTEIN.

WE KNOW FROM IMMUNOFLOURESENCE

THAT IN CELL SPECIFIC EXPRESSION

IS FOUND IN THE NUCLEUS AND THE

CYTOPLASM BUT THAT IS ALL WE

KNOW PRETTY MUCH AT THIS POINT

AS TO HOW IT IS WORKING.

WE STARTED LOOKING AT THE

LIFETIME OF THE PROTEIN AND SOME

ISSUES ABOUT HOW IT IS

TRANSLATED AND THAT IS YET TO BE

DETERMINED.

AT THIS POINT, OUR MODEL IS THAT

YES, THERE IS A LARGE COMPLEX

THAT IS MEDIATING THE

RESTRICTIONS.

IT IS SOMETHING THAT IS LARGELY

EXPRESSED IN MANY CELLS BUT THAT

WHEN 809 IS EXPRESSED, THIS

COMPLEX WAS TETHERED TO THE

CRITICAL PBSs AND IT MUST THEN

ACT TO SILENCE THE GENOME.

WE KNOW THAT THERE MIGHT BE, WE

KNOW THERE ARE POSSIBLY OTHER

ZINC FINGER GUYS THAT RECOGNIZE

OTHER PBSs.

WE THEREIN IS ONE IN MOUSE CELLS

RECOGNIZED AS VIRUS THAT IS USE

LYSINE TRNA.

WE EXPECT THERE ARE MORE.

SO I THINK OUR CURRENT NOTION IS

THAT THERE IS PROBABLY AN ARRAY,

A HANDFUL OF ZINC FINGER

PROTEINS THAT SPLICE EINVOLVED

PRESUMABLY OTHERS THAT HUMANS

HAVE EVOLVED THAT WILL TARGET

THIS MACHINERY TO MEDIATE THE

SILENCING OF MANY BUT NOT ALL

RETROVIRUSES THAT ARE

CHALLENGING US TO INVADE OUR

CELLS.

AND WE CONTINUE TO ASK A LOT OF

QUESTIONS ABOUT HOW THE

SILENCING HAPPENS.

WE KNOW THERE WILL BE OTHER

PLAYERS.

ONE THAT WE KNOW IS IMPORTANT IS

A PROTEIN CALLED HP1.

HETEROCHROMATIN PROTEIN GAMMA.

THERE ARE ALPHA BETA GAMMA HP

1S.

IT IS A KEY PROTEIN AT

HETEROCHROMATIN AND ITS PRESENT

AT TEAL MEERS AND SOME

UCHROMATIC SITES WHERE GENES ARE

SILENCED.

IT'S STABLEDIDES TOW SILENCE

CHROMATIN BY H3K9 DIMETHYL MARKS

ON THE HISTONE.

AND IS IT KNOWN TO BE A KEY

COMPONENT THAT BRINGS WITH IT

DNA METHYL METHYL TRANSFERASES.

WE THINK OF ITS IS A BRIDGING

MOLECULE TO GUYS LIKE THE DNA

METHYL TRANSFERASE.

IT'S AN INTERESTING PROTEIN

BECAUSE IT MEDIATES HERITABLE

SILENCING ON A TARGETED GENE

EVEN AFTER THE HP1 ITSELF HAS

BEEN REMOVED, SO THE MARKS

REMAIN.

THE HISTONE MARKS AND THE DNA

MARKS CAN PERSIST OVER MANY

GENERATIONS TO CONTINUE TO

SILENCE THE GENE THAT IT HAS

ACTED UPON.

WE KNOW IT'S ABSOLUTELY CRITICAL

FOR THE EMBYRONIC STEM CELL

SILENCING BECAUSE THERE EXISTS

MUTANTS OF TRIM 28 THAT HAVE

BEEN ENGINEERED BY THE GROUPS.

THESE ARE VERY ELABORATELY

ENGINEERED CELLS, CELLS IN WHICH

ALLELES HAVE BEEN KNOCKED OUT

AND ANOTHER HAS BEEN REPLACED BY

TRIM 28 THAT SPECIFICALLY

INTERACTS WITH HP1.

AND THESE HAVE BEEN MADE IN F9EC

CELLS, PERFECTLY FOR US.

SO ALL WE HAVE TO DO WAS MEASURE

THE FOLD RESTRICTION IN THOSE

CELLS AS COMPARED TO NORMAL

CELLS.

HERE IS THAT RATIO OF SILENCING

OF THE TWO MARKER REPORTER

GENOMES.

THOSE ARE NOT SILENCED.

THEY ARE EQUALLY EXPRESSED IN

RAT FIBROBLASTS.

IN NORMAL HETEROZYGOAT CELLS

WHERE ONE ASHEOL NIL AND THE

OTHER IS WILDTYPE, THERE IS

PLENTY OF TRIM 28 FOR SILENCING

BUT IF WE HAVE TESTS IN THE

CELLS THAT HAVE ONE ALLELE GONE

AND THE OTHER MUTANT, WE LOSE

ALL THE RESTRICTIONS.

SO HP1 INTERACTION WITH TRIM 28

IS CRITICAL, TOTALLY CRITICAL

FOR THE SILENCING.

AND WE THINK AND WE KNOW THAT

THERE WILL BE OTHER NEW PROTEINS

INVOLVED AS WELL.

SO WE LEARNED A VARIETY OF

PROTEOMICS SCREENS TO LOOK FOR

WHAT OTHER PROTEINS ARE BOUND TO

EACH PLAYERS.

WE FOUND THAT ONE WE LIKE A LOT

IS A POORLY-STUDIED PROTEIN,

EBP1 INVOLVED IN SIGNAL

TRANSDUCTION NAMED AFTER ITS

SUPPOSED ROLE IN BINDING ERB3.

