Showing papers on "Bromodomain published in 2002"
TL;DR: These new findings demonstrate that bromodomain/acetyl‐lysine recognition can serve as a pivotal mechanism for regulating protein–protein interactions in numerous cellular processes including chromatin remodeling and transcriptional activation and reinforce the concept that functional diversity of a conserved protein modular structure is achieved by evolutionary changes of amino acid sequences in the ligand binding site.
770 citations
TL;DR: The substantial diversification of HATs and HDACs that has occurred since the divergence of plants, animals and fungi suggests a surprising degree of evolutionary plasticity and functional diversification in these core chromatin components.
Abstract: Sequence similarity and profile searching tools were used to analyze the genome sequences of Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster for genes encoding three families of histone deacetylase (HDAC) proteins and three families of histone acetyltransferase (HAT) proteins. Plants, animals and fungi were found to have a single member of each of three subfamilies of the GNAT family of HATs, suggesting conservation of these functions. However, major differences were found with respect to sizes of gene families and multi-domain protein structures within other families of HATs and HDACs, indicating substantial evolutionary diversification. Phylogenetic analysis identified a new class of HDACs within the RPD3/HDA1 family that is represented only in plants and animals. A similar analysis of the plant-specific HD2 family of HDACs suggests a duplication event early in dicot evolution, followed by further diversification in the lineage leading to Arabidopsis. Of three major classes of SIR2-type HDACs that are found in animals, fungi have representatives only in one class, whereas plants have representatives only in the other two. Plants possess five CREB-binding protein (CBP)-type HATs compared with one to two in animals and none in fungi. Domain and phylogenetic analyses of the CBP family proteins showed that this family has evolved three distinct types of CBPs in plants. The domain architecture of CBP and TAFII250 families of HATs show significant differences between plants and animals, most notably with respect to bromodomain occurrence and their number. Bromodomain-containing proteins in Arabidopsis differ strikingly from animal bromodomain proteins with respect to the numbers of bromodomains and the other types of domains that are present. The substantial diversification of HATs and HDACs that has occurred since the divergence of plants, animals and fungi suggests a surprising degree of evolutionary plasticity and functional diversification in these core chromatin components.
667 citations
TL;DR: It is shown that stable promoter occupancy by SWI/SNF and SAGA in the absence of transcription activators requires the bromodomains of the Swi2/Snf2 and Gcn5 subunits, respectively, and nucleosome acetylation.
568 citations
TL;DR: It is demonstrated that the acetylated residues in Smad7 also are targeted by ubiquitination and that acetylation of these lysine residues prevents subsequent ubiquitinations.
349 citations
TL;DR: These results provide the first genetic evidence for an in vivo role in mammals for a member of the Fsh/Brd family and suggest that Brd4 is likely to correspond to the Brd-like element of the mediator of transcriptional regulation isolated by Y. W. Jiang.
Abstract: In a gene trap screen we recovered a mouse mutant line in which an insertion generated a null allele of the Brd4 gene. Brd4 belongs to the Fsh/Brd family, a group of structurally related proteins characterized by the association of two bromodomains and one extraterminal domain. Members of this family include Brd2/Ring3/Fsrg1 in mammals, fs(1)h in Drosophila, and Bdf1 in Saccharomyces cerevisiae. Brd4 heterozygotes display pre- and postnatal growth defects associated with a reduced proliferation rate. These mice also exhibit a variety of anatomical abnormalities: head malformations, absence of subcutaneous fat, cataracts, and abnormal liver cells. In primary cell cultures, heterozygous cells also display reduced proliferation rates and moderate sensitivity to methyl methanesulfonate. Embryos nullizygous for Brd4 die shortly after implantation and are compromised in their ability to maintain an inner cell mass in vitro, suggesting a role in fundamental cellular processes. Finally, sequence comparisons suggest that Brd4 is likely to correspond to the Brd-like element of the mediator of transcriptional regulation isolated by Y. W. Jiang, P. Veschambre, H. Erdjument-Bromage, P. Tempst, J. W. Conaway, R. C. Conaway, and R. D. Kornberg (Proc. Natl. Acad. Sci. USA 95:8538-8543, 1998) and the Brd4 mutant phenotype is discussed in light of this result. Together, our results provide the first genetic evidence for an in vivo role in mammals for a member of the Fsh/Brd family.
298 citations
TL;DR: It is shown that the previously characterized transcriptional repressor complex NuRD (nucleosomeremodeling and deacetylase) binds to the histone H3 N-terminal tail and that methylation at lysine 4, but not lysin 9, prevents binding.
