Ernest Martinez

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 inSaccharomyces 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 TAFII-containing complex], and STAGA [SPT3-TAFII31-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.

A Novel Human SRB/MED-Containing Cofactor Complex, SMCC, Involved in Transcription Regulation

A novel human complex that can either repress activator-dependent transcription mediated by PC4, or, at limiting TFIIH, act synergistically with PC4 to enhance activator-dependent transcription has been purified. This complex contains homologs of a subset of yeast mediator/holoenzyme components (including SRB7, SRB10, SRB11, MED6, and RGR1), homologs of other yeast transcriptional regulatory factors (SOH1 and NUT2), and, significantly, some components (TRAP220, TRAP170/hRGR1, and TRAP100) of a human thyroid hormone receptor-associated coactivator complex. The complex shows direct activator interactions but, unlike yeast mediator, can act independently of the RNA polymerase II CTD. These findings demonstrate both positive and negative functional capabilities for the human complex, emphasize novel (CTD-independent) regulatory mechanisms, and link the complex to other human coactivator complexes.

Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression.

The nuclear hormone receptor superfamily is characterized by an impressive functional diversity of its members despite a remarkable overall structural unity. A variety of ligands bind specifically to them and these receptors control gene networks that have profound effects on growth, development, and homeostasis. The ligand‐receptor complexes recognize transcriptional enhancer DNA sequences, the hormone response elements, resulting in induction or repression of gene activity. The similarity between all these hormone response enhancer elements, as well as between the receptors themselves, indicates a conserved general strategy for the hormonal control of transcription by steroids. The activated receptors bind to responsive promoters and most likely mediate the assembly of stage‐ and tissue‐specific transcription factor complexes that stimulate or inhibit gene expression.—Wahli, W.; Martinez, E. Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression. FASEB J. 5: 2243–2249; 1991.

Cloning of an Inr- and E-box-binding protein, TFII-I, that interacts physically and functionally with USF1

The transcription factor TFII‐I has been shown to bind independently to two distinct promoter elements, a pyrimidine‐rich initiator (Inr) and a recognition site (E‐box) for upstream stimulatory factor 1 (USF1), and to stimulate USF1 binding to both of these sites. Here we describe the isolation of a cDNA encoding TFII‐I and demonstrate that the corresponding 120 kDa polypeptide, when expressed ectopically, is capable of binding to both Inr and E‐box elements. The primary structure of TFII‐I reveals novel features that include six directly repeated 90 residue motifs that each possess a potential helix–loop/span–helix homology. These unique structural features suggest that TFII‐I may have the capacity for multiple protein–protein and, potentially, multiple protein–DNA interactions. Consistent with this hypothesis and with previous in vitro studies, we further demonstrate that ectopic TFII‐I and USF1 can act synergistically, and in some cases independently, to activate transcription in vivo through both Inr and the E‐box elements of the adenovirus major late promoter. We also describe domains of USF1 that are necessary for its independent and synergistic activation functions.

The estrogen-responsive element as an inducible enhancer : DNA sequence requirements and conversion to a glucocorticoid-responsive element

The estrogen‐responsive element (ERE) present in the 5′‐flanking region of the Xenopus laevis vitellogenin (vit) gene B1 has been characterized by transient expression analysis of chimeric vit‐tk‐CAT (chloramphenicol acetyltransferase) gene constructs transfected into the human estrogen‐responsive MCF‐7 cell line. The vit B1 ERE behaves like an inducible enhancer, since it is able to confer estrogen inducibility to the heterologous HSV thymidine kinase (tk) promoter in a relative position‐ and orientation‐independent manner. In this assay, the minimal B1 ERE is 33 bp long and consists of two 13 bp imperfect palindromic elements both of which are required for the enhancer activity. A third imperfect palindromic element is present further upstream within the 5′‐flanking region of the gene but is unable to confer hormone responsiveness by itself. Similarly, neither element forming the B1 ERE can alone confer estrogen inducibility to the tk promoter. However, in combinations of two, all three imperfect palindromes can act cooperatively to form a functional ERE. In contrast a single 13 bp perfect palindromic element, GGTCACTGTGACC, such as the one found upstream of the vit gene A2, is itself sufficient to act as a fully active ERE. Single point mutations within this element abolish estrogen inducibility, while a defined combination of two mutations converts this ERE into a glucocorticoid‐responsive element.

A Human SPT3-TAFII31-GCN5-L Acetylase Complex Distinct from Transcription Factor IID

In yeast, SPT3 is a component of the multiprotein SPT-ADA-GCN5 acetyltransferase (SAGA) complex that integrates proteins with transcription coactivator/adaptor functions (ADAs and GCN5), histone acetyltransferase activity (GCN5), and core promoter