IT HAS SOME ANTIVIRAL HISTORIES.

THERE IS NOT MUCH KNOWN ABOUT

IT.

BUT WE FOUND IT AS BINDING ON

ZINC FINGER 809 DIRECTLY.

WE FOUND THAT BY RNAI

KNOCKDOWNS, WE LOSE MOST OF THE

SILENCING.

IT'S NOT AS PROFOUND AS THE LOSS

OF TRIM 28 OR 809 ITSELF, BUT A

VERY LARGE PORTION OF THE

SILENCING IS LOST WHEN YOU KNOCK

DOWN EBP1.

SO WE ARE NOW EMBARKING ON THAT.

LET ME SUMMARIZE THIS PART OF

THE STORY, THIS PROGRESS REPORT.

WE KNOW THAT SILENCING IS

CRITICAL TO THE LIFE OF THE

EMBYRONIC STEM CELL.

TRIM 28 ITSELF KNOCKOUT IS

LETHAL IN THE MOUSE.

THAT LETHALITY IS NOT SIMPLY DUE

TO THE FAILURE TO SILENCE

INCOMING VIRUS OR EVEN PROBABLY

ENDOGENOUS RETROVIRUSES.

MANY GENES ARE NOT SILENCED.

BUT CERTAINLY AT THE IS TRUE

WHEN YOU KNOCK OUT TRIM 28 EARLY

IN EMBRYO GENESIS, THE MAP OF

INDUCTION OF EXPRESSION, VERY

BAD THINGS HAPPEN WITHOUT TRIM

28.

WE THINK THE SILENCING INVOLVED

HISTONE METHYLTRANSFERASES AND

HISTONE DEACETYLASES NOW.

THAT'S ALL PART OF THE

SILENCING.

WE THINK THIS WHOLE BUSINESS IS

A VERY GENERAL FACT OF LIFE FOR

STEM CELLS.

IT'S A PARDON OF STEMNESS.

AND THERE IS SOME ONGOING WORK

TO SHOW PRETTY CLEARLY IN IS

PART OF THE SILENCING OF HIV

PROVIRUSES IN ITS RESERVOIRS

WHERE IT LIVES IN SILENT CELLS.

AND JONATHAN CAR AND OTHERS

SHOWN THAT KNOCKDOWN OF HP1

ACTIVATES LATENT HIV1 GENOME TO

THE EXTENT THAT THOSE REPRESENT

TRUE MODELS OF THE CELLS IN

WHICH HIV1 HIDES IN THE AFFECTED

GENOME.

ALL THE WORK ON THE SILENCING I

TOLD YOU ABOUT WAS DONE BY DAN

WOLF AND THE WORK CONTINUES NOW

IN MY LAB WITH THE VERY TALENTED

CHROMATIN POSTDOC, SHARON AND A

GRADUATE STUDENT, GARY.

WE DO DNA METHYLATION WORK IN

COLLABORATION WITH TIM AND WE

ARE BEHOLD EN TO THE FRENCH

GROUP FOR PROVIDING US THESE

CELL LINES AND EVERYTHING IN THE

LAB.

SO I'LL STAND THERE AND

HOPEFULLY WE'LL OPEN UP TO

QUESTIONS LATER ABOUT OUR

EFFORTS.

NOW I WANT TO TURN TO THE SECOND

TOPIC I HOPE WE CAN SQUEEZE IN

MERE.

RESTARTED AS A FUND SIDE PROJECT

SO I SHOULD TRY EXPLAIN HOW THIS

STARTED.

SO BASIC I GOT A PHONE CALL ONE

DAY FROM CAROL A PERSON WHO

WORKED MOST OF HER LIFE AT

WOOD -- AND SHE GOT MY NAME

BECAUSE SHE WAS GOOD FRIENDS

WITH ANNE GIFFORD AND GIFFORD

WAS THE TECHNICIAN IN THE LAB AT

MIT WHEN I WAS A POSTDOC AND

THEY REMEMBERED ME 30 YEARS

LATER.

AND CAROL HAD ASKED, WE HAVE AN

INTERESTING LEUKEMIA, AND WE

WONDER IF MAYBE IT HAS A VIRAL

ETIOLOGY.

AND WHO WOULDN'T?

SO SHE CALLED ME UP AND TOLD ME

THIS STORY.

SO I'LL TELL WHERE YOU WE HAVE

TAKEN THE STORY IN THE LAST

COUPLE OF YEARS.

SO THE ORGANISM IS OF INTEREST

NOT ONE YOU PROBABLY THINK ABOUT

A LOT.

IT'S THE SOFT SHELL CLAM, THE

STEAMER, TO BE DISTINGUISHED

FROM THE HARD SHELL CLAM.

I REALLY LIKE SOFT SHELL CLAMS.

AND SO I WAS INTRIGUED.

SO IT'S A MOLLUSC.

IT'S NATIVE TO

THE -- RESTRICTIVE TO THE NORTH

AMERICAN EAST COAST AND NEW

ENGLAND, LONG ISLAND SOUND,

CHESAPEAKE BAY.

IT'S PRETTY MUCH EXTENT FROM THE

CHESAPEAKE.

IT'S A COMMERCIALLY MODERATELY

VALUABLE HARVEST OF 10-20

MILLION DOLLARS A YEAR.

IT'S SUBJECT TO A NEOPLASTIC

DISEASE THAT IS A RAPIDLY

INCREASING PREVALENCE OVER THE

YEARS.

AND THE DISEASE HAS BEEN CALLED

DISSEMINATED NEOPLASIA.

WE HAVE HAEMIC NEOPLASIA.