272 citations
TL;DR: Almo-terminal acetylation occurs on the bulk of eukaryotic proteins and on regulatory peptides, whereas lysine acetylations occurs at different positions on a variety of proteins, including histones, transcription factors, nuclear import factors, and α-tubulin.
Abstract: Acetylation of proteins, either on various amino-terminal residues or on the e-amino group of lysine residues, is catalyzed by a wide range of acetyltransferases. Amino-terminal acetylation occurs on the bulk of eukaryotic proteins and on regulatory peptides, whereas lysine acetylation occurs at different positions on a variety of proteins, including histones, transcription factors, nuclear import factors, and α-tubulin.
248 citations
TL;DR: The three-dimensional solution structure of the PCAF bromodomain in complex with a lysine 50-acetylated Tat peptide provides the structural basis for the specificity of this molecular recognition and reveals insights into the differences in ligand selectivity of bromidomains.
238 citations
TL;DR: The present study suggests that Brd4 regulates cell cycle progression in part by interacting with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication.
Abstract: Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G(1) upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G(1) to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.
161 citations
TL;DR: It is demonstrated that acetylation of Tat establishes a novel protein–protein interaction domain at the surface of Tat that is necessary for the transcriptional activation of the HIV promoter.
Abstract: The human immunodeficiency virus (HIV) Tat protein plays an essential role in promoting efficient transcriptional elongation of viral transcripts. We report that the transcriptional co-activator PCAF and Tat interact and synergize to activate the HIV promoter. The binding of Tat and PCAF in vitro and in vivo is dependent on the acetylated state of Lys50 of Tat and on the PCAF bromodomain. Structural analysis of the acetylated Tat peptide bound to the PCAF bromodomain defined amino acids Y47 and R53 in Tat and V763, Y802, and Y809 in PCAF as critical interaction points between the two proteins. Mutation of each of these residues in either Tat or PCAF inhibited in a cumulative manner the Tat-PCAF interaction in vitro and in vivo, and abrogated the synergistic activation of the HIV promoter by both proteins. These observations demonstrate that acetylation of Tat establishes a novel protein-protein interaction domain at the surface of Tat that is necessary for the transcriptional activation of the HIV promoter.
142 citations
TL;DR: This study establishes for the first time a transcriptional role for ProTα in recruitment or stabilization of coactivator p300, as well as other basal transcription factors, at the nucleosomes for regulation of transcription.
Abstract: The Epstein-Barr virus nuclear antigen 3C (EBNA3C), encoded by Epstein-Barr virus (EBV), is essential for mediating transformation of human B lymphocytes. Previous studies demonstrated that EBNA3C interacts with a small, nonhistone, highly acidic, high-mobility group-like nuclear protein prothymosin alpha (ProTα) and the transcriptional coactivator p300 in complexes from EBV-infected cells. These complexes were shown to be associated with histone acetyltransferase (HAT) activity in that they were able to acetylate crude histones in vitro. In this report we show that ProTα interacts with p300 similarly to p53 and other known oncoproteins at the CH1 amino-terminal domain as well as at a second domain downstream of the bromodomain which includes the CH3 region and HAT domain. Similarly, EBNA3C also interacts with p300 at regions which include the CH1 and CH3/HAT domains, suggesting that ProTα and EBNAC3C may interact in a complex with p300. We also show that ProTα activates transcription when targeted to promoters by fusion to the GAL4 DNA binding domain and that this activation is enhanced by the addition of an exogenous source of p300 under the control of a heterologous promoter. This overall activity is down-modulated in the presence of EBNA3C. These results further establish the interaction of cellular coactivator p300 with ProTα and demonstrate that the associated activities resulting from this interaction, which plays a role in acetylation of histones and coactivation, can be regulated by EBNA3C. Furthermore, this study establishes for the first time a transcriptional role for ProTα in recruitment or stabilization of coactivator p300, as well as other basal transcription factors, at the nucleosomes for regulation of transcription.
TL;DR: A genetic screen to isolate mutant alleles of the histones H3 and H4 genes that would “lock” telomeric marker genes into a silenced state discovered that each of these mutants had a dramatic reduction in the level of acetylation at lysine 12 within the histone H4 tail, which is proposed to serve as a “memory mark” for propagating the expression state of a telomersic gene.
