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