Enhancers Located in Heavy Chain Regulatory Region (hs3a, hs1,2, hs3b, and hs4) Are Dispensable for Diversity of VDJ Recombination

Rouaud, Pauline; Vincent-Fabert, Christelle; Fiancette, Rémi; Cogné, Michel; Pinaud, Eric; Denizot, Yves

doi:10.1074/jbc.m112.341024

Cited by 34 publications

(40 citation statements)

References 22 publications

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“…Bone marrow cells were enriched for Hardy's fractions C9 and D (mostly large and small pre-B cell populations) expressing CD25 after magnetic sorting (Miltenyi Biotec). DNA was extracted and IgV H repertoire diversity was examined using the Immun'Ig test (ImmunID Technologies, Grenoble, France) as described in Rouaud et al (22). Multiplex PCR assays were performed with forward primers specific for all functional members of a given V H family and reverse primers specific for a given J H segment (http://www.immunid.com).…”

Section: Flow Cytometrymentioning

confidence: 99%

The Eμ Enhancer Region Influences H Chain Expression and B Cell Fate without Impacting IgVH Repertoire and Immune Response In Vivo

Marquet

Garot

Bender

et al. 2014

The Journal of Immunology

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The IgH intronic enhancer region Eμ is a combination of both a 220-bp core enhancer element and two 310–350-bp flanking scaffold/matrix attachment regions named MARsEμ. In the mouse, deletion of the core-enhancer Eμ element mainly affects VDJ recombination with minor effects on class switch recombination. We carried out endogenous deletion of the full-length Eμ region (core plus MARsEμ) in the mouse genome to study VH gene repertoire and IgH expression in developing B-lineage cells. Despite a severe defect in VDJ recombination with partial blockade at the pro–B cell stage, Eμ deletion (core or full length) did not affect VH gene usage. Deletion of this regulatory region induced both a decrease of pre–B cell and newly formed B cell compartments and a strong orientation toward the marginal zone B cell subset. Because Igμ H chain expression was decreased in Eμ-deficient pre–B cells, we propose that modification of B cell homeostasis in deficient animals was caused by “weak” pre–B cell and BCR expression. Besides imbalances in B cell compartments, Ag-specific Ab responses were not impaired in animals carrying the Eμ deletion. In addition to its role in VDJ recombination, our study points out that the full-length Eμ region does not influence VH segment usage but ensures efficient Igμ-chain expression required for strong signaling through pre–B cells and newly formed BCRs and thus participates in B cell inflow and fate.

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Section: Flow Cytometrymentioning

confidence: 99%

The Eμ Enhancer Region Influences H Chain Expression and B Cell Fate without Impacting IgVH Repertoire and Immune Response In Vivo

Marquet

Garot

Bender

et al. 2014

The Journal of Immunology

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“…17, 18). Thus, the prevailing notion is that the 3′RR-mediated activation of GL transcription preceding CSR is restricted to mature B cells (17)(18)(19). This leaves it unknown whether the 3′RR is programmed to activate GL promoters of the IgH constant domain only after activation of B cells or whether it can do so in a developmentally regulated, constitutive manner before induction.…”

mentioning

confidence: 99%

Inducible CTCF insulator delays the IgH 3′ regulatory region-mediated activation of germline promoters and alters class switching

