Structural basis for activation of SAGA histone acetyltransferase Gcn5 by partner subunit Ada2

The Gcn5 histone acetyltransferase (HAT) subunit of the SAGA transcriptional coactivator complex catalyzes acetylation of histone H3 and H2B N-terminal tails, posttranslational modifications associated with gene activation. Binding of the SAGA subunit partner Ada2 to Gcn5 activates Gcn5’s intrinsically weak HAT activity on histone proteins, but the mechanism for this activation by the Ada2 SANT domain has remained elusive. We have employed Fab antibody fragments as crystallization chaperones to determine crystal structures of a yeast Ada2/Gcn5 complex. Our structural and biochemical results indicate that the Ada2 SANT domain does not activate Gcn5’s activity by directly affecting histone peptide binding as previously proposed. Instead, the Ada2 SANT domain enhances Gcn5 binding of the enzymatic cosubstrate acetyl-CoA. This finding suggests a mechanism for regulating chromatin modification enzyme activity: controlling binding of the modification cosubstrate instead of the histone substrate.

POWERDRESS interacts with HISTONE DEACETYLASE 9 to promote aging in Arabidopsis

Leaf senescence is an essential part of the plant lifecycle during which nutrients are re-allocated to other tissues. The regulation of leaf senescence is a complex process. However, the underlying mechanism is poorly understood. Here, we uncovered a novel and the pivotal role of Arabidopsis HDA9 (a RPD3-like histone deacetylase) in promoting the onset of leaf senescence. We found that HDA9 acts in complex with a SANT domain-containing protein POWERDRESS (PWR) and transcription factor WRKY53. Our genome-wide profiling of HDA9 occupancy reveals that HDA9 directly binds to the promoters of key negative regulators of senescence and this association requires PWR. Furthermore, we found that PWR is important for HDA9 nuclear accumulation. This study reveals an uncharacterized epigenetic complex involved in leaf senescence and provides mechanistic insights into how a histone deacetylase along with a chromatin-binding protein contribute to a robust regulatory network to modulate the onset of plant aging.

The SANT Domain of p400 ATPase Represses Acetyltransferase Activity and Coactivator Function of TIP60 in Basal p21 Gene Expression

ABSTRACT The TIP60 histone acetyltransferase plays diverse roles in DNA damage responses, DNA double-strand break repair, and transcriptional regulation. TIP60 resides within a multisubunit complex that has been shown to be targeted by transcription factors and to be involved in histone acetylation and transcriptional activation. p400, an SWI2/SNF2-related ATPase that serves as an ATP-dependent chromatin remodeling enzyme, exists as an integral subunit of a TIP60 complex but also resides within a distinct complex that presumably lacks TIP60 and appears to be involved in the transcriptional repression of basal p53 target gene expression. Here, we describe a TIP60-containing p400 complex population in which the acetyltransferase activity of TIP60 is repressed by interactions with p400. We further show that an SWI3-ADA2-N-CoR-TFIIIB (SANT) domain of p400 binds directly to the histone acetyltransferase (HAT) domain of TIP60 and blocks both its enzymatic activity and its coactivator function in regulating basal p21 gene expression. Our results thus suggest that p400 represses basal p21 gene expression through dual mechanisms that include the direct inhibition of TIP60 enzymatic activity described here and the previously described ATP-dependent positioning of H2A.Z at the promoter.

Nucleosome Remodeling and Transcriptional Repression Are Distinct Functions of Isw1 in Saccharomyces cerevisiae

ABSTRACT The SANT domain is a nucleosome recognition module found in transcriptional regulatory proteins, including chromatin-modifying enzymes. It shows high functional degeneracy between species, varying in sequence and copy number. Here, we investigate functions in vivo associated with two SANT motifs, SANT and SLIDE, in the Saccharomyces cerevisiae Isw1 chromatin-remodeling ATPase. We show that differences in the primary structures of the SANT and SLIDE domains in yeast and Drosophila melanogaster reflect their different functions. In yeast, the SLIDE domain is required for histone interactions, while this is a function of the SANT domain in flies. In yeast, both motifs are required for optimal association with chromatin and for formation of the Isw1b complex (Isw1, Ioc2, and Ioc4). Moreover, nucleosome remodeling at the MET16 locus is defective in strains lacking the SANT or SLIDE domain. In contrast, the SANT domain is dispensable for the interaction between Isw1 and Ioc3 in the Isw1a complex. We show that, although defective in nucleosome remodeling, Isw1 lacking the SANT domain is able to repress transcription initiation at the MET16 promoter. Thus, chromatin remodeling and transcriptional repression are distinct activities of Isw1.

