Bruton's Tyrosine Kinase Regulates Immunoglobulin Promoter Activation in Association with the Transcription Factor Bright

Abstract

Bright (B-cell regulator of immunoglobulin heavy chain transcription) binding to immunoglobulin heavy chain loci after B-cell activation is associated with increased heavy chain transcription. Our earlier reports demonstrated that Bright coimmunoprecipitates with Bruton's tyrosine kinase (Btk) and that these proteins associate in a DNA-binding complex in primary B cells. B cells from immunodeficient mice with a mutation in Btk failed to produce stable Bright DNA-binding complexes. In order to determine if Btk is important for Bright function, a transcription activation assay was established and analyzed using real-time PCR technology. Cells lacking both Bright and Btk were transfected with Bright and/or Btk along with an immunoglobulin heavy chain reporter construct. Immunoglobulin gene transcription was enhanced when Bright and Btk were coexpressed. In contrast, neither Bright nor Btk alone led to activation of heavy chain transcription. Furthermore, Bright function required both Btk kinase activity and sequences within the pleckstrin homology domain of Btk. Bright was not appreciably phosphorylated by Btk; however, a third tyrosine-phosphorylated protein coprecipitated with Bright. Thus, the ability of Bright to enhance immunoglobulin transcription critically requires functional Btk.Bright (B-cell regulator of immunoglobulin [Ig] heavy chain transcription) is a B-cell-restricted transcription factor that binds specific A-T-rich sequences. The protein consists of an acidic amino-terminal domain, a DNA-binding A-T-rich interaction domain, a putative transactivation domain, and a protein interaction domain (18). The carboxyl-terminal domain of Bright currently has no assigned function. Bright was originally identified in an antigen-specific B-cell line, BCg3R-1d, as a mobility-shifted complex induced after stimulation with interleukin-5 (IL-5) and antigen (54). Binding sites for Bright were originally identified 5Ј of the basal promoter of the V1 S107 gene but also exist within the matrix association regions on either side of the intronic enhancer (53, 55). Bright binding to the 5Ј-flanking sequences of the V1 S107 variable heavy chain (V H ) promoter correlated with two-to sixfold increases in heavy chain mRNA levels in response to 55). Deletion of Bright binding sites flanking the V1 promoter resulted in lack of antigen-and IL-5-stimulated heavy chain transcription (55). Bright expression is tightly regulated in normal murine lymphocytes, occurring in pre-B cells and late stages of B-cell differentiation (58). However, Bright is not present in detectable amounts in immature B cells, suggesting that it may not play a role in maintenance of Ig expression (58). On the other hand, Bright activity is induced in B cells activated in response to lipopolysaccharide (LPS), CD40 ligand stimulation, and anti-CD38 (55, 59). These data suggest that Bright enhances Ig heavy chain transcription above basal levels following B-cell activation.Our earlier results revealed that Bruton's tyrosine kinase (Btk) associates with Brig...

Induction of Immunoglobulin Heavy-Chain Transcription through the Transcription Factor Bright Requires TFII-I

“…However, Bright itself was not appreciably phosphorylated (44). These data led to the hypothesis that a third protein, a Btk substrate, was associated with the Bright/ Btk complex (32).…”

“…The Ig reporter construct contained the S107 V1 heavy-chain genomic sequence from Ϫ574 base pairs 5Ј of the transcription start site through the leader sequence, first intron, and 146 bases of the coding sequence, along with a 1-kb XbaI fragment containing the intronic enhancer (32). The fulllength human Bright expression construct was described previously (28).…”

“…Preliminary evidence from a hamster cell line demonstrated that a 107-kDa protein that coprecipitated with transiently transfected Bright/ Btk also cross-reacted with anti-TFII-I antibody (32). We therefore sought to determine if Bright associated with endogenous TFII-I in B lymphocytes and whether such an association explained the requirement for a third protein in our Ig model system.…”

“…Recently, an in vitro model system using an Ig reporter gene was developed to determine if Btk contributed to Bright function (32). In this model system, Btk kinase activity was critically required for Bright-dependent transactivation of the Ig heavychain promoter.…”

“…Our previous studies suggested that the Bright/Btk complex required a third protein for transcription activation (32) and that this protein was likely to be a Btk substrate. Preliminary evidence from a hamster cell line demonstrated that a 107-kDa protein that coprecipitated with transiently transfected Bright/ Btk also cross-reacted with anti-TFII-I antibody (32).…”