Regulation of clustered gene expression by cofactor of BRCA1 (COBRA1) in breast cancer cells.

Eucaryotic genes that are coordinately expressed tend to be clustered. Furthermore, gene clusters across chromosomal regions are often upregulated in various tumors. However, relatively little is known about how gene clusters are coordinately expressed in physiological or pathological conditions. Cofactor of BRCA1 (COBRA1), a subunit of the human negative elongation factor, has been shown to repress estrogen-stimulated transcription of trefoil factor 1 (TFF1 or pS2) by stalling RNA polymerase II. Here, we carried out a genome-wide study to identify additional physiological target genes of COBRA1 in breast cancer cells. The study identified a total of 134 genes that were either activated or repressed upon small hairpin RNA-mediated reduction of COBRA1. Interestingly, many COBRA1-regulated genes reside as clusters on the chromosomes and have been previously implicated in cancer development. Detailed examination of two such clusters on chromosome 21 (21q22) and chromosome X (Xp11) reveals that COBRA1 is physically associated with a subset of its regulated genes in each cluster. In addition, COBRA1 was shown to regulate both estrogen-dependent and -independent transcription of the gene cluster at 21q22, which encompasses the previously identified COBRA1-regulated TFF1 (pS2) locus. Thus, COBRA1 plays a critical role in the regulation of clustered gene expression at preferred chromosomal domains in breast cancer cells.

Contributions of UP elements and the transcription factor FIS to expression from the seven rrn P1 promoters in Escherichia coli.

The high activity of the rrnB P1 promoter in Escherichia coli results from a cis-acting DNA sequence, the UP element, and a trans-acting transcription factor, FIS. In this study, we examine the effects of FIS and the UP element at the other six rrn P1 promoters. We find that UP elements are present at all of the rrn P1 promoters, but they make different relative contributions to promoter activity. Similarly, FIS binds upstream of, and activates, all seven rrn P1 promoters but to different extents. The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each rrn P1 promoter. Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most rrn P1 promoters compared to rrnB P1. Our studies indicate that the overall activities of the seven rrn P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter. These studies have implications for the control of gene expression of unlinked multigene families.

Aromatic amino acids in region 2.3 of Escherichia coli sigma 70 participate collectively in the formation of an RNA polymerase-promoter open complex.

Formation of an initiation-competent RNA polymerase-promoter complex involves DNA melting over a region of about 12 base-pairs, which includes the start site of transcription, thus enabling the template strand to base-pair with the initiating nucleoside triphosphates. By studying the effects of alanine substitutions, we have investigated the role of the aromatic amino residues in the Escherichia coli sigma(70) conserved region 2.3 in promoter strand separation. The resulting mutants were assessed for their activity in vivo in the context of a sigma(70)/sigma(32) hybrid sigma factor that could be targeted to a specific hybrid promoter in the cell. All substitutions lead to an at least twofold reduction in expression of the hybrid promoter-driven reporter gene. The in vitro assay of single substitutions indicated cold sensitivity similar to that previously observed with analogous substitutions in Bacillus subtilis sigma(A). Kinetic assays showed that these substitutions slowed the rate of open complex formation at 37 degrees C as well. RNA polymerase reconstituted with a sigma(70) containing multiple alanine substitutions readily binds to promoter DNA, but then proceeds slowly beyond the first intermediate complex on the pathway to formation of the transcription-competent complex. These data demonstrate that together the aromatic residues in region 2.3 of E. coli sigma(70) ensure that DNA strand separation proceeds efficiently, even if no individual residue may be essential for accomplishment of the process.

Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase alpha subunit.

