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>> GOOD AFTERNOON, EVERYONE.
WELCOME TO THE WEDNESDAY
AFTERNOON LECTURE AND TO
PRESENTATION BY DISTINGUISHED
RESEARCHER AND THE LEADER IN THE
WHOLE AREA OF THE SCIENCE AND
SCIENCE POLICIES, KEITH
YAMAMOTO.
HE IS CURRENTLY THE EXECUTIVE
VICE TEEN AND VICE CHANCELLOR
FOR RESEARCH AS WELL AS
PROFESSOR OF CELLULAR AND
MOLECULAR PHARMACOLOGY AT UCSF
AND YES, DESPITE ALL OF THOSE
RULES, CONTINUESES TO DO REALLY
INTERESTING SCIENCE RELATED TO
THE GROUP OF CORTICOID RECEPTOR.
HE GOT HIS UNDERGRADUATE DEGREE
FROM OHIO STATE AND PH.D. FROM
PINS TON AND LANDED AT UCSF
WHICH HE MUST HAVE LIKED RATHER
WILL.
THAT IS WHERE HE HAS BEEN
WITHOUT INTERRUPTION BUT WITH
LOTS OF DIFFERENT POSITIONS
SINCE 1973.
AND NOW, HAVING ARISEN TO THIS
POSITION OF VICE CHANCELLOR FOR
RESEARCH, HAS A CRITICAL ROLE IN
STEERING THAT REMARKABLE
INSTITUTION AT A REMARKABLE
TIME.
HE HAS ALSO BEEN ELECTED TO THE
NATIONAL ACADEMY OF SCIENCES, TO
THE INSTITUTE OF MEDICINE AS
WELL, AND IS A FELLOW OF THE
AMERICAN ASSOCIATION FOR THE
ADVANCEMENT OF SCIENCE.
KEITH IS ALSO ONE OF THE MOST
DEDICATED CITIZENS OF THE
BIOMEDICAL RESEARCH ENTERPRISE
IN THE WAY IN WHICH HE SERVES
NIH.
HE HAS BEEN ON NUMEROUS STUDY
SECTIONS AND CHAIRED SEVERAL OF
THEM.
HE HAS SERVED US MOST RECENTLY
ON THE BIOMEDICAL WORKFORCE AND
DIVERSITY IN THE BIOMEDICAL
WORKFORCE WORKING GROUPS OF THE
ADVISORY COMMITTEE TO THE
DIRECTOR AND HE IS GREATLY
APPRECIATED AND WISE ADVICE IN
THOSE AREAS.
HE IS CURRENTLY ON THE ADVISORY
COUNCIL TO THE SENATE FOR
SCIENTIFIC REVIEW.
HE HAS PLAYED MANY IMPORTANT
ROLLS IN OUR DELIBERATIONS ABOUT
HOW TO MAKE OUR PEER REVIEW
SYSTEM, WHICH WE THINK IS THE
BEST IN THE WORLD BUT IT'S NOT
PERFECT, SO WE CONTINUE TO TRY
TO MAKE IT BETTER AND HE HAS
BEEN VERY INFLUENTIAL IN THAT
REGARD.
HE ALSO HAS PLAYED A REALLY
CRITICAL ROLE IN TERMS OF TRYING
TO GET THE WORD OUT TO EVERYBODY
IN THE COMMUNITY THERE IS THE
VALUE OF MEDICAL RESEARCH AND HE
SERVES AS THE CHAIR OF THE
COMMITTEE ON LIFE SCIENCES.
THIS IS A COALITION OF
SCIENTIFIC SOCIETY GROUPS THAT
MAKES THAT CASE VERY EFFECTIVELY
AND THIS IS THE TIME WHERE THAT
CASE NEEDS TO BE MADE,
PARTICULARLY EFFECTIVELY.
SO USUALLY WHEN KEEK IS HERE, WE
ARE HAVING SOME -- KEITH IS
HERE, WE ARE HAVING INTENSE
DEBATE ABOUT SCIENCE POLICY,
ABOUT FUNDING, BUT IT IS A
DELIGHT TO BE ABLE TO INVITE HIM
TO COME TO THE PODIUM TODAY TO
TALK ABOUT SCIENCE.
AND THE SCIENCE THAT HE DOES
MOST ABLY.
I'M SURE YOU'RE GOING TO ENJOY
WHAT HE HAS TO SAY.
FOLLOWING HIS PRESENTATION,
WE'LL HAVE TIME FOR A FEW
QUESTIONS AND THERE ARE PEOPLE
LISTENING ON THE WEB SO WE'LL
ASK THE QUESTIONERS TO COME TO
THE MICROPHONE AND THEN THERE
WILL BE A RECEPTION IN THE
LIBRARY WHERE YOU CAN CONTINUE
THE CONVERSATION.
HIS TITLE IS COMBINATORIALITY,
SINGLEALIGRATION AND ALLOSTERY
AND DETERMINES OF SPECIFIC
TRANSCRIPTIONAL REGULATION BY
THE GLUCOCORTICOID RECEPTOR.
PLEASE JOIN ME IN WELCOMING
KEITH YAMAMOTO.
[ APPLAUSE ]
>> GLAD TO BE HERE TO TALK ABOUT
SCIENCE FOR A CHANGE AND I
REALLY APPRECIATE THE
OPPORTUNITY.
SO THANK YOU.
FRANCIS IS TRYING TO DISRUPT
THINGS.
HE GAVE ME A LETTER JUST BEFORE
HE CAME UP AND INTRODUCED ME.
WE WERE TALKING AND HE SOMEHOW
GOT OUT OF A PERMANENT FILE, A
LETTER WHEN WE WERE COMPETING
OVER A BIT OF SCIENCE IN THE
70S.
I CAN'T REMEMBER IF THIS HAS
BEEN RESOLVED OR NOT.
ALL RIGHT.
SO, THANKS.
I'M GOING TO TALK TODAY ABOUT
THIS -- AND I APOLOGIZE FOR THIS
TITLE.
I THINK THAT ANYBODY WHO TURNS
IN A THREE-LINE TITLE WITH A
COLON IN THE MIDDLE IS
DISPLAYING THEIR INSECURITIES OR
SOMETHING.
[ LAUGHS ]
SO I APOLOGIZE FOR THAT.
BUT, WHAT I WANT TO TELL YOU
ABOUT IS A SERIES OF STUDIES
THAT WE HAVE BEEN CARRYING OUT
OVER A LONG TIME IN THE LAB THAT
WE ARE EXCITED ABOUT.
I THINK THEY GIVE US SOME HINTS
AND CLUES ABOUT MET ZERO AN
REGULATION THAT HOPEFULLY WILL
BE GENERAL AND NOT ONLY BE
INFORMATIVE AT THE SCIENTIFIC
LEVEL, BUT GIVE US INSIGHTS THAT
COULD BE USEFUL IN VARIOUS
APPLICATIONS AND I'LL MENTION A
COUPLE AS WE GO ALONG.
I WANT TO START WITH SOMETHING
WE KNEW SOMETHING ABOUT.
I CALL IT THE GOOD OLE' DAYS OF
WHAT WE THOUGHT ABOUT WHEN WE
WERE LOOKING AT CONTROLLING
REGULATION IN BACTERIA.
IT'S NOTABLE BECAUSE IT IS THE
BASIS FOR A LOT OF THINGS WE
THINK ABOUT HERE AT THE NIH OF
COURSE A TREMENDOUS AMOUNT OF
SEMINOLE WORK IN THIS AREA WAS
DONE BACK THEN.
TRANSCRIPTIONAL REGULATION WORKS
BY AFFECTING THE CAPACITY OF US
TO BIND TO PROMOTORS AND THE
REGULATION IS RELATIVELY SIMPLE.
ONE OR TWO SIGNAL RESPONSE
FACTORS EFFECTS THE BINDING
ACTIVITY AND THEN THE LACK OF
PRESSURE AND THEN THE CYCLIC A&P
BINDING CHARACTERIZING HERE IN
THE LAB.
AND THIS GAVE US WAY WAY TO
THINK ABOUT THINGS.
I'LL SAY THE MECHANISMS ASIDE
FROM OCCLUSION OF POLYMERASE
BINDING BY WHICH POSITIVE
REGULATORY FACTORS WORK TO DO
SOMETHING TO MAKE IT WORK
BETTER, STILL HAS NOT BEEN
MECHANISTICALLY SOLVED.
WE HAVE A PARADOX FOR THINKING
ABOUT WHAT IS GOING ON, BUT IF
ONE WANTED TO UNDERSTAND THE
MECHANISMS OF TRANSCRIPTIONAL
REGULATION, THERE IS STILL A
HUGE AMOUNT OF INFORMATION TO BE
MIND FROM THESE SIMPLE SYSTEMS.
SO WE BEGIN TO THINK ABOUT WHAT
HAPPENS IN MET ZERO ANS AND HOW
TRANSCRIPTION REGULATION WORKS.
WE CAN DRAW A MODEL THAT LOOKS
SIMILAR.
SO WE KNOW THE GLUCOCORTICOID
RECEPTOR IS SOLVED IN THE
ABSENCE OF HORMONE AND
ASSOCIATES WITH HORMONE CHANGES
WITH PROPERTIES SO IT GOES INTO
THE NUCLEUS AND BINDS TO
SPECIFIC SITES ON THE GENOME
GLUCOCORTICOID RESPONSE
ELEMENTS, GREs AND REGULATES
TRANSCRIPTION EITHER POSITIVELY
OR NEGATIVELY.
SO, IT LOOKS LIKE THE SAME
FRAMEWORK WE HAVE BEEN LOOKING
AT BUT, WE KNOW THAT
PROKARYOTIC-LIKE MODEL, CAN'T BE
THAT SIMPLE BECAUSE
GLUCOCORTICOID RECEPTOR ACTION,
UNLIKE INEE COAL I IS CONTACT
SPECIFIC.
CELLS ARE SPECIFIC.
IT'S NOT ALL ONE E.COLI CELL
AFTER ANOTHER.
THE WAY THE RECEPTOR WORKS IN
THE DIFFERENT SETTINGS IS
DIFFERENT.
SO IF YOU LOOK AROUND OR WALK
AROUND THIS WHEEL, HERE IS A
SERIES OF, IN THIS CASE, TISSUES
OR ORGANS IN WHICH THE
GLUCOCORTICOID RECEPTOR WORKS.
AND AS YOU READ THE LITTLE
CAPTIONS, YOU CAN SEE IN THOSE
DIFFERENT SETTINGS, RECEPTORS
ARE DOING DIFFERENT THINGS
PHYSIOLOGICALLY AND WE KNOW AT
THE MOLECULAR LEVEL AS WELL THAT
IS TRUE.
SO THE SAME REGULATORY FACTOR
BINDING TO THE SAME SMALL
MOLECULE ECHOLESTEROL-DERIVED
STARED HORMONE IS HAVING THESE
VERY DIFFERENT ACTIONS AND IT IS
DOING THAT BY REGULATING
DIFFERENT GENES IN THE CELLS.
