Comparative analysis of mouse and human Igh 3' regulatory regions identifies conserved structural features and species-specific regulation for enhancer orthologues
Sepulveda Trujillo, Manuel Alejandro
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In B cells, the Igh gene locus contains several DNase I hypersensitive (hs) sites with enhancer activity. Known regulators include the 3' regulatory region, which is located downstream of the Calpha gene(s) in both mouse and human. In vivo experiments have implicated the last two murine 3' enhancers, hs3B-hs4, in class switching and somatic hypermutation. Our laboratory has previously reported that murine hs4 was regulated by NFkappaB, octamer binding proteins and Pax5. My thesis work showed that human hs4 is regulated differently. Electrophoretic mobility shift assays (EMSA) and Western Blot analysis of normal B cells showed a complex binding pattern formed by NFkappaB, YY1, Oct-1 and Oct-2 (but not by Pax5). Similar EMSA patterns were detected in mature human B cell lines (BL-2, Ramos, HS-Sultan) and in diffuse large B cell lymphoma (DLBCL) cell lines, although the latter had an additional distinctive slow migrating complex containing YY1 associated with Rel-B. We have confirmed by endogenous co-immunoprecipitation an association of YY1 with Rel-B, but not with other NFkappaB family members. In addition, we confirmed the in vivo association of YY1, NFkappaB, and octamer family members with the human hs4 enhancer in B cells by chromatin immunoprecipitation assays (ChIP). Transient transfection assays showed robust hs4 enhancer activity in the mature B cell lines, which was dependent on synergistic interactions between NFkappaB and Oct-2. In contrast to the regulation of mouse hs4, we find that human hs4 does not involve Pax5. These differences mirror the limited sequence homology between mouse and human 3' enhancers. A comprehensive genomic analysis of the regions downstream of Calpha from human and mouse showed that the 3' regulatory regions share some unique structural (but not sequence) features. These include an extensive palindrome flanking the hs1,2 enhancer together with families of locally repetitive sequences outside the enhancer elements. Interestingly, in the human 3' regulatory region these repeats resemble switch-sequences. We speculate that the structural features in which the 3 ' enhancers are situated are an intrinsic part of this complex regulatory region and are required for proper long-distance activity in vivo.
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