Although the acetylation of histones has a well-documented regulatory role in transcription, its role in other chromosomal functions remains largely unexplored. Here we show that distinct patterns of histone H4 acetylation are essential in two separate pathways of double-strand break repair. A budding yeast strain with mutations in wild-type H4 acetylation sites shows defects in nonhomologous end joining repair and in a newly described pathway of replication-coupled repair. Both pathways require the ESA1 histone acetyl transferase (HAT), which is responsible for acetylating all H4 tail lysines, including ectopic lysines that restore repair capacity to a mutant H4 tail. Arp4, a protein that binds histone H4 tails and is part of the Esa1-containing NuA4 HAT complex, is recruited specifically to DNA double-strand breaks that are generated in vivo. The purified Esa1-Arp4 HAT complex acetylates linear nucleosomal arrays with far greater efficiency than circular arrays in vitro, indicating that it preferentially acetylates nucleosomes near a break site. Together, our data show that histone tail acetylation is required directly for DNA repair and suggest that a related human HAT complex may function similarly.

The specific post-translational modifications to histones influence many nuclear processes including gene regulation, DNA repair and replication. Recent studies have identified effector proteins that recognize patterns of histone modification and transduce their function in downstream processes. For example, histone acetyltransferases (HATs) have been shown to participate in many essential cellular processes, particularly those associated with activation of transcription. Yeast SAGA (Spt-Ada-Gcn5 acetyltransferase) and SLIK (SAGA-like) are two highly homologous and conserved multi-subunit HAT complexes, which preferentially acetylate histones H3 and H2B and deubiquitinate histone H2B. Here we identify the chromatin remodelling protein Chd1 (chromo-ATPase/helicase-DNA binding domain 1) as a component of SAGA and SLIK. Our findings indicate that one of the two chromodomains of Chd1 specifically interacts with the methylated lysine 4 mark on histone H3 that is associated with transcriptional activity. Furthermore, the SLIK complex shows enhanced acetylation of a methylated substrate and this activity is dependent upon a functional methyl-binding chromodomain, both in vitro and in vivo. Our study identifies the first chromodomain that recognizes methylated histone H3 (Lys 4) and possibly identifies a larger subfamily of chromodomain proteins with similar recognition properties.

A number of transcriptional coactivator proteins have been identified as histone acetyltransferase (HAT) proteins, providing a direct molecular basis for the coupling of histone acetylation and transcriptional activation. The yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex requires the coactivator protein Gcn5 for HAT activity. Identification of protein subunits by mass spectrometry and immunoblotting revealed that the TATA binding protein-associated factors (TAF(II)s) TAF(II)90, -68/61, -60, -25/23, and -20/17 are integral components of this complex. In addition, TAF(II)68 was required for both SAGA-dependent nucleosomal HAT activity and transcriptional activation from chromatin templates in vitro. These results illustrate a role for certain TAF(II) proteins in the regulation of gene expression at the level of chromatin modification that is distinct from the TFIID complex and TAF(II)145.

In an effort to better understand the mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin, we employed an iterative search of human expressed sequence tags to identify novel basic-helix-loop-helix-PAS (bHLH-PAS) proteins that interact with either the Ah receptor (AHR) or the Ah receptor nuclear translocator (ARNT). We characterized five new "members of the PAS superfamily," or MOPs 1-5, that are similar in size and structural organization to the AHR and ARNT. MOPs 1-4 have N-terminal bHLH and PAS domains and C-terminal variable regions. MOP5 contained the characteristic PAS domain and a variable C terminus; it is possible that the cDNA contains a bHLH domain, but the entire open reading frame has yet to be completed. Coimmunoprecipitation studies, yeast two-hybrid analysis, and transient transfection experiments demonstrated that MOP1 and MOP2 dimerize with ARNT and that these complexes are transcriptionally active at defined DNA enhancer sequences in vivo. MOP3 was found to associate with the AHR in vitro but not in vivo. This observation, coupled with the fact that MOP3 formed tighter associations with the 90-kDa heat shock protein than the human AHR, suggests that MOP3 may be a conditionally active bHLH-PAS protein that requires activation by an unknown ligand. The expression profiles of the AHR, MOP1, and MOP2 mRNAs, coupled with the observation that they all share ARNT as a common dimeric partner, suggests that the cellular pathways mediated by MOP1 and MOP2 may influence or respond to the dioxin signaling pathway.

