bcr editing and transcriptional regulationutminers.utep.edu/raguilera/14-advimmunol lecture...

Advanced Immunology –Aguilera Lecture 2/18/2014

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BCR Editing and BCR Editing and Transcriptional RegulationTranscriptional Regulation

ProfessorProfessor AguileraAguilera

AID is essential for Switching, Hypermutation and Gene ConversionAID is essential for Switching, Hypermutation and Gene Conversion


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Current Model for Switch RecombinationCurrent Model for Switch Recombination

Current Model for Somatic HypermutationCurrent Model for Somatic Hypermutation


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CD21

CD21 bind the complement system and activates B cells

CD19

Co-ligation leads to phosphorylation of CD19

leading to signaling

Ig receptor and coIg receptor and co--receptors needed for activationreceptors needed for activation

B-cell

(BL2)

anti-IgM anti-CD19 and anti-CD21

CrossCross--linking of all three receptors leads to activation of linking of all three receptors leads to activation of somatic hypermutation somatic hypermutation

Look at mutation of Look at mutation of IgIg V region or C regionV region or C regionby sequencing PCR productsby sequencing PCR products

analyze after 90 minanalyze after 90 min

Nature Immunology 3, 815 - 821 (2002) AID-dependent somatic hypermutation occurs as a DNA

single-strand event in the BL2 cell line


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Note that little mutation is generally observed Note that little mutation is generally observed at nearby CH region at nearby CH region ––important structureimportant structure


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Similar mutation rates were detected at G1 and G2/MSimilar mutation rates were detected at G1 and G2/MBut not during active DNA replication (S)But not during active DNA replication (S)

Somatic Mutation occurs on one strand in G1 phase of cell cycleSomatic Mutation occurs on one strand in G1 phase of cell cycle


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Mutation does not require de novo mRNA synthesisMutation does not require de novo mRNA synthesis

Disruption of the AID gene to test role of AID in Somatic MutationDisruption of the AID gene to test role of AID in Somatic Mutation


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Mutation of AID disrupts Somatic MutationMutation of AID disrupts Somatic Mutation


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cc--mycmycIg geneIg gene

Rearraged AllelesRearraged Alleles

Aberrant switch recombination events can lead to chromosome translocationsAberrant switch recombination events can lead to chromosome translocations

In mice the 12;15 In mice the 12;15 translocation is translocation is

detected in >90%detected in >90%of lymphocyte tumorsof lymphocyte tumors


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Deletion of the cDeletion of the c--mycmyc oncogeneoncogene promotorpromotor stops translocationsstops translocations


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14;18 Translocation is the most common rearrangement in Human Lymphomas 14;18 Translocation is the most common rearrangement in Human Lymphomas

(all Follicular Lymphomas)(all Follicular Lymphomas)

BclBcl--2 (B2 (B--cell lymphoma 2)cell lymphoma 2)

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Receptor EditingReceptor Editing


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Re-rearrangement of κκκκ-light chainresults in new receptor specificity

Clonal Deletion


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Vκ1

κκκκ light-chain variable region gene recombination

Vκ2

1st rearrangement

V

Jκ1 Jκ2 Jκ3 Jκ4 Jκ5

V

CκEnh

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Secondary LightSecondary Light--Chain RearrangementsChain Rearrangements

Vκ2Vκ3 VJκ1

CκEnh2nd rearrangement

RS

Different specificity

The organization of the κκκκ light chain locus allows for secondaryrearrangements that can remove a previously rearranged VJκκκκ

Jκ5

self-reactive

Apparently signaling through the Ig receptor can modulateRAG re-expression depending on strength of signal


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V gene replacement through V gene replacement through weak weak RSS homologiesRSS homologies

Can replace one Can replace one V with another)V with another)

Front Immunol. 2014 Jan 30;5:10.Trials and Tribulations with VH Replacement.

