SUMMARY Bromodomain-containing protein 4 (Brd4) is an epigenetic reader and transcriptional regulator recently identified as a cancer therapeutic target for acute myeloid leukemia, multiple myeloma, and Burkitt's lymphoma. Although chromatin targeting is a crucial function of Brd4, there is little understanding of how bromodomains that bind acetylated histones are regulated, nor how the gene-specific activity of Brd4 is determined. Via interaction screen and domain mapping, we identified p53 as a functional partner of Brd4. Interestingly, Brd4 association with p53 is modulated by casein kinase II (CK2)-mediated phosphorylation of a conserved acidic region in Brd4 that selectively contacts either a juxtaposed bromodomain or an adjacent basic region to dictate the ability of Brd4 binding to chromatin and also the recruitment of p53 to regulated promoters. The unmasking of bromodomains and activator recruitment, concurrently triggered by the CK2 phospho switch, provide an intriguing mechanism for gene-specific targeting by a universal epigenetic reader.

Abstract-Arsenic is atherogenic, carcinogenic, and genotoxic. Because atherosclerotic plaque has been considered a benign smooth muscle cell tumor, we have studied the effects of arsenite on DNA integrity of human vascular smooth muscle cells. By using single-cell alkaline electrophoresis, apparent DNA strand breaks were detected in a 4-hour treatment with arsenite at a concentration above 1 mol/L. DNA strand breaks of arsenite-treated cells were increased by Escherichia coli formamidopyrimidine-DNA glycosylase and decreased by diphenylene iodinium, superoxide dismutase, catalase, pyruvate, DMSO, or D-mannitol. Extract from arsenite-treated cells showed increased capacity for producing superoxide when NADH was included in the reaction mixture; however, addition of arsenite to extract from untreated cells did not increase superoxide production. The superoxide-producing ability of arsenite-treated cells was also suppressed by diphenylene iodinium, 4,5-dihydroxy-1,2-benzenedisulfonic acid disodium salt (Tiron), or superoxide dismutase. Superoxide production and DNA strand breaks in arsenite-treated cells were also suppressed by transfecting antisense oligonucleotides of p22phox, an essential component of NADH oxidase. Treatment with arsenite also increased the mRNA level of p22phox. These results suggest that arsenite activates NADH oxidase to produce superoxide, which then causes oxidative DNA damage. The result that arsenite at low concentrations increases oxidant levels and causes oxidative DNA damage in vascular smooth muscle cells may be important in arsenic-induced atherosclerosis. (Circ Res. 2000;86:514-519.)Key Words: arsenite Ⅲ oxidative DNA damage Ⅲ NADH oxidase Ⅲ atherosclerosis A rsenic, an element present in the earth's crust, comes to the surface through mining and utilization of groundwater. Serious contamination by inorganic arsenic occurs through long-term ingestion of high concentrations of arsenic in drinking water. Chronic exposure to arsenic has been related to increased incidences of skin, lung, bladder, liver, and kidney cancers. 1,2 Arsenic exposure is also associated with various vascular disorders, including angiosarcomas, 3 atherosclerotic plaques, 4 and hypertension in humans. 5 The epidemiological evidence for shared risk factors for cancer and atherosclerosis has been reviewed by Hansen. 6 Exposure to carcinogenic environmental agents is associated with an increased risk of atherosclerosis. Therefore, somatic mutation and cell proliferation may play a role in the pathogenesis of atherosclerotic plaques. The predominant cell type in plaques is the vascular smooth muscle cell (VSMC). Proliferation of VSMCs is essential for plaque formation and development. By examining the isoenzymes of glucose-6-phosphate dehydrogenase, human atherosclerotic plaques were shown to be monoclonal in origin. 7 Furthermore, DNA samples from human coronary artery plaques were demonstrated to transform NIH3T3 cells. 8 These observations suggest that atherosclerotic plaques are presumably benign smooth muscle...

Bromodomain-containing protein 4 (BRD4) is an emerging epigenetic drug target for intractable inflammatory disorders. The lack of highly selective inhibitors among BRD4 family members has stalled the collective understanding of this critical system and the progress toward clinical development of effective therapeutics. Here we report the discovery of a potent BRD4 bromodomain 1 (BD1)-selective inhibitor ZL0590 (52) targeting a unique, previously unreported binding site, while exhibiting significant anti-inflammatory activities in vitro and in vivo. The X-ray crystal structural analysis of ZL0590 in complex with human BRD4 BD1 and the associated mutagenesis study illustrate a first-in-class nonacetylated lysine (KAc) binding site located at the helix αB and αC interface that contains important BRD4 residues (e.g., Glu151) not commonly shared among other family members and is spatially distinct from the classic KAc recognition pocket. This new finding facilitates further elucidation of the complex biology underpinning bromodomain specificity among BRD4 and its protein–protein interaction partners.