I GREW UP DIGGING THESE CLAMS IN

MY HOME IN MASSACHUSETTS.

BY KNEW THEM VERY WELL AND I WAS

SORRY TO HEAR THEY WERE BEING

WIPED OUT.

A NUMBER OF BEDS ARE PRETTY MUCH

EMPTY.

A LOT OF THE BEDS IN

MASSACHUSETTS ARE CLOSED TO

FISHING BECAUSE OF DISEASE.

SO IT'S WORRISOME.

ANY A SO THIS IS WHAT THESE

ORGANISM LOOK LIKE.

THEY ARE BASICALLY A LIVING

FILTER.

SO THEY PUMP WATER OVER THESE

GILLS AND EXTRACT NUTRIENTS FROM

THE WATER.

BUT BEYOND THAT PART OF THEIR

LIVES, THEY HAVE PRETTY NORMAL

ORGANS.

THEY HAVE A HEART.

THEY HAVE A CIRCULATION.

IN PARTICULAR, THEY HAVE

HEMOSITES THAT LOOK LIKE OUR

LYMPHOCYTES.

THE DISEASE LOOKS A LOT LIKE A

LEUKEMIA 90% OF THE ANIMALS ARE

SICK OR DYING.

IT IS SEASONAL.

NOBODY KNOWS WHY.

BUT THE KEY IS THAT THE DISEASE

IS CHARACTERIZED BY THIS MASS UP

HYPERPLASIA OF LEUKEMIA-LIKE

CELLS.

SO IF YOU STICK A NEEDLE INTO

THE CLAM AND BLEED THEM, IT IS

IMMEDIATELY OBVIOUS BECAUSE THE

HEME LIMP IS MILKY TO THE NAKED

EYE AS OPPOSED TO BEING CLEAR

AND THE CELL COUNTY ARE IN THE

REALM OF 10 TO THE EIGHTH

MERMILL.

THEY ARE ENLARGED AND MY TO

THEICALLY ACTIVE.

VERY LITTLE IS KNOWN  --

MY TO THEICALLY.

THERE IS A SURFACE PROTEIN

IDENTIFIED AS ONLY THE ANTIGEN

OF ANTIBODY THAT CAROL RAISED

AGAINST THE LEUKEMIA BEST

DIAGNOSIS IS BY CELL CYTOMETRY.

SO THEY ARE CLEARLY UNDERGONE

PRETTY DRAMATIC -- THINK OF THEM

AS ONCOGENIC CHANGES.

SO NEARLY HEMOSITES PLATED ON A

GLASS SIDE LOOKS LIKE THAT.

THEY LOOK HAPPY.

BUT THE LEUKEMIA CELLS ARE ROUND

AND MITOTICALLY VERY ACTIVE AND

DIVIDING LIKE CRAZY.

ONE OF CAROL'S FAVORITE THINGS

TO DO IS TAKE SAMPLES FROM THE

SLIDES AND SHOW THEM TO CLINICAL

PATHOLOGISTS AND SAY, WHAT DO

YOU THINK?

AND THE PATHOLOGIST WILL SAY

THIS IS REALLY SERIOUS BLAST.

CLEARLY IN PERSON IS VERY ILL.

AND THEN SHE'LL SAY OKAY, BY THE

WAY THERE IS A CLAM.

NOT A HUMAN.

THE PATHOLOGIST WILL BE

IMMEDIATELY VERY OFFENDED AND

SAID HE WASN'T TOLD.

BUT THE POINT IS THAT HE CAN'T

TELL THAT THIS IS NOT A

MAMMALIAN OR HUMAN LEUKEMIA.

SO, TO THE NAKED EYE THESE CLAMS

SEEM TO HAVE LEUKEMIA.

AND THERE IS NO REAL SERIOUS

UNDERSTANDING OF THE CAUSE.

IT IS THOUGHT TO BE ASSOCIATED

WITH ENVIRONMENTAL POLLUTION.

AND A LOT OF THE WORK IN THE

FIELD HAS TRIED TO SUPPORT THAT

IDEA.

THERE IS CERTAINLY SUPPORT FOR

IT.

IT'S KNOWN NOW THAT THERE ARE

MUTATIONS IN p53.

YES, THEY HAVE p53.

THEY HAVEELIVATED EXPRESSION.

p53 SEQUESTERED INTO THE

CYTOPLASM.

SO A LOT OF INTERESTING THINGS

ARE GOING ON WITH RESPECT TO

THAT.

MOST IMPORTANTLY FROM OUR POINT

OF VIEW, THERE IS EVIDENCE THAT

THIS IS TRANSMISSIBLE BETWEEN

ANIMALS, EITHER BY THE LOU

KEIMIC CELLS OR FILTRATE BUT THE

SAWS OF THAT ISN'T CLEAR.

VIRUS-LIKE PARTICLES HAVE BEEN

REPORTED BY EM AND THOSE REPORTS

ARE PRETTY MUCH DISMISSED NOW.

THERE ARE REPORTS OF REVERSE

TRANSCRIPTASE AND VERY RECENTLY

IN THE LAST MONTH, THERE ARE

REPORTED VERY LARGE INCREASES IN

VIRUS-LIKE MRNAs DETECTED WITH

PRIMERS THAT WERE DESIGNED

GENATIONALALLY FOR RETRO

TRANSPOSONS.

WE SUSPECT THIS IS WHAT WE ARE

TALKING ABOUT TODAY.

SO, CAROL SENT A SAMPLE OF THESE

TO LOOK AT.

WE SAID, WE'LL DO REVERSE

TRANSCRIPTASE ASSAYS AND WE DO

THOSE ROUTINELY WITH VERY CROWD

SAMPLES.

