Structure-functional analyses of Bright, a B cell regulator of immunoglobulin heavy chain transcription

Bright is a B cell specific trans-activator that regulates IgH gene transcription by binding promoter and enhancer-associated MARs within the IgH gene locus. Domains important for Bright function include the ARID, which mediates sequence-specific binding to DNA and REKLES, a highly conserved but less well understood region within Bright and its two paralogues, Bdp (Bright and dri-like protein) and Bright-like. This thesis further explores features of Bright with respect to the role of these domains in cellular localization, protein-protein interactions, and transcriptional activation. Unexpectedly for a transcription factor, Bright accumulates to significant levels within the cytoplasm as well as the nucleus. Bright appears to be actively exported in a CRM-1 dependent manner. The nuclear localization of Bright is mediated by residues in the N-terminus of the REKLES domain (REKLESa), whereas nuclear export is mediated by the C-terminal region of RECKLES (REKLESb) along with the C-terminal 19 amino acids. Bright accumulates in the nucleus as a result of the loss of its nuclear export activity. This nuclear accumulation also correlates with the cell cycle arrest at G2/M phase. The REKLESb domain also mediates self-association of Bright or heteromeric association of Bright and Bdp. Point mutations in the REKLESb domain abolish the DNA-binding and nuclear export activity of Bright. In addition, Bdp appears to interact with Bright and to facilitate the nuclear localization and retention Bright. DNA binding and trans-activation studies indicate that the promoter-associated MARs repress IgH transcription in non-B cells, and Bright alleviates this effect. Promoter-MAR mediated Bright trans-activation is antagonized by direct competition of MAR DNA binding by the ubiquitously expressed repressor complex NF-mNR. However, it was found that NF-mNR includes Bright in B cells but not in non-B cells. The binding activity of NF-mNR and Bright in B cells is reciprocally altered during the cell division cycle and by the B cell mitogen LPS. LPS treatment had no effect on Bright localization but increased the total amount of Bright in the nucleus and cytoplasm. The increased level of Bright appears to displace NF-mNR from the MARs and to facilitate IgH gene transcription.