Isolation and characterization of BEN, a member of the TFII-I family of DNA-binding proteins containing distinct helix–loop–helix domains

The restriction of immunoglobulin variable region promoter activity to B lymphocytes is a well known paradigm of promoter specificity. Recently, a cis-element, located downstream of the transcription initiation site of murine heavy chain variable promoters, was shown to be critical for B cell activity and specificity. Here we show that mutation of this element, termed DICE (Downstream Immunoglobulin Control Element), reduces in vivo activity in B cells. Gel mobility shift assays show that DICE forms B cell-specific complexes that were also sensitive to DICE mutation. DICE mutation strongly reduces the ability of a distal immunoglobulin heavy chain intronic enhancer to stimulate transcription. We also identify a DICE-interacting factor: a TFII-I-related protein known as BEN (also termed Mus-TRD1 and WBSCR11). Dominant-negative and RNA i -mediated knockdown experiments indicate that BEN can both positively and negatively regulate IgH promoter activity, depending on the cell line.During development, an organism must exclusively express some genes in particular tissues or cell types. A number of genes and their promoters have been studied in detail in order to obtain general principles from their mechanism of regulation. Among the most intensely studied are the immunoglobulin variable (V) 1 region promoters. Ig heavy chain (IgH) and light chain (Ig ) genes are selectively expressed in B lymphocytes, and their respective V region (V H and V ) promoters are normally inactive in all other tissues (reviewed in Ref. 1). The IgH and Ig loci are sequentially activated during B lymphocyte maturation. Activation includes three principal events. Transcription initiates at various positions within each locus, including the V H and V promoters (2, 3). Recombination of a V region gene segment with a joining (J) gene segment, in the case of the chain, and a diversity (D) and J segment, in the case of the heavy chain, forms an Ig variable region exon of a given specificity (4). Hyperacetylation of chromatin within the Ig loci also makes it less compact and more accessible to transacting factors (5, 6). The relative timing of these three events is unclear; however, it has been shown that non-coding (germ line) transcription precedes recombination for some V H segments (3, 7). The direct relationship between these events is also unclear; however, in the heavy chain locus, V-DJ recombination brings a downstream intronic enhancer into proximity with the V H promoter. The relocation of the enhancer greatly stimulates expression of the recombined IgH (reviewed in Ref. 8). These multiple interdependent layers of regulation serve to tightly control Ig expression and prevent activation outside of the normal B cell maturation pathway.Studies from a number of laboratories have determined that in isolation, V H and V promoters are preferentially active in B lymphocytes (9, 10). A feature of most V H and V promoters is the octamer motif (5Ј-ATGCAAAT-3Ј), located 10 -25 nucleotides upstream of the TATA box (and inverted in the case of V ) (9,(1...

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Regulation of Immunoglobulin Promoter Activity by TFII-I Class Transcription Factors

Tantin

Tussie-Luna

Roy

et al. 2004

“…To corroborate the results from the SELEX analysis of R4, we tested the interactions between wild-type and mutated versions of oligonucleotides derived from the TnI SURE (844/823) and from the EGF site of the HOXc8 enhancer that was demonstrated previously to bind GTF3/BEN (12). As shown in Fig.…”

Section: Delineation Of the Consensus Binding Sequence For Gtf3-r4 -Gmentioning

confidence: 97%

“…Together, these data suggest that GTF3 is involved in the down-regulation of the TnI slow gene in developing fast muscles. Like GTF3, BEN was isolated in a yeast onehybrid screen as a factor that binds to the EFG site of the Hoxc8 enhancer (12).…”

mentioning

confidence: 99%

Multiple GTF2I-like Repeats of General Transcription Factor 3 Exhibit DNA Binding Properties

Vullhorst

Buonanno

2005

A hallmark of general transcription factor 3 (GTF3) is the presence of multiple GTF2I-like repeats that were suggested to mediate protein-protein interactions. However, we have recently demonstrated that repeat 4 is necessary and sufficient for binding of GTF3 to the bicoid-like motif of the Troponin I slow enhancer. Given the sequence similarity between different GTF2I-like repeats we hypothesized that DNA binding might be a common property of this domain type. We subjected five repeats of GTF3 to random oligonucleotide selection (SELEX) to assess their DNA binding potentials. We delineated the consensus sequence G TC G A GATTA G BG A for repeat 4 and showed that binding sites for GTF3 in enhancers for Troponin I and homeobox c8 (HOXc8) are in very good agreement with this motif. SELEX selections for repeats 5 and 2 enriched for oligonucleotides that were also bound by R4, suggesting that they share common sequence preferences, whereas repeat 3 exhibited relaxed sequence requirements for DNA binding. No binding was observed for repeat 1. We also show that GTF2I-like repeats 4 and 6 of transcription factor II-I (TFII-I) exhibit modest DNA binding properties. Lastly, we identified several amino acids of GTF3 repeat 4 required for high affinity protein-DNA interaction. Based on the ability of many repeats to bind DNA in vitro, we suggest that GTF2I-like domains evolved by duplication and diversification of a prototypic DNA-binding ancestor.

“…The N-terminal end of the protein contains the leucine zipper motif (4) that is also present in all the isoforms of human and mouse TFII-I (8,19,20). Moreover, several TFII-I-related proteins contain this motif approximately in the same position (21)(22)(23)(24)(25)(26). 2 Based on our observations, we propose that the ⌬-isoform remains in a "closed" conformation until the LZ motif becomes available to interact with other partners: either another molecule of ⌬ or another molecule of ␤ (Fig.…”

Section: Discussionmentioning

confidence: 99%

Structure-Function Analysis of TFII-I

Cheriyath¹,

Roy

2001

The transcription factor TFII-I can bind specifically to several DNA sequence elements and is implicated in both basal and activated transcription. There are four alternatively spliced isoforms of TFII-I, all characterized by the presence of six I-repeats, R1-R6, each containing a potential helix-loop-helix motif implicated in protein-protein interactions. These isoforms exhibit both homomeric and heteromeric interactions that lead to nuclear localization. In this study we mapped two distinct regions in TFII-I that affect its DNA binding. Deletion of either of these regions led to abrogation of DNA binding and transcriptional activation from both the V␤ and c-fos promoters. The I-repeats, as expected, were capable of mediating homomeric interactions either individually or in combination. Unexpectedly, an additional homomeric interaction domain was found within the N-terminal end of TFII-I that includes a putative leucine zipper motif. These data suggest a model in which TFII-I undergoes regulated homomeric interaction mediated by both the N-terminal end and the I-repeats.