Elucidation of IgH intronic enhancer functions via germ-line deletion

Abstract

Studies of chimeric mice demonstrated that the core Ig heavy chain (IgH) intronic enhancer (iE) functions in V(D)J and class switch recombination at the IgH locus. To more fully evaluate the role of this element in these and other processes, we generated mice homozygous for germ-line mutations in which the core sequences of iE (cE) were either deleted (cE ⌬/⌬ mice) or replaced with a pgk-Neo R cassette (cE N/N mice). The cE ⌬/⌬ mice had reduced B cell numbers, in association with impaired D to JH and VH to DJH rearrangement, whereas cE N/N mice had a complete block in IgH V(D)JH recombination, confirming that additional cis elements cooperate with iE to enforce D to JH recombination. In addition, developing cE ⌬/⌬ and cE N/N B lineage cells had correspondingly decreased levels of germ-line transcripts from the JH region of the IgH locus (0 and I transcripts); although both had normal levels of germ-line VH transcripts, suggesting that cE may influence IgH locus V(D)J recombination by influencing accessibility of JH proximal regions of the locus. Consistent with chimera studies, peripheral cE ⌬/⌬ B cells had normal surface Ig and relatively normal class switch recombination. However, cE ⌬/⌬ B cells also had relatively normal somatic hypermutation of their IgH variable region genes, showing unexpectedly that the cE is not required for this process. The availability of mice with the iE mutation in their germ line will facilitate future studies to elucidate the roles of iE in VH(D)JH recombination in the context of IgH chromatin structure and germ-line transcription.germ-line transcription ͉ immunoglobulin heavy chain ͉ somatic hypermutation ͉ VDJ recombination ͉ class switch recombination T he mouse Ig heavy chain (IgH) locus can be divided into two principal regions: an Ϸ2.2-Mb 5Ј region containing clusters of variable (V H ), diversity (D H ) and joining (J H ) segments and an Ϸ200-kb region downstream region harboring the sets of constant region exons (C, C␦, C␥3, C␥1, C␥2b, C␥2a, C, and C␣) referred as C H genes. Generation of an Ig heavy chain protein, which is first expressed in B cell development, involves assembly of a V H DJ H exon upstream of C via the V(D)J recombination reaction (1). In antigen-stimulated peripheral B cells, expression of downstream IgH isotypes (such as IgG, IgE, or IgA) can occur via a class switch recombination (CSR) reaction that replaces C with a downstream set of C H exons (2). Somatic hypermutation (SHM) of the variable region exon, which allows selection of higher affinity antibodies, also occurs in antigen-stimulated B cells (3). V(D)J recombination is initiated by the RAG endonuclease, whereas CSR and SHM are initiated by activation induced deaminase (4). The overall mechanisms that control these processes in the context of B cell development and activation are still being elucidated; however, many studies have implicated a role for transcriptional control elements (5). Major known control elements in the IgH locus include the transcriptional enhancer that lies within the intr...

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

“…Our study thus validates at the DNA level the hypothesis that during CSR, the 3 0 RR especially targets the S acceptor region rather than the S m donor region. Although the Em enhancer could be proposed to support S m accessibility when the 3 0 RR is deleted, it is noteworthy that it is not itself mandatory for CSR 3 . The 3 0 RR-independence of S m is also somehow striking comparing with the known 3 0 RR-dependence of pV H promoters and rearranged VDJ regions with regards to AID recruitment and SHM 8 .…”

“…1a). 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 .…”

Transcription of a Productively Rearranged Ig VDJCα Does Not Require the Presence of HS4 in the Igh 3′ Regulatory Region

“…1). The first of these, E , is found in an intron separating the V region and C region coding sequences and, although initially discovered because of its ability to enhance transcription from the promoter of an assembled IgH gene, it has since been shown to be essential for efficient V-D-J recombination (3)(4)(5)(6)(7)(8)(9). The other four enhancers lie at the far 3Ј end of the IgH locus and have been shown to contribute substantially to IgH gene transcription in Ig-secreting cells as well as play an important role in CSR (10,11).…”

YY1 helps to bring loose ends together: Figure 1.

“…When recombination of a stably transfected recombination substrate was studied, iEµ was both required and sufficient for recombination (Oltz et al 1993). More importantly, when endogenous iEµ was deleted in mice, a partial block in D H -J H recombination was observed and a more substantial, but still partial, block in V H -D H J H was observed, establishing the importance of iEµ in heavy-chain recombination, especially V H -D H J H rearrangement (Perlot et al 2005).…”

“…The mechanism(s) by which iEµ enhances V H -D H J H recombination is not established and analysis of mice harboring the germline deletion (Perlot et al 2005) did not include an analysis of IgH chromosome condensation or looping. Many transcriptional regulators bind specifically to functional sites in iEµ (for review, see Calame and Sen 2004).…”