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Fig. 6.7

Important Genes/Proteins Expressed During BImportant Genes/Proteins Expressed During B--cell Differentiationcell Differentiation

RecombinationRecombination

Surrogate light chainSurrogate light chain

Signal TransductionSignal Transduction

TranscriptionalTranscriptionalregulationregulation

FunctionFunction

time of expressiontime of expression

BSAPBSAP

RAGRAG--11

RAGRAG--22


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Progenitor ProProgenitor Pro--B PreB Pre--B Naïve B GC B PlasmaB Naïve B GC B Plasma

Ikaros PU.1 E2A BSAP NFIkaros PU.1 E2A BSAP NF--κκκκκκκκB OctB Oct--2 STAT2 STAT--6 Ets6 Ets--11EBF BclEBF Bcl--6 TFE3 Blimp6 TFE3 Blimp--11

OCAOCA--B C/EBPB C/EBPβ β β β β β β β NFNF--κκκκκκκκB B

Transcription Factors Known to Regulate Critical Steps in BTranscription Factors Known to Regulate Critical Steps in B--cell Differentiationcell Differentiation

Approximate time when a particular factor is critical:Approximate time when a particular factor is critical:

active NFactive NF--κκκκκκκκBB

E2A, EtsE2A, Ets--1, Oct1, Oct--2,2,OCAOCA--B, TF3E, BSAPB, TF3E, BSAP

PU.1PU.1

IkarosIkaros

IdId

EBF EBF

Approximate Expression Pattern:Approximate Expression Pattern:

Henderson and Calame (1998). Annu.Rev.Immunol. 16:163Henderson and Calame (1998). Annu.Rev.Immunol. 16:163--200.200.

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ProPro--B and PreB and Pre--B cellsB cells

Targeted disruption of BSAP disrupts BTargeted disruption of BSAP disrupts B--lymphopoiesis and Vlymphopoiesis and V--DD--J joiningJ joining

Morrison, A.M. et al., (1998). Loss- and gain-of-function mutations reveal an important role of BSAP(Pax-5) at the start and end of B cell differentiation. Semin. Immunol.10, 133-42

BSAPBSAP--//--

wtwtVH DJ

BSAP BSAP --//--

BSAPBSAP--//-- propro--B cellsB cells


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Receptor Editing and Marginal Zone B Cell DevelopmentAre Regulated by the Helix-Loop-Helix Protein, E2A

Melanie W. Quong,1 Annica Martensson,2 Anton W. Langerak,3 Richard R. Rivera,1 David Nemazee,2 and Cornelis Murre 1

J. Exp. Med. Volume 199, Number 8, April 19, 2004 1101–1112

E2A Transcription factor promotesE2A Transcription factor promotes λ λ λ λ λ λ λ λ light chain rearrangementlight chain rearrangement

3-83 Ig transgene reacts against self (MHC H-2Kkb)And transgenic mice have monoclocal B cells

in the permissive background (H-2Kd)

When crossed to the non-permissive background B cells undergo receptor editing to replace light

chains with another kappa or a lambda


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3-83TgIgHa/IgLκκκκκκκκ

B10D2Not autoreactive

Non-deletingbackground

Deleting MHC (Deleting MHC (KKdd))


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Most lymphocyteMost lymphocyte--specific genes are regulated by a complex specific genes are regulated by a complex

array of array of ciscis regulatory elementsregulatory elements

exon 1 exon 2

mRNA

TATA/Inr

e s

promoter elementspromoter elements silencersilencerenhancerenhancer

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PromotersPromoters

ciscis--acting DNA elements acting DNA elements are position and orientation dependentare position and orientation dependent

~ 10 ~ 10 -- 50 bp upstream of gene50 bp upstream of gene

EnhancersEnhancerspositive actingpositive acting

Enhancers and silencersEnhancers and silencers areare ciscis--acting acting elements that can exert effect from a elements that can exert effect from a

large distance and are orientation large distance and are orientation independentindependent

SilencersSilencersnegative actingnegative acting

regulation of Ig and TCR genesregulation of Ig and TCR genesElements involved in the transcriptional Elements involved in the transcriptional


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TATA-Box or Initiator

TATA

Eukaryotic Promoter/Transcription Initiation ComplexEukaryotic Promoter/Transcription Initiation Complex

