The Distal VH Gene Cluster of the Igh Locus Contains Distinct Regulatory Elements with Pax5 Transcription Factor-Dependent Activity in Pro-B Cells

Abstract

V(H)-DJ(H) recombination of the immunoglobulin heavy chain (Igh) locus is temporally and spatially controlled during early B cell development, and yet no regulatory elements other than the V(H) gene promoters have been identified throughout the entire V(H) gene cluster. Here, we discovered regulatory sequences that are interspersed in the distal V(H) gene region. These conserved repeat elements were characterized by the presence of Pax5 transcription factor-dependent active chromatin by binding of the regulators Pax5, E2A, CTCF, and Rad21, as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B cell development. The pro-B cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal V(H)-DJ(H) recombination at the Igh locus.

Tcragene recombination is supported by aTcraenhancer- and CTCF-dependent chromatin hub

“…At Igh and Igk, CTCF sites are generally intergenic except for those in the proximal one-third of the V H array, which are downstream of V H RSSs; moreover, CTCF sites are not associated with the major Igh and Igk enhancer elements (25,26,(48)(49)(50). At these loci, CTCF promotes long-distance interactions between CTCFbinding elements (24)(25)(26), but these looping interactions appear primarily to restrict or insulate the activities of the major enhancer elements.…”

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

“…Furthermore, we observed that the level of Pax5 mRNA is twofold lower in YY1 −/− pro-B cells, and binding of Pax5 to the PAIR promoters also reduced. Because Pax5 is essential for antisense transcription from PAIR elements (19), this could also contribute to the reduction in PAIR-supported antisense transcription. In addition, we demonstrated that YY1 −/− pro-B cells showed greatly diminished interactions of PAIR elements with Eμ.…”

“…These 14 PAIR elements contain binding motifs for Pax5, CTCF, and E2A. Transcription from PAIR elements is dependent on the transcription factor Pax5, which only binds to these elements in pro-B cells (19). Data from our laboratory demonstrated that CTCF acts as negative regulator of transcription from PAIR elements because the knockdown of CTCF resulted in increased antisense transcription from PAIR elements in pro-B cells (15).…”

“…The timing of both sense and antisense germ-line transcription precisely correlates with the timing of rearrangement, and thus has been proposed to make the regions accessible for rearrangement (2,3). The regulatory elements and transacting factors for the antisense transcripts are not known, other than the recently described Pax5-activated intergenic repeat (PAIR) elements (19). These 14 PAIR elements contain binding motifs for Pax5, CTCF, and E2A.…”

Preparing Targets for V(D)J Recombinase: Transcription Paves the Way

“…Indeed, in the Pillars article, Yancopoulos and Alt (8) foreshadowed a major issue that remains unresolved today: the identity of cis elements that drive V H -glt and presumably recombination of the complex V H cluster (8). Progress has been slow, but a recent report that certain mouse V H clusters have PAX5 target elements harboring intrinsic enhancer activity may provide a much-needed boost (27). Beyond chromatin accessibility, it is now clear that the three-dimensional structure of AgR loci plays an important role in facilitating long-range recombination, including V to DJ H (28,29).…”