Noncoding transcription within the Igh distal V _H region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells

Abstract

Noncoding sense and antisense germ-line transcription within the Ig heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eμ, the start site of Iμ germ-line transcription, in a lineage-and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1 −/− pro-B cells are greatly impaired in distal V H gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat-shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal V H genes close to the DJ H rearrangement which is adjacent to Eμ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal V H genes to undergo efficient rearrangement.A ntigen receptors in lymphocytes are assembled in the highly regulated lineage-specific process of V(D)J recombination, which creates a diverse repertoire of Ig and T-cell receptors. In each precursor lymphocyte, one each of the many V, D, and J gene segments at the appropriate receptor loci are juxtaposed to create a V(D)J exon encoding the variable region of the antigen receptor. In B-lineage progenitors, rearrangement occurs first at the Ig heavy chain (Igh) locus, where D H to J H rearrangement occurs first on both alleles, followed by V H to DJ H rearrangement (1). After successful rearrangement of the Igh locus, rearrangement at the Igk light chain locus begins. These successive stages of rearrangement have been proposed to be regulated by differential accessibility of different portions of the loci at the appropriate time for rearrangement (2). Early indications of this stage-specific and lineage-specific accessibility came from the observation that unrearranged gene segments underwent noncoding transcription at the stage immediately preceding their rearrangement (3, 4).The murine Igh locus spans ∼2.8 Mb, of which ∼2.4 Mb contains the 195 V H gene segments (5). The V H genes are divided i...

A structural hierarchy mediated by multiple nuclear factors establishes IgH locus conformation

“…First, YY1 deficiency results in locus decompaction and preferential recombination of the proximal V H genes (Liu et al 2007). This phenotype closely resembles that of Pax5-deficient pro-B cells, yet the major YY1-binding sites are located within the 3 ′ IgH domain far away from Pax5-binding PAIR elements (Verma-Gaur et al 2012). While YY1 function may depend on its weak binding to PAIR4, PAIR6, and PAIR8 (Medvedovic et al 2013), an alternative possibility is that YY1 is required to increase association between the structured V H region and the 3 ′ IgH domain.…”

“…This phenomenon is referred to as locus compaction (or contraction), and its functional significance is inferred from the strong correlation between reduced locus compaction and restricted use of D H -distal V H gene segments in pro-B cells of mice bearing several genetic mutations that affect B-cell development. The most prominent of these are deletions of genes encoding the transcription factors Pax5 (Fuxa et al 2004) and YY1 (Liu et al 2007;Verma-Gaur et al 2012) and "cis" mutations within the IgH locus that delete the intronic enhancer Eμ (Guo et al 2011a). A compacted IgH locus structure may also minimize hazardous DNA translocation events during V(D)J recombination.…”

“…The role of Eµ and the transcription factor YY1 in this model is not clear. One possibility could be that YY1 binds to a subset of PAIR elements and regulates antisense transcripts at PAIR4, PAIR6, and PAIR8 (Verma-Gaur et al 2012). We had previously proposed a two-step model for generating IgH locus conformation (Guo et al 2011a).…”

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

“…Within the distal VH domain, two distinct regions were notable: (i) The region encompassing the proximal J558 genes (region II) had a nucleosomal density intermediate between the VH proximal domain (region III) and the distal J558 segments (region I), and (ii) the distal J558 region (region I) was marked by its high level of nucleosome occupancy, encompassing all but one of the PAIR elements believed to be involved in the regulation of IgH rearrangement (43,47). Between these two regions was a short region of transition, with background levels of nucleosome density that were neither enriched nor depleted (Fig.…”

“…1). V(D)J recombination in the distal domain is known to be dependent on several transcription factors, including Pax5 (42, 43), Ikaros (44), EZH2 (45), and YY1 (46,47), as well as the cytokine IL7 (48), but recombination of at least some proximal gene segments occurs in their absence (42,44,46,49). Within the distal domain, a further subdivision has been noted as mentioned above, between the tightly clustered J558 gene segments closer to DH (proximal J558) and those further away (distal J558), with the 3609 gene segments interspersed among the distal segments (40, 50).…”