WE WORK WITH TISSUE CULTURES IN

THE MEDIA DIRECTLY, THROW THEM

INTO COCKTAIL AND THEY WORK.

THAT'S OUR STANDARD WAY OF

MEASURING VIRUS REPLICATION.

SO WE USE THOSE STANDARD WAYS TO

LOOK AT HEMOLYMPH FROM DISEASE,

AND NORMAL CLAMS.

RIGHT AWAY THERE WERE VERY HIGH

LEVELS OF REVERSE TRANSCRIPTASE

IN THE DECEASED ANIMALS.

MEDIUM LEVELS IN THE

INTERIMMEDIATE LEVELS AND

NOTHING IN THE NORMAL.

SO WE WERE IMMEDIATELY EXCITED

BY THIS THAT THERE WAS SOME KIND

OF -- THIS WAS CELL-FREE

ACTIVITY IN THE HEMOLYMPH.

AND IF WE CULTURED THE HEMOSITE

IN-VITRO FOR A FEW DAYS, WE GOT

EVEN HIGHER LEVELS.

THE CULTURE SITUATION FOR THESE

CELLS ARE NOT TOO BAD.

THEY GROW IN THE NEM WITH 10%

CAP SERUM.

THEY DO GROW AT 10 DEGREES

INSTEAD OF 37.

AND THE ONLY OTHER FUNNY THING

IS YOU HAVE TO ADD HALF MOLAR

SALT TO THE CULTURE MEDIUM.

THAT'S SEA WATER BASICALLY BUT

THEY GROW.

AND THEY DON'T GROW FOR LONG,

THEY GROW FOR A FEW DAYS AND

THEN POOP OUT.

MORE OR LESS LIKE ANY OTHER

PRIMARY CELL.

WE WOULD DETECT OUR ACTIVITY

HERE AS INCORPORATED ON

HOMOPOLYMER TEMPLATES SUBSTRATES

AS WE ALWAYS DO UNDER THE

CONDITIONS THAT WE USE NORMALLY

PROM LONE REVERSE TRANSCRIPT

ACE.

SO WE SAID, IF THIS IS REAL, WE

NEED TO CLEAN THE DNA FROM THE

ELEMENT THAT IS CAUSE THIS

REVERSE TRANSCRIPTASE ACTIVITY.

WE EXTRACTED RNA FROM THE

HOTTEST SAMPLES WE COULD GET.

AND WE SENT IT OFF FOR

DESEQUENCING.

WE DID THIS WITH OUR

COLLABORATOR ACROSS THE STREET,

LIP KIN, WHOA S. A VIRUS HUNTER

GUY IN THIS DAY AND AGE.

HE SENT US BACK ABOUT 200,000

READS FROM THE SAMPLES AND WE

LOOKED HARD THEN AT THESE FOR

RETROVIRAL RELATEDNESS: THERE

WERE INDEED SEQUENCE RUNS THAT

HAD HEME APOLOGIES TO VARIOUS

PARTS OF RETROVIRUSES.

MOST WERE PAUL.

SO LOOKING BACK THEN WITH THE

NEW PRIMERS DESIGNED BASED ON

THE SEQUENCES WE AMPLIFIED, IN

THAT PAULS, WE WENT BACK TO THE

RNA AGAIN, USED NOW THE BETTER

PRIMERS INSTEAD OF THE RANDOM

PRIMING WE HAD DONE BEFORE AND

GOT VERY NICE FRAGMENTS THAT

WERE CLEARLY RETROVIRAL-LIKE AND

WE SEQUENCED THEM AND PATCHED

THEM TOGETHER AND WE NOW HAVE

ABOUT 5KV OF THE CONDIG THAT WE

THINK REPRESENTS A PORTION, NOT

ALL, OF THE ELEMENTS IN OUR

PLAN.

AND WE JUST STARTED GOING BACK

AND LOOKING AT GENOMIC DNA AS

WELL.

SO, THESE WERE SOME OF THOSE

AMPLIFICATIONS.

SO I ALIGNED THEM HERE ON THE

FINAL SEQUENCE THAT WE HAVE GOT

SO THE INITIAL PRIMER HITS WERE

THESE FORWARD AND THEN REVERSE

PRIMERS.

WE WERE ABLE TO AMPLIFY THE LONG

PRODUCTS.

MOST WHEN WE HAD JUST TINY

LITTLE FRAGMENTS AND WE HAD

SEQUENCE PRIMERS BESIDES THOSE,

WHEN WE COULD SPAN ALL THE WAY

FROM ONE TO THE OTHER, WE KNEW

WE HAD SOMETHING IN THAT

PRESENCE ON THE SAME RNA

MOLECULE.

SO THESE PRODUCTS WERE IN THE

REALM OF TWO KB OR SO AND SO NOW

WE WERE GETTING SIGNIFICANT SEE

CONVENIENCE READS.

SO PUTTING EVERYTHING TOGETHER.

WE NOW HAVE A SEQUENCE TO AT

LEAST LOOK AT TO GAZE AT.

THESE ARE SOME OF THE FEATURES

THAT I CAN TELL YOU ABOUT.

SO WE ARE SPANNING ROUGHLY FROM

AND ALIGNING THESE HERE WITH

MALONEY.

WHAT WE SUDDEN WE ARE GOING FROM

THE NUCLEAR CASA GENE THROUGH

PROTEASE REVERSE TRANSCRIPT ACE,

RNAH AND INTERGREATS.

SO THE OBVIOUS FEATURES IN THIS

GUY IS IT IS ONE LONG ORF.

NO STOP CODE ON IT BETWEEN GAG

AND POL.

NO FRIENDSHIP.

THESE ARE ALL INTERESTING.