Braikia

Oudinet

Haddad

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to switch from IgM expression to the expression of downstream isotypes. CSR is preceded by inducible germline (GL) transcription of the constant genes and is controlled by the 3′ regulatory region (3′RR) in a stimulus-dependent manner. Why the 3′RR-mediated upregulation of GL transcription is delayed to the mature B-cell stage is presently unknown. Here we show that mice devoid of an inducible CTCF binding element, located in the α constant gene, display a marked isotype-specific increase of GL transcription in developing and resting splenic B cells and altered CSR in activated B cells. Moreover, insertion of a GL promoter downstream of the CTCF insulator led to premature activation of the ectopic promoter. This study provides functional evidence that the 3′RR has a developmentally controlled potential to constitutively activate GL promoters but that this activity is delayed, at least in part, by the CTCF insulator, which borders a transcriptionally active domain established by the 3′RR in developing B cells. E xpression of complex loci is developmentally programmed or induced by specific stimuli and is often controlled by distant regulatory elements within relatively large chromatin domains. Transcriptional and architectural factors play an important role in the establishment and maintenance of these domains and facilitate long-range interactions between regulatory elements and target promoters (1, 2). The Ig heavy chain (IgH) locus is expressed in a lineage-and developmental stage-dependent manner. Various cis-acting elements including promoters, enhancers, and insulators control IgH locus expression and are engaged in multiple long-range interactions (3, 4).Factors such as YY1, PAX5, IKAROS, CTCF, and Cohesin play important roles in various aspects of long-range events at the IgH locus, including V(D)J recombination, CSR, and promoter/enhancer and enhancer/enhancer interactions (3-6). Multiple CTCF binding elements (CBEs) were reported along the IgH locus. The majority of these CBEs lie within the variable domain (7), and two CBEs were identified within the V H -D intergenic region (7-9). At the 3′ end of the locus, ∼10 CBEs were identified downstream of the 3′RR and are thought to delineate the 3′ border of the IgH locus (10). More recently, a discrete CBE was identified within the α constant gene (11), but its role in vivo is presently unknown.Upon antigen challenge, mature B cells can undergo CSR that allows B cells to change the heavy-chain constant domain of an IgM to IgG, IgE, or IgA. CSR to a particular isotype is induced by specific external stimuli, including antigens, mitogens, cytokines, and intercellular interactions. CSR is mediated by highly repetitive sequences called switch (S) sequences located upstream of the constant exons and is preceded by germline (GL) transcription of the S sequences that originates from GL promoters, named I promoters (12).The 3′RR is composed of four enhance...

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“…Among 5 0 elements, the intronic Em enhancer is reported as a master control element of V(D)J recombination 3,4 . The IgH 3 0 regulatory region (3 0 RR), that encompasses the four transcriptional enhancers hs3a, hs1,2, hs3b and hs4, controls m transcription in mature B-cells 5 , and is the master element controlling conventional class switch recombination (CSR) 6,7 and somatic hypermutation (SHM) 8 but without role on V(D)J recombination 9,10 . CSR is a cis-deletion or trans-recombination process that allows the C m gene to be replaced in mice with one of the downstream C g3 , C g1 , C g2b , C g2a , C e or C a gene.…”

mentioning

confidence: 99%

Elucidation of IgH 3′ region regulatory role during class switch recombination via germline deletion

et al. 2015

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In mature B cells, class switch recombination (CSR) replaces the expressed constant Cm gene with a downstream C H gene. How the four transcriptional enhancers of the IgH 3 0 regulatory region (3 0 RR) control CSR remains an open question. We have investigated IgG 1 CSR in 3 0 RR-deficient mice. Here we show that the 3 0 RR enhancers target the S g1 acceptor region (and poorly the S m donor region) by acting on epigenetic marks, germline transcription, paused RNA Pol II recruitment, R loop formation, AID targeting and double-strand break generation. In contrast, location and diversity of S m -S g1 junctions are not affected by deletion of the 3 0 RR enhancers. Thus, the 3 0 RR controls the first steps of CSR by priming the S acceptor region but is not implicated in the choice of the end-joining pathway.

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Enhancers Located in Heavy Chain Regulatory Region (hs3a, hs1,2, hs3b, and hs4) Are Dispensable for Diversity of VDJ Recombination

Cited by 34 publications

References 22 publications

The Eμ Enhancer Region Influences H Chain Expression and B Cell Fate without Impacting IgVH Repertoire and Immune Response In Vivo

The Eμ Enhancer Region Influences H Chain Expression and B Cell Fate without Impacting IgVH Repertoire and Immune Response In Vivo

Inducible CTCF insulator delays the IgH 3′ regulatory region-mediated activation of germline promoters and alters class switching

Elucidation of IgH 3′ region regulatory role during class switch recombination via germline deletion

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