Abstract: Telomeric position effect in Saccharomyces cerevisiae is a chromatin-mediated phenomenon in which telomere proximal genes are repressed (silenced) in a heritable, but reversible, fashion. Once a transcriptional state (active or silenced) is established, however, there is a strong tendency for that state to be propagated. Twenty-five years ago, H. Weintraub and colleagues suggested that such heritability could be mediated by posttranslational modification of chromatin [Weintraub, H., Flint, S. J., Leffak, I. M., Groudine, M. & Grainger, R. M. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 401–407]. To identify potential sites within the chromatin that might act as sources of “memory” for the heritable transmission, we performed a genetic screen to isolate mutant alleles of the histones H3 and H4 genes that would “lock” telomeric marker genes into a silenced state. We identified mutations in the NH2-terminal tail and core of both histones; most of the amino acid changes mapped adjacent to lysines that are known sites of acetylation or methylation. We developed a method using MS to quantify the level of acetylation at each lysine within the histone H4 NH2-terminal tail in these mutants. We discovered that each of these mutants had a dramatic reduction in the level of acetylation at lysine 12 within the histone H4 tail. We propose that this lysine serves as a “memory mark” for propagating the expression state of a telomeric gene: when it is unacetylated, silent chromatin will be inherited; when it is acetylated an active state will be inherited.
TL;DR: It is suggested that acetylation of lysine 92 negatively modulates IRF7 DNA binding and that over-expression of PCAF led to decreasedIRF7 activity.
TL;DR: A systematic mutational analysis of Acf1 was performed to elucidate the functions of specific subregions of the protein and revealed DNA- and ISWI-binding regions that are important for the chromatin assembly and ATPase activities of ACF.
Abstract: ACF is a chromatin-remodeling complex that catalyzes the ATP-dependent assembly of periodic nucleosome arrays. This reaction utilizes the energy of ATP hydrolysis by ISWI, the smaller of the two subunits of ACF. Acf1, the large subunit of ACF, is essential for the full activity of the complex. We performed a systematic mutational analysis of Acf1 to elucidate the functions of specific subregions of the protein. These studies revealed DNA- and ISWI-binding regions that are important for the chromatin assembly and ATPase activities of ACF. The DNA-binding region of Acf1 includes a WAC motif, which is necessary for the efficient binding of ACF complex to DNA. The interaction of Acf1 with ISWI requires a DDT domain, which has been found in a variety of transcription and chromatin-remodeling factors. Chromatin assembly by ACF is also impaired upon mutation of an acidic region in Acf1, which may interact with histones during the deposition process. Lastly, we observed modest chromatin assembly defects on mutation of other conserved sequence motifs. Thus, Acf1 facilitates chromatin assembly via an N-terminal DNA-binding region with a WAC motif, a central ISWI-binding segment with a DDT domain, and a C-terminal region with an acidic stretch, a WAKZ motif, PHD fingers, and bromodomain.
TL;DR: The observations suggested that Fsrg1 may be involved in the transcriptional activities of genes involved in proliferation of the mammary epithelia during pregnancy and in orchestrating postlactation involution and apoptosis.
Abstract: Fsrg1 (female sterile homeotic-related gene 1) is the mouse homolog of the human RING3 protein, which has been shown to associate with the E2 promoter binding factor (E2F) transcription factor and to have a possible role in cell cycle-linked transcriptional regulation. The Fsrg1 protein is 60% identical in sequence to the RNA polymerase II mediator subunit Fsrg4, another member of this subfamily of double bromodomain-containing proteins that are homologs of Drosophila female sterile homeotic. Antibodies against murine Fsrg1 were generated and used in immunoblot and immunoprecipitation experiments to identify proteins interacting with Fsrg1 and RING3. In the presence of acetylated but not nonacetylated histone H3 and H4 peptides, RING3 was shown to interact with E2F, mediator components cyclin-dependent kinase 8 and thyroid receptor-associated protein 220, and the RNA polymerase II large subunit. Fsrg1 mRNA had been previously shown to be expressed at high levels in the epithelium of the adult mouse mammar...
TL;DR: The identification and characterization of human CIA/hASF1 (identified previously as a histone chaperone) as an interactor of two tandem bromodomain modules of human (h)TAFII250/CCG1, the largest subunit of TFIID is shown.
