Abstract
Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus. We found that these CTCF sites share a consensus motif distinct from other CTCF sites in the mouse genome. Additionally, we could divide these CTCF sites into three categories: intergenic sites remote from any coding element, upstream sites present within 8 kb of the VH-leader exon, and recombination signal sequence (RSS)-associated sites characteristically located at a fixed distance (∼18 bp) downstream of the RSS. We noted that the intergenic and upstream sites are located in the distal portion of the VH locus, whereas the RSS-associated sites are located in the DH-proximal region. Computational analysis indicated that the prevalence of CTCF-binding sites at the IgH locus is evolutionarily conserved. In all species analyzed, these sites exhibit a striking strand-orientation bias, with >98% of the murine sites being present in one orientation with respect to VH gene transcription. Electrophoretic mobility shift and enhancer-blocking assays and ChIP–chip analysis confirmed CTCF binding to these sites both in vitro and in vivo.
Highlights
Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains
Some of these CTCF sites have been functionally analyzed via targeted deletion: a portion of the 3Ј-regulatory region (3Ј-RR) of the immunoglobulin heavy chain (IgH) locus that contains several CTCF- and Pax5-binding sites modestly affects V(D)J recombination and contraction of the locus [19]; and a regulatory region located between the VH and DH gene clusters that contain two CTCF-binding elements contributes to the developmental regulation of V(D)J recombination [20, 21]
Does CTCF bind directly to particular DNA sequences in the IgH locus when these sequences become accessible, or is it being recruited indirectly via protein–protein interactions with other DNA-binding proteins, such as Yin Yang 1 (YY1) [12, 25], cohesin [18, 45], or Pax5 [9, 10]? Here, we find that the majority of CTCF-occupied sites overlap with a computationally-identifiable sub-consensus motif, mVH–CTCF (5Ј-GACCAGCAGGGGGC-3Ј), that is distinct from the generic CTCF consensus motif that is found elsewhere in the mouse genome
Summary
Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. CTCF, a ubiquitously-expressed nuclear protein that is involved in many cellular processes, is a interesting transcription factor that has been localized to numerous sites across the murine immunoglobulin heavy chain (IgH) locus [9, 17, 18] Some of these CTCF sites have been functionally analyzed via targeted deletion: a portion of the 3Ј-regulatory region (3Ј-RR) of the IgH locus that contains several CTCF- and Pax5-binding sites modestly affects V(D)J recombination and contraction of the locus [19]; and a regulatory region located between the VH and DH gene clusters that contain two CTCF-binding elements contributes to the developmental regulation of V(D)J recombination [20, 21]. Within the VH domains of both the human and mouse IgH loci, the CTCF-binding sites all share the same orientation
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