RESUMO
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. Histone modifications were similar in resting splenic B cells and in splenic B cells induced by lipopolysaccharide to undergo CSR. From the pro-B-cell stage onward, the approximately 11-kb region immediately downstream of hs4 displayed H3 and H4 modifications indicative of open chromatin. This region contained newly identified DNase I-hypersensitive sites and several CTCF target sites, some of which were occupied in vivo in a developmentally regulated manner. The open chromatin environment of the extended 3' RR in mature B cells was flanked by regions associated with dimethylated K9 of histone H3. Together, these data suggest that 3' RR elements are located within a specific chromatin subdomain that contains CTCF binding sites and developmentally regulated modules.
Assuntos
Região 3'-Flanqueadora/genética , Linfócitos B/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas Repressoras/metabolismo , Acetilação/efeitos dos fármacos , Animais , Linfócitos B/imunologia , Medula Óssea/imunologia , Medula Óssea/metabolismo , Fator de Ligação a CCCTC , Cromatina/genética , Cromatina/imunologia , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Desoxirribonuclease I/metabolismo , Elementos Facilitadores Genéticos/genética , Histonas/genética , Histonas/imunologia , Switching de Imunoglobulina/efeitos dos fármacos , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Lipopolissacarídeos/farmacologia , Região de Controle de Locus Gênico/genética , Camundongos , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Baço/imunologia , Baço/metabolismo , Transcrição Gênica/genéticaRESUMO
Immunoglobulin heavy chain (Igh) locus rearrangements are controlled in part by an approximately 30 b complex 3' regulatory region located 3' of C alpha: this region contains several enhancers. We report here the comparison of the genomic sequences of the 3' regulatory region and further downstream sequences from mouse, rat, human and chimpanzee. Only short segments of homology were detected in the 3' regulatory region, and these were located in the vicinity of the known 3' enhancers. The nearest highly conserved segment is the nearest non-Igh gene, hole, which is located approximately 62 kb downstream of mouse C alpha. Analysis of murine 3' Igh sequences by single nucleotide polymorphism (SNP) and restriction fragment length polymorphism (RFLP) detected a transition region (high to low SNP or RFLP density) approximately 120 kb downstream of mouse C alpha. Although there is only limited sequence identity between rodent and primate 3' Igh regulatory regions, all of these regulatory regions contain a palindrome and locally repetitive elements. Locally repetitive elements in primates comprise blocks of "switch-like" sequences that differ from the families of inverted and tandem repeats that are present in rodents. We propose that together with enhancers, these "conserved" structural features are essential for the activity of the 3' Igh regulatory region in vivo.