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

Abstract

Bright/ARID3a/Dril1, a member of the ARID family of transcription factors, is expressed in a highly regulated fashion in B lymphocytes, where it enhances immunoglobulin transcription three-to sixfold. Recent publications from our lab indicated that functional, but not kinase-inactive, Bruton's tyrosine kinase (Btk) is critical for Bright activity in an in vitro model system, yet Bright itself is not appreciably tyrosine phosphorylated. These data suggested that a third protein, and Btk substrate, must contribute to Bright-enhanced immunoglobulin transcription. The ubiquitously expressed transcription factor TFII-I was identified as a substrate for Btk several years ago. In this work, we show that TFII-I directly interacts with human Bright through amino acids in Bright's protein interaction domain and that specific tyrosine residues of TFII-I are essential for Bright-induced activity of an immunoglobulin reporter gene. Moreover, inhibition of TFII-I function in a B-cell line resulted in decreased heavy-chain transcript levels. These data suggest that Bright functions as a three-component protein complex in the immunoglobulin locus and tie together previous data indicating important roles for Btk and TFII-I in B lymphocytes.The transcription factor Bright (B-cell regulator of immunoglobulin H [IgH] transcription)/ARID3a/Dril1 is a B-cellspecific protein first discovered in a mature mouse B-cell line, BCg3R1-d, as a mobility-shifted protein complex that caused three-to sixfold increases in heavy-chain mRNA levels in response to stimulation with a T-dependent antigen and interleukin-5 (42, 43). Bright binds to AϩT-rich regions of the intronic heavy-chain enhancer previously identified as matrix association regions and to regions 5Ј of some variable heavychain (V H ) promoters, including the V1 S107 family gene (15,43). The cDNA for Bright was isolated in 1995, and the protein was shown to interact with DNA as a multimeric complex that included multiple copies of Bright (15). The Bright protein structure consists of an acidic N-terminal domain of unknown function, a DNA-binding AϩT-rich interaction domain (ARID), a putative transactivation domain, a protein interaction domain containing a helix-turn-helix region, and a small carboxyl-terminal domain with no assigned function.Earlier studies indicated that Bruton's tyrosine kinase (Btk), the defective enzyme in X-linked immunodeficiency disease in both mice and humans, is a component of the Bright DNAbinding complex (28, 44). X-linked immunodeficiency disease in mice, or X-linked agammaglobulinemia (XLA) in humans, results in blocks in B-lymphocyte development that ultimately lead to a deficient production of serum antibodies (9,33,40). Patients with XLA are unable to fight normal bacterial infections without frequent intravenous Ig treatments. Although Btk was identified as the genetic defect in XLA many years ago, the mechanism by which Btk deficiencies lead to early blocks at the pro-B-to pre-B-lymphocyte stages in humans remains unclear.Recently, an in vitro mode...

Anti-Nuclear Antibody Production and Autoimmunity in Transgenic Mice That Overexpress the Transcription Factor Bright

“…Several groups have demonstrated that B cells from mice deficient in Btk do not proliferate normally at the T1 immature stage and that T2 transitional stage xid B cells fail to respond to signals through the surface IgR (9,49). Our data, using an in vitro model system, suggested that Btk is critically required for Bright-mediated up-regulation of transcription of at least some H chains (21,22). We hypothesize that Bright and Btk function coordinately in transitional B cells to mediate further differentiation through this important tolerance checkpoint.…”

“…Sera from nonimmunized Bright-transgenic mice contained slightly increased levels of total Ig compared with nontransgenic littermates. Expression of the V1 gene, previously shown to be regulated by Bright in vitro (21,22), was also enhanced in transgenic spleen cells relative to littermate controls. Because the V1 gene is used predominantly in the antiself response against phosphorylcholine (PC) (25)(26)(27), Ag-specific responses reactive with this hapten were also examined.…”

“…Several Bright-binding sites were identified 5Ј of the basal promoter of the V1 S107 family gene, and these sites were required for IL-5 and Ag-induced H chain transcription by Bright in vitro (16,20). More recent data indicate that Bright-enhanced transcription of a V1 reporter construct critically requires both the kinase activity of Bruton's tyrosine kinase (Btk) and the Btk substrate, TFII-I (21,22). These data suggest that Bright and Btk share common pathways in some cases, but may also have independent functions.…”

“…Abs purchased from BD Biosciences were: FITC-conjugated CD19 (1D3), CD21 (7G6), and CD4 (RM4-4); PE-conjugated CD8 (53-6.7), CD3 (145-2C11), CD43 (57), CD40 (1C10), CD69 (Hi.2F3), CD80 (1G10), CD86 (GL1), and CD23 (B3B4); allophycocyanin-conjugated CD45R/B220 (RA3-6B2), CD93/C1qRp (AA4.1); and PerCP-conjugated CD45R/B220(RA3-6B2). FITC-IgM, PE-IgD (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), goat anti-mouse IgM-and 1-A9, MHC class II (KH116)-biotin, appropriate isotype controls and streptavidin conjugated-allophycocyanin were from BD Biosciences or Southern Biotechnology Associates. Cells were stained as previously described (23) and fixed in 0.2% paraformaldehyde overnight.…”

“…cDNA was generated in reactions containing RNA from ϳ7.5 ϫ 10 3 cells as previously described (32). Levels of S107 family V H -specific IgM and GAPDH mRNA were assessed as described (22). Samples were electrophoresed through 1.0% agarose gels, transferred to GeneScreen Plus hybridization membrane (PerkinElmer Life Science Products), and hybridized with a [ 32 P]V1 cDNA probe.…”