Showing papers on "Bromodomain published in 1999"

Structure and ligand of a histone acetyltransferase bromodomain

Abstract: Histone acetylation is important in chromatin remodelling and gene activation1,2,3,4. Nearly all known histone-acetyltransferase (HAT)-associated transcriptional co-activators contain bromodomains, which are ∼110-amino-acid modules found in many chromatin-associated proteins5,6,7,8,9. Despite the wide occurrence of these bromodomains, their three-dimensional structure and binding partners remain unknown. Here we report the solution structure of the bromodomain of the HAT co-activator P/CAF (p300/CBP-associated factor)10,11. The structure reveals an unusual left-handed up-and-down four-helix bundle. In addition, we showby a combination of structural and site-directed mutagenesis studies that bromodomains can interact specifically with acetylated lysine, making them the first known protein modules to do so. The nature of the recognition of acetyl-lysine by the P/CAF bromodomain is similar to that of acetyl-CoA by histone acetyltransferase. Thus, the bromodomain is functionally linked to the HAT activity of co-activators in the regulation of gene transcription.

p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage.

Abstract: The p53 tumor suppressor protein is a sequence-specific transcription factor that modulates the response of cells to DNA damage. Recent studies suggest that full transcriptional activity of p53 requires the coactivators CREB binding protein (CBP)/p300 and PCAF. These coactivators interact with each other, and both possess intrinsic histone acetyltransferase activity. Furthermore, p300 acetylates p53 to activate its sequence-specific DNA binding activity in vitro. In this study, we demonstrate that PCAF also acetylates p53 in vitro at a lysine residue distinct from that acetylated by p300 and thereby increases p53's ability to bind to its cognate DNA site. We have generated antibodies to acetylated p53 peptides at either of the two lysine residues that are targeted by PCAF or p300 and have demonstrated that these antibodies are highly specific for both acetylation and the particular site. Using these antibodies, we detect acetylation of these sites in vivo, and interestingly, acetylation at both sites increases in response to DNA-damaging agents. These data indicate that site-specific acetylation of p53 increases under physiological conditions that activate p53 and identify CBP/p300 and PCAF as the probable enzymes that modify p53 in vivo.

Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction

Abstract: SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5D spt3D and gcn5D spt8D) causing loss of a member of each of the moderate classes have severe phenotypes, similar to spt7D, spt20D ,o rada1D mutants. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription.

ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly

Abstract: The assembly of core histones and DNA into periodic nucleosome arrays is mediated by ACF, an ISWI-containing factor, and NAP-1, a core histone chaperone, in an ATP-dependent process. We describe the isolation of Drosophila acf1 cDNA, which encodes the p170 and p185 forms of the Acf1 protein in ACF. Acf1 is a novel protein that contains two PHD fingers, one bromodomain, and two new conserved regions. Human WSTF, which is encoded by one of multiple genes that is deleted in Williams syndrome individuals, is the only currently known mammalian protein with each of the conserved motifs in Acf1. Purification of the native form of Acf1 led to the isolation of ACF comprising Acf1 (both p170 and p185 forms) and ISWI. Native Acf1 did not copurify with components of NURF or CHRAC, which are other ISWI-containing complexes in Drosophila. Purified recombinant ACF, consisting of Acf1 (either p185 alone or both p170 and p185) and ISWI, catalyzes the deposition of histones into extended periodic nucleosome arrays. Notably, the Acf1 and ISWI subunits function synergistically in the assembly of chromatin. ISWI alone exhibits a weak activity that is ~3% that of ACF. These results indicate that both Acf1 and ISWI participate in the chromatin assembly process and suggest further that the Acf1 subunit confers additional functionality to the general ‘motor’ activity of ISWI.

The bromodomain: a chromatin-targeting module?

Abstract: It has recently been demonstrated that bromodomains — motifs found in several eukaryotic transcription factors — bind to acetyl-lysine, a modification of histones that is important for transcription. This finding suggests that the regulatory effects of histone acetylation may be exerted by bromodomain-containing proteins.

TIF1γ, a novel member of the transcriptional intermediary factor 1 family

Abstract: We report the cloning and characterization of a novel member of the Transcriptional Intermediary Factor 1 (TIF1) gene family, human TIF1γ. Similar to TIF1α and TIF1β, the structure of TIF1β is characterized by multiple domains: RING finger, B boxes, Coiled coil, PHD/TTC, and bromodomain. Although structurally related to TIF1α and TIF1β, TIF1γ presents several functional differences. In contrast to TIF1α, but like TIF1β, TIF1γ does not interact with nuclear receptors in yeast two-hybrid or GST pull-down assays and does not interfere with retinoic acid response in transfected mammalian cells. Whereas TIF1α and TIF1β were previously found to interact with the KRAB silencing domain of KOX1 and with the HP1α, MOD1 (HP1β) and MOD2 (HP1γ) heterochromatinic proteins, suggesting that they may participate in a complex involved in heterochromatin-induced gene repression, TIF1γ does not interact with either the KRAB domain of KOX1 or the HP1 proteins. Nevertheless, TIF1γ, like TIF1α and TIF1β, exhibits a strong silencing activity when tethered to a promoter. Since deletion of a novel motif unique to the three TIF1 proteins, called TIF1 signature sequence (TSS), abrogates transcriptional repression by TIF1γ, this motif likely participates in TIF1 dependent repression.