THE NUCLEAR CAPS ACE HAS TWO

APPARENT BIG FINGERS.

NO STOP CODON.

RTs ARE MARKED BY A PARTICULAR

LITTLE SEQUENCE, YXTD BOX SO THE

ACTIVE SITE OF THE BOX.

THIS GUY HAS AN IADD AT THIS

BOX.

SO NONCANON KEL.

THE INTEGRATE HAS A BIG FINGER.

VERY TRADITIONAL.

IT HAS A CATALYTIC CLUSTERS OF

THE INTEGRATION.

WE HAVE NOT HIT AN ENVELOPE.

DON'T FINISH WE ARE GOING TO AND

WE HAVE NOT YET GOTTEN PROBABLY

ALL OF THE GAG GENE.

SO THIS IS WHERE WE ARE.

SO IT'S A SEEMINGLY AUTHENTIC

RETROELEMENT, RETROELEMENT

SEQUENCE.

SO WITH THAT IN HAND, WE THOUGHT

WE SHOULD LOOK BACK AT THE

EXPRESSION OF THIS ELEMENT OR

VIRUS IN CLAMS.

SO WE HAVE DONE THAT.

SO THESE ARE OUR TPCR LEVELS OF

RNAs EXTRACTED FROM A NEW

PANEL OF NORMAL CLAMS, DIAGNOSIS

NOT BEING PERFECT.

OR TRANSITIONALAL OR

INTERMEDIATELY SIT CLAMS.

AND YOU SEE PRETTY GOOD

CORRELATION.

SO THE NORMAL CLAMS AS A RULE

HAVE NO OR LOW REFRESHING.

THERE ARE SAMPLES IN HERE THEY

ARE REALLY ZERO.

I SHOULD SAY FOR THOSE WHO OF

WHO WORRY, I WENT TO THE

SUPERMARKET AND BOUGHT A BUNCH

OF CLAMS FROM FAIRWAY AND THEY

ARE ALL COMPLETELY ZERO.

SO NO EXPRESSION IN YOUR GARDEN

VARIETY CLAMS IN THE STORE.

A NUMBER OF CLAMS THAT ARE

DIAGNOSED AS BEING PARTIAL AND

MOST OF THE CLAMS THAT ARE SICK

HAVE ENORMOUSLY HIGH LEVELS OF

EXPRESSION OF THIS ELEMENT.

SO THAT'S SUMMARIZED HERE OVER A

LARGER NUMBER.

SO, NORMAL CLAMS AS A RULE HAVE

REALLY NO EXPRESSION AND THERE

IS A HUGE RANGE BUT OFTEN

EXTREMELY HIGH LEVELS OF

EXPRESSION PRESENT.

AND THAT'S NOT UNLIKE RETROVIRAL

EXPRESSION IN A NUMBER OF OTHER

DISEASES, INCLUDING HUMAN

DISEASES WHERE THERE IS JUST

ENORMOUS RANGE BUT OFTEN HIGH

LEVEL OF EXPRESSION IN DISEASED

INDIVIDUALS.

AND THE CLAMS ARE NOT ONLY

EXPRESSING RNA, BUT THAT RN

SAMPLE MEDIATING ENORMOUS DNA

EXPANSION AS WELL.

BECAUSE THE TOTAL DNA EXTRACTED

FROM THE MOST DECEASED CLAMS

HAVE INCREDIBLY HIGH LEVELS OF

DNA DETECTED BY SOUTHERN BLOT

HERE.

SO NORMAL CLAMS HAVE ESSENTIALLY

NONE.

THERE ARE DIGESTS GENOMIC DNA.

THERE ARE FAKE BANDS THAT YOU

MAY BE ABLE TO SEE.

WE THINK NORMAL CLAMS MAY HAVE

ABOUT ONE COPY OF AN ELEMENT.

BUT THE DISEASED CLAMS HAVE

ENORMOUS 100 FOLD OR MORE

EXPANSION OF DNA AS WELL SO

THESE ARE NOT -- NEITHER THE

VIRUS OR ELEMENT IS UNDERGOING

ACTIVE TRANSCRIPTION TO GENERATE

HIGH LEVELS OF DNA.

WE ARE USING PROBES FOR

DIFFERENT PARTS OF THE POL GENE

AND WE ARE DIGESTING THE DNA A

VARIETY OF DIFFERENT RESTRICTION

ENZYMES ALLOWING US TO PATCH

TOGETHER A MAP OF THE GENOME

TOTALLY CONSISTENT WITH THE

SEQUENCE WE HAVE, AND EXTENDING

OUTWARD.

VERY OFTEN WE WILL SEE FRAGMENTS

THAT ARE CORRECTLY PREDICTED

INTERNAL FRAGMENTS BUT WHEN WE

ARE SEEING ALSO FLANKING

SEQUENCES FRAGMENTS OUTSIDE THIS

INDICATES WE ARE HAVING RANDOM

INTEGRATION INTO ADJACENT DNA OF

THIS AD DNA.

WE NEVER SEE AN UNINTEGRATED

FULL-LENGTH FREE FREESTANDING COPY.

WE ARE ONLY SEEING INTEGRATED

FORM.

ANDENING OUR MINDS, WE ARE NOT

SEEING CLONAL INTEGRATION.

NOT SEEING IN THE TUMOR IN THE

DECEASED CLAMS EVER -- DISEASED

CLAMS THAT WOULD ACCOUNT FOR HOT

INTEGRATION EVENT.

WE ARE SEEING FULLY

HETEROGENEOUS INTEGRATION SO WE

DON'T GUESS THIS IS ACTING

THROUGH THE MYC GENE.