The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is essential for transcription of genes coding for the snRNAs (U1, U2, etc.). In Drosophila melanogaster, the heterotrimeric DmSNAPc recognizes a 21-bp DNA sequence, the proximal sequence element A (PSEA), located approximately 40 to 60 bp upstream of the transcription start site. Upon binding the PSEA, DmSNAPc establishes RNA polymerase II preinitiation complexes on U1 to U5 promoters but RNA polymerase III preinitiation complexes on U6 promoters. Minor differences in nucleotide sequence of the U1 and U6 PSEAs determine RNA polymerase specificity; moreover, DmSNAPc adopts different conformations on these different PSEAs. We have proposed that such conformational differences in DmSNAPc play a key role in determining the different polymerase specificities of the U1 and U6 promoters. To better understand the structure of DmSNAPc-PSEA complexes, we have developed a novel protocol that combines site-specific protein-DNA photo-cross-linking with site-specific chemical cleavage of the protein. This protocol has allowed us to map regions within each of the three DmSNAPc subunits that contact specific nucleotide positions within the U1 and U6 PSEAs. These data help to establish the orientation of each DmSNAPc subunit on the DNA and have revealed cases in which different domains of the subunits differentially contact the U1 versus U6 PSEAs.The Drosophila melanogaster small nuclear RNA (snRNA)-activating protein complex (DmSNAPc) is a heterotrimeric transcription factor (21) that is required for the synthesis of the U1, U2, U4, U5, and U6 spliceosomal snRNAs (2, 22, 31). Homologous protein complexes are required for snRNA gene expression in humans (1,11,12,18,(33)(34)(35) and for spliced leader RNA synthesis in trypanosomes (6,7,30). This indicates that SNAPc appeared early in eukaryotic evolution and continues in contemporary times to be utilized for the transcription of important noncoding RNA molecules in diverse organisms. DmSNAPc binds sequence-specifically to an essential, conserved ϳ21-bp promoter element termed the proximal sequence element A (PSEA) that is located approximately 40 to 60 bp upstream of the transcription start site of fly snRNA genes (8,13,19,23).In animals, the U1 to U5 snRNA genes are transcribed by RNA polymerase II (Pol II), but U6 snRNA genes are transcribed by RNA Pol III (4,5,10,14,19,24,29,31). Surprisingly, the primary determinant of the RNA polymerase specificity of the D. melanogaster snRNA genes is the precise sequence of the PSEA. A few conserved nucleotide differences in the 3Ј half of the 21-bp PSEA are sufficient to determine the polymerase specificity of the fly snRNA genes in vitro and to restrict the polymerase specificity in vivo (2,19,20,26).The three subunits of DmSNAPc are termed DmSNAP190, DmSNAP50, and DmSNAP43 so that the names correspond to the most widely used nomenclature for the three orthologous human SNAPc subunits, SNAP190, SNAP50, and SNAP43. These three human subunits are also known as PTF␣, PTF␤, an...

this paper, we have become aware of a mistake in the identity of the photo-cross-linking probe used to map the domain of DmSNAP190 that cross-linked to phosphate position 24 of the U1 proximal sequence element A (PSEA). The probe used to generate the data presented in Fig. 2 (lanes 1 to 3) and 3C (lanes 13 to 18) was in fact not a position 24 probe; instead, a U1 PSEA phosphate position 12 probe was used in error. Reperformance of these experiments with the correct probe revealed that phosphate position 24 of the U1 PSEA cross-linked to a region of DmSNAP190 located between amino acid residues 169 and 247 rather than between residues 306 and 358. None of the other conclusions of the publication were affected. We sincerely regret this error and any inconvenience that may have resulted to our colleagues.Page 2414, Fig. 2, lanes 1-3: Although this panel was generated with the misidentified probe, the results of the experiment were the same when performed with the correct probe for U1 PSEA position 24 (data not shown). The conclusion that phosphate position 24 cross-linked to the N-terminal half of DmSNAP190 was not affected.Page 2416: Figure 3C, lanes 13-18, should appear as shown below. These results indicate that position 24 of the U1 PSEA cross-linked to DmSNAP190 C terminal of amino acid residue 169 but N terminal of residue 247 inclusive.