Attenuation of IL-7 Receptor Signaling Is Not Required for Allelic Exclusion

Will, Wynette M.; Aaker, Joshua D.; Burchill, Matthew A.; Harmon, Ian R.; O’Neil, Jennifer J.; Goetz, Christine; Hippen, Keli L.; Farrar, Michael A.

doi:10.4049/jimmunol.176.6.3350

Cited by 8 publications

(7 citation statements)

References 25 publications

(30 reference statements)

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“…7c). Flow cytometric analysis of mice heterozygous for two allotypically marked Igh alleles indicated, however, that allelic exclusion was not violated in splenic B cells of caStat5 mice (Supplementary Fig 8 online) similar to 3′E κ -/- mice39,40.…”

Section: Resultsmentioning

confidence: 93%

RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci

et al. 2009

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Coordinated recombination of homologous antigen receptor loci is thought to be important for allelic exclusion. Here, we show that homologous Ig alleles pair in a stage-specific manner that mirrors the recombination patterns of these loci. The frequency of homologous Ig pairing was substantially reduced in the absence of the RAG1-RAG2 recombinase and was rescued in Rag1-/- developing B cells with a transgene expressing a RAG1 active site mutant that supports DNA binding but not cleavage. The introduction of DNA breaks on one Ig allele induced ATM-dependent repositioning of the other allele to pericentromeric heterochromatin. ATM activated by the cleaved allele acts in trans on the uncleaved allele to prevent bi-allelic recombination and chromosome breaks or translocations.

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Section: Resultsmentioning

confidence: 93%

RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci

et al. 2009

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“…Alternatively, increased IRF4 expression in pre-B cells might override repressive effects of STAT5, as was previously observed in bone marrow cultures even in the presence of high levels of IL7 (14). To explore this issue, we assessed Igκ rearrangement in pre-B cells isolated from either wild-type mice, or mice containing a constitutively active STAT5 transgene (44). Isolated DNA was subjected to PCR to detect Vκ-Jκ rearrangement using degenerate primers for various Vκ sequences in association with a primer downstream of the Jκ region.…”

Section: Resultsmentioning

confidence: 99%

A Developmentally Controlled Competitive STAT5–PU.1 DNA Binding Mechanism Regulates Activity of the Ig κE3′ Enhancer

Hodawadekar

Park

Farrar

et al. 2012

The Journal of Immunology

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Stage-specific rearrangement of immunoglobulin heavy and light chain genes poses an enigma because both processes utilize the same recombinational machinery, but the heavy chain locus is accessible at the pro-B cell stage, while the light chain loci become accessible at the pre-B cell stage. Transcription factor STAT5 is a positive-acting factor for rearrangement of distal Vh genes but attenuation of IL7 signaling and loss of activated STAT5 at the pre-B cell stage corresponds with Igκ locus accessibility and rearrangement, suggesting that STAT5 plays an inhibitory role at this locus. Indeed, loss of IL7 signaling correlates with increased activity at the Igκ intron enhancer. However, the κE3’ enhancer must also be regulated as this enhancer plays a role in Igκ rearrangement. We show here that STAT5 can repress κE3’ enhancer activity. We find that STAT5 binds to a site that overlaps the κE3’ PU.1 binding site. We observed reciprocal binding by STAT5 and PU.1 to the κE3’ enhancer in primary bone marrow cells, STAT5 and PU.1 retrovirally transduced pro-B cell lines, or embryonic stem cells induced to differentiate into B lineage cells. Binding by STAT5 corresponded with low occupancy of other enhancer binding proteins, whereas PU.1 binding corresponded with recruitment of IRF4 and E2A to the κE3’ enhancer. We also find that IRF4 expression can over-ride the repressive activity of STAT5. We propose a novel PU.1/STAT5 displacement model during B cell development, and this, coupled with increased IRF4 and E2A activity regulates κE3’ enhancer function.

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“…Surface staining, intracellular staining and cell cycle staining using Hoechst have all been described previously (16, 24). Antibodies specific for murine CD19 (1D3), IL-7R alpha-chain (A7R34), c-kit (2B8), CD25 (PC61), IgM(II/41), and pY695 STAT5 were purchased from BD Pharmingen.…”

Section: Methodsmentioning

confidence: 99%

IL-7 Functionally Segregates the Pro-B Cell Stage by Regulating Transcription of Recombination Mediators across Cell Cycle

Johnson

Chaumeil

Micsinai

et al. 2012

The Journal of Immunology

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Antigen receptor diversity involves the introduction of DNA double stranded breaks during lymphocyte development. To ensure fidelity, cleavage is confined to the G0/G1 phase of cell cycle. One established mechanism of regulation is through periodic degradation of the RAG2 recombinase protein. However, there are additional levels of protection. Here we show that cyclical changes in the IL-7R signaling pathway functionally segregate pro-B cells according to cell cycle status. In consequence, the level of a downstream effector of IL-7 signaling, phospho-STAT5, is inversely correlated with cell cycle expression of Rag, a key gene involved in recombination. Higher levels of phopho-STAT5 in S/G2 correlate with decreased Rag expression and Rag relocalization to pericentromeric heterochromatin (PCH). These cyclical changes in transcription and locus repositioning are ablated upon transformation with v-Abl, which renders STAT5 constitutively active across the cell cycle. We propose that this activity of the IL-7R/STAT5 pathway plays a critical protective role in development, complementing regulation of RAG2 at the protein level, to ensure that recombination does not occur during replication. Our data, suggesting that pro-B cells are not a single homogeneous population explain inconsistencies in the role of IL-7 signaling in regulating Igh recombination.

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Attenuation of IL-7 Receptor Signaling Is Not Required for Allelic Exclusion

Cited by 8 publications

References 25 publications

RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci

RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci

A Developmentally Controlled Competitive STAT5–PU.1 DNA Binding Mechanism Regulates Activity of the Ig κE3′ Enhancer

IL-7 Functionally Segregates the Pro-B Cell Stage by Regulating Transcription of Recombination Mediators across Cell Cycle

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