BUT THE LEVEL OF AMPLIFICATION

IS ENORMOUS.

THIS IS INTERNAL FRAGMENT AND IT

ALLOWS US TO SEE THAT THERE ARE

A COPY NUMBER IN A NORMAL CLAM

THAT CAN BE AMPLIFIED WITH 10 OR

100 FOLD IN THE DISEASED CLAM.

AND THAT'S REALLY WHERE WE ARE.

SO WE KNOW ONLY THAT THERE ARE

HIGH LEVELS OF REVERSE

TRANSCRIPTASE OFTEN PRESENT.

AND HIGH LEVELS AGAIN IN THE

CULTURE MEDIUM FROM CULTURED

HEMOSITES.

WE THAN RT ACTIVITY IN THE

MEDIUM IS PALITTABLE BUT HAVE

NOT VISUALIZED THE VIRUS AS THEY

EXISTS YET.

WE KNOW ALSO THERE ARE VERY HIGH

LEVELS OF RNA OF THIS ELEMENT IN

THE DISEASED ANIMAL AND IT'S THE

LEVELS CORRELATE PRETTY MUCH THE

DIAGNOSIS, WE KNOW THAT THE

SEQUENCE OF THE POL REGION AND A

BIT MORE AND IT REALLY LOOKS

LIKE A RETROELEMENT OR

RETROVIRUS IN 99 WAYS.

AND THAT ARE LOW COPY NUMBERS,

MAYBE ONE OF THE GENOME OF

HEALTHY NUMBERS AND ENORMOUSLY

HIGH LEVELS IN THE ANIMALS THAT

UNDERGOING ACTIVE TRANSCRIPTION.

SO, WE WANT TO KNOW IF IT IS IN

OFFICIALS.

CAN WE TRANSMIT IT IN CULTURE?

WE ARE TRYING TO DO THAT.

WE NEED THE COMPLETE GENOME?

WE DON'T HAVE IT.

WE DON'T KNOW IT'S FULL

REPERTOIRE OF RNAs AND

PROTEINS.

WE DON'T KNOW ITS GEOGRAPHIC

RANGE AND DON'T KNOW ITS SPECIES

HOST RANGE EVEN.

DISEASES LIKE THIS ARE FOUND IN

OTHER HOLUX.

SO -- MOLLUSCS.

MAYBE THIS GUY ITSELF IN A

VARIETY OF OTHER SPECIES, SO

CAROL AND HER FRIENDS ARE

COLLECTING FOR US OTHER DISEASED

SPECIES TO LOOK AT.

AND WE DON'T REALLY FINISH IT'S

AN EXOGENOUS VIRUS AND DON'T

KNOW,CAUSES DISEASE.

SO WE ARE HAVING FUN.

ALL OF THIS WORK IN MY LAB ON

THE CLAMS OF DONE BY ONE

POSTDOC, GLORIA, AND IS GOING

BACK TO HER NATIVE CHILE IN A

COUPLE OF MONTHS.

THE PEOPLE OUT IN THE FIELD

INCLUDE JAMES SHAREY AND HIS

STUDENTS AND WORKS WITH CAROL

AND ALL THE SEQUENCING WORK WAS

DONE WITH THE HELP OF IAN LIP

KIN.

I WILL STOP THERE AND TAKE

QUESTIONS.

THANK YOU.

[ APPLAUSE ]

>> ANY QUESTIONS?

I JUST WANT TO SAY THAT THERE IS

A RECEPTION AFTER THE TALK THAT

I FORGOT TO MENTION DURING MY

INTRODUCTION.

SO PLEASE JOIN US AFTER THE

TALK.

>> SO, STEVE, EXCELLENT TALK.

SO, IN TERMS OF YOUR FIRST PART

ABOUT SILENCING, YOU WOULD ARGUE

THAT VIRUSES THAT CONTAIN TRNA

PROLINE PBS WOULD BE SILENCED IF

THEY FOR EXAMPLE, INFECTED

EMBYRONIC CELLS OR STEM CELLS.

BUT WE ACTUALLY HAVE THE -- AND

I THINK YOU ILLUSTRATED ONE OF

THE EXAMPLES OF HTLB1 WITH

PROLINE.

WE KNOW THAT HTLB1 CAN IN

FACT -- HUMAN EMBYRONIC STEM

CELLS AND ALSO HUMAN

HEMATOPOIETIC CD34 POSITIVE

CELLS WITHOUT ANY EVIDENCE OF

SILENCING OR RESTRICTIONS.

SO I WAS WONDERING WHETHER YOU

WOULD SPECULATE ON THAT.

>> SO EVERYTHING I SHOWED YOU IN

THE HISTORY IS ON THE MOUSE.

EMBYRONIC STEM CELLS.

WE JUST STARTED LOOKING AT HUMAN

CELLS.

THERE ISN'T VERY MUCH KNOWN.

WE DO NOT YET KNOW IF THE

TRADITIONAL EMBYRONIC STEM CELLS

WILL COLLECTIVELY SILENCE

PROLINE TRNA UTILIZING

RETROVIRUSES AT THIS POINT.

WE ARE LOOKING AT THAT.

WE HAVE ONLY RECENTLY GOTTEN

PERMISSION.

IT WAS THE BUSH ERA PROBLEMS.

WE STARTED COLLABORATIONS WITH

GEORGE DAILY TO LOOK AT THIS.

THE HUMAN EMBYRONIC STEM CELL

HAS NOT GOTTEN MUCH OVERLAP WITH

THE RETROVIRUS WORLD, TO OUR

SURPRISE.

SO WE ARE STARTING TO LOOK AT

THIS AND THERE IS REALLY VERY

LITTLE HISTORY IN HUMAN

ES-CELLS.