SO IT LOOKS LIKE A SERIOUS
DEPARTURE FROM WHAT WE ARE
FAMILIAR WITH IN THE CASE OF.
SO THE QUESTION THEN ARISES, HOW
CAN A SINGLE REELINGTORY FACTOR
OF THE GLUCOCORTICOID RECEPTOR
FUNCTION IN THIS WAY SO THAT AS
YOU GO FROM SETTING TO SETTING,
WHAT HAPPENS IN A LIVER IS VERY
PRECISE.
AND IT'S VERY DIFFERENT FROM
WHAT HAPPENS IN THE KIDNEY.
HOW DO YOU GET BOTH PRECISION IN
STAMPING OUT THE SAME SET OF
REGULATORY GENES AND REGULATED
NETWORKS IN A GIVEN SETTING OR
ORGAN, AND YET BE FLEXIBLE
ENOUGH SO WHEN YOU GO TO THE
NEXT ORGAN OVER, EVERYTHING
CHANGES?
CHANGES ARE RATHER DRAMATIC IN
TERMS OF THE FRACTION OF GENES
THAT ARE DISTINCT IN ONE SETTING
OR ANOTHER.
NOW, OF COURSE THAT'S NOT ALL.
THIS IS NOT JUST SORT OF A PARTY
TRICK FOR MOLECULAR BIOLOGISTS,
BUT ALL OF THESE CHANGES ARE
THEMSELVES COORDINATED INTO
PHYSIOLOGICALLY SENSIBLE HOLE SO
YOU END UP HAVING THE STARED
HORMONE PUT INTO THE BLOODSTREAM
SIGNALING A PHYSIOLOGIC STATE
THAT THE BODY CAN RESPOND TO IN
A LOGICAL AND ORDER LEEWAY.
SO IF THERE THERE IS A NETWORK
OF INFORMATION THAT IS BEING
SOMEHOW HANDLED IN THIS WAY, SO
THAT YOU END UP WITH PRECISE
GENE REGULATION YET FLEXIBLE
FROM ONE SETTING TO ANOTHER.
SO THAT PARADOX, IN A SENSE,
THAT WE WOULD LIKE TO STRUGGLE
WITH HERE.
NOW, WHAT DO WE KNOW ABOUT WHAT
IS GOING ON IN MEATZOAN?
OR EUKARYOTES IN THIS CASE?
TEXTBOOK LEVEL INFORMATION.
ALL HELL BREAKS LOOSE.
THINGS ARE COMPLICATED.
YOU DON'T HAVE BINDING SITES FOR
ONE OR TWO FACTORS, HAVE YOU
THEM FOR MANY.
THEY ARE KIND OF SCATTERED ALL
OVER IN THE REGION OF THE GENES,
SORT OF.
A LOT OF DIFFERENT FACTORS ARE
BINDING, AT FLEET SOME CASES
SHOWN TO AGGREGATE TOGETHER IN
THIS SORT OF WAY.
THIS IS THE SORT OF CARTOON THAT
SENDS PEOPLE SCREAMING FOR THE
EXITS, YOU THINK YOU'RE FREE TO
LEAVE BUT YOU'RE NOT.
THAT DON'T WORK ON
TRANSCRIPTIONAL REGULATION AND
THEY SAY, WHEN YOU FIGURE IT
OUT, PLEASE COME BACK AND LET US
KNOW, BUT IN THE MEANTIME,
PLEASE DON'T BOTHER US.
SO THE QUESTION IS, IS THERE A
WAY TO THINK ABOUT THIS IN WHICH
WE CAN MOVE FORWARD?
SO I'M GOING TO PRESENT ONE THAT
I THINK IS A SIMPLIFYING
ASSUMPTION THAT I THINK ACTUALLY
IS BASED AT LEAST CONCEPTUALLY
IN FACT, AND I'M CERTAIN IT'S
BASED ON FACT IN GENERAL.
TO SORT OF DECONVOLUTE IN OUR
THINKING, CONSIDER THE NOTION
THAT THIS BIG GLOB OF STUFF IS
IN FACT CAN BE THOUGHT OF AS A
SINGLE FACTOR.
AND SO, I WOULD SAY THEN IT'S
THE COMBINATION OF GENE
REGULATORY PROTEINS, THE
COMBINATION OF GENE REGULATORY
PROTEINS THAT COME TOGETHER AND
THAT COMBINATION CAN BE THOUGHT
OF AS A SINGLE DISTINCT FACTOR.
IT HAS MULTIPLE POINTS AND A
SINGLE FACTOR.
AND IT IS BOUND SPECIFICALLY TO
A COMPOSITE RESPONSE ELEMENT,
DIFFERENT PATCHES OF DNA.
SEQUENCE TO WHICH SOME FACTORS
ASSOCIATE TO THEN GIVE RISE TO
THIS ASSEMBLED COMPLEX THAT CAN
BE VIEWED AS A SINGLE DISTINCT
FACTOR AND THE COMBINATION THAT
MAKES A DIFFERENCE.
NOW THIS TOO IS NOT A NEW IDEA.
BRITAIN AND DAVIDSON IN THE MID
60S, MAYBE EARLY 60S, BEGAN TO
WRITE ABOUT THE NOTION OF
SOLVING THE PROBLEM OF HOW WE
GAIN SPECIFICITY FOR REGULATION
BY USING JUST COMBINATORIAL
ARITHMETIC TO SOLVE THE
OTHERWISE ENDLESS LOOP OF
SAYING, IF YOU PROPOSE A
SELF-SPECIFIC FRACTOR, FOR
EXAMPLE, YOU NEED TO EXPLAIN HOW
YOU MAKE A CELL SPECIFIC FACTOR
AND WHAT YOU WOULD DO TO INVOKE
A CELL-SPECIFIC FACTOR.
THAT DOESN'T HELP.
THIS ALLOWS YOU TO ESCAPE THAT
PROBLEM.
WE CAN TALK ABOUT IT IN MORE
DETAIL BUT IT'S OB GAT THAT
SOMETHING LIKE THIS, THIS
COMBINATORIAL NOTION IS IN PLAY.
SO NOW YOU CAN SEE THAT THIS
LOOKS LIKE A SINGLE FACTOR,
ASSOCIATING MERE, A PROMOTOR,
AND REGULATING ITS EXPRESSION IN
A SPECIFIC WAY.
SO, COMBINATORIAL REGULATION,
GENE, CELL, PATH PHYSIOLOGY,
SPECIFIC COMPLEXES THAT ARE
BUILT FROM DIFFERENT
COMBINATIONS FACTORS THAT ARE
NOT UNIQUE BUT BROADLY EXPRESSED
BUT DIFFERENT SUBSETS FROM A
LONG MENU OF FACTORS THAT ARE
PRODUCED IN A GIVEN CELL.
THIS IS NICE.
BUT OF COURSE IT RAISES THE NEXT
LEVEL OF QUESTION.
WHAT SPECIFIES THE CONTEXT
SPECIFIC ASSEMBLY OF THIS UNIQUE
COMBINATION?
IT'S GREAT YOU CAN EVOKE ANY
COMBINATION.
WHAT SPECIFIES IT?
I'M GOING GIVE YOU TWO LEVELS OF
ANSWER TO THAT QUESTION OR TWO
WAYS AT LEAST TO CLOSE IN ON A
WAY OF THINKING ABOUT WHAT THE
ANSWER IS.
SO, THE FIRST IS THIS, A
COMBINATORIALITY.
BUT TO ANSWER THE SECOND
QUESTION, IS WHAT SPECIFIES THIS
ASSEMBLY OF UNIQUE COMBINATION
OF FACTORS IS THE SECOND LEVEL
ANSWER.
AND THAT IS, IT IS SIGNALING.
THAT SIGNAL, SPECIFICALLY SIGNAL
INTEGRATION, THE INTEGRATION OF
MULTIPLE BITS OF IN COMING
SIGNALING INFORMATION, THAT NIN
CASE, IN LINK ON A SINGLE
REGULATORY FACTOR.
HERE IS THE ANATOMICALLY CORRECT
VERSION OR DESCRIPTION OF THE
GLUCOCORTICOID RECEPTOR FROM M
TERMINUS TO C TERMINUS.
AF STANDS FOR ACTIVATION
FUNCTION SO THESE ARE REG --
REGULATORY DOMAINS.
THERE IS A DNA BINDING OR GENOME
BINDING IN THE MIDDLE AND C
TERMINIS IS THE REGION THAT
ASSOCIATES WITH THE HORMONES.
SO, I SAY THAT THERE ARE
MULTIPLE SIGNALS IMPINGING ON
THIS.
WE KNOW ONE A HORMONE.
AND SO, I THINK THE INTERESTING
THING ABOUT THESE MULTIPLE
SIGNALS I'M GOING RECOUNT FOR
YOU, AND WE'LL TALK ABOUT FOUR,
FOUR CLASSES OF SIGNALS, IS THAT
IN AGGREGATE, THEY PROVIDE
SIGNALING INFORMATION,
INFORMATION TO THE RECEPTOR THAT
ACCOUNTS FOR THIS NEED TO BE
GENE, CELL AND PHYSIOLOGY
SPECIFIC.
THE LIGAND IS A PHYSIOLOGICALLY
SPECIFIC SIGNAL.
THERE IS A NEUROENDOCRINE SIGNAL
THAT SAYS MAKE CORTISOL AND
DRIVES THE SIGNAL TO THE ADRENAL
CORTEX.
CORTEX DUMPS CORTISOL INTO THE
BLOODSTREAM AND SAYS THE
PHYSIOLOGICAL STATE OF THIS
ORGANISM IS SUCH THAT IT IS
UNDER STRESS AND TRYING TO GIVE
A SEMINAR AT THE NIH AND SO,
MAKES MORE GLUCORTICOIDS.
SO IT DOES THAT.
SO HERE IS A PHYSIOLOGICAL
STATUS SIGNAL FOR THE RECEPTOR
AND IT BINDS IN THE C TERMINUS.
RECEPTOR ALSO BINDS DNA
SPECIFICALLY IT BINDS TO WHAT WE
CALL A GBS, GR BINDING SEQUENCE
THAT SEQUENCE IS A SET OF BIG
SET OF VARIANTS OF THIS 15 MER,
WHICH YOU RECOGNIZE HAS
PALINDROMIC HEXAMER THAT IS ARE
SEPARATED BY THREE BASE PAIRS.
SO THE GR BINDING SEQUENCE NOW
IS A GENE-SPECIFIC SIGNAL, A
SIGNAL PROVIDING THE RECEPTOR
WITH INFORMATION ABOUT WHERE IT
IS ON THE GENOME, IT'S BINDING
SPECIFICALLY TO THE SEQUENCE.
THE GRE THAT I REFERRED TO, THE
GLUCOCORTICOID RESPONSE ELEMENT
IS A REGION OF THE GENOME THAT
IS ABLE TO CONFER A PARTICULAR
FLAVOR CONTEXT-SPECIFIC FLAVOR,
OF THE GLUCOCORTICOID RESPONSE
AND EMBODIES THE GBS.