Effects of the SANT Domain of Tension-Induced/Inhibited Proteins (TIPs), Novel Partners of the Histone Acetyltransferase p300, on p300 Activity and TIP-6-Induced Adipogenesis

ABSTRACT We previously identified a set of transcription regulators, referred to as TIPs (tension-induced/inhibited proteins), with a role in myogenic versus adipogenic differentiation. Here we report that the TIP family comprises eight isoforms, all bearing a SANT (switching-defective protein 3, adaptor 2, nuclear receptor corepressor, and transcription factor IIIB) domain and some of them presenting S-adenosyl-l-methionine (SAM) and nuclear receptor box (NRB) motifs, all characteristic of histone-modifying enzymatic complexes. TIPs have SANT-dependent, p300-mediated histone acetyltransferase (HAT) activity. Ectopic TIP-6 (SANT+ SAM− NRB−) but not TIP-6ΔSANT induced de novo PPARγ2-mediated adipogenic gene expression in NIH 3T3 cells and promoted preadipocyte differentiation into fat cells. TIP-6 was also involved in mediating hormonally/biochemically induced adipogenic differentiation of 3T3-L1 cells. Furthermore, TIP-6 was identified in adipose tissue in vivo. TIP-6 bound directly and indirectly to p300 and histone H4 (H4). Deletion of the SANT domain did not abolish TIP-6 interaction with p300 and H4 but eliminated direct TIP-6 binding to p300. Chromatin immunoprecipitation assays showed the recruitment of TIP-6, TIP-6ΔSANT, and p300 to the PPARγ2 promoter, but H3/H4 acetylation occurred only when p300 was directly associated with TIP-6. These studies demonstrated the importance of TIPs in the recruitment of p300 to specific promoters and in the regulation of p300 HAT activity through the involvement of the SANT domain. Furthermore, we identified TIP-6 as a new member of the adipogenic cascade.

Host Cell Factor and an Uncharacterized SANT Domain Protein Are Stable Components of ATAC, a Novel dAda2A/dGcn5-Containing Histone Acetyltransferase Complex in Drosophila

ABSTRACT Gcn5 is a conserved histone acetyltransferase (HAT) found in a number of multisubunit complexes from Saccharomyces cerevisiae, mammals, and flies. We previously identified Drosophila melanogaster homologues of the yeast proteins Ada2, Ada3, Spt3, and Tra1 and showed that they associate with dGcn5 to form at least two distinct HAT complexes. There are two different Ada2 homologues in Drosophila named dAda2A and dAda2B. dAda2B functions within the Drosophila version of the SAGA complex (dSAGA). To gain insight into dAda2A function, we sought to identify novel components of the complex containing this protein, ATAC (Ada two A containing) complex. Affinity purification and mass spectrometry revealed that, in addition to dAda3 and dGcn5, host cell factor (dHCF) and a novel SANT domain protein, named Atac1 (ATAC component 1), copurify with this complex. Coimmunoprecipitation experiments confirmed that these proteins associate with dGcn5 and dAda2A, but not with dSAGA-specific components such as dAda2B and dSpt3. Biochemical fractionation revealed that ATAC has an apparent molecular mass of 700 kDa and contains dAda2A, dGcn5, dAda3, dHCF, and Atac1 as stable subunits. Thus, ATAC represents a novel histone acetyltransferase complex that is distinct from previously purified Gcn5/Pcaf-containing complexes from yeast and mammalian cells.