Prior to ligand activation, the unactivated aryl hydrocarbon receptor (AhR) exists in a heterotetrameric 9S core complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and an unknown subunit. Here we report the purification of an ϳ38-kDa protein (p38) from COS-1 cell cytosol that is a member of this complex by coprecipitation with a FLAG-tagged AhR. Internal amino acid sequence information was obtained, and p38 was identified as the hepatitis B virus X-associated protein 2 (XAP2). The simian ortholog of XAP2 was cloned from a COS-1 cDNA library; it codes for a 330-amino-acid protein containing regions of homology to the immunophilins FKBP12 and FKBP52. A tetratricopeptide repeat (TPR) domain in the carboxy-terminal region of XAP2 was similar to the third and fourth TPR domains of human FKBP52 and the Saccharomyces cerevisiae transcriptional modulator SSN6, respectively. Polyclonal antibodies raised against XAP2 recognized p38 in the unliganded AhR complex in COS-1 and Hepa 1c1c7 cells. It was ubiquitously expressed in murine tissues at the protein and mRNA levels. It was not required for the assembly of an AhR-hsp90 complex in vitro. Additionally, XAP2 did not directly associate with hsp90 upon in vitro translation, but was present in a 9S form when cotranslated in vitro with murine AhR. XAP2 enhanced the ability of endogenous murine and human AhR complexes to activate a dioxin-responsive element-luciferase reporter twofold, following transient expression of XAP2 in Hepa 1c1c7 and HeLa cells.

The SAGA complex is a conserved histone acetyltransferase-coactivator that regulates gene expression in Saccharomyces cerevisiae. SAGA contains a number of subunits known to function in transcription including Spt and Ada proteins, the Gcn5 acetyltransferase, a subset of TATA-binding-protein-associated factors (TAF II s), and Tra1. Here we report the identification of SLIK (SAGA-like), a complex related in composition to SAGA. Notably SLIK uniquely contains the protein Rtg2, linking the function of SLIK to the retrograde response pathway. Yeast harboring mutations in both SAGA and SLIK complexes displays synthetic phenotypes more severe than those of yeast with mutation of either complex alone. We present data indicating that distinct forms of the SAGA complex may regulate specific subsets of genes and that SAGA and SLIK have multiple partly overlapping activities, which play a critical role in transcription by RNA polymerase II.The histone components of chromatin are subjected to several posttranslational modifications including acetylation, phosphorylation, ubiquitination, methylation, and ADP-ribosylation that regulate their function and that of the corresponding DNA sequences (16,56). Of these modifications the acetylation and deacetylation of the ε-amino groups of the conserved lysine residues present in the amino terminal tails of all four core histones have long been linked to transcriptional activity and have been the most intensively studied histone modification. The acetylation of histones is catalyzed by histone acetyltransferases (HATs), which are often found to be associated with large multisubunit protein complexes that contain components with identity or homology to known regulators of transcription (17,47).The first transcription-related type A HAT to be identified was the Tetrahymena p55 protein, a homologue of Saccharomyces cerevisiae Gcn5 (8). Gcn5 was originally identified through genetic screens as a transcriptional coactivator in yeast (23) and thus provided a strong foundation for the direct coupling of histone acetylation and transcriptional stimulation. Gcn5 has subsequently been identified as a component of high-molecular-weight acetyltransferase/transcriptional adaptor complexes from both yeast and human cells (19,25,37,40,43,45,49,50). The association of Gcn5 in multiprotein complexes potentiates its nucleosomal HAT activity (3,19,43,49,58,59), important for the transcriptional stimulatory activity of these complexes from chromatin templates in vitro and in vivo (31,54,61). In addition this association bestows upon Gcn5 an expanded lysine specificity (3, 20, 52, 65). Thus, a common feature of HAT proteins with proposed coactivator function is their frequent association with other subunits that potentiate catalytic function and/or mediate interactions with activators or basal factors (7).We have previously identified Gcn5 as a component of the 1.8-MDa transcriptional regulatory complex SAGA (Spt-AdaGcn5-acetyltransferase). In this complex Gcn5 is associated with the Ada1 to Ada3 member...

Previous studies have shown that the transcriptional coactivator protein Gcn5 functions as a catalytic histone acetyltransferase (HAT). In this work, we examine the roles of the Ada2 and Ada3 coactivator proteins that are functionally linked to Gcn5. We show that yeast Ada2, Ada3, and Gcn5 form a catalytic core of the ADA and Spt-Ada-Gcn5-acetyltransferase HAT complexes, which is necessary and sufficient in vitro for nucleosomal HAT activity and lysine specificity of the intact HAT complexes. We also demonstrate that Ada3 is necessary for Gcn5-dependent nucleosomal HAT activity in yeast extracts. Our results suggest that Ada2 potentiates the Gcn5 catalytic activity and that Ada3 facilitates nucleosomal acetylation and an expanded lysine specificity.

The SAGA histone acetyltransferase/transcriptional adaptor complex is composed of multiple transcriptional regulators including Ada, Spt, and TAFII proteins. Here we identify an additional novel subunit of the complex, Tra1, an ATM/PI-3-kinase-related homolog of the human TRRAP cofactor, which is essential for c-Myc and E2F-mediated oncogenic transformation. Mass spectrometry, immunoblotting, and immunoprecipitation experiments confirm the stable association of this protein within SAGA. In addition, the Tra1 protein is a component of at least two other histone acetyltransferase protein complexes. These results indicate a role for Tra1 in the regulation of transcriptional activation through the recruitment of HAT activity to an activator-bound promoter.