WHAT A GRS A LARGER REGION OF
THE GENOME THAT INCLUDES NOT
ONLY THE GBS SEQUENCES, BUT
BINDING SITES FOR OTHER NON-GR
TRANSCRIPTIONAL REGULATORY
FACTORS AS WELL.
SO EVERY GRE IS A COMPOSITE
ELEMENT THAT CONTAINS BINDING
ELEMENTS FOR THE GLUCOCORTICOID
RECEPTOR AND OTHER NONRECEPTOR
TRANSCRIPTIONAL REGULATORY
FACTORS.
SO THE GRE AND THE GBS ARE GENE
SPECIFIC SIGNALS TO THE RECEPTOR
AND THEY SAY HERE IS WHERE YOU
ARE ON THE GENOME AND BEHAVE
ACCORDINGLY.
SO SOMEHOW THE SEQUENCE IS
CONFERRING SOME SIGNALING
INFORMATION AS WELL.
NOW YOU RECOGNIZE THAT THE
FACTORS THAT THEN ASSOCIATE ARE
GOING TO GIVE SOME
CELL-SPECIFIC, CELL CONTEXT
INFORMATION TO THE RECEPTOR
BECAUSE IT IS GOING TO BE THE
MENU OF FACTORS AND THE LEVELS
OF THEIR ACTIVITIES THAT ARE IN
FACT CELL SPECIFIC.
SO WHILE THERE ARE VERY FEW
FACTORS THAT ARE MADE ON ONLY
ONE CELL TYPE, THE RELATIVE
LEVELS OF THE MENU OF FACTORS
DOES VARY IN CELL SPECIFIC WAYS
AND SO THE AVAILABLE AND LEVELS
OF ACTIVITY OF THE NONGR FACTORS
WHO CAN PROVIDE CELL-SPECIFIC
INFORMATION TO THE RECEPTOR.
AND IF I'MY, THE FOURTH CLASS,
IS POSTTRANSLATIONAL
MODIFICATION.
RECEPTOR ITSELF IS MODIFIED IN A
BEVY OF DIFFERENT WAYS.
AND EACH OF THESE, EACH OF 7 OR
8 OR 9 PHOSPHORYLATION SITES,
FOR EXAMPLE, ON THE RECEPTOR IS
AN END POINT OF ONE SIGNAL
TRANSDUCTION PATHWAY COMING FROM
OUTSIDE OF THE CELL AND THEN
ACTIVATE A KINASE THAT
EVENTUALLY KINASE THAT REACHES
RECEPTOR AND DECORATES A
SPECIFIC RESIDUE.
THAT'S JUST ONE EXAMPLE IT'S ONE
KIND OF SIGNAL.
SO EACH OF THOSE THEN IS
PROVIDING PHYSIOLOGICALLY
SPECIFIC SIGNALING INFORMATION
TO THE RECEPTOR BY DECORATING
SPECIFIC SITES ALONG THE WAY
WITH PARTICULAR CHEMICALS.
SO NOW, YOU CAN SEE THAT IF YOU
THEN DO THE COMBINATORIAL
ARITHMETIC ON, THIS THE NUMBER
OF COMBINATIONS THAT COULD BE IN
AGGREGATE, BEING INPUT
INFORMATION INTO THE RECEPTOR TO
SAY, HERE IS YOUR STATUS CELL
SPECIFIC AND GENE SPECIFIC, AND
RESPOND ACCORDINGLY.
SO THAT IS THE IDEA THEN.
AND IT IS HAVING DONE THAT THEN,
THAT COREGULATORS ASSOCIATE AND
BEGIN TO BUILD OUT A REGULATORY
COMPLEX THAT IS BUILT OF MANY
PROTEINS, PROBABLY ON THE ORDER
OF 100 POLYPEPTIDE CHAINS AND
GROWN OUT SPECIFIC
TRANSCRIPTIONAL REGULATORY
COMPLEX.
NOW THERE ARE LOTS OF ACTION,
MUST HAVE MUCH OF THE REALLY
EXCITING WORK AT THE NIH ON THE
DYNAMICS OF THESE COMPLEXES.
SO THE ASSEMBLY IS ONE THING.
BUT WHAT WE KNOW IS THAT THE
WAYS THAT THEY ARE PRODUCED, HOW
STABLE THEY ARE, THE NECESSITY
FOR DECONSTRUCTING THESE
COMPLEXES ONCE THEY ARE BUILT, A
LOT OF ACTION HERE ABOUT WHICH
THERE IS GROWING BODY OF
INFORMATION AS I SAID, SOME OF
THIS HERE IN THE LABS AND THAT
ARE GOING TO BE VERY IMPORTANT.
IT'S IMPORTANT TO KNOW.
AND THERE ARE LOTS OF KINETIC
ACTIVITY THAT WILL NEED TO BE
CONSIDERED IN THE WAY WE THINK
ABOUT THESE COMPLEXES AND HOW
THEY WORK.
THEY ARE NOT JUST STABLE GLOBS
ON THE DNA THE WAY MOLECULAR
BIOLOGISTS LIKE TO DRAW THEM.
NOW WE HAVE A WAY OF THINKING OF
SAYING, THE RECEPTOR CAN BE MADE
DIFFERENT IN VERY UNIQUE WAYS
THAT RESPOND TO PHYSIOLOGIY AND
CELL TYPE AND GENE, TO THEN BE
ABLE TO DO DIFFERENT THINGS.
HOW DOES THAT WORK?
AND HYPOTHESIZED CHANGING THE
SHAPE OF RECEPTORS IN SPECIFIC
WAYS TO DRIVE THE ASSEMBLY OF
UNIQUE COMPLEXES OF FACTORS.
AND SO IT COULD PRODUCE GR
SURFACE THAT IS RECRUIT DISTINCT
COMBINATIONS OF FACTORS.
AND DRIVES YOUR THINKING IN A
COUPLE OF INTERESTING WAYS.
AT LEAST IT DRIVES MY THINKING.
FIRST OF ALL, IT SAYS THAT THE
REGULATORY COMPLEX COMPOSITION
AND ACTIVITY AND STRUCTURE
DEPEND ON THESE DIFFERENT
CONTENTS.
NOTHING HARD WIRED ABOUT THE
SHAPE OF THE RECEPTOR AND WHAT
IT IS GOING TO ASSOCIATE WITH.
THE SECOND IS IMPORTANT TOO.
THE SIGNALS, AND THE RECEPTOR
ITSELF, BUT IN THIS CASE, THE
SIGNALS, LACK INTRINSIC
ACTIVITIES.
IF YOU OPEN A PHARMACOLOGY
TEXTBOOK, YOU WILL BE ABLE TO
LOOK AT A SET OF STRUCTURE THAT
SAY THIS IS A STRONG AGONIST, A
STRONG ANTAGONIST AND SO FORTH.
THAT'S TRUE IN SOME CONTEXT BUT
THAT STRONG AGONIST IN A
DIFFERENT CONTEXT MAY BE
ANTAGONIST IT'S ALL CONTEXT
SPECIFIC AND THIS NOTION THAT
YOU CAN IDENTIFY THINGS WITH A
GIVEN KIND OF ACTIVITY AND GIVEN
IN A DEFINED CONTEXT.
SO I'M GOING TO SPEND THE REST
OF THE TALK TALKING ABOUT THESE
TWO QUESTIONS.
VERY ASYMMETRICALLY.
ONE WITH NOTIONS IN PLACE, HOW
CAN WE THINK ABOUT HOW TO
ACHIEVE THIS PRECISION OF
FLEXIBILITY?
AND I'M GOING TO SCRUNCH -- ONE
POSTDOC'S A HUGE AMOUNT OF WORK
INTO 4 OR 5 SLIDES TO SUMMARIZE
THAT AND THEN SPEND THE REST OF
THE TIME WITH A SECOND QUESTION
OF THIS IDEA, THE HYPOTHESES
THAT THE SIGNALS ARE ACTING IN
AN ALLOSTERIC WAY CHANGING THE
SHAPE OF THE RECEPTOR AND
THEREFORE, SPECIFYING THE
SURFACES TO WHICH OTHER FACTORS
CAN APPEND AND THEN BUILDING OUT
SPECIFIC REGULATORY COMPLEXES
FROM THERE.
SO, A FANTASTIC POSTDOC CAME TO
THE LAB AND SAID, THIS PRECISION
FLEXIBILITY THING IS COOL.
LET'S DO SOMETHING TO FIGURE IT
OUT.
HIS IDEA WAS, HE SAID WHAT WE
SHOULD DO IS REFINE THE QUESTION
AND LOOK IN TWO DIFFERENT CELL
TYPES AT A GENE THAT IS
GLUCOCORTICOID REGULATED IN CELL
TYPES AND BY DIFFERENT
MECHANISMS.
AND I SAID, YOU'RE RIGHT.
THAT WILL BE GREAT SIMPLIFYING
WAY TO LOOK AT THINGS.
HOW ABOUT YOU LOOK AT A GENE
THAT IS REGULATED BY THE SAME
FACTOR GR IN 2 CELLS TYPES AND
IN THIS CELL?
AND HE SAID, NOT A PROBLEM.
HE WENT AWAY AND CAME BACK.
AND SAID NOT A PROBLEM, WE'LL
LOOK FOR GENES THAT ARE
ACTIVATED BY GR IN ONE CELL AND
WE PRESSED -- REPRESSED BY GR IN
ANOTHER GENE.
I SAID, I KNEW THAT.
I WAS TRYING TO FIGURE OUT IF
YOU WOULD THINK OF IT.
[ LAUGHS ]
SO HE SORTED THROUGH OUR
EXPRESSION ARRAYS AND IN TWO
CELL TYPES, HUMAN CELL TYPES,
A549 FOR LUNG AND U2O S FOR
BONE, OSTEOSARCOMA, ACTUALLY.
AND OUT OF THE SEVERAL THOUSAND
GENES THAT ARE REGULATED IN
THOSE CELLS, I THINK ABOUT 2000
OF THEM OVERLAP.
OUT OF THOSE, 31 WERE REGULATED
IN OPPOSITE DIRECTIONS.
SO HERE IS AN EXAMPLE OF FOUR OF
THEM.
AND YOU CAN SEE IN THIS CASE,
REPRESSING THEY ARE OPPOSITE
HERE AND SO FORTH.
LOOKING ACROSS THE TIME
COORDINANTS.
SO THAT WAS INTERESTING.
SO HE HAD A SET OF THINGS TO
LOOK AT SO HE COULD ASK, WHERE
IS THE FLEXIBILITY COME FROM TO
BE ABLE TO SWITCH MECHANISMS
LIKE THIS?
IN THIS PROFOUND WAY OF LOOKING
AT POSITIVE AND NEGATIVE GENES?
HOW ARE PRECISION AND
FLEXIBILITY ACHIEVED AT THESE
GREs THAT CAN END UP ON ONE
SIDE OF THE FENCE OR THE OTHER
WITH RESPECT TO ACTIVATION OR
OPPRESSION?
