Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex.

Abstract

The transcriptional adaptor protein Gcn5 has been identified as a nuclear histone acetyltransferase (HAT). Although recombinant yeast Gcn5 efficiently acetylates free histones, it fails to acetylate histones contained in nucleosomes, indicating that additional components are required for acetylation of chromosomal histones. We report here that Gcn5 functions as a catalytic subunit in two high-molecular-mass native HAT complexes, with apparent molecular masses of 0.8 and 1.8 megadalton (MD), respectively, which acetylate nucleosomal histones. Both the 0.8-and 1.8-MD Gcn5-containing complexes cofractionate with Ada2 and are lost in gcn5A, ada2A, or ada3A yeast strains, illustrating that these HAT complexes are bona fide native Ada-transcriptional adaptor complexes. Importantly, the 1.8-MD adaptor/HAT complex also contains Spt gene products that are linked to TATA-binding protein (TBP) function. This complex is lost in spt20/ada5A and spt7A strains and Spt3, Spt7, Spt20/Ada5, Ada2, and Gcn5 all copurify with this nucleosomal HAT complex. Therefore, the 1.8-MD adaptor/HAT complex illustrates an interaction between Ada and Spt gene products and confirms the existence of a complex containing the TBP group of Spt proteins as demonstrated by genetic and biochemical studies. We have named this novel transcription regulatory complex SAGA (_Spt-Ada-Gcn5-Acetyltransferase). The function of Gcn5 as a histone acetyltransferase within the Ada and SAGA adaptor complexes indicates the importance of histone acetylation during steps in transcription activation mediated by interactions with transcription activators and general transcription factors (i.e., TBP).[Key Words." Acetyltransferase; nucleosome; transcription; Spt; Ada; Gcn5] Received March 28, 1997; revised version accepted May 15, 1997.Chromatin structure has an intricate role in the regulation of eukaryotic gene transcription. Nucleosomes suppress basal transcription initiation in vivo and in vitro increasing the dependence of transcription on the function of sequence-specific activator proteins (for review, see Grunstein 1990; Owen-Hughes and Workman 1994). Chromatin structures are remodeled before or during transcription activation generating DNase I hypersensitive regions (DHSs) at transcription control elements (Hager et al. 1995;Steger and Workman 1996;Svaren and Horz 1996). Multiprotein complexes have been impliSCorresponding authors.

Developmental potential of Gcn5−/− embryonic stem cells in vivo and in vitro

“…Gcn5 acetylates histones H3 and H2B (Grant et al, 1997(Grant et al, , 1999. H3 acetylation is decreased approximately twofold in undifferentiated Gcn5 Ϫ/Ϫ cells (data not shown), indicating that Gcn5 is a major H3 HAT activity in these cells, but it is not the only one.…”

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“…SAGA (SPT/ADA/GCN5/Acetyltransferase) is a 1.8 MDa complex containing approximately 20 proteins which have been implicated in transcriptional regulation, primarily through genetic screens in yeast (Grant et al, 1997). However, several recent studies have demonstrated that in addition to TRRAP, many other components of the SAGA complex are also highly conserved from yeast to humans (Martinez et al, 1998;Ogryzko et al, 1998;Smith et al, 1998a).…”

Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and Piccolo NuA4 histone acetyltransferase complexes

“…In contrast, their SAGA and NuA4 parent complexes acetylate both histones and nucleosomes [5,6]. Since Ada2, Ada3 and Gcn5 form a triple complex [7,8], we hypothesized that this Ada2/Ada3/Gcn5 complex would acetylate nucleosomes.…”