Upstream A-tracts stimulate transcription from a variety of bacterial promoters, and this has been widely attributed to direct effects of the intrinsic curvature of A-tract-containing DNA. In this work we report experiments that suggest a different mechanism for the effects of upstream A-tracts on transcription. The similarity of A-tract-containing sequences to the adenine- and thymine-rich upstream recognition elements (UP elements) found in some bacterial promoters suggested that A-tracts might increase promoter activity by interacting with the alpha subunit of RNA polymerase (RNAP). We found that an A-tract-containing sequence placed upstream of the Escherichia coli lac or rrnB P1 promoters stimulated transcription both in vivo and in vitro, and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP alpha subunit. The A-tract sequence was protected by wild-type RNAP but not by alpha-mutant RNAPs in footprints. The effect of the A-tracts on transcription was not as great as that of the most active UP elements, consistent with the degree of similarity of the A-tract sequence to the UP element consensus. A-tracts functioned best when positioned close to the -35 hexamer rather than one helical turn farther upstream, similar to the positioning optimal for UP element function. We conclude that A-tracts function as UP elements, stimulating transcription by providing binding site(s) for the RNAP alphaCTD, and we suggest that these interactions could contribute to the previously described wrapping of promoter DNA around RNAP.

Escherichia coli promoters with UP elements of different strengths: modular structure of bacterial promoters.

The alpha subunit of Escherichia coli RNA polymerase (RNAP) participates in promoter recognition through specific interactions with UP element DNA, a region upstream of the recognition hexamers for the sigma subunit (the -10 and -35 hexamers). UP elements have been described in only a small number of promoters, including the rRNA promoter rrnB P1, where the sequence has a very large (30- to 70-fold) effect on promoter activity. Here, we analyzed the effects of upstream sequences from several additional E. coli promoters (rrnD P1, rrnB P2, lambda pR, lac, merT, and RNA II). The relative effects of different upstream sequences were compared in the context of their own core promoters or as hybrids to the lac core promoter. Different upstream sequences had different effects, increasing transcription from 1.5- to approximately 90-fold, and several had the properties of UP elements: they increased transcription in vitro in the absence of accessory protein factors, and transcription stimulation required the C-terminal domain of the RNAP alpha subunit. The effects of the upstream sequences correlated generally with their degree of similarity to an UP element consensus sequence derived previously. Protection of upstream sequences by RNAP in footprinting experiments occurred in all cases and was thus not a reliable indicator of UP element strength. These data support a modular view of bacterial promoters in which activity reflects the composite effects of RNAP interactions with appropriately spaced recognition elements (-10, -35, and UP elements), each of which contributes to activity depending on its similarity to the consensus.

Mutations in sigma factor that affect the temperature dependence of transcription from a promoter, but not from a mismatch bubble in double-stranded DNA.

Specificity of promoter utilization in bacterial RNA polymerases is imparted by a class of proteins referred to as sigma factors. Conserved region 2.3 of these proteins is thought to play a role in the strand separation process that occurs during the formation of an initiation-competent RNA polymerase-promoter complex. We have used a heterologous system consisting of Escherichia coli core RNA polymerase and Bacillus subtilis sigma A to probe the effects of amino acid substitutions in region 2.3. In agreement with previous work [Juang & Helmann (1994) J. Mol. Biol. 235, 1470-1488] we observe that several amino acid substitutions exacerbate the deleterious effect of low temperature on promoter-dependent initiation. On the other hand, no such enhanced cold sensitivity is found with double-stranded templates that contain short "bubbles" of single-stranded DNA, indicating that the DNA-melting defect imposed by these mutant sigma factors can be suppressed by the use of such bubble templates. These results support the involvement of region 2.3 in the strand separation process that accompanies open complex formation at promoters.

A mismatch bubble in double-stranded DNA suffices to direct precise transcription initiation by Escherichia coli RNA polymerase.

Formation of a transcription-competent "open" complex between Escherichia coli RNA polymerase and a promoter, where base pairing is disrupted over a region of 12 base pairs including the start site of transcription, is a complex process involving at least three steps: recognition of specific DNA sequences, a conformational change in RNA polymerase, and DNA melting. By using synthetic constructs devoid of promoter-specific sequences, we show here that a mismatch bubble of 12 base pairs suffices to direct transcription initiation in divergent directions from its edges, reflecting the absence of polarity determinants for RNA polymerase binding. Bubble transcription is obtained with both core polymerase and holoenzyme, but efficient formation of heparin-resistant initiation complexes requires the sigma (specificity) factor. Based on these results it is likely that the sigma factor blocks access of the heparin to a site on the holoenzyme.