WHEN HE BEGAN TO CHARACTERIZE
THESE GENES, HE GOT A SURPRISE
WITH ONE OF THEM.
IT'S HERE.
HERE IS DOSE RESPONSE STUDIES
AND YOU CAN SEE THAT THERE IS
SOME BEHAVIORS.
SO THAT IS THE CONCENTRATION AT
WHICH HE PICKED UP THESE 31
GENES, WAS 10 TO THE MINUS
7th MOLAR AND YOU CAN SEE
ACTIVATED 549, REPRESSED IN U2OS
AND VICE VERSA AT THE STANDARD
CONCENTRATION.
BUT WHEN HE DID THE DOSE
RESPONSE, LOOK WHAT HAPPENED.
THIS GENE ANCHORED 1, GOES
THROUGH THIS DISCONTINUITY.
AND SO, THE GENE IS INDEED
REPRESSED 10 TO THE MINUS
7th MOLAR AT STARRED
CONCENTRATION BUT IN LOWER
CONCENTRATIONS IT'S ACTIVATED
AND THEN CRASHES AND BECOMES
REPRESSED.
SO WE PUZZLED OVER THAT.
AND HE SAID HE DIDN'T KNOW HOW
TO BEEN WHAT HE WAS GOING TO DO.
HE WAS GOING TO TRY DIFFERENT
WAYS TO VARY THE LEVELS OF
ACTIVITY OF THEIR RECEPTORS.
SO HE WASN'T THINKING OF THIS AS
THE CONCENTRATION.
HE WAS THINKING OF IT AS A WAY
TO ACHIEVE A LOW-LEVEL OF GR
ACTIVITY IN THE CELL.
WE HAVE OTHER TWICE ACHIEVE
THAT.
WE HAVE LIGANDS AREN'T AS
STRONG.
WE HAVE AT LEAST IN SOME
CONTEXT, WE HAVE COFACTORS THAT
THE GR DEPENDS ON THAT WE CAN
PLAY WITH.
WE CAN LOOK AT RECEPTOR LEVELS.
WE CAN PUT IN MUTATIONS AND SO
WE BASICALLY DID ALL OF THEM.
AND SO, THAT'S WHAT HE DID.
HE WANTED TO MODULATE THE GR
ACTIVITY BY CHANGING ALL THIS
OTHER STUFF.
I'M NOT GOING TO DRAG YOU
THROUGH THAT TO SHOW YOU JUST
ONE CARTOON THAT MAKES AN
IMPORTANT POINT.
NO YOU'RE LOOKING AT THE TWO
DIMENSIONAL HEAT MAP.
I KNOW YOU CAN'T REED THE LABELS
FROM BEYOND THE FIRST ROW.
THIS IS TIME AND HORMONE DOSE.
AND YOU CAN SEE FROM THE SHAPE
OF THE TWO DIMENSIONAL HEAT MAPS
THAT EACH OF THE FOUR GENES IS
DIFFERENT.
MORE IMPORTANTLY, WHAT CAN YOU
SEE IS THAT THERE ARE CONDITIONS
IN WHICH LOW LEVELS OF ACTIVITY,
EACH OF THE GENES IS ACTIVATED
AND THEN REPRESSED.
IF IT'S NOT REPRESSED DOWN TO
THE BASELINE, IT ENDS UP BEING
ACTIVATION F IT IS REPRESSED
BELOW THE BASELINE IT'S THAT
REPRESSION.
BUT ALL DISPLAY THE SAME
DISCONTINUOUS BEHAVIOR.
SO WHAT THAT TOLD HIM IS THAT
THERE MIGHT BE A CONSTANT
TRANSCRIPTIONAL REGULATORY MOTIF
THAT IS STAMPED OUT FOR EACH OF
THESE GENES.
WHETHER THERE IS SOMETHING
FANCY, I DON'T KNOW.
AT LEAST IN EACH CASE, HE CAN
FIND CONDITIONS WHERE HE CHANGES
THE LEVEL OF RECEPTOR ACTIVITY
AND END UP WITH THIS SURPRISING
BEHAVIOR.
IT'S SURPRISING UNLESS YOU'RE
STUDYING TRANSCRIPTION NETWORK
MOTIFS BECAUSE THAT IS WHAT HE
HAS BEEN DOING FOR *** AIDS BY
THE TIME WE DID THESE STUDIES --
DECADES.
PUBLISHED IN 2003, A SERIES OF
THESE TRANSCRIPTION NETWORK
MOTIFS, BEHAVIORS AND EACH OF
THESE GENES, THEIR BEHAVIORS
COULD BE ACCOUNTED FOR WITH SUCH
NETWORK MOTIF THAT ALANHAD GIVEN
THIS CHARMING NAME OF IN
COHERENT TYPE ONE FEED FAR LOOP.
IT ROLLS OFF THE TONGUE, I
THINK.
WHAT CHARACTERIZES THIS TYPE ONE
FFL IS THIS.
THAT IS THAT AT LOW REGULATORY
FACTORS, HE DID THESE STUDIES IN
BACTERIA AND THEN ARREST AND
THEN MORE RECENTLY -- AND THEN
AT LOW LEVELS OF GR.
THE TARGET GENE IS ACTIVATED.
BUT AS GR ACTIVITY INCREASES, IT
PASSES THROUGH AN UNDEFINED,
MOLECULARLY UNDEFINED MODE,
CALLED INHIBITORY THRESHOLD
WHERE THE RECEPTOR SWITCHES FROM
BEING ACTIVATOR TO REPRESSOR.
AT A TIME OR DOSE OR SOMETHING
AFFECTING RECEPTOR ACTIVITY
DEPENDENT WAY, THE GENE SWITCHES
FROM BEING ACTIVATED TO BEING
OPPRESSED.
SO, SIGNAL SPECIFIC AFFECTS FOR
DIFFERENT SIGNALS AND GENES ARE
AFFECTED IN DIFFERENT WAYS AND
GIVES RISE TO DIFFERENT
PATTERNS.
INHIBITORY ARMS OF THIS NETWORK
COULD PRODUCE PRECISION AND
FLEXIBILITY AND GIVE RISE TO
ACTIVATION, GENE REPRESSION OR
NO NET ACTIVITY IF THEY ARE
BALANCED.
SO THERE ARE TWO WAYS TO NOT
HAVE A RESPONSIVE GENE.
ONE IS TO BALANCE ACTIVATION AND
REPRESSION AND THE OTHER IS TO
NOT HAVE THE RECEPTOR IN PLAY.
AND SO, BY AFFECTING THE
RELATIVE AMOUNT OF ATTENTION
THAT EACH OF THESE ARMS GET, YOU
WOULD END UP WITH A CONDITION OR
CONTEXT DEPENDENT ACTIVATION OR
OPPRESSION IN THE CASE WE ARE
LOOKING AT, CELL TYPE DEPENDENT.
NOW, THIS NOTION HAS INTERESTING
APPLICATIONS FOR THE COMPOSITION
AND STRUCTURE AND ACTIVITIES
THAT ONE MIGHT FIND IN
REGULATORY COMPLEXES AS YOU LOOK
ON ONE SIDE OR THE OTHER OF THE
ACTIVATION VERSUS REPRESSION
DOSE.
THOSE ARE THE KINDS OF
EXPERIMENTS THAT ARE NOW ONGOING
IN THE LAB.
OKAY.
SO, THE MOST PROVOCATIVE NOTION
ON THIS, AND THE ONE THAT WE ARE
EXCITED ABOUT IN OPERATING ON
RIGHT NOW, IS THIS NOTION THAT
THIS SIGNAL REGULATED V4 NETWORK
MACHINERY MIGHT BE RESIDENT
WITHIN THE REGULATORY COMPLEX
ITSELF.
AND IN VARIOUS INDIRECT
EXPERIMENT REASONS FOR BELIEVING
THAT IS THE CASE.
IF YOU READ ALON'S PAPERS, HE
HAS THE DIFFERENT ARMS OF THE
NETWORK, ACTUALLY BEING
ACTIVATION OF DIFFERENT GENES,
DIRECT ACTIVATION OF THE TARGET
GENE AND THEN ACTIVATION OF
ANOTHER GENE WHICH BECOMES A
REPRESSOR ON THE TARGET GENE.
THAT WORKS TOO BUT WE HAVE DONE
EXPERIMENTS TO SUGGEST THAT IT
MAY NOT BE THAT COMPLICATED AND
A MORE ELEGANT SOLUTION WOULD BE
THE SWITCH IS RESIDING WITHIN
THE REGULATORY COMPLEX ITSELF
AND THAT IS THE MOLECULAR
CHANGES THAT ARE INVOLVED IN
THROWING THAT SWITCH THAT
PROVIDES FLEXIBILITY, MAY BE
QUITE SIMPLE.
WE'LL SEE.
BUT THAT IS THE IDEA.
THAT THIS SET OF NETWORK MOTIFS
AND THERE ARE OTHERS THAT HAVE
BEEN INFERRED, IF NOT
IDENTIFIED, IN THE CASE OF GR,
OTHER THAN THE TYPE I FEED
FORWARD LOOP THAT WE WERE
LOOKING FOR.
AND SIGNAL INTEGRATION BY
REGULATORY COMPLEXES THEMSELVES
COULD INFER THIS.
SO I'M GOING LIFE THAT AND I'LL
COME BACK TO IT AT THE END AND
MOVE ON TO THE SECOND
MECHANISTIC QUESTION.
AND THAT IS THE NOTION OF GR
ALLOSTERY DRIVING THIS
REGULATORY SPECIFICITY.
THAT NOTION HAD BEEN AROUND THE
LAB FOR A LONG TIME IN VARIOUS
FORMS BUT A SET OF EXPERIMENTS
BY LISA WATSON IN THE LAB GOT US
ON TRACK TO BEGIN TO DO SOME
DIRECT STRUCTURAL ANALYSIS.
SIMPLIST IRK.
TAKE THE 15 BASE PAIR GBS
SEQUENCE AND TAKE A HALF DOZEN
DIFFERENT ONES, FOR THOSE WHO
ARE CLOSE ENOUGH CAN SEE THESE
AREN'T VERY DIFFERENT.
ONE HEXAMER IS ALWAYS THE SAME.
THE SPACER SEQUENCES VARY A
LITTLE BIT AND THE RIGHT HAND
HEXAMER VARIES A LITTLE BIT.
THAT'S IT.
THEY ARE VERY SIMILAR TO ATTACK
ONE ELEMENT INTO A SIMPLE
REPORTER PLASMID AND TRANSFECT
THE CELLS.
AND LISA ASKED TWO QUESTIONS.
ONE IS, ARE SEQUENCE VARIANTS OF
THE 15 MERES ENOUGH TO LEAD TO
DIFFERENCES IN TRANSCRIPTIONAL
REGULATION?
YOU SEE THEY ARE.
ACTIVITY VARIES OVER A 4-FOLD
RANGE WHEN ALL YOU HAVE DONE IS
TO CHANGE THE SPACER SEQUENCE.
