Human STAGA Complex Is a Chromatin-Acetylating Transcription Coactivator That Interacts with Pre-mRNA Splicing and DNA Damage-Binding Factors In Vivo

Abstract

GCN5 is a histone acetyltransferase (HAT) originally identified in Saccharomyces cerevisiae and required for transcription of specific genes within chromatin as part of the SAGA (SPT-ADA-GCN5 acetylase) coactivator complex. Mammalian cells have two distinct GCN5 homologs (PCAF and GCN5L) that have been found in three different SAGA-like complexes (PCAF complex, TFTC [TATA-binding-protein-free TAF_II-containing complex], and STAGA [SPT3-TAF_II31-GCN5L acetylase]). The composition and roles of these mammalian HAT complexes are still poorly characterized. Here, we present the purification and characterization of the human STAGA complex. We show that STAGA contains homologs of most yeast SAGA components, including two novel human proteins with histone-like folds and sequence relationships to yeast SPT7 and ADA1. Furthermore, we demonstrate that STAGA has acetyl coenzyme A-dependent transcriptional coactivator functions from a chromatin-assembled template in vitro and associates in HeLa cells with spliceosome-associated protein 130 (SAP130) and DDB1, two structurally related proteins. SAP130 is a component of the splicing factor SF3b that associates with U2 snRNP and is recruited to prespliceosomal complexes. DDB1 (p127) is a UV-damaged-DNA-binding protein that is involved, as part of a complex with DDB2 (p48), in nucleotide excision repair and the hereditary disease xeroderma pigmentosum. Our results thus suggest cellular roles of STAGA in chromatin modification, transcription, and transcription-coupled processes through direct physical interactions with sequence-specific transcription activators and with components of the splicing and DNA repair machineries.

ACKNOWLEDGMENTS

We thank G. Chu for pBJ5-FLAG-p125/DDB1, S. Berger for hADA2 antiserum, J. Manley for CPSF160 antibodies, R. Lamb for pGEX-5X-3-DDB1, S. Linn for p48 (DDB2) antibodies, S. McMahon and M. Cole for TRRAP antiserum, B. Slagle for XAP1 (DDB1) peptide antibodies, A. Krutchinsky for valuable contributions, and J. Fu and C. Bhattacharyya for technical assistance. We are also grateful to Cedric S. Wesley for the help in establishment of the large population ofDrosophila flies.

This work was supported by grants from the NIH to R.G.R. (CA42567) and B.T.C. (RR00862).

ADDENDUM

Similar to the results reported here for STAGA, studies published while this article was under review have shown that TFTC is recruited to UV-damaged DNA (9) and that the PCAF complex facilitates transcription of chromatin in vitro (49a).