Transcriptional Activators

TFsTFs

POL II

IIFIIE

IIHIIJTBP

IIB

TAFs

IIA

LymphocyteLymphocyte--Specific transcription factors (TFs) are Specific transcription factors (TFs) are required to activate several lymphocyterequired to activate several lymphocyte--specific genesspecific genes

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me me

closed closed -- inactive chromatininactive chromatin

PromoterPromoter me EnhancerEnhancer

is heavily methylated (CpG)is heavily methylated (CpG)

Promoters and Enhancers Collaborate for Optimal TissuePromoters and Enhancers Collaborate for Optimal Tissue--Specific ExpressionSpecific Expression

me

“open” chromatin

A model of interaction of Promoter and Enhancer Elements

Ig gene

PromoterPromoter

EnhancerEnhancer

me

me

looping?looping?


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PV Ds Js Cµµµµ

Unrearranged VUnrearranged V--genes are generally not expressedgenes are generally not expressed

1000x increase in1000x increase inexpressionexpression

Why?

V D J

V

No or low level sterile transcripts

Cµµµµ

Enhancer within CEnhancer within Cµµµµµµµµ intronintron

Active only in B cellsActive only in B cells

no enhancerno enhancer----no expressionno expressionSimilar to viral enhancers but BSimilar to viral enhancers but B--cell specificcell specific

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VVHH

PromoterPromoter

EnhancerEnhancer

Cµµµµ

transcription startstranscription starts

Ernst and Smale (95)

Multiple lymphocyte and nonMultiple lymphocyte and non--lymphocyte specificlymphocyte specificfactors interact with the Ig promoter and enhancer regionsfactors interact with the Ig promoter and enhancer regions

Cαααα 3’ Enh

Not all TFs are lymphocyteNot all TFs are lymphocyte--specificspecific


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RAG Gene RegulationRAG Gene Regulation

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Cloning of the Cloning of the RAGRAG--11 and and RAGRAG--22 Promoter RegionsPromoter Regions

RAG Cosmid Clone~10 kb~10 kb ~5 kb~5 kb


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RAG-luciferase

reporter plasmid

Analysis of Promoter RegionsAnalysis of Promoter Regions

Transfect cell lines

test cell extractfor luciferase activity

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0

10

20

30

40

50

60

Pre-B

Pre T

Fibroblast

-279 -91 -83 -76 -68 -57

The The RAGRAG--22 promoter exhibits cellpromoter exhibits cell--type specificitytype specificity

core promoter regioncore promoter region

Inr InrG-rich BSAP →→→→

→→→→

Fo

ld L

ucif

era

se A

cti

vit

y


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AAGACAGTCATTTTTTGTGGGTTTTCTAAAATGTTTTTAATTTTAACAAATCGA AAGCTT - -220

IkarosGTTTCAATTAGACCTTGTCTTTACAAATGACTGGTATCTCGGGACTTAACATTTTCAATC -160

E-box

EBSTGTCAGTAATGAAAAGGTTAAACTTGAGAGGAACACCCTGGTGGGTTTCTGTCTCCCTCA -100

GG--richrich

ACCATCACAGGGGTGAGGGGTGCACAGGGAGGGGGGGGAGGGGGAAGGAAGGAGAGGTCACAGTCAGTTACTCCCGTTACACCA-40

GCACACCAGTGCTCACTGGAACATCTGTAGGCACACAAACACTCTACCCTGCAGCCTTCA+20EBS E-box

Ikaros/EBS EBSGCTTGGCACAAACTAAACAGTGACTCTTCCCCAAGTGCCGAGTTTAATTCCTGGCTTGGC

→→→→Ikaros 5' splice site

CGAAAGGATTCAGAGAGGGATAAGCAGCCCCTCTGGCCTTCAGGTAGGTCCAATACCTTC

→→→→→→→→+1

RAGRAG--22 Promoter RegionPromoter Region

BSAP(PAXBSAP(PAX--5)5)