THE SPACER SEQUENCE IS ALMOST 20
ANGSTROMS FROM THE NUCLEOTIDES
AND SPACER ARE ALMOST 20
ANGSTROMS AWAY FROM THE NEAREST
PART OF THE PROTEIN.
CHANGE THE SPACER SEQUENCE AND
YOU CAN GET A 4-FOLD VARIATION
IN THE LEVEL OF GENE ACTIVATION.
THE SEQUENCE IS DOING SOMETHING
IMPORTANT AND WE LIKE TO KNOW
WHAT IT IS.
THE KEY SECOND EXPERIMENT THAT
REALLY SAID THAT SHOULD YOU
THINK ABOUT STRUCTURE,
CONFIRMATION, IS THAT THERE IS
NOT A CORRELATION BETWEEN THE
TRANSCRIPTIONAL REGULATORY
ACTIVITY AND THE AFFINITY WITH
WHICH THE RECEPTOR BINDS TO THE
SEQUENCES.
IT'S ALMOST THE OPPOSITE.
SO THE MOST TIGHTLY BINDING ONES
REGULATE THE LEAST WILD AND VICE
VERSA.
NOTHING ELSE IS GOING ON THAT IS
MOST INTERESTING.
THAT GOT US THINKING ABOUT THE
STRUCTURE.
WE HAVE BEEN THINKING ABOUT
STRUCTURE FOR SOMETIME.
AND WE HAD DONE SOME EXPERIMENTS
TO FIGURE THIS OUT EARLIER, THAT
HELPED TO SORT OF DRIVE THE
DIRECTION, THE EXPERIMENTAL
DIRECTION THAT LISA WATSON
PURSUED.
WHAT NAY HAD SHOWN OR HAD DONE,
WAS TO CRYSTALLIZE ON TO
DIFFERENT 15 MERES AND THEN ASK
IF THE CONFIRMATIONS OF CRYSTAL
GRAPHICS OF THE RECEPTORS WERE
DIFFERENT WHEN THEY HAD
DIFFERENT SEQUENCES.
AND THE ANSWER WAS FROM ONE OF
THESE SORT OF, SORT OF ANSWERS.
AND THAT IS THAT THERE WAS ONE
REGION OF THE DNA BINDING DOMAIN
THAT WE CALL THE LEVER ARM, FOR
HISTORICALLY FORGETTABLE
REASONS, THAT CHANGED THE
CONFIRMATION.
AND SO IT WAS SORT OF LIKE THE
HALF ANSWER.
WHAT DO WE DO WITH THIS ANSWER?
THIS IS AGAIN THE DELIVERY ARM
FAR FROM THE DNA.
HOW IS IT GETTING INFORMATION?
WE ARE TRYING TO -- SHOULDN'T
THERE BE CHANGES HERE?
WHAT IS GOING ON?
WE KNEW FROM EARLIER CRYSTAL
GRAPH ANALYSIS THAT ONLY SIX
NUCLEOTIDES OF 15 WERE CONTACTED
IN A NUCLEOTIDE-SPECIFIC WAY BY
THE RECEPTOR.
SO, THESE GUYS IN RED ARE
ACTUALLY TOUCHED BY THIS
RECOGNITION HELIX AND TOUCHES
THE EDGES OF THE BASES.
SO, DOES THE LEVER ON
CONFIRMATION IMPLY SOME SORT OF
ALLOSTERIC PATH?
WE PLAYED IT UP BIG IN SCIENCE
PAPERS BUT WE DIDN'T KNOW.
AND WHAT IS GOING ON?
ONLY NINE OF THE 15GBS
NUCLEOTIDES ARE NOT CONTACT AND
WE KNEW FROM LISA'S EXPERIMENTS
THAT SHE COULD CHANGE THE
NUCLEOTIDES IN DIFFERENT GBSs
THAT WERE NOT TOUCHED BY THE
RECEPTOR AND YET THE ACTIVITY
TRANSCRIPTIONAL ACTIVITIES WERE
CHANGING.
SO BOTH OF THOSE QUESTIONS VERY
RELEVANT.
NOW FROM STARING HARD AT THE
CRYSTAL GRAPHIC WORK, MILES AND
LISA WERE ABLE TO DRAW ONE
CONCLUSION FROM ONE PAIR OF
GBSs, ABOUT WHAT COULD BE
GOING ON AS YOU CHANGE THE
SPACER SEQUENCE.
THAT'S THIS PAIR.
AND AGAIN, THOSE OF YOU YOU
CLOSE ENOUGH TO SEE WILL SEE THE
HEXAMERS ARE IDENTICAL IN THESE
TWO GBSs AND ALL THAT IS
DIFFERENT IS THAT THE SPACER
CHANGES FROM Gs TO As.
NOW WE KNEW FROM OLD WORK, THAT
HAVE SHOWN WHEN YOU HAVE STRANDS
LIKE THIS AND HAVE As ON ONE
STRAND, YOU TEND TO CONSTRICT
THE MINOR GROOVE AND THE CRYSTAL
GRAPHIC STRUCTURE SHOWS THAT WAS
TRUE.
SO THIS GRAY GUY THAT HAS A RUN
OF As HAS A NAYOER MINOR
GROOVE THAN THE ONE WITH THE
Gs.
SO THAT WAS INTERESTING.
BECAUSE IT INDICATED THAT THE
SPACER SEQUENCE JUST CONFIRMED
THAT THE SPACER SEQUENCE WAS
CHANGING THE DNA SHAPE AND A
HARD LOOK AT THE SITE CHANGE IN
THAT NEIGHBORHOOD REVEALED THERE
WAS A LYSINE RESIDUE AT 490 THEY
FLIPPED ON WHETHER THE MINOR
GROUP WAS NARROW OR NORMAL WHEN
WAS: WHEN IT WAS NOR, THE SITE
CHAIN CAME DOWN AND MADE CONTACT
WITH THIS STRAND OF THE DNA AND
WHEN THE GROOVE WAS NARROW, THIS
CHAIN FLIPPED AND REACHED OVER
AND MADE CONTACT WITH THE OTHER
STRAND.
WE COULD TRY TO PUT TOGETHER A
HAND WAVING ARGUMENT THAT ARGUED
WHEN THE SITE CHAIN FLIPPED, IT
WOULD EFFECT THE CONFIRMATION OF
THE LEVER ARM THAT IS MAYBE THE
CASE, BUT AT FLEET THIS CASE, WE
COULD ARGUE WITH SOME REASON
THAT THE DNA SHAPE WAS BEING
READ BY THE SPACER SEQUENCE.
BEING AFFECTED BOY THE SPACER
SEQUENCE AND BEING READ BY THE
REST OF THE RECEPTOR IN SOME WAY
THAT MIGHT INVOLVE THIS FLIP.
SO, THAT IS ALL NICE AND IT WAS
A WAY TO BEEN AT LEAST THE
SPACER SEQUENCE CHANGES.
HERE WE DON'T HAVE GOOD WAYS TO
THINK ABOUT THE OTHER ONES IN
THE ANALYSIS WE HAVE DONE.
SO IT REMAINS AN OPEN QUESTION.
BUT IT LEFT STILL OPEN THE
BIGGER QUESTION OF, IS THERE ANY
EVIDENCE FOR SOME KIND OF
ALLOSTERIC PATH TO GO FROM THAT
HELIX REACHING INTO THE MAJOR
GROOVE AND TOUCHING THE EDGES OF
THE BASES AND SENSING THE
SEQUENCE DIFFERENCES AND DRIVING
OUT TO CONFIRMATIONAL CHANGES IN
THE REST OF THE DNA BINDING
DOMAIN THAT COULD TRANSMIT
INFORMATION TO THE REST OF THE
RECEPTOR?
SO, LISA SAID, WELL, SOMETHING
THAT WE WERE WORRIED ABOUT WHEN
WE STARTED CRYSTAL GRAPHIC
ANALYSIS, WE WERE PROBABLY GOING
TO BE LOOKING FOR CONFIRMATIONAL
CHANGE THAT IS REQUIRED SUCH
SMALL CHANGES IN DELTA G THAT IT
WOULD BE EASY TO IMAGINE THE
CRYSTAL PACKING FORCES COULD
MASK THOSE CHANGES AND MAYBE
THAT WAS WHY WE WERE SEEING
CHANGES IN THE LEVER ARM.
SO LISA WENT OFF TO LEARN NMR.
AND SO WHAT YOU'RE LOOKING AT
HERE IS A TWO DIMENSIONAL
HETERONUCLEAR SINGLE QUANTUM
CORRELATION HSQC SPECTRUM BY
NMR.
AND IF YOU DON'T KNOW ANYTHING
ABOUT NMR, ALL YOU NEED TO KNOW
IS IT IS SCORING THE CHEMICAL
ENVIRONMENT OF THE AMYLOID BONDS
AT EAST AMINO ACID IN THIS 89
AMINO ACID FRAGMENT.
THERE IS 89 SPOTS AND EACH IS
REPORTING ON THE CHEMICAL
ENVIRONMENT DISPLAYED OUT IN TWO
DIMENSIONS OF THE BOND AT EACH
AMINO ACID.
LISA WENT TO THE HARD WORK OF
ASSIGNING EACH SPOT, AND SHE
COULD THEREFORE ASK HOW DOES THE
BINDING SEQUENCE AFFECT THE
CONFIRMATION?
SO WHAT YOU'RE LOOKING AT HERE
IS THE DNA BINDING FRAGMENT
ALONE.
WHAT HAPPENS WHEN YOU PUT IT ON
TO DIFFERENT DNA SEQUENCES?
THAT IS WHAT IS SHOWN HERE.
SO NOW HAVE YOU SEEN THE OVERLAY
AT THREE DIFFERENT SEQUENCES AND
EACH OF THE COLORS REFLECTS A
DIFFERENT GBS AND YOU CAN SEE
EACH FROM THE BACK OF THE ROOM
THAT SOME SPOTS ARE COMPLETELY
COINCIDENT AND THAT OTHERS ARE
NOT.
A BOXED THREE OF THESE POSITIONS
BECAUSE I'M GOING TO BE TALKING
ABOUT THEM AS WE GO THROUGH THE
REST OF THE TALK.
POSITION 477 AND 478 ARE IN THE
DIMERIZATION REGION.
WE WILL LOOK HARDER AT THAT AND
POSITION 470 IS IN THE LEVER
ARM.
SO ALREADY WE CAN BEGIN TO
EXTRACT INFORMATION FROM THESE
BY ASKING, WHAT GR SURFACES ARE
BEING REFLECTED BY REFERRING THE
NMR STRUCTURE BACK TO THE
CRYSTAL STRUCTURE TO SEE WHERE
WE ARE IN THE MOLECULE AS IT IS
SITTING ON THE DNA?
