Developmental control of chromatin at the 3' end of the Igh locus

Abstract

The Igh locus is subject to many processes that occur during B-cell development, including V(D)J recombination, class switching, and somatic hypermutation. 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 switching (CSR) to several isotypes through its control of germline transcription. The expression of non-Igh genes, beginning ∼33kb downstream of hs4, does not correlate with the expression pattern of the Igh locus. We hypothesize that the Igh locus lies within a delimited chromosomal domain that allows for the containment of specific DNA processes. We further propose that the locus has chromatin subdomains containing active elements (genes and regulators). RFLP and DNA methylation analysis determined that high sequence polymorphism and progressive DNA demethylation during B cell development are not limited to known elements of the locus. By measuring levels of acetylated histones H3 and H4 and of di-me K4 H3 with chromatin immunoprecipitation assays, we found that early in B cell development, chromatin encompassing the enhancers of the 3' RR acquires step-wise modifications characteristic 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 pattern of histone modifications was similar in resting splenic B cells and in splenic B cells induced by LPS to undergo CSR. From the pro-B cell stage onward, the ∼11 kb region immediately downstream of hs4 also displayed H3/H4 modifications typical of open chromatin. This region contained newly identified DNase I hypersensitive sites (hs5 and hs6) and several target sites for CTCF, a protein associated with mammalian insulators. The open chromatin environment of the extended 3' RR in mature B cells was flanked by regions associated with di-methylated K9 of histone H3. Together, these data suggest that 3' RR elements are located within a specific chromatin sub-domain that contains CTCF binding sites and developmentally regulated modules.