The Igh locus undergoes massive changes in its DNA during B cell maturation, first at the 5' end of the locus via VDJ joining, and then downstream to involve Ch sequences in CSR. These DNA changes are focused on the Igh locus, sparing downstream adjacent sequences, and limiting mutagenic effects that could, otherwise, result in malignant transformation. The prime cis-regulators of the Igh locus are E and a complex 3' Igh regulatory region that is the major focus of this proposal. 3' Igh enhancers have been shown to influence CSR at distances of 40-150 kb. Developmental changes in replication patterns and nuclear sublocalization of the Igh locus also occur during B cell maturation and a domain of Igh replication terminates considerably downstream of the most 3' known enhancer. These observations suggest multiple levels of regulation at the 3' end of the Igh locus. Chromatin immunoprecipitation analysis will be used to identify features of chromatin architecture that mark the limits of the Igh replicative domain. Using has identified a 5-7 kb extension of the 3' regulatory region that becomes associated with markers of active chromatin early in B cell differentiation, followed by step-wise association of other modules of this region. This proposal will assay the analymodules of the 3' regulatory region that acquire a step-wise association with active chromatin during B cell differentiation, and evidence for other epigenetic domains of 3' Igh sequences will be sought. The interaction of modules of the 3' regulatory region with transcription factors that foster changes in chromatin will be analyzed. This proposal is focused on analysis of a 5-7 kb region downstream of hs4, the most 3' known enhancer, which is associated with open chromatin in pro-B cells. Other modules of the 3' regulatory region appear to gain step-wise association with active chromatin during B cell differentiation. Early in B cell differentiation, DNA rearrangement activity first occurs at the 5' end of the Igh locus to generate the variable region. With commitment to the B cell stage, DNA rearrangements shift to downstream constant region genes, via class switching. Only two major cis regulatory elements are known, i.e. the intronic enhance r and a complex w' regulatory region containing four known enhancers-a major target.
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