SO FOR EXAMPLE, LISA WAS ABLE TO
LEARN QUICKLY THAT CHANGES IN
THE SPACER, HERE IS A SET OF
THEM, DIFFERENT SPACER
SEQUENCES, BUT NOT IN HAVE SITE
POSITIONS 13 AND 15 THAT WE
CHOSE FOR SPECIFIC REASONS THAT
THEY ARE NOT CONTACTED BUT
DIRECTLY BY THE GR, CHANGES IN
POSITION 13 AND 15 AFFECT THE
DIMER INTERFACE.
THE SPACER SEQUENCES AS YOU
CHANGE SPACERS, YOU CAN SEE THE
SPOTS ARE IN DIFFERENT PLACES
FOR 477 OR 478.
BUT AS YOU CHANGE THE HALF
SITES, THEY ARE NOT IN DIFFERENT
PLACES.
SO SHE CAN SAY, AH, YOU CHANGED
THE SPACER SEQUENCE, YOU CHANGE
THE INSTRUCT NUR THIS REGION OF
THE DNA BINDY DOMAIN.
BUT IF YOU CHANGE THE HALF SITE,
YOU DON'T.
THERE IS SOME SPECIFIC MAP THAT
RELATES CHANGES IN A PARTICULAR
PART OF THE GBS SEQUENCE TO
CHANGES IN CONFIRMATION.
NOT ONLY THAT, BY LOOKING AT IN
THIS HEAT MAP LEPSENTATION,
LOOKING AT THE RESIDUES THAT
CHANGE IN THEIR CONFIRMATION AS
A FUNCTION OF THE GBS SEQUENCE,
SHE CAN NOW SEE THAT THERE ARE
CHANGES THAT ARE STANDARD FROM
DNA RING LETS ALL THE WAY
THROUGH THE LIVER ARM OUT TO THE
DIMERIZATION REGION AND SHE CAN
AT LEAST INFER AN ALLOSTERIC
PATH.
SO THE DNA SIGNALS APPEAR TO BE
TRANSMITTED FROM THE DNA READING
HEAD OUT TO AT LEAST TWO, IF NOT
THROUGH, THE DIMER INTERFACE.
AND IT LED TO THIS FUNCTIONAL
QUESTION THEN, DOES THAT MATTER?
DOES THE DIMER INTERFACE
CONTRIBUTE IN SOME WAY TO THIS
SPECIFICITY OF TRANSCRIPTIONAL
REGULATION BY THE RECEPTOR?
SO, THE WAY TO TEST THAT IS TO
MAKE A MUTATION IN THE DIMER
INTERDAYS AND ASK WHAT THE
CONSEQUENCES ARE STRUCTURALLY
AND WITH RESPECT TO THE
ACTIVITY.
SO LISA DID THAT AND SHE CHOSE
THIS RESIDUE FOR A PARTICULAR
REASON.
AND THAT IS THAT MUTATIONS AT
THAT SITE -- SO IN 1991, WHEN WE
CRYSTALIZED THE DNA BINDING
DOMAIN RECEPTOR ON A GENERIC
GBS, WE WERE ABLE TO SEE A SET
OF FOUR CONTACTS THAT BROUGHT
THE TWO MONOMER DOMAINS
TOGETHER.
AND ONE OF THOSE SITES, A477T
GOT FAMOUS BECAUSE A MUTATION
WAS MADE OF THEM AND CARRIED
THAT MUTATION THROUGH A LOT OF
DIFFERENT PHYSIOLOGICAL SETTINGS
THEMSELVES AND NOW ALL THE WAY
INTO MICE.
AND IS ABLE TO SEE PHENOTYPES
ALL THE WAY AT THE ANIMAL LEVEL
OF CHANGING THAT RESIDUE.
SO WE KNEW THAT IT WAS A
FUNCTIONAL RESIDUE IN SOME WAYS.
WHETHER IT IS MORE IMPORTANT
THAN OTHERS, NO OTHER SITES HAVE
BEEN EXPLORED TO THAT DEPTH.
AT LEAST WE KNEW FROM THE WORK
DERIVED BY OTHERS THAT HAVE
LOOKED AT THIS MUTATION, THAT
SOMETHING FUNCTIONAL WAS
OCCURRING WHEN YOU MADE THIS
MUTATION NOT JUST A MOLECULAR
BIOLOGICAL TOOL.
SO THIS REGION THEN MAKES A
CONTACT AND YOU CAN ASK WHAT
HAPPENS WHEN YOU DISRUPT THE
DIMERIZATION REGION.
HOW DOES ALTERING THE DIMER
INTERFACE AFFECT CONFIRMATION?
YOU CAN SEE IN THE OVERLAY THERE
ARE CHANGES FROM THE HEAT MAP
AND THOSE THAT ORIGINATED IN THE
DIMER INTERFACE THEN EXTEND ALL
THE WAY BACK TO THE DNA
INTERFACE.
SO THE DIMER INTERFACE MUTATION
EFFECTS THE DNA BINDING
INTERFACE.
ALLOSTERIC COUPLING APPEARED TO
BE BIDIRECTIONAL.
AGAIN, DYNAMICALLY YOU PREDICT
THAT TO BE THE CASE.
AND THIS IS VERY GENETIC SUPPORT
FOR THAT IDEA.
I'M NOT GOING TO TALK AT ALL
ABOUT A BEAUTIFUL SERIES OF
STUDIES THAT WAS DONE.
THERE IS A NATURAL PLACE VARIANT
IN THE GR CALLED GR GAMMA AND IT
MOVES A SITE IN THE A WAY THAT
EXTENDS THE MESS GER RNA FOR THE
GR BY THREE NUCLEOTIDES.
THE THREE NUCLEOTIDES CODE FOR
ARGININE RESIDUE THAT SITS RIGHT
IN THE MIDDLE OF THE LEVER ARM
SO WE ARE INTERESTED TO SEE WHAT
THE CONSEQUENCES ARE FOR
STRUCTURE AND ACTIVITY.
WE HAVE DONE A BEAUTIFUL SERIES
OF STUDIES THAT AGAIN SHOWED
THAT WHEN YOU MUTATE IN THIS
CASE, THE MIDDLE OF THE INFERRED
PATH WAY, YOU SEE CHANGE THAT IS
GO OUT IN BOTH DIRECTIONS THAT
ARE AFFECTING BOTH STRUCTURE AND
ACTIVITY.
SO PRETTY SERIOUS EXPERIMENTS OF
HIS.
AND OF COURSE THIS ALSO HAS
BROAD IMPLICATIONS FOR
COMBINATORIAL SIGNALING.
NOW, HAVING THIS EFFECT GO OUT
FROM THE INTERFACE, DNA
INTERFACE OUT TO THE DIMER, AT
LEAST LED TO A QUESTION OF
WHETHER THE ALLOSTERY GOES
BEYOND.
THERE ISN'T JUST A BEYOND, THERE
IS A LOW FRAGMENT OF THE GR.
BUT IT IS A DIMER.
SO YOU COULD ASK DOES THE
SIGNALING FROM ONE HEXAMER, ONE
MONOMER'S CONTACTING, GO UP
THROUGH THAT MONOMER, EXTEND
THROUGH THE DIMER INTERFACE INTO
THE OTHER MONOMER?
THAT WOULD BE INTERESTING.
SO, LISA ASKED THAT QUESTION.
HERE JUST TO SIMPLIFY THINGS,
SHE IS LOOKING ONLY AT THIS ONE
RESIDUE IN THE LEVER ARM.
WE KNEW THERE IS A LOT OF
SENSITIVITY CONFIRMATIONAL AND
FUNCTIONAL SENSITIVITIY THERE.
SO LOOKING AT GLYCIN 470, HERE
IS A GBS IN WHICH THE HEXAMER
SEQUENCES ARE DIFFERENT.
SO IT STANDS TO REASON THAT THE
LEVER ARM RESIDUE IN THE TWO
MONOMERS WOULD BE IN DIFFERENT
CHEMICAL ENVIRONMENTS AND YOU
SEE TWO SPOTS.
IN THIS RESPONSE, IN THIS GBS,
THE HEXAMERS ARE THE SAME.
STOW STANDS TO REASON THAT THE
G470s IN THE TWO LEVER ARMS IN
THOSE TWO CASES WOULD BE IN THE
SAME CHEMICAL ENVIRONMENT AND
THERE IS ONLIY A SING E8 SPOT.
THAT IS COOL.
BUT THAT'S NOT THE INTERESTING
RESULT.
THE INTERESTING RESULT IS HERE.
THAT IS WHEN YOU OVERLAY THEM.
THIS SINGLE SPOT DOESN'T ALIGN
WITH EITHER OF THE SPOTS HERE
DESPITE THE FACT THAT THIS
SEQUENCE IS THE SAME AS THIS
SEQUENCE AND THIS SEQUENCE.
SO THE INFERENCE IS THAT THE
SPOT HERE IS DIFFERENT THAN
THESE GUYS BECAUSE THEY ARE
CROSS INFORMING EACH OTHER AND
SAYING TO EACH OTHER, WE HAVE
DIMERIZATION SITUATION HERE
WHERE THE TWO MONOMERS ARE
DIFFERENT STATES AND FEEDING
FROM ACROSS THEIR DIMER
INTERFACES.
THAT'S THE IDEA.
AND LISA EXTENDED THIS, TO LOOK
AT OTHER GBSs, IT WAS
SUPPORTED.
SO NOW SHE IS LOOKING AT FOUR
DIFFERENT GBSs AND YOU CAN SEE
THAT THERE ARE FOUR SPOTS.
SO IN EVERY CASE, THE
INFORMATION IS DIFFERENT.
SO WHAT HAPPENS THEN IF YOU
MUTATE THE DIMER INTERFACE, THE
REGION SHE IS INFERRING IS THE
FEEDER OF INFORMATION FROM ONE
MONOMER TO ANOTHER?
MUTATE THAT ONE RESIDUE AND
EVERYTHING COLLAPSES INTO A NEW
POSITION, NOT THE SAME AS ANY OF
THE OTHERS.
AND IT SAYS, THEY STOPPED GIVING
EACH OTHER INFORMATION AND WHEN
THEY DO THAT, THEN THE
INFORMATION JUST COLLAPSES TO
ONE KIND AND THE RECEPTOR IS
UNABLE TO DISTINGUISH DIFFERENT
GBSs.
THAT'S THE MODEL.
THAT IN THE WILDTYPE DIMER
INTERFACE, THE DIFFERENT SUBUNIT
STATES ARE FEEDING INFORMATION
TO THE OTHER SUBUNIT IN THE
DISRUPTIVE DOIMER INTERFACE, YOU
END UP WITH A YET DIFFERENT
CONFIRMATION AND A LOSS OF THAT
FEEDING OF INFORMATION.
THAT THE GR4717 MUTATION FAILS
TO TRANSMIT THE DNA SIGNALS TO
THE PARTNER MONOMER AND FAILS TO
DISCRIMINATE BETWEEN DIFFERENT
GBSs.