Abstract: General transcription initiation factor IID (TFIID) plays a central and critical role in transcription initiation from both naked and chromatin templates. Although interaction between several DNA-binding proteins and TFIID were identified and well characterized, functional linkage between TFIID and chromatin factors has remained to be elucidated. Here we show the identification and characterization of human CIA/hASF1 (identified previously as a histone chaperone) as an interactor of two tandem bromodomain modules of human (h)TAFII250/CCG1, the largest subunit of TFIID. Although yeast (y)TAFII145, a homologue of hTAFII250/CCG1 in Saccharomyces cerevisiae, lacks bromodomains, glutathione S-transferase pull-down and immunoprecipitation assays revealed that Asf1p (antisilencing function 1), the counterpart of CIA in S. cerevisiae, interacts with Bdf1p (bromodomain factor 1), which is reported to serve as the missing bromodomain in yTAFII145. Furthermore, yeast strain lacking the BDF1 gene shows the Spt phenotype that is shown also by the ASF1 gene disruptant, and a double-knockout strain of both genes shows synthetic lethality, indicating that ASF1 genetically interacts with bromodomains associated with yTFIID. We also found that Asf1p coprecipitates with yTFIID subunits from yeast whole-cell extract, and overexpression of yTFIID subunits suppress the Spt phenotype caused by gene disruption of the ASF1. This study describes the functional linkage between TFIID and a histone chaperone.
TL;DR: A temperature‐sensitive mutant allele of NPS1, nps1‐13, which has amino acid substitutions within the bromodomain is isolated and this mutation perturbed the interaction between the RSC components and enhanced the sensitivity of the cells to several DNA‐damaging treatments at the permissive temperature.
TL;DR: It is proposed that the BrD plays a critical role in vivo by directing transregulators to their cognate location on nucleosomal DNA.
TL;DR: The cDNA cloning of the human polybromo-1 ( hPB1 ) gene that encodes a 1634-amino acid polypeptide containing six bromodomains shows 91% identity to the previously identified chicken polybrosa-1 with conservation of the bromidomains and other characteristic features.
Abstract: The bromodomain is suggested as a chromatin-targeting domain involved in regulation of gene expression and chromatin structure. We here report the cDNA cloning of the human polybromo-1 ( hPB1 ) gene that encodes a 1634-amino acid polypeptide containing six bromodomains. The hPB1 polypeptide shows 91% identity to the previously identified chicken polybromo-1 with conservation of the bromodomains and other characteristic features. Northern blot analysis detected the ubiquitous expression of hPB1 mRNA in a variety of human tissues. Four alternative splicings were found within the hPB1 coding region: three making in-frame deletions and one resulting in a C-terminal truncation. The hPB1 gene is located on chromosome 3p21, where the tumor suppressor genes for breast, lung and kidney cancers have been mapped.
TL;DR: It is shown that the bromodomain of PCAF acetylase specifically binds to acetylated Tat and leads to dissociation of Tat from TAR, a transactivation response RNA element in nascent HIV-1 transcripts.
Journal Article•
TL;DR: BRD-7 of bromodomain protein is similar to three proteins containing known structural bromidomain motif by bio-informatics analysis, suggesting the bromadomain of BRD- 7 should belong to co-activator subgroup and may have similar function that can selectively interact with acetylated histone peptide.
Abstract: BACKGROUND & OBJECTIVE BRD-7 is a novel gene (AF: 152604), containing a bromodomain, was cloned in our lab. Previous studies showed that BRD-7 plays an obviously suppressive role on NPC cell growth. In order to clarify the function mechanism of this gene, we investigate an important motif of BRD-7, the bromodomain. METHODS The bromodomain of BRD-7 was analyzed by homology-based amino sequence and secondary structure analysis. In addition, we constructed a prokaryotic expression vector of bromodomain. Western blot analysis was used to confirm the expression of the bromodomain protein in Escherichia coli. RESULTS Homology-based sequence analysis revealed that the bromodomain of BRD-7 possibly contains four alpha helices (Z, A, B and C), and a hydrophobic pocket which is an important structure to recognize acetylated histone peptide. This bromodomain encoding a 12.8 kD protein, was introduced into Escherichia coli using the pGEX-4T-2 expression vector. After isopropyl beta-D-thiogalactopyranoside(IPTG) induction, a new anticipated protein of 38.8 kD appeared on SDS-PAGE and the result was confirmed by Western blot analysis. CONCLUSION BRD-7 of bromodomain protein is similar to three proteins containing known structural bromodomain motif by bio-informatics analysis, suggesting the bromodomain of BRD-7 should belong to co-activator subgroup and may have similar function that can selectively interact with acetylated histone peptide.