allelic exclusion and role of chromatin in …utminers.utep.edu/raguilera/14-utep-lect4.pdf2014...

2014 Aguilera Lecture #32014 Aguilera Lecture #3

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Allelic Exclusion and Allelic Exclusion and Role of Chromatin in RecombinationRole of Chromatin in Recombination

Professor Aguilera

Lecture 3


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GFP+ =pluripotent stem cells?


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Required reading for all-so need to download the paper –cannot post it but can send it to

you via email if you cannot get it

One question will come out of this paper for midterm


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B/T Lymphocytes

Big-Bang Theory of Immunology

RAGs and V-(D)-J recombination appeared early in vertebrate evolution

450 x 106 years ago

Exon 1 Exon 2

Did transposable elements create the first antigen receptor genes?Did transposable elements create the first antigen receptor genes?

RAG Transposon

Capture of Rec.Signals by Primitive Receptor Gene


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Plasterk , R. (1998). “Ragtime jumping”. Nature, 394:718

Target DNA

RAG transposition strongly resembles transposon transposition

Signal Joining InactivatesTransposition


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RAG transposition probably created the antigen receptor genes

Positive and Negative Control of RecombinationPositive and Negative Control of Recombination

�� Positive control is necessary to activate the RAG genes Positive control is necessary to activate the RAG genes

at a specific stage of lymphocyte differentiationat a specific stage of lymphocyte differentiation--thetheProPro--B/ProB/Pro--T cell stage T cell stage

�� Negative control is essential to maintain integrity of Negative control is essential to maintain integrity of

functionally rearranged genes by preventing functionally rearranged genes by preventing rearrangements that could destroy functional genes rearrangements that could destroy functional genes or lead to aberrant rearrangementsor lead to aberrant rearrangements--oncogene activationoncogene activation

�� Negative control is also necessary to prevent the Negative control is also necessary to prevent the

production of lymphocytes with more than one specificproduction of lymphocytes with more than one specificreceptorreceptor--to prevent antito prevent anti--self reactivityself reactivity-- Allelic ExclusionAllelic Exclusion


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Phenomenon of Allelic ExclusionPhenomenon of Allelic Exclusion

IgH a/b

Produces antibodies of a or b type only

+

IgH b/bIgH a/a

IgH a/a B-cells IgH b/b B-cells

IgH a/b B-cellsFig. 5.11

Allelic Exclusion Prevents Production of Multiple Receptors from One Cell Allelic Exclusion Prevents Production of Multiple Receptors from One Cell


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Ordered rearrangement and Allelic Exclusion

RAG expression

Heavy-chain V-D-J

Ig signaling

rapid cell

division

Light-chain V-J

Stem cell Early Late Large Small Immature B Mature Bpro-B pro-B pre-B pre-B

Down regulation coincides with pre-B cell receptor and expression of functional Ig on surface of B cells

Ig signaling

Ohmori & Hikida, 1998

Nat. Immunol. Review (2003)


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Pro BPro B

RAGRAG--1/2 “on”1/2 “on”

VDJ+VDJ+

start DJstart DJHH

Heavy chain +Heavy chain +surrogate chainsurrogate chain

Allelic Exclusion is Regulated by Signal TransductionAllelic Exclusion is Regulated by Signal Transduction

HeavyHeavy--Chain Allelic ExclusionChain Allelic Exclusion

Stop recombination of Stop recombination of heavy chain genesheavy chain genes

Activate Activate κκκκκκκκ locuslocusVVκκκκκκκκ--JJκκκκκκκκ

LightLight--Chain Allelic ExclusionChain Allelic Exclusion

κκκκκκκκ first then first then λλλλλλλλImmature B cellImmature B cell

RAGRAG-- 1/21/2“off”“off”

IgMIgMµ µ µ µ µ µ µ µ + + κκκκκκκκ

chainschains

λλλλλλλλ5 an VpreB do not rearrange5 an VpreB do not rearrange••On same chromosome as light chainsOn same chromosome as light chains••Expressed only in BExpressed only in B--cellscells••Highly evolutionarily conservedHighly evolutionarily conserved••Thought to recognize invariant selfThought to recognize invariant self--antigensantigens