SO THAT DIMER INTERFACE IS
CRITICAL FOR DEFINING AN
ALLOSTERIC PATH THAT ACTUALLY IS
AMPLIFYING THE AMOUNT OF
INFORMATION THAT COMES FROM THE
GBS BECAUSE THE TWO HEXAMERS
HALF SITES ARE NOT ONLY FORMING
THE MONOMER STRUCTURE THAT IS
BOUND TO IT, BUT ALSO THE DIMER
PARTNER ON THE OTHER SIDE.
SO THEN THE FUNCTIONAL QUESTION
AGAIN, YOU CAN SEE THE WAY WE DO
THESE EXPERIMENTS IN THE LAB.
THE FUNGAL QUESTION, THAT IS
GREAT.
DOES 477T MUTATION AFFECT THE
ACTIVITY OF THE STRUCTURE?
AND IT DOES.
SO AGAIN WITHOUT DRAGGING YOU
THROUGH EACH EXAMPLE, IF WE JUST
LOOK AT THESE TWO WHERE VGK AND
SGKGG WERE THE SPACER SEQUENCES
ALTERED BUT OTHERWISE THE
HEXAMERS WHILE DIFFERENT FROM
EACH OTHER ARE DIFFERENT ON THE
TWO HALVES, ARE THE SAME ACROSS
THE TWO GBSs.
AND YOU CAN SEE THAT WITH
WILDTYPE GR, THE ACTIVITY, THE
TRANSCRIPTIONAL ACTIVITY IS VERY
MUCH SPACER SEQUENCE DEPENDENT.
AND IN THE MUTATION IT LEWIS
THAT IS DISCRIMINATION.
SO, THE A4717 DISCRIMINATES
POORLY BETWEEN THE GBA SEQUENCES
JUST AS THE STRUCTURE COLLAPSED
DOWN TO A SINGLE STRUCTURE
DISTINCT FROM THAT WILDTYPE.
I'M NOT GOING TO DWELL ON THIS
EXCEPT TO SAY THAT LISA DID A
VERY SENSITIVE SERIES OF
CHARACTERIZATIONS OF THE
WILDTYPE A477.
THE MUTATION WAS IN PART BECAUSE
THERE HAD BEEN A DOGMA THAT HAD
BUILT-UP IN THE FIELD ABOUT THIS
PARTICULAR MUTATION THAT HAD
ARGUED THAT IT FAILS TO BIND TO
DNA AND THAT IT LOSES COOPERATE
ACTIVITY AND DOESN'T DIMERIZE
ANYMORE.
AND WE THINK THAT IT IS VERY
CLEAR FROM THESE STUDIES THAT
NEITHER OF THOSE THINGS ARE
TRUE.
IT CLEARLY BINDS DNA AND
REGULATES AND ACTIVATES IT AND
IMPRESSES AND DIMER ICES AND
PARTLY BINDS COOPERATIVELY.
I WON'T TAKE YOU THROUGH THE GEL
SHIFT DATA AND RESONANCE
STUDIES, EXCEPT TO DAZZEL YOU
WITH HOW BEAUTIFUL THESE
EXPERIMENTS ARE.
AND TO GIVE YOU THE CONCLUSION
POINTS THAT THE BINDING IS THE
SAME.
YOU ALWAYS GET DIMERS OF A477T
MUTATION.
YOU LOSE A LOT OF COOPERATIVE
ACTIVITY BUT NOT -- IT'S STILL
VERY COOPERATIVE.
THE WILDTYPE HAS A COEFFICIENT
OF 1.7, WHICH IS VERY HANDSOME
AND THE MUTANT HAS A COEFFICIENT
OF 1.4, WHICH IS STILL VERY
GOOD.
SO MAKING THAT ONE CHANGE OF THE
DIMER INTERFACE AND COMPROMISES
COOPERATE ACTIVITY BUT CLEARLY,
DID DOESN'T LOSE IT.
YOU LOSE ABOUT AN ORDER OF
MAGNITUDE BECAUSE YOU INCREASE
THE ASSOCIATION RATE BY AN ORDER
OF 92 AND THERE IS VERY POOR
DISCRIMINATION BETWEEN GBSs
AND PHYSICAL CHARACTERIZATIONS
AS WE HAVE SEEN POOR
DISCRIMINATION IN STRUCTURE AND
POOR DISCRIMINATION IN
REGULATORY ACTIVITY.
SO THIS JUST IS SORT OF A FINAL
STRUCTURAL CARTOON THAT TRIES TO
MAKE THE POINT, AS YOU LOOK AT
IT FROM AFAR OF THE HEAT MAPS
THAT THERE IS A -- OR THAT
PARTICULAR CHANGES NGBS SEQUENCE
LEAD TO CHANGES IN DIFFERENT
REGIONS OF GBS.
SO IN PRINCIPLE, ONE COULD LEARN
ENOUGH TO BECOME PREDICTIVE
ABOUT THIS.
AND MAKE SPECIFIC PREDICTIONS
ABOUT THE RENALONS OF THE DNA
BINDING DOMAIN THAT ARE
SENSITIVE TO OR PREDICTED TO
CHANGE, MAYBE BETTER SAID UPON
CHANGES OF THE SPECIFIC
NUCLEOTIDE WITHIN THE GBS AND
WHETHER THAT WILL END UP BEING
FUNCTIONALLY INFORMATIVE IN THE
SPECIFIC CONTEXT, IS ONE OF THE
DIRECTIONS THAT WE ARE PUSHING.
SO THE GBS'S AFFECT THE GR
CONFIRMATION AT THE DISTINCT
SURFACES.
FINALLY, AGAIN, RETURN TO THE
STRUCTURE OR THE ACTIVITY
QUESTIONS.
AND REALLY IN A SENSE, THIS IS A
GENETIC STAND-IN FOR SOMETHING
WE ARE WILL BE LACKING
STRUCTURALLY.
I TOLD YOU WE ARE LOOKING ONLY
THE FRAGMENT OF THE GR OF THE
DNA BINDING DOMAIN IS A SENSIBLE
PLACE TO START BECAUSE IF YOU'RE
LOOKING FOR SEQUENCE DEPENDENT
SIGNALING, YOU WANT TO ASK, WHAT
EMANATES FROM THE SEQUENCE AND
YOU START WITH THE READING HEAD.
BUT OF COURSE THERE IS ALSO
DISSATISFYING BECAUSE WE CAN'T
SAY WHAT HAPPENS TO ALL OF THOSE
OUTBORROWED REMOTE DOMAINS OF
THE RECEPTORS THAT ARE SO
IMPORTANT FOR FORMING THE BRIDGE
ON TO SURFACES OR FORMING
SURFACES WHICH WE KNOW THAT SOME
FACTORS, AND WE WOULD INFER MANY
FACTORS ASSOCIATE.
SO HERE IS A GENETIC EXPERIMENT
THAT IMPLIES AT LEAST TO US,
THAT DIFFERENT GRDNA BINDING
DOMAIN CONFIRMATIONS CORRELATE
WITH CHANGES IN CONFIRMATION
HERE READ BY DIFFERENT
UTILIZATION OF PARTICULAR
SURFACES OF RECEPTOR AND IN THIS
REMOTE POSITION.
SO HERE IS THE EXPERIMENT.
YEARS AGO, IN THE LAB, A SET OF
CELL LINES WERE CONCONSTRUCTED
THAT CONTAIN EITHER THE WILDTYPE
GR OR GR CARRYING MUTATIONS THAT
WERE CLEAN KNOCKOUTS OF THESE
DOMAINS AF1 OR THE DIMERIZATION
REGION OR AF2.
SIMPLE POINT MUTATION HERE,
SIMPLE POINT MUTATION HERE AND A
TRIPLE POINT MUTATION HERE THAT
JORGE REALLY HAD MADE IN THE LAB
SOME YEARS BEFORE.
CLEAN KNOCKOUTS OF THOSE
INDIVIDUAL DOMAINS LEAVING THE
24TH DOMAINS IN TACT.
NOW SHE HAS STABLE CELL LINES
THAT EXPRESS WILDTYPE FOR EACH
OF THOSE THREE KNOCKOUT
MUTATIONS AND ALLOWED THEM TO
THEN ASK, WHAT HAPPENS WITH THE
ACTIVITY OF THESE -- COMING FROM
THESE DIFFERENT GBSs WHEN YOU
MAKE MUTATIONS OF A PARTICULAR
DOMAIN?
ANOTHER WAY TO SAY IT IS, HOW
MUCH DOES THE ACTIVITY COME FROM
A GIVEN GBS DEPEND ON THE
ACTIVITY COMING FROM ONE OF
THOSE DOMAINS AS TESTED BY
MUTATING THEM?
HERE ARE TWO EXAMPLES.
GBS AND FKBB5 AND THE WILDTYPE
AND THE BLACK BAR AND YOU CAN
SEE WHEN YOU MUTATE THE
DIMERIZATION DOMAIN THAT THE
GBS, ACTIVITY GOES UP.
MUTATE IT HERE AND THE ACTIVITY
GOES DOWN.
DIFFERENT DEPENDENCE ON THIS
REGION.
THE PAL GBS IS ALMOST ENTIRELY
DEPENT END FOR ACTIVITY ON AF2.
YOU KNOCK THAT OUT AND IT'S
DEAD.
IT'S PRETTY DEPENDENT IN THE
CASE OF FKBB5 AS WELL.
HERE IS AN INTERESTING ONE.
AF1, YOU MUTATE AF1, PAL DOESN'T
CARE IT'S NOT USING IT TO MOUNT
THE REGULATION.
YOU KNOCK OUT AF1 HERE AND IT
CARES A LOT.
SO IT DEPENDS ON AF2, NOT ON
AF1.
AND IT IS A GAIN OF FUNCTION.
FKBP5 SPACER USES ALL DOMAINS,
AF2 THE MOST, AF1 NEXT AND THEN
THEY ARE ABOUT THE SAME.
IT IMPLIES THAT THE GBSs ARE
PUTTING THESE OUT BOARD DOMAINS
ALSO INTO DIFFERENT CONFIRMATION
BEING SCORED ADDS DIFFERENT
DEPENDENCES OF THE RECEPTOR ON
THE DOMAINS FOR THE REGULATORY
ACTIVITY.
MAYBE THE SURFACES ARE UTILIZED
VERY STRONGLY IN THIS CASE, BUT
NOT IN THIS CASE, FOR EXAMPLE.
THE ITCH INDICATION THEN IS THAT
THIS ALLOSTERY EXTEND FROM THE
DNA BINDING DOMAIN INTO OTHER
PARTS OF THE RECEPTOR WHERE WE
AND OTHERS ARE STRUGGLING WITH
DIFFERENT KIND OF STRUCTURAL
ANALYSIS TO ASK THAT QUESTION
DIRECTLY.
OKAY.
SO, WHERE HAVE WE BEEN?
COMBINATORIAL SIGNALING IS A
LONG TITLE.
CONCLUSIONS AND PERSPECTIVES AND
PECKALATIONS.
I'M NOT GOING TO TELL YOU WHICH
IS WHICH.
COMBINATORIALITY.
