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1.
J Biol Chem ; 290(48): 28997-9009, 2015 Nov 27.
Article in English | MEDLINE | ID: mdl-26468280

ABSTRACT

Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus specific transcription. GCN5 (KAT2A) is a member of the GNAT (Gcn5-related N-acetyltransferase) family of HATs. In metazoans this enzyme is found in two functionally distinct coactivator complexes, SAGA (Spt Ada Gcn5 acetyltransferase) and ATAC (Ada Two A-containing). These two multiprotein complexes comprise complex-specific and shared subunits, which are organized in functional modules. The HAT module of ATAC is composed of GCN5, ADA2a, ADA3, and SGF29, whereas in the SAGA HAT module ADA2b is present instead of ADA2a. To better understand how the activity of human (h) hGCN5 is regulated in the two related, but different, HAT complexes we carried out in vitro HAT assays. We compared the activity of hGCN5 alone with its activity when it was part of purified recombinant hATAC or hSAGA HAT modules or endogenous hATAC or hSAGA complexes using histone tail peptides and full-length histones as substrates. We demonstrated that the subunit environment of the HAT complexes into which GCN5 incorporates determines the enhancement of GCN5 activity. On histone peptides we show that all the tested GCN5-containing complexes acetylate mainly histone H3K14. Our results suggest a stronger influence of ADA2b as compared with ADA2a on the activity of GCN5. However, the lysine acetylation specificity of GCN5 on histone tails or full-length histones was not changed when incorporated in the HAT modules of ATAC or SAGA complexes. Our results thus demonstrate that the catalytic activity of GCN5 is stimulated by subunits of the ADA2a- or ADA2b-containing HAT modules and is further increased by incorporation of the distinct HAT modules in the ATAC or SAGA holo-complexes.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Histone Acetyltransferases/metabolism , Multiprotein Complexes/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , DNA-Binding Proteins , Histone Acetyltransferases/genetics , Humans , Multiprotein Complexes/genetics , Sf9 Cells , Spodoptera , Transcription Factors/genetics
2.
PLoS One ; 8(7): e70035, 2013.
Article in English | MEDLINE | ID: mdl-23894581

ABSTRACT

The SGF29 protein binds to tri-methylated lysine-4 of histone H3 (H3K4me3), which is a histone modification associated with active promoters. Human SGF29 is a subunit of the histone acetyltransferase module of the SAGA (Spt-Ada-Gcn5 acetyltransferase) and ATAC (Ada-Two-A-containing 2A) co-activator complexes. Previous work revealed that the SAGA complex is recruited to endoplasmic reticulum (ER) stress target genes and required for their induction. Here, we report the involvement of SGF29 in the survival of human cells from ER stress. SGF29 knockdown results in impaired transcription of the ER stress genes GRP78 and CHOP. Besides histone H3K14 acetylation, we find that SGF29 is also required for the maintenance of H3K4me3 at these genes, which is already present prior to ER stress. Reduced levels of H3K4me3 in the absence of SGF29 correlate with a decreased association of ASH2L, which is a core component of the SET1/MLL complexes, to GFP78 and CHOP. In conclusion, our results suggest that the H3K4me3-binding protein SGF29 plays a central and dual role in the ER stress response. Prior to ER stress, the protein coordinates H3K4me3 levels, thereby maintaining a 'poised' chromatin state on ER stress target gene promoters. Following ER stress induction, SGF29 is required for increased H3K14 acetylation on these genes, which then results in full transcriptional activation, thereby promoting cell survival.


Subject(s)
Acetyltransferases/metabolism , Endoplasmic Reticulum Stress/physiology , Histones/metabolism , Acetylation , Acetyltransferases/biosynthesis , Acetyltransferases/genetics , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Survival/physiology , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/genetics , Gene Knockdown Techniques , Humans , Lysine/metabolism , Methylation , Osteosarcoma/genetics , Osteosarcoma/metabolism , Osteosarcoma/pathology , Promoter Regions, Genetic , Transcription Factor CHOP/biosynthesis , Transcription Factor CHOP/genetics , Transcription Factor CHOP/metabolism , Transcription, Genetic
3.
EMBO J ; 29(14): 2381-94, 2010 Jul 21.
Article in English | MEDLINE | ID: mdl-20562830

