Silencer elements controlling the B29 (Igbeta) promoter are neither promoter- nor cell-type-specific

Abstract

The murine B29 (Igbeta) promoter is B cell specific and contains essential SP1, ETS, OCT, and Ikaros motifs. Flanking 5' DNA sequences inhibit B29 promoter activity, suggesting this region contains silencer elements. Two adjacent 5' DNA segments repress transcription by the murine B29 promoter in a position- and orientation-independent manner, analogous to known silencers. Both these 5' segments also inhibit transcription by several heterologous promoters in B cells, including mb-1, c-fos, and human B29. These 5' segments also inhibit transcription by the c-fos promoter in T cells suggesting they are not B cell-specific elements. DNase I footprint analyses show an approximately 70-bp protected region overlapping the boundary between the two negative regulatory DNA segments and corresponding to binding sites for at least two different DNA-binding proteins. Within this footprint, two unrelated 30-bp cis-acting DNA motifs (designated TOAD and FROG) function as position- and orientation-independent silencers when located directly 5' of the murine B29 promoter. These two silencer motifs act cooperatively to restrict the transcriptional activity of the B29 promoter. Neither of these motifs resembles any known silencers. Mutagenesis of the TOAD and FROG motifs in their respective 5' DNA segments eliminates the silencing activity of these upstream regions, indicating these two motifs as the principal B29 silencer elements within these regions.

Figure 1

B29 minimal promoter activity is modulated by 5′ flanking sequences. Transient transfections to detect transcriptional expression of sequential B29 promoter deletion constructs were carried out in the M12 B cell line. Deletion constructs are identified by nucleotide numbers with respect to the major start site of transcription (+1). The activity of each deletion construct is expressed as a percentage of the CAT activity obtained with the B29 minimal (−164) promoter construct (100%).

Figure 2

B29 gene 5′ regions act as position- and orientation-independent silencers. Transient transfections to detect transcriptional expression of B29 promoter/silencer region constructs were carried out in the M12 B cell line. The silencer fragments and their position and orientation relative to the B29 minimal (−164) promoter are as indicated. The activity of each construct is expressed as a percentage of the CAT activity obtained with the B29 minimal (−164) promoter construct (100%). Deletion constructs −164, −354, and −565 are identified by nucleotide numbers with respect to the major start site of transcription (+1) and are shown for comparison.

Figure 3

B29 5′ silencer elements interact with DNA binding proteins. (A) The endpoint of the DNase I footprints are identified by nucleotide numbers with respect to the start site of transcription (+1). Lanes containing reactions incubated with either 10 μg or 20 μg M12 B cell crude nuclear extract are indicated (lanes 3 and 4). Lanes containing reactions incubated without extract are indicated (lanes 2 and 5). Reactions were electrophoresed alongside a G+A ladder of the probe (lanes 1 and 6). (B) Double-stranded oligonucleotides corresponding to the sequence from positions −349 to −321 (TOAD motif, lanes 1–4) and −381 to −356 (FROG motif, lanes 5–8) were end-labeled. Lanes 1 and 5 contain TOAD and FROG motif probes alone, respectively. The TOAD motif probe was incubated with 5 μg of M12 B cell nuclear extract (lanes 2–4) and the FROG motif probe was incubated with 5 μg of M12 B cell nuclear extract (lanes 6–8). Probes were incubated in the presence of a 1,000-fold excess of unlabeled TOAD motif competitor (lanes 3 and 8) or were incubated in the presence of a 1,000-fold excess of unlabeled FROG motif competitor (lanes 4 and 7). The specifically formed TOAD and FROG complexes are indicated as such with arrows.

Figure 4

Silencing activity in the B29 5′ gene regions corresponds to TOAD and FROG DNA binding motifs. Transient transfections to detect transcriptional expression of B29 promoter/silencer motif constructs were carried out in the M12 B cell line. The TOAD and FROG silencer motifs, positions −349 to −321 and −381 to −356, respectively, and their positions and orientations relative to the B29 minimal (−164) promoter are as indicated. The activity of each construct is expressed as a percentage of the CAT activity obtained with the B29 minimal (−164) promoter construct (100%). Deletion constructs −164, −354, and −565 are identified by nucleotide numbers with respect to the major start site of transcription (+1) and are shown for comparison.

Figure 5

Site-directed mutagenesis of the TOAD and FROG motifs disrupts silencing activity. Transient transfections to detect transcriptional expression of deletion constructs −354 and −565 with introduced point mutations in the TOAD and FROG motifs, respectively, were carried out in the M12 B cell line. Mutagenized nucleotides are indicated by underlined lowercase type. The activity of each construct is expressed as a percentage of the CAT activity obtained with the B29 minimal (−164) promoter construct (100%). Deletion constructs −164, −354, and −565 are identified by nucleotide numbers with respect to the major start site of transcription (+1) and are shown for comparison.