DISTINCT COMBINATIONS OF
REGULATORY FACTORS AND
COREGULATORS ASSEMBLE INTO GENE,
CELL AND PHYSIOLOGY SPECIFIC
REGULATORY COMPLEXES OF GREs.
I CAN SEE THE SLIDES A LOT
BETTER HERE.
REELINGTORY LOGIC.
WE ARE INFERRING THAT FROM
EXPERIMENTS MORE THAN WHAT I
TOLD BUT, THAT THE REGULATORY
COMPLEX MACHINERY MAY BE RIGGED,
OPTIMIZED, TO BE ABLE TO CARRY
OUT THIS PRECISION AND
FLEXIBILITY PARADOX.
THAT WOULD BE INTERESTING IF IT
WAS ALL RESIDENT WITHIN THE
REGULATORY COMPLEXES AND LITTLE
CHANGES OVER ENOUGH TO FLIP IT
IN THIS CASE FOR ACTIVATION.
SIGNAL INTEGRATION.
GR RECEIVES MULTIPLE CHASESES OF
SIGNALING INPUTS WHICH TOGETHER
PRODUCE CONTEXT-SPECIFIC EFFECTS
ON GR CONFIRMATION AND THAT
SIGNAL-DRIVEN CONFIRMATIONS
TRIGGER THE DEPLOYMENT OF
DISTINCT COMBINATIONS OF GR
FUNCTIONAL DOMAINS THAT IN TURN
PROVOKE THE ASSEMBLY OF DISTINCT
REGULATORY COMPLEXES.
THERE ARE OTHER INFERENCES THAT
GO BEYOND THIS AND IN FACT N-A
SENSE, THESE STUDIES I THINK
PROVIDE ONE KIND OF INTERESTING
POINT OF ACCESS TO UNDERSTANDING
ONE OF THE SORT OF PILLARS OF
BIOLOGICAL REGULATION OF
ALLOSTERY.
IT'S BEEN WITH US FOR A VERY
LONG TIME.
AND THE DETAILED MECHANISMS HAVE
MOVED STRUCTURAL INFORMATION
THROUGH THE CORE OF A PERSON
REMAIN PRETTY MYSTERIOUS.
SO YOU CAN THINK OF THEN
CHANGING THE SEQUENCE OF THE GBS
AS DOING LIGAND CHEMISTRY, AND
PRETTY EASY LIGAND CHEMISTRY
THAT THEN ALLOWS YOU TO ACCESS
THE CONSEQUENCES OF PARTICULAR
CHANGES IN THOSE LIGANDS ON
CONFIRMATION.
I THINK IT MAY END UP FEEDING IT
SOME INFORMATION.
THERE IS A LARGER HIGHER
DIMENSION QUESTION AS WELL THAT
IS INTERESTING.
AND THAT IS, IF THERE IS SOME
RELATIONSHIP BETWEEN THE
REGULATORY MECHANISM AT A GIVEN
PLACE, AND THE PHYSIOLOGICAL END
POINT THAT YOU ARE SHOOTING FOR,
SO IS THERE SOME LINK BETWEEN
PHYSIOLOGICAL NETWORK AND
TRANSCRIPTIONAL REGULATORY
NETWORKS THAT WE CAN SEE?
A WAY OF THINKING ABOUT THIS IS
WHETHER ONE CAN BEGIN TO LINK
REGULATORY MECHANISM WITH
PHYSIOLOGICAL OUTPUT.
IN BACTERIA, YOU CAN SORT OF DO
THIS.
YOU CAN LOOK AT A OPERON, THE
ARRANGEMENT OF THE REGULATORY
ELEMENTS, AND SAY I BET THIS IS
A SUGAR METABOLISM OPERON.
THIS IS REPRESSOR BINDING SITE
NEXT TO THE RNA -- BLOCKING THE
POLYMERASE AND THERE IS A CYCLIC
A.
AND P BINDING SITE UP STREAM.
IF YOU HAD A DOMAIN THAT HAD AN
OPEN READING FRAME THAT READ OUT
A SERIES OF REPEATED CODONS FOR
A GIVEN AMINO ACID AND COULD
EXTEND INTO THE OPERON ITSELF,
YOU COULD SAY, THIS LOOK LOOKS
LIKE A OPERON.
SO A LINK BETWEEN THE REGULATORY
MECHANISM AND THE PHYSIOLOGICAL
NETWORK.
IR BET THAT IS THE CASE HERE BUT
HARDER TO SEE BECAUSE OF THE
SIGNALING INPUTS.
IF THAT IS TRUE, THEN IT OPENS
AN INTERESTING POSSIBILITY.
THAT IS THAT MAYBE WE CAN DEFINE
CONDITIONS WHERE WE CAN REDUCE
COMPLEX PHYSIOLOGICAL READ OUTS,
OSTEOPOROSIS, HOW IS THAT?
OR HOW ABOUT DEPRESSION?
AND MOLECULAR CHANGE IN THE
RECEPTOR, BEHAVIOR OF A SURFACE
AND THE BEHAVIOR OF A GIVEN
COFACTOR TO ASSOCIATE,
MODIFICATIONS STATES THAT HAS
ONE ACTIVITY IN ONE CASE AND NOT
IN ANOTHER.
THAT WOULD CHANGE A LOT OF
THINGS.
CERTAINLY IT WOULD CHANGE THE
WAY WE THINK ABOUT DOING
PHARMACEUTICAL TESTING, FOR
EXAMPLE.
TO BE FREE OF ANIMALS AND THE
TIME AND EXPENSE OF DOING ANIMAL
ASSAYS TO WELL VADATE THIS IN A
BAY THAT WAS PREDICTED.
THIS LED US TO BE ON A BIG
SEARCH AND I'M NOT GOING THROUGH
THE DISEASE AREAS THAT WE ARE
INTERESTED IN.
BUT FOR THIS NOTION OF CAUSATIVE
PRIMARY REGULATING GENES,
CAUSATIVE MEANING THAT THERE IS
A GENE WHOSE FUNCTION OR
RESPONSE IS ESSENTIAL FOR THE
HORMONAL SIGNALING.
YOU LOSE THE HORMONAL RESPONSE
AND EXPRESS IN THE ACK SENSE OF
HORMONE GIVES YOU THE PHENOTYPE
EVEN WITHOUT THE HORMONE AND SO
FORTH.
PRIMARY MEANING THAT THERE IS
GRE NEARBY.
A GRE THAT IS NOT ONLY
RESPONSIBLE FOR MEDIATING
HORMONAL RESPONSE BUT HAS A
RECEPTOR BOUND TO IT AS WELL
BECAUSE THAT'S OUR MOLECULAR
LEVERAGE.
SO WE ARE LOOKING.
I THINK THAT THERE WILL BE A
LINK HERE AND IF IS TRUE IT WILL
BE QUITE INFORMATIVE.
SO, EXPERIMENTS DONE BY
FANTASTIC WONDERFUL GROUP OF
COLLEAGUES, SHEN HONG CHEN AND
SAMANTHA COOPER.
I DIDN'T TELL YOU ABOUT THE
BIOINFORMATICS INPUT OF THESE
TWO WONDERFULLY TALENT THE
GRADUATE STUDENTS, SAM COOPER
AND BEN SCHILLER WHO DID A LOT
OF CODE WRITING.
LISA WATSON AND SEBASTIAN IN HIS
OWN LAB IN BERLIN AND MILES IN
HIS OWN LAB IN IOWA.
I'M NOT AN NMR BY ANY STRETCH OF
THE IMAGINATION.
I CAN SPELL NMR, BUT JOHN GROSS
IS AT UCS.
AND HAS GIVEN US LOTS OF ADVICE.
AND CHRIS, A POSTDOC
COLLABORATED WITH LISA TO DO
THESE STUDIES.
I THANK YOU FOR YOUR ATTENTION.
[ APPLAUSE ]
>> THANK YOU.
I KNOW WE ARE A LITTLE PAST THE
HOUR AND PEOPLE WHO HAVE TO GO
SHOULD FEEL LIKE YOU CAN JUMP
UP.
I'M HOPING MAYBE THERE MIGHT BE
ONE OR TWO QUESTIONS FOR THOSE
WHO HAVE THE TIME TO STAY AND
ENGAGE.
IF YOU HAVE A QUESTION, WE HAVE
MICROPHONES IN THE AISLES SO
PEOPLE WHO ARE LISTENING ON THE
WEB CAN HEAR THE CONVERSATION.
YES?
>> SO FOR THE COMPLEXES THAT YOU
MENTIONED DIFFERENT
PHYSIOLOGICAL CONTEXT, HAVE YOU
OR ANYONE ELSE DONE, FOR
EXAMPLE, MASS SPEC ANALYSIS TO
LOOK AT ALL OF THESE DIFFERENT
COMPLEX IN DIFFERENT CONTEXT?
>> HAVE WE LOOKED AT THE -- YOU
MEAN JUST LOOK AT A GIVEN
PARTICULAR COMPLEX IN DIFFERENT
CONTEXT?
I THINK A LOT OF PEOPLE ARE
WORKING ON THAT.
WE CERTAINLY ARE.
AND WE ARE LOOKING IN PARTICULAR
CELL STATES AND IN A COUPLE OF
DISEASE STATES PUTTING SOME
FOCUS RIGHT NOW ON LYMPHOBLASTIC
LEUKEMIAS, DISEASES BIG
CHILDHOOD CANCERS, BOTH T-CELL
AND B-CELL THAT ARE VERY WELL
TREATED WITH GLUCORTICOIDS
EXCEPT THEY HAVE BECOME RESIST
WANT.
SO WE ARE LOOKING TO SEE IN THE
SENSE OF RESIST ASSIST ENT CASES
WHAT THE CHANGES ARE.
>> YOU SAID SOMETHING ABOUT
BEING PERHAPS AS MANY AS 100
PROTEINS INVOLVED IN THESE
TRANSCRIPTIONAL COMPLEXES THAT
INCLUDE GLUCOCORTICOID RECEPTOR.
WHERE DOES THAT NUMBER COME
FROM?
HOW SOLID IS IT?
AND HOW VARIABLE IS IT?
>> I SORT OF MADE IT UP.
[ LAUGHS ]
IS THAT A PROBLEM?
[ LAUGHS ]
SO WE KNOW TRANSFERASE COMPLEXES
AND CHROMATIN MODELING COMPLEXES
THAT CONTAIN ON THE ORDER OF 20
PROTEINS ADDS UP FAST.
WHETHER YOU END UP OR HAVE A
COMPLEX THAT HAS 100 POLYPEPTIDE
CHAINS SITTING THERE, DON'T
KNOW.
>> SOUND A LITTLE SCARY.
WELL, FOR THOSE WHO WOULD LIKE
TO CONTINUE THE CONVERSATION,
KEITH WILL BE IN THE LIBRARY
WHERE THERE WILL BE ALSO
REFRESHMENTS.
SO PLEASE COME AND JOIN AND
THANK YOU AGAIN KEITH, LET'S
THANK OUR SPEAKER ONE MORE TIME.
[ APPLAUSE ]