ABSTRACT

All DNA-related processes rely on the degree of chromatin compaction. The highest level of chromatin condensation accompanies transition to mitosis, central for cell cycle progression. Covalent modifications of histones, mainly deacetylation, have been implicated in this transition, which also involves transcriptional repression. Here, we show that the Gcn5-containing histone acetyl transferase complex, Ada Two A containing (ATAC), controls mitotic progression through the regulation of the activity of non-histone targets. RNAi for the ATAC subunits Ada2a/Ada3 results in delayed M/G1 transition and pronounced cell division defects such as centrosome multiplication, defective spindle and midbody formation, generation of binucleated cells and hyperacetylation of histone H4K16 and alpha-tubulin. We show that ATAC localizes to the mitotic spindle and controls cell cycle progression through direct acetylation of Cyclin A/Cdk2. Our data describes a new pathway in which the ATAC complex controls Cyclin A/Cdk2 mitotic function: ATAC/Gcn5-mediated acetylation targets Cyclin A for degradation, which in turn regulates the SIRT2 deacetylase activity. Thus, we have uncovered an essential function for ATAC in regulating Cyclin A activity and consequent mitotic progression.


Subject(s)
Acetyltransferases/metabolism , Mitosis/physiology , Acetyltransferases/genetics , Amino Acid Sequence , Animals , Cyclin A/metabolism , Cyclin-Dependent Kinase 2/metabolism , HeLa Cells , Humans , Mice , Molecular Sequence Data , NIH 3T3 Cells , RNA Interference , Spindle Apparatus/metabolism , Tubulin/metabolism , p300-CBP Transcription Factors/genetics , p300-CBP Transcription Factors/metabolism
4.
Cell Mol Life Sci ; 67(4): 611-28, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19936620

ABSTRACT

Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus-specific transcription. The GCN5 HAT was identified as a subunit of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) multiprotein complex. Vertebrate cells express a second HAT, PCAF, that is 73% identical to GCN5. Here, we report the characterization of the mammalian ATAC (Ada-Two-A-Containing) complexes containing either GCN5 or PCAF in a mutually exclusive manner. In vitro ATAC complexes acetylate lysine 14 of histone H3. Moreover, ATAC- or SAGA-specific knock-down experiments suggest that both ATAC and SAGA are involved in the acetylation of histone H3K9 and K14 residues. Despite their catalytic similarities, SAGA and ATAC execute their coactivator functions on distinct sets of inducible target genes. Interestingly, ATAC strongly influences the global phosphorylation level of histone H3S10, suggesting that in mammalian cells a cross-talk exists linking ATAC function to H3S10 phosphorylation.


Subject(s)
Gene Expression Regulation , Histone Acetyltransferases/metabolism , Multienzyme Complexes/metabolism , p300-CBP Transcription Factors/metabolism , Acetylation , Animals , Catalysis , Drosophila , Gene Knockdown Techniques , HeLa Cells , Histone Acetyltransferases/genetics , Histones/metabolism , Humans , Multienzyme Complexes/genetics , Phosphorylation , p300-CBP Transcription Factors/genetics
5.
Mol Cell Biol ; 29(6): 1649-60, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19114550

ABSTRACT

One of the central questions in eukaryotic transcription is how activators can transmit their signal to stimulate gene expression in the context of chromatin. The multisubunit SAGA coactivator complex has both histone acetyltransferase and deubiquitination activities and remodels chromatin to allow transcription. Whether and how SAGA is able to regulate transcription at specific loci is poorly understood. Using mass spectrometry, immunoprecipitation, and Western blot analysis, we have identified human SPT20 (hSPT20) as the human homologue of the yeast Spt20 and show that hSPT20 is a bona fide subunit of the human SAGA (hSAGA; previously called TFTC/STAGA/PCAF) complex and that hSPT20 is required for the integrity of the hSAGA complex. We demonstrate that hSPT20 and other hSAGA subunits, together with RNA polymerase II, are specifically recruited to genes induced by endoplasmic reticulum (ER) stress. In good agreement with the recruitment of hSAGA to the ER stress-regulated genes, knockdown of hSTP20 hampers ER stress response. Surprisingly, hSPT20 recruitment was not observed for genes induced by another type of stress. These results provide evidence for a direct and specific role of the hSPT20-containing SAGA complex in transcriptional induction of ER stress-responsive genes. Thus, hSAGA regulates the transcription of stress-responsive genes in a stress type-dependent manner.


Subject(s)
Endoplasmic Reticulum/metabolism , Transcription Factors/metabolism , p300-CBP Transcription Factors/metabolism , Acetylation , Amino Acid Sequence , Gene Expression Regulation , HeLa Cells , Humans , Molecular Sequence Data , Protein Subunits/genetics , Protein Subunits/metabolism , RNA Polymerase II/metabolism , Transcription Factors/genetics , Transcription, Genetic , p300-CBP Transcription Factors/genetics
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