THERE ARE ENDOGENOUS HUMAN

RETROVIRUSES.

THERE ARE THOSE THAT UTILIZE

ESSENTIALLY THE WHOLE ARRAY OF

TRNAs.

AS KNOW THEY ARE ALL NAMED ON

THE BASIS OF THE TRNA THAT THEY

UTILIZE SO HRKK USES LYSINE AND

SO ON.

ALL OF THAT BEING SAID, WE DON'T

KNOW IF HUMANS HAVE IN THEIR

REPERTOIRE ZINC FINGER THAT

WOULD BE THE EQUIVALENT OF WHAT

WE KNOW THE MOUSE CAN DO.

IT'S AN INTERESTING THOUGHT THAT

ALTHOUGH THERE ARE MOUSE

RETROVIRUSES THAT TRNAs THAT

ARE SILENCED AND THAT ARE NOT.

THEY ARE BOTH OUT THERE IN THE

WORLD AND THAT THE GENOMES OF

MICE ARE LITTERED WITH VIRUSES

THAT ARE SILENCED AS A RULE.

AND WE HAVE ALL OF THOSE

SILENCED RETROVIRUSES IN OUR

GENOMES AS WELL BUT HOW THEYER

SILENCED ISN'T CLEAR.

>> SO IF I COULD JUST HAVE A

FOLLOW-UP.

IF YOU COULD CONTRAST YOUR

PROTEIN-BASED SILENCING OF

ENDOGENOUS RETROVIRUS ELEMENTS,

VERSUS THE EMERGING FIELD OF

HIGH RNA-BASED SILENCING, I WAS

WONDERING, DO YOU THINK ONE

CONTRIBUTES 50% AND THE OTHER

CONTRYSTS 30%?

WHAT IS YOUR FEELING ON THAT?

>> I WOULDN'T PUT NUMBERS ON

THAT.

I WOULD SAY THE PROLINE-BASED

SILENCING IN MOUSE IS REALLY,

REALLY POTENT.

I THINK IT'S PROBABLY 95% OR

MORE OF THE TOTAL SILENCING.

BUT AS YOU SAY, THERE IS THE RNA

ONE AND WE ARE VERY INTERESTED

IN KIND OF A SECOND LEVEL OR A

SECOND LEVEL PROTEIN BASED

SILENCING THAT WE KNOW ALSO GOES

ON.

SO EVEN THE GLUTAMINE VIRUS

FAMILY ARE SILENCED IN MOUSE

STEM CELLS.

MORE SLOWLY, LESS POTENTLY, AND

WE THINK UTILIZING OTHER

ELEMENTS IN THE LPR SO PEOPLE

HAVE KNOWN FOR A LONG TIME THERE

ARE NEGATIVE REGULATORY ELEMENTS

AS THEY ARE CALLED IN OTHER

PARTS OF THE RETROVIRAL LTRs

THAT ARE TARGETED FOR SILENCING.

WE HAVE SOME IDEAS FOR THE

PROTEIN THAT IS MIGHT DEVIATE

THAT.

THEY WERE CLEARLY BACK UP PBS

SILENCING MACHINERY BOTH PROTEIN

AND NONPROTEIN SOURCES.

AND I GUESS THE RELEVANT

EFFICIENCIES OF THOSE WILL BE

DIFFERENT WITH DIFFERENT VIRUSES

AND DIFFERENT SPECIES.

>> ARE IPS CELLS RESTRICTIVE?

>> WE ARE LOOKING AT THAT WITH

GEORGE DAILY.

THE EARLY RESULTS ARE THAT WE

DON'T KNOW.

YES, PROBABLY.

AND THEN WITH THAT BEING SAID,

THE NEXT ISSUE WOULD BE EXACTLY

WHEN?

SO AS GEORGE TELLS US, THERE ARE

STAGES OF IPS CELLS FROM

STARTING WITH MORE

DIFFERENTIATED CELLS.

WHEN DOES IT KICK IN AS WE GO IN

THAT DIRECTION BACK TOWARDS STEM

CELLS, THIS IS WHAT WE ARE VERY

KEEN TO KNOW.

WE KNOW MORE GOING THE OTHER WAY

IN MOUSE WHEN YOU TAKE A SILENT

EMBYRONIC STEM CELL AND INDUCE

DIFFERENTIATION.

WE KNOW WHEN THE VIRUSES COME ON

BUT I THINK THIS WILL BE AN

INTERESTING STORY TO FIND OUT

WHEN IT IS ENCODED WHEN YOU'RE

GOING BACKWARD AND

DIFFERENTIATION.

THERE ARE OTHER THINGS TO SAY

ABOUT THE STORY BUT THOSE ARE

VERY INTERESTING QUESTIONS.

>> SO ENTERTAINED BY THE TALK, I

HAVE ONE QUESTION FOR EACH TALK.

THE FIRST QUESTION IS, HOW DOES

THE EMBYRONIC STEM CELL AVOID

SHUTTING OFF ITS OWN ENDOGENOUS

TRNAs?

>> THERE IS A VERY GOOD ANSWER,

I THINK TO THIS QUESTION, WHICH

IS THAT THE SILENCING ELEMENT

HERE, THE PBS, IS INCLUDING THE

18 NUCLEOTIDES OF THE THREE

PRIME END OF THE TRNA.

SO THAT INCLUDES SPECIFICALLY

THE TCA BASIS AT THE 3-PRIME END

OF THE TRNA.

AND THOSE ARE IMPORTANT.

IF YOU DON'T HAVE THOSE, THE PBS

IS NOT RECOGNIZED FOR SILENCING.