+80

+140

Coding Exon ~6 kb downstreamCoding Exon ~6 kb downstream

Pro/Pre-B B Plasma Pre-T mature-T Thymus

B-cells T-cells

BSAP

BSAPBSAP

Two distinct factors interact with the BSAP binding siteTwo distinct factors interact with the BSAP binding site

AGGAGGTCACAGTCAGTTACTCCCGTTACA

TCCTCCAGTGTCAGTCAATGAGGGCAATGTWT

?

c-Myb


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00

1100

2200

3300

4400

5500

6600

PrePre--B cellB cell

WT BSAPmWT BSAPm

Mutation of the core BSAP site leads to a decrease Mutation of the core BSAP site leads to a decrease in promoter activityin promoter activity

RAG-2 promoter construct

decrease decrease

BSAP and SP1 synergistically activate the RAGBSAP and SP1 synergistically activate the RAG--2 promoter2 promoter

Fo

ld P

rom

ote

r a

cti

vit

yF

old

P

rom

ote

r a

cti

vit

y

0

20

40

60

80

100

120

140

+Sp1

++MybMybvector

+Sp1RAG-2 +BSAP +Myb +Sp1

+BSAPalone

0

20

40

60

80

100

120

140

+Sp1

++MybMybvector

+Sp1RAG-2 +BSAP+BSAP ++MybMyb +Sp1

+BSAP+BSAP

RAG-2 promoter vector

alone

RAG-2 promoter GA-mutant vector

Transcription factor Transcription factor cDNAscDNAs vectors vectors transfectedtransfected into fibroblast cell lineinto fibroblast cell line


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2) The GA2) The GA--box plays a critical role in the regulation of the promoter box plays a critical role in the regulation of the promoter

for Bfor B-- and Tand T--cell expressioncell expression

4) BSAP and 4) BSAP and Sp1 synergistically Sp1 synergistically activate the promoteractivate the promoter

5) c5) c--MybMyb transcription factor interacts within BSAP binding site transcription factor interacts within BSAP binding site

and also collaborates with Sp1 to activate the promoterand also collaborates with Sp1 to activate the promoter

Miranda, G. A., Villalvazo, M., Galic, Z., Alva, J., Abrines, R., Yates, Y., Evans, C.J. and

Aguilera, R.J. (2002). Combinatorial regulation of the murine RAG-2 Promoter by Sp1

and distinct lymphocyte-specific transcription factors. Molecular Immunology 38:1151-

1159.

Summary of features of the Summary of features of the RAGRAG--22 promoter promoter ::

1) BSAP interacts with and activates the promoter in B1) BSAP interacts with and activates the promoter in B--cellscells

3) Sp1 and Sp3 bind and activate the promoter 3) Sp1 and Sp3 bind and activate the promoter viavia the GAthe GA--boxbox

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BSAPSP1

LCR or Enhancer

Regulation of the RAGRegulation of the RAG--2 promoter is mediated by multiple control elements2 promoter is mediated by multiple control elements

Inr

cc--MybMyb

POL IITAFs


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OFF ON

Control RegionsControl Regions

Apart from promoters, additional elements are required for RAG expressionApart from promoters, additional elements are required for RAG expression

A putative LCR region controls lymphocyteA putative LCR region controls lymphocyte--specific expression of both promoters specific expression of both promoters

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Analysis of trangenic mice with RAG region deletions

B/T cells

B cells

no B/T cells


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Upstream region affects the expression of both RAG genesUpstream region affects the expression of both RAG genes

E

Yu et al., 1999

LoopingLooping--out modelout modelfor coordinated regulationfor coordinated regulation

Using these transgenic systems, two groups identified Using these transgenic systems, two groups identified essential elements required for lymphocyteessential elements required for lymphocyte--specific specific expression of both RAGexpression of both RAG--1 and RAG1 and RAG--2 in mice2 in mice

silencersilencer

antisilencerantisilencer

In abscense of anti-silencer, silencer is dominant

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(2004) Vol 5:443


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Nat. Immunol. (2004) Vol 5:443

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