Specific Acetylation of Chromosomal Protein HMG-17 by PCAF Alters Its Interaction with Nucleosomes

Abstract: Nonhistone chromosomal proteins HMG-14 and HMG-17 are closely related nucleosomal binding proteins that unfold the higher-order chromatin structure, thereby enhancing the transcription and replication potential of chromatin. Here we report that PCAF, a transcription coactivator with intrinsic histone acetyltransferase activity, specifically acetylates HMG-17 but not HMG-14. Using mass spectrum sequence analysis, we identified the lysine at position 2 as the predominant site acetylated by PCAF. Lysine 2 is a prominent acetylation site in vivo, suggesting that this PCAF-mediated acetylation is physiologically relevant. Experiments with HMG-17 deletion mutants and competition studies with various protein fragments indicate that the specific acetylation of HMG-17 is not determined solely by the primary sequence near the acetylation site. By equilibrium dialysis we demonstrated that acetylation reduces the affinity of HMG-17 to nucleosome cores. In addition, we found that the binding of HMG-14 and HMG-17 to nucleosome cores inhibits the PCAF-mediated acetylation of histone H3. Thus, the presence of HMG-14 and HMG-17 affects the ability of PCAF to acetylate chromatin, while the acetylation of HMG-17 reduces its binding affinity to chromatin. Conceivably, in HMG-17-containing chromatin, acetylation of HMG-17 precedes the acetylation of histones.

The Activity of Mammalian brm/SNF2α Is Dependent on a High-Mobility-Group Protein I/Y-Like DNA Binding Domain

Abstract: The mammalian SWI-SNF complex is a chromatin-remodelling machinery involved in the modulation of gene expression. Its activity relies on two closely related ATPases known as brm/SNF2alpha and BRG-1/SNF2beta. These two proteins can cooperate with nuclear receptors for transcriptional activation. In addition, they are involved in the control of cell proliferation, most probably by facilitating p105(Rb) repression of E2F transcriptional activity. In the present study, we have examined the ability of various brm/SNF2alpha deletion mutants to reverse the transformed phenotype of ras-transformed fibroblasts. Deletions within the p105(Rb) LXCXE binding motif or the conserved bromodomain had only a moderate effect. On the other hand, a 49-amino-acid segment, rich in lysines and arginines and located immediately downstream of the p105(Rb) interaction domain, appeared to be essential in this assay. This region was also required for cooperation of brm/SNF2alpha with the glucocorticoid receptor in transfection experiments, but only in the context of a reporter construct integrated in the cellular genome. The region has homology to the AT hooks present in high-mobility-group protein I/Y DNA binding domains and is required for the tethering of brm/SNF2alpha to chromatin.

1H, 15N and 13C resonance assignments for the bromodomain of the histone acetyltransferase P/CAF.

Abstract: Bromodomains are modules of about 110 amino acid residues that are found in a large number of chromatin-associated proteins, many of which are involved in transcriptional activation (Haynes et al., 1992; Brownell and Davis, 1996a). Particularly, the bromodomains are present in nearly all known histone acetyltransferase (HAT)-associated transcriptional coactivators, which play a central role in the regulation of nucleosome remodeling and gene activation via histone acetylation (Brownell et al., 1996b; Jeanmougin et al., 1997). It has been suggested on the basis of its modular nature that bromodomain may be involved in protein–protein interactions (Jeanmougin et al., 1997). However, no conformational studies and binding partners of the bromodomain family are available. We have recently initiated structural studies of a bromodomain from the human HAT coactivator P/CAF (p300/CBP-associated factor), using heteronuclear multidimensional NMR spectroscopy techniques. Here, we report the nearly complete sequencespecific backbone and side-chain 1H, 15N, and 13C resonance assignments of the P/CAF bromodomain.

Nucleic acids encoding BAZ1α transcriptional regulator and methods of use

Abstract: Genes each encoding a novel transcriptional regulator having a bromodomain have been successfully isolated from a human testis cDNA library using primers prepared based on an EST sequence found using the bromodomain sequence of the transcriptional regulator. These genes are structurally analogous to each other.

Clinging to Histones

Abstract: Structural biologists have taken a close look at a protein region, or "domain," that may guide proteins to specific sites on chromatin, the complex of DNA and proteins making up the chromosomes. The region in question is the bromodomain, a conserved sequence containing roughly 100 amino acids found in some 30 chromatin-associated proteins. Using nuclear magnetic resonance, the researchers have determined the structure of the bromodomain and shown that it contains a cleft that specifically recognizes acetylated histones. Because acetyl addition to histones helps open up the chromatin so gene expression can occur, the finding suggests that the bromodomain helps bring in the proteins necessary for gene activation.