Surrogate Light Chain of the PreSurrogate Light Chain of the Pre--B cell ReceptorB cell Receptor

HomologousHomologoustoto VVλλλλλλλλ

VpreBVpreB

λλλλλλλλ55HomologousHomologous

toto CCλλλλλλλλ

IgIgαααααααα/Ig/Igββββββββ areare signaling signaling

components of Igcomponents of Ig

see Fig. 5.3see Fig. 5.3


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Signaling through the Ig surface receptorSignaling through the Ig surface receptor

B220B220

Tyrosine kinasesTyrosine kinases

Protein Phosphorylation, etc

Ig crosslinking by AgIg crosslinking by Ag

see Fig. 3.31

Transcriptional Activation of Specific GenesTranscriptional Activation of Specific Genes

signals end up in the nucleussignals end up in the nucleus

Pro-B

IgH+κκκκ light chain

Immature B

Signaling is necessary for B cell DevelopmentSignaling is necessary for B cell Development

Stem cell

�� Mutation of IgH, surrogate-light chain (λλλλ5) or signaling

components (Igββββ) blocks B-cell differentiation

IgMλλλλ5, Igββββ

TargetedTargetedmutationsmutations


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Functionally rearranged Ig or TCR genesFunctionally rearranged Ig or TCR genes

isolate B/T cells

Testing Allelic Exclussion in Transgenic MiceTesting Allelic Exclussion in Transgenic Mice

VV DD JJ

Ig/TCR Transgenic AnimalIg/TCR Transgenic Animal

Analysis of B/T developmentand status of endogenous genes

Test for “allelic exclusion”

Profound block of endogenousProfound block of endogenousH & L chain rearrangementsH & L chain rearrangements

thusthusTrangene exerts “allelic exclusion”Trangene exerts “allelic exclusion”

Prevents other allele from being expressedPrevents other allele from being expressed

all B cells have functional Igall B cells have functional Ig

IgM Transgenic AnimalIgM Transgenic Animal

Heavy and Light Ig genesHeavy and Light Ig genes

Most endogenous genes were in germlineMost endogenous genes were in germlineconfiguration or DJ but almost no VDJ or Vconfiguration or DJ but almost no VDJ or VκκκκκκκκJJκκκκκκκκ


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Introduction of functional heavy chain geneIntroduction of functional heavy chain geneinto PreB cells leads to dramaticinto PreB cells leads to dramatic

induction ofinduction of VVκ κ κ κ κ κ κ κ JJκκκκκκκκ RecombinationRecombination

Unexpected effect of Ig transgeneUnexpected effect of Ig transgene

• Heavy chain exerts a positive effect on L-chain rearrangement and a negative effect on endogenousheavy chain gene recombination

Conclussion:

Fig. 4.29

TCR TCR ββββββββ--genesgenes

Rearrangement of TCR GenesRearrangement of TCR Genes

Fig. 4.37

TCR TCR γγγγγγγγ--genesgenes

α/δα/δα/δα/δ-chains

δδδδ αααα

TCR TCR αααααααα//δδδδδδδδ LocusLocus

δδδδδδδδ locus within locus within αααααααα--TCR locusTCR locus

gets deleted in TCRgets deleted in TCRαβαβαβαβαβαβαβαβ TT--cellscells

TCRTCRδδδδδδδδ

TCRTCRαααααααα


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ββββ

αααα

Nat. Immunol. Review (2003)

V DJββββ

Vββββ rearrangement

TcRαααα beginsrearrangement

βαβαβαβα1bad

Ordered rearrangement of TOrdered rearrangement of T--Cell Antigen Receptor GenesCell Antigen Receptor Genes

MHC Class IIpresenting cell

βαβαβαβα2good

RAG 1/2RAG 1/2“OFF“OFF”

CD4+

βαβαβαβα2

Positively selected goes to the periphery

RAG 1/2 RAG 1/2 “ON”“ON”

self-reactive

VDJββββ

Pre-Tαααα

TCRββββ


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CD3 complexCD3 complex

ε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δ

Y

YY

Y

Y

Y

Y

Y

Y

Y

Y

Y

YY

YY

YY

YY

Y

Y

Y

Y

Signal transduction mediated by TCR requires CD3 complex Signal transduction mediated by TCR requires CD3 complex

