Chromatin Architecture near a Potential 3′ End of the Igh Locus Involves Modular Regulation of Histone Modifications during B-Cell Development and In Vivo Occupancy at CTCF Sites

Abstract

The murine Igh locus has a 3 regulatory region (3 RR) containing four enhancers (hs3A, hs1,2, hs3B, and hs4) at DNase I-hypersensitive sites. The 3 RR exerts long-range effects on class switch recombination (CSR) to several isotypes through its control of germ line transcription. By measuring levels of acetylated histones H3 and H4 and of dimethylated H3 (K4) with chromatin immunoprecipitation assays, we found that early in B-cell development, chromatin encompassing the enhancers of the 3 RR began to attain stepwise modifications typical of an open conformation. The hs4 enhancer was associated with active chromatin initially in pro-and pre-B cells and then together with hs3A, hs1,2, and hs3B in B and plasma cells. The immunoglobulin heavy chain locus (Igh) (see Fig. 1) is arguably the most complex mammalian locus. B-cell development is marked by the progressive expression of Igh genes (via VDJ recombination, somatic hypermutation, and class switch recombination [CSR]). These regulated processes, some involving substantial DNA rearrangements and deletions, are required for the generation of antibody diversity (reviewed in references 47 and 57). Spanning ϳ3 Mb, the murine Igh locus contains a limited number of enhancers, including the intronic enhancer (E), which has been implicated in V to DJ joining and chain expression (reviewed in reference 47), and a complex 3Ј regulatory region (3Ј RR) located immediately downstream of C␣, the last constant region gene. The human Igh locus contains two 3Ј RRs, each located downstream of one of the duplicated C␣ genes (9, 49, 62).The murine 3Ј RR spans ϳ28 kb downstream of C␣ and comprises four enhancers, each associated with DNase I hypersensitivity (hs3A, hs1,2, hs3B, and hs4) (reviewed in reference 33). hs3A and hs3B are located at the termini of a 25-kb inverted repeat flanking hs1,2 (7, 66), and these three enhancers acquire DNase I hypersensitivity at later stages of B-cell development (66). The hs4 enhancer is the only 3Ј enhancer with activity and DNase I hypersensitivity throughout B-cell development (19, 48), although no role for hs4 or any of the 3Ј enhancers in early B-cell development has been shown.Targeted deletions of the 3Ј RR enhancers have shown that hs3A and hs1,2 are individually dispensable for B-cell development and Igh expression (46), while the combination of hs3B and hs4 is critical for the process of CSR (63). The 3Ј RR has also been proposed to regulate Igh expression in plasma cells (23,40,48,63,70). In accord with earlier suggestions that it functions as a locus control region (43), the 3Ј RR shows synergistic activity and position-independent regulation when tested in transgenic model systems (8,33,59). However, copynumber-dependent expression has not been observed (8). In addition to a function within the Igh locus, the 3Ј RR is likely to be responsible for dysregulation of c-myc expression resulting from certain Igh::c-myc translocations in mouse plasmacytoma cells as well as in human Burkitt's lymphoma and multiple myeloma cells (30,36,43).Th...

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

“…Furthermore, additional control elements not yet identified might lie among or downstream of the known ones. Interestingly, in collaboration with others we have recently identified a region with insulator activity downstream of hs4, and this region was not included in previous minilocus constructs but is present in BAC 141e18 (22).…”

“…In transient transfection assays, hs4 is the most robust of the 3Ј IgH enhancers in mature B cell lines and plasmacytoma lines and the only one to show activity in pre-B cell lines (17,19,20). It is also the only one of the 3Ј IgH enhancers that shows DNase I hypersensitivity in both pro-B and pre-B cells; the other enhancers become DNase I hypersensitive later, at the surface Ig ϩ cell stage (17,19,21,22). Second, there was reason to doubt that hs3b was required for either CSR or Igh transcription, because the deletion of the highly homologous element hs3a (97% identity) had no effect on either process (18,23).…”

“…The DNase I hypersensitivity assay was performed as described previously (22). Briefly, cell nuclei were isolated from cell lines (9921,137.1, 7.1, and 6.1) by Dounce homogenization and treated with increasing amounts of DNase I (2 ϫ 10 7 cells per sample; Worthington Biochemical) for 5 min at 37°C.…”

Spi-C Has Opposing Effects to PU.1 on Gene Expression in Progenitor B Cells

“…Chromatin immunoprecipitation was performed as described previously (41). Briefly, 10 8 cells were grown as described above and collected by centrifugation, resuspended in IL-7-containing IMDM, and subjected to cross-linking with formaldehyde for 10 min at 37°C.…”

“…07-212). Real-time PCR for regions of the 3Ј regulatory region, IgH intronic enhancer, and IL-7R␣ was performed as described previously (41). Primer pairs used to amplify these regions were described previously (41).…”