SUMMARY The bromodomain protein 4 (BRD4) is an atypical kinase and histone acetyl transferase (HAT) that binds to acetylated histones and contributes to chromatin remodeling and early transcriptional elongation. During transcription, BRD4 travels with the elongation complex. Since most alternative splicing events take place co-transcriptionally, we asked if BRD4 plays a role in regulating alternative splicing. We report that distinct patterns of alternative splicing are associated with a conditional deletion of BRD4 during thymocyte differentiation in vivo. Similarly, the depletion of BRD4 in T cell acute lymphoblastic leukemia (T-ALL) cells alters patterns of splicing. Most alternatively spliced events affected by BRD4 are exon skipping. Importantly, BRD4 interacts with components of the splicing machinery, as assessed by both immunoprecipitation (IP) and proximity ligation assays (PLAs), and co-localizes on chromatin with the splicing regulator, FUS. We propose that BRD4 contributes to patterns of alternative splicing through its interaction with the splicing machinery during transcription elongation.
Escherichia coli K-12 contains nine paralogs of CspA, namely CspA-CspI. In spite of considerable sequence similarity among these genes, the individual members of this family show significant differences in their expression regulation. Among these nine members, cspA, B, G and I have been reported to be cold-induced. The unusually long 5'-untranslated region (5'-UTR) of these four and other cold-induced genes has often been associated with their inducibility. Sequence analysis of the cspE upstream region revealed two promoter-like motifs having high scores. We identified the promoter site and established that cspE has a much shorter 5'-UTR compared to other cold-induced genes. Our results showed that cspE is induced to about threefold at both the transcript and the protein level in response to cold-shock. Its transcript half-life increases significantly upon cold-shock. Furthermore, we demonstrated that RNase E, a key endonuclease responsible for mRNA degradation in E. coli, regulates cspE transcript stability, possibly through the assembly of a degradosome. In silico analysis of the cspE 5'-UTR revealed alternative secondary structures at 37 and 15 degrees C. A point mutation that was predicted to relax the secondary structure of the 5'-UTR at 15 degrees C showed considerable reduction in transcript stability, indicating that alternative transcript secondary structures might be the cause of the differential stability.
cspD, a member of cspA family of cold shock genes in Escherichia coli, is not induced during cold shock. Its expression is induced during stationary phase. CspD inhibits DNA replication, and a high level of the protein is toxic to cells. Recently, CspD was proposed to be associated with persister cell formation in E. coli. Here, we show that cyclic AMP receptor protein (CRP) upregulates cspD transcription. Sequence analysis of the cspD upstream region revealed two tandem CRP target sites, CRP site-I (the proximal site centered at ؊83.5 with respect to the transcription start) and CRP site-II (the distal site centered at ؊112.5). The results from electrophoretic mobility shift assays showed that CRP indeed binds to these two target sites in PcspD. The promoter-proximal CRP target site was found to play a major role in PcspD activation by CRP, as studied by transcriptional fusions carrying mutations in the target sites. The results from in vitro transcription assays demonstrated that CRP activates PcspD transcription in the absence of additional factors other than RNA polymerase. The requirement for activating region 1 of CRP in PcspD activation, along with the involvement of the 287, 265, and 261 determinants of the ␣-CTD, suggest that CRP activates by a class I-type mechanism. However, only moderate activation in vitro was observed compared to high activation in vivo, suggesting there might be additional activators of PcspD. Overall, our findings show that CRP, a global metabolic regulator in E. coli, activates a gene potentially related to persistence. E scherichia coli K-12 contains nine paralogs of CspA, CspACspI, collectively known as the CspA family of cold shock proteins (CSPs). Among the nine members, cspA, cspB, cspE, cspG, and cspI are induced in response to a temperature downshift (1, 2). cspD, however, is not induced during cold shock. It is found to be induced during stationary-phase, glucose starvation (3) and oxidative stress (4). The CspD protein forms a homodimer, localizes to the nucleoid in stationary-phase cells (5), and inhibits DNA replication presumably by nonspecific binding to the opened, single-stranded regions at replication forks (6). cspD-null mutants are viable, but the overproduction is shown to be toxic (6, 7). Recently, Kim et al. (4,8) have shown that CspD toxin is associated with biofilms and persister cell formation. Bacterial persisters are the small number of slow-growing antibiotic tolerant cells among populations of rapidly growing cells which arise in biofilms and in stationary-phase cultures (9).The induction of CspD during stationary phase is independent of S , the stationary-phase sigma factor (3). Cyclic AMP (cAMP) receptor protein (CRP), also known as catabolite activator protein (CAP) is one of the global regulatory factors involved in stationary-phase induction of large groups of genes in E. coli (reviewed in reference 10). CRP modulates transcription initiation by RNA polymerase (RNAP) by binding to a 22-base consensus target DNA sequence contained in the gene prom...
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