PrePre--TCRTCRαααααααα

Pre-T cell receptor is also associated with CD3 complex

TCRTCRββββββββ

CD3 complexCD3 complex

ε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δε, ζ, γ, δ

Y

YY

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

PrePre--T cell receptor is composed of TCRT cell receptor is composed of TCRββββββββ chain and Prechain and Pre--TTαααααααα chainchainand is required for normal Tand is required for normal T--cell developmentcell development


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CD45CD45

LckLck

TCR TCR α/βα/βα/βα/βα/βα/βα/βα/β

CD4/CD8CD4/CD8

Signaling molecules associated with TCR affect Allelic ExclusionSignaling molecules associated with TCR affect Allelic Exclusion

intracellular signalingintracellular signaling

��

rearrangerearrange ββββββββ

prepre--T cell receptorT cell receptorα/βα/βα/βα/βα/βα/βα/βα/βTCRTCR

sIgMsIgM

rearrange rearrange ΗΗΗΗΗΗΗΗ rearrangerearrange κ κ κ κ κ κ κ κ oror λλλλλλλλ

TT--cell lineagecell lineage

BB--cell lineagecell lineageRAG deficient mice are blockedRAG deficient mice are blocked

rearrange rearrange αααααααα

prepre--B cell receptorB cell receptor

Similarities between T & B cell developmentSimilarities between T & B cell development


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Two old questions that are finally being addressed:Two old questions that are finally being addressed:

How come the Ig lightHow come the Ig light--chains don’t rearrange at chains don’t rearrange at same time as heavysame time as heavy--chain V genes?chain V genes?

andand

How come T cells don’t rearrange Ig genes and B How come T cells don’t rearrange Ig genes and B cells don’t rearrange TCR genes?cells don’t rearrange TCR genes?

VVHsHs

DDHHJJHH

“Closed” or inactive chromatin “Closed” or inactive chromatin

“Open” Chromatin“Open” Chromatin

“Recombinase”“Recombinase”

Gene accessibility controls recombination Gene accessibility controls recombination in lymphoid and nonin lymphoid and non--lymphoid cellslymphoid cells

Ig enhancerIg enhancer

In ProIn Pro--B cells, lightB cells, light--chain loci and TCR loci would be in inactive chromatin stagechain loci and TCR loci would be in inactive chromatin stage


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bone marrow stromal cellsbone marrow stromal cells

What Signals Activate VWhat Signals Activate V--(D)(D)--J Recombination?J Recombination?

Activation of Transcription FactorsActivation of Transcription Factors

by external stimuliby external stimuli

RAGs “on” or “off”RAGs “on” or “off”

ILIL--77other factorsother factors

Early steps in DifferentiationEarly steps in Differentiation

ILIL--7R7R ILIL--7 signals activate V7 signals activate V--(D)(D)--JJ

Activation of chromatin at RAGActivation of chromatin at RAG

and Ig/TCR lociand Ig/TCR loci

Activation of accessory factorsActivation of accessory factors

necessary for gene expression andnecessary for gene expression and

signal transductionsignal transduction

VsDs

Js

“Closed” Chromatin

“Open” Chromatin

“Recombinase”

Enhancer

Chromatin Status Controls Chromatin Status Controls Accessibility to Variable (VAccessibility to Variable (V--DD--J) Region GenesJ) Region Genes


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HistoneHistone AcetylationAcetylation has an effect on has an effect on recombination efficiencyrecombination efficiency

Pp 485-498

δδδδδδδδ αααααααα

Mouse TCR Mouse TCR αααααααα//δδδδδδδδ LocusLocus

ααααααααss δδδδδδδδss δδδδδδδδ δδδδδδδδ δδδδδδδδ δδδδδδδδ

δδδδδδδδ

ααααααααss αααααααα ααααααααss

11--5%5% 8080--90%90%

1010--15%15%

BlockedBlockedBlockedBlocked


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HuTCRHuTCRδδδδδδδδ

Express and rearrange the human transgene appropriatelyExpress and rearrange the human transgene appropriately

Normal V to DNormal V to Dδ δ δ δ δ δ δ δ joining but no VD to Jjoining but no VD to Jδδδδδδδδ