AND IN FACT, THE GENES FOR

TRNAs DON'T HAVE THOSE BECAUSE

THEY ARE PADDED CLOSE

TRANSCRIPTIONALLY TO THE TRNA.

SO THAT'S A REMARKABLE FACT.

THE SYSTEM HAPPENS OR MAYBE WAS

EVOLVED EVEN TO BE ABLE TO DO

THAT DISCRIMINATION.

SO THE PRO VIRAL PBS VERSIONS

ARE SILENCABLE AND RECOGNIZED

TRNAs ARE NOT.

SO THAT'S THE BASIS FOR THE

DISCRIMINATION.

>> IS IT A VERY ELEGANT GUESS.

SO THE SECOND QUESTION MAYBE

MORE OF INJECTION.

RETROVIRUSES OFTEN MAKE A

CIRCLE.

WONDER IF YOU GO THOUGHT OF

HUNTING THE CIRCULAR DNA FOR

THOSE CELLS?

>> SO I GUESS IF I'M

UNDERSTANDING YOU RIGHT, YOU'RE

SAYING WHAT IS THE STATE OF THE

DNA IN THOSE?

>> NO, IT'S MORE THAN IF THE DNA

IS CIRCULAR, YOU CAN USE THE

AWESOME POWER OF

-- [ INDISCERNIBLE ]

[ LOW AUDIO ]

>> TRUE.

BUT WHEN THE CASE WHERE YOU

DON'T HAVE ANY PRIMERS TO USE

AND SEQUENCE INFORMATION, THAT

WOULD BE --

>> YOU DON'T HAVE TO CARE.

>> BUT WE THINK WE DO HAVE

PRIMERS FOR OUR CLAM GUYS.

SO WE ARE JUST GOING THAT WAY.

BUT WE DON'T KNOW THE STATE OF

THE DNA AND WHEN IT'S BEING

AMPLIFIED.

OUR GUESS WOULD HAVE BEEN THAT

HEY, IT'S JUST DRAWING A

LINEAR -- THAT WOULD BE THE MOST

ABUNDANT DNA IN THE EXPANSION.

BUT IT COULD BE CIRCULAR.

I THINK THE OTHER POSSIBLE

ANSWER WILL BE THAT IT'S HOPPING

AND THAT MOST OF THE STUFF WE

ARE SEEING IS INTEGRATED.

BUT I DON'T KNOW.

>> I WAS GOING TO ASK YOU A

QUESTION ABOUT TRNA EXPRESSION

BUT I WON'T.

>> THEY ARE NOT SILENCED.

>> WHAT ABOUT THE NEIGHBORING

GENES?

MLV INTEGRATES AND ACTIVELY

TRANSCRIBED GENES NEAR THE

PROMOTORS.

IS THIS A PROMOTOR KILLER?

>> SO I DON'T GUESS WE REALLY

KNOW TO WHAT EXTENT THAT HAPPENS

IN INFECTED ES-CELLS.

OF COURSE IT'S ONLY THERE IT

WOULD HAPPEN.

NOTHING MUCH NEW TO SAY ABOUT

THAT.

SO IN THE EARLY DAYS OF THE

DISCOVERING OF THE SILENCING

BUSINESS, WHEN PEOPLE LOOKED AT

THE SPREAD, IT'S ALL ARTIFICIAL.

SO LOOKING TO NODES, THE

SILENCING WAS SPREADING OVER A

LONG THE KB.

SO PRESUMABLY THAT'S THE CASE.

>> THERE ARE SO MANY ANTIVIRAL

DRUGS -- [ INDISCERNIBLE ]

>> TO USE THEM TO TREAT CLAMS OR

THAT THING?

NO.

SO THAT WAS AN INTERESTING

THOUGHT.

I DON'T GUESS WE CAN TREAT CLAMS

WITH AZT BUT ONE OF THE EARLY

THINGS WE WANT TO DO IS EXPRESS

THE RT.

MAKE A LOT OF IT AND THEN REALLY

CHARACTERIZE IT THE WAY WE WOULD

CHARACTERIZE ANY RETROVIRAL RT.

WE ARE JUST STARTING THAT AND

AMONG THE CHARACTERIZATIONS

WOULD BE TO ASK WHAT DRUGS WORK

ON -- IF WE WERE REALLY SERIOUS

ABOUT SOLVING THE PROBLEM OF

THIS DISEASE, IF IT IS VIRAL IN

ORIGIN, WE DON'T KNOW THIS YET,

THERE WOULD BE A PATH.

THE PATH WOULD BE IN MY VIEW, TO

MAKE A RESISTANT CLAM AND THAT

IS NOT TOTALLY CRAZY TO IMAGINE

BECAUSE THEY ARE ACTUALLY PRETTY

AMENABLE TO MANIPULATION AT THE

DNA LEVEL.

YOU CAN -- THEY MAKE A LOT OF

SPERM A LOT OF EGGS AND YOU CAN

AT THAT LEVEL, WORK WITH THEM

GENETIC ORGANISMS.

THEY ARE NOT THE FASTEST

ORGANISMS IN THE WORLD BUT THEY

ARE WORKABLE.

IF THERE WERE A RECEPTOR, FOR

EXAMPLE, FOR THE VIRUS, I COULD

IMAGINE GENERATING A RECEPTOR

NEGATIVE CLAM AND THAT WOULD

SAVE THE WORLD.

I DON'T FINISH THAT WILL HAPPEN.

>> ON THE THOUGHT OF SAVING THE

CLAMS, PLEASE JOIN US TO

CONTINUE THIS CONVERSATION WITH

STEVE AT THE RECEPTION.

THANK YOU FOR COMING AND THANK

YOU FOR A GREAT TALK.

[ APPLAUSE ]