A novel 3' IgH enhancer element regulated at multiple stages of B cell development

Abstract

Multiple cis-acting elements are involved in the regulation of IgH gene expression and rearrangement events, including the intronic enhancer, E{dollar}\mu,{dollar} and a complex regulatory region located downstream of the IgH cluster, which has recently been termed a locus control region (LCR). We have identified and characterized a novel component of the downstream control region, and have studied the regulation of this element during B cell development.;Investigation of a DNase I hypersensitivity site present in pre B and plasma cells led to the identification of the novel enhancer, {dollar}3\sp\prime\alpha{dollar}-hs4. The activity of {dollar}3\sp\prime\alpha{dollar}-hs4 was localized to an 800 bp segment, cloned from both BALB/c and C57BL/6 mouse strains. {dollar}3\sp\prime\alpha{dollar}-hs4 activity is restricted to cells of the B lineage. Reflecting its pattern of DNase I hypersensitivity, {dollar}3\sp\prime\alpha{dollar}-hs4 is active at multiple stages of B cell development, as assessed in transient transfection assays. {dollar}3\sp\prime\alpha{dollar}-hs4 is the only 3{dollar}\sp\prime{dollar} regulatory element to be identified that is active early in B cell differentiation. Given its activity profile, it is a candidate for regulating multiple IgH-processes, such as V(D)J recombination, isotype class switching and somatic mutation.;To study the regulation of {dollar}3\sp\prime\alpha{dollar}-hs4, an electromobility shift assay (EMSA) was employed as a means of identifying enhancer binding proteins. Multiple binding sites for BSAP (B-cell Specific Activator Protein) and Oct-1 were localized within {dollar}3\sp\prime\alpha{dollar}-hs4. BSAP and octamer binding proteins are also regulators of the previously identified 3{dollar}\sp\prime{dollar} IgH enhancer, 3{dollar}\sp\prime\alpha{dollar}E, suggesting that the two elements may be subject to coregulation. In addition, the first example of an NF-{dollar}\kappa{dollar}B-like complex bound to an IgH enhancer was mapped to {dollar}3\sp\prime\alpha{dollar}-hs4. This finding is significant since an interaction with the 3{dollar}\sp\prime{dollar} IgH enhancers may represent a mechanism by which {dollar}\kappa{dollar}B complexes function in regulating heavy chain class switching. Mutational analysis of {dollar}3\sp\prime\alpha{dollar}-hs4 was performed to assess the contribution of these factors to enhancer activity during B cell development. These experiments revealed that {dollar}\kappa{dollar}B complexes contribute to enhancer activation at multiple stages of development; that Oct-1 positively regulates {dollar}3\sp\prime\alpha{dollar}-hs4 in pre B and B cells; and that BSAP is a repressor in pre B cells but an activator of the enhancer in a B cell line. We speculate that {dollar}3\sp\prime\alpha{dollar}-hs4 functions, in conjunction with other 3{dollar}\sp\prime{dollar} regulatory elements, to control processes involved in IgH gene rearrangements and expression both at early and late stages of B cell development.