The Mouse Immunoglobulin Heavy Chain V-D Intergenic Sequence Contains Insulators That May Regulate Ordered V(D)J Recombination

Abstract

V(D)J recombination of the multigene antigen receptor loci is essential for the generation of a diverse antigen receptor repertoire. Recombination is strictly regulated, occurring only in lymphocytes due to restricted expression of the recombination activating gene enzymes, RAG1 and RAG2, therein. Further, T cell receptors only recombine in T cells, B cell receptors only recombine in B cells, and the loci only recombine at specific stages in lymphocyte differentiation. In B cells, the Igh recombines before the Ig light chains. Finally some antigen receptor loci (e.g. the Igh) have two ordered recombination events. A D gene first recombines with a J gene on both alleles, followed by recombination of a V gene to the DJ recombined segment. Once a productive VDJ rearrangement has been generated, further V to DJ recombination is prevented on the second allele, a process termed allelic exclusion, which in B cells ensures that each B cell expresses a monoclonal IgH (1).Ordered recombination is crucial for antigen receptor integrity, but key questions remain: how is recombination order achieved, and how is it regulated? Numerous studies have suggested that order is achieved through alterations in the chromatin conformation of individual gene domains at sequential stages of lymphocyte development (2). In the mouse Igh locus, the D-J-C region acquires histone post-translational modifications characteristic of open chromatin before the V region (3, 4). Non-coding RNA transcripts, including I, generated from E, located 3Ј of the J genes (5), and o, transcribed from the promoter of the most 3Ј D gene, DQ52, occur on germ line alleles (6). Following D to J recombination, non-coding transcripts are generated from the V genes (7,8). Furthermore, extensive antisense intergenic transcription occurs throughout the D and J domains before D to J, and then throughout the V domain before V to DJ recombination (9, 10). Nuclear positioning may also play a role in ordered V(D)J recombination. The Igh locus is tethered at the nuclear periphery via the V region in non-B cells (11,12). Relocation toward euchromatic regions occurs preferentially from the DJC end, favoring D to J recombination. Furthermore, locus compaction through DNA looping is required for distal V gene recombination (13,14). Several transcription factors, including Pax5 (13), YY1 (15), and Ikaros (16), play a role in looping, and in their absence, only the D-proximal V genes recombine. Following productive V(D)J recombination and cell surface expression of an IgH polypeptide, several of the above processes are reversed to silence V to DJ recombination of the second allele by allelic exclusion. Both Igh V regions decontract, V region germ line transcription is lost, and the second Igh allele is recruited to pericentric heterochromatin via the D-distal V genes (1). In contrast, both DJC regions remain transcriptionally active (9, 17). Thus, there is differential chromatin regulation of both activation and inactivation of the DJC versus V regions of the Igh locus.

Regulated large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination

“…S1), and the DNA derived from the mononucleosome fraction arising from each (50). The IgH 3′ regulatory region (3′RR, green), Eμ enhancer (Eμ, green), CTCF binding elements (CBE1/2, red) (70,71), and PAX5-dependent regulatory (PAIR) elements (43) are indicated below the locus. The approximate sizes of the proximal and distal VH domains are indicated in the figure. concentration was pooled, hybridized to the arrays, and analyzed as described (Materials and Methods).…”

Elements between the IgH variable (V) and diversity (D) clusters influence antisense transcription and lineage-specific V(D)J recombination

“…The large intergenic region between the most 3′ functional V H segment (V H 81X) (11) and the most 5′ D segment (DFL16.1) (27) has a number of properties suggestive of a potential regulatory function, including multiple DNase I hypersensitivity sites, differential histone modifications in V H and D regions in pro-B versus pro-T cells, and two defined CTCF binding sites located several kilobases upstream of DFL16.1 (25,(28)(29)(30). To evaluate potential regulatory properties of the V H to D intergenic region, we used the Velocigene technology (31) to create an IgH allele termed ΔV H -D that harbors a 109.8-kb deletion that encompasses the region from 6.4 kb 5′ of V H 81X (V H 7183.a2.3) to 847 bp 3′ of DFL16.1 ( Fig.…”

“…In addition, although developing T cells generate low levels of DJ H rearrangements, they do not append V H segments to the DJ H complexes or generate V H to D rearrangements, implicating V H to DJ H rearrangement as the regulated step with respect to B versus T lineage specificity (14). In these contexts, the ≈100-kb intergenic region between the mouse V H and D clusters is a likely candidate for harboring sequences that might regulate V H to DJ H rearrangement (15,24,25). Correspondingly, insertion of a V H segment just upstream of the most 5′ D segment deregulated its rearrangement both in terms of order and lineage specificity (26).…”

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

“…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)(8)(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).…”