OK blocked

HuTCRHuTCRδ δ δ δ δ δ δ δ with deleted with deleted δδδδδδδδ enhancer do not fully rearrangeenhancer do not fully rearrange

δδδδ αααα

Transgenic animals with a deletion of ETransgenic animals with a deletion of Eαααααααα result in inhibition result in inhibition of Vof V--JJα α α α α α α α and elimination of hyperand elimination of hyper--acetylation of regionacetylation of region


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CHIP Assay

Does the Status of Histone Acetylation Affect Recombination?Does the Status of Histone Acetylation Affect Recombination?

cleave chromatin into mono-nucleosomes

add Anti-acetylated H3

AcAc

Ac

Ac

Ac

AA

A

Immunoprecipate withProtein-A beads

Serial dilutions of bound and unbound fractions aresubjected to PCR with

specific primers

Separate products on gel

(chromatin immunoprecipitation)

Detected byDetected by

Southern withSouthern with

specific probesspecific probes

(no antibody)

Normal ControlNormal Control

plus enhancerplus enhancer

more unboundmore unbound--not acetylatednot acetylated

less bound less bound --acetylated acetylated

Looking for differences between transgenic linesLooking for differences between transgenic linesIn the amount of acetylated vs unacetylated histonesIn the amount of acetylated vs unacetylated histones

Missing Missing

TCR TCR αα--enhancerenhancer


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wild-type Eδδδδ (RAG-2-/-) mutant Eδδδδ (RAG-2-/-)

Octamer

(B-cell specific gene)

Mutation of EMutation of Eδδδδδδδδ (delta enhancer) results in large decrease (delta enhancer) results in large decrease in histone acetylationin histone acetylation

V and Ds not V and Ds not affected as muchaffected as much

Enhδδδδ

δδδδδδδδ δδδδ δδδδ

ac

ety

lati

on

ac

ety

lati

on

Normal V to DNormal V to Dδ δ δ δ δ δ δ δ joining but no VD to Jjoining but no VD to Jδδδδδδδδ

OK blocked

A Tail of Histone Acetylation and DNA Recombination. M. S. Schlissel. Science 287, 438-440

Enh dependentEnh dependentEnh independentEnh independent

HAT = HistoneHAT = Histone

acetyltransferaseacetyltransferase

TF = Transcription Factor(s)TF = Transcription Factor(s)

VV--DDδδδδδδδδ occurs first VDoccurs first VD--JJδδδδδδδδ secondsecond

Rearrangement controlled by enhancer dependent Rearrangement controlled by enhancer dependent and independent pathwaysand independent pathways

Fig. LegendGetting chromatin to relax. Regulation of V(D)J recombination by histone acetylation. (Top) A transcription factor (TF)binds to the enhancer for the TCRδδδδ gene locus and recruits HAT activity to the locus. HAT acetylates local histones, altering chromatin structure and allowing regulatory proteins to gain access to the gene locus.


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Histone acetylation and deacetylation appears to controlHistone acetylation and deacetylation appears to controlaccessibility of RAGaccessibility of RAG--recombinase complexrecombinase complex

RAGs

deacetylasedeacetylase

acetylaseacetylase

(A) Acetylation of histone proteins may alter the structure of individual nucleosomes, allowing recombinase enzymes to recognize recombination signal sequences (RSSs) that flank gene segments encoding antigen receptors. (B) Acetylated tails of H3/H4 histones may serve as the binding site for chromatin remodeling complexes, resulting in nucleosome sliding or displacement. (C) Acetylation may alter interactions between adjacent nucleosomes, leading to an unraveling of the 30-nm chromatin fiber and enhanced accessibility of recombination signal sequences to recombinase. Each of these effects can be reversed by histone deacetylase activity (HDAC).

Figure legend:


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What does this have to do with “allelic exclusion”?What does this have to do with “allelic exclusion”?

Control of accessibility of recombinase is essentialControl of accessibility of recombinase is essentialfor the model of allelic exclusionfor the model of allelic exclusion

Only way to explain why one set of genes stops toOnly way to explain why one set of genes stops torearrange while other are “turnedrearrange while other are “turned--on”on”

allelic exclusion and role of chromatin in …utminers.utep.edu/raguilera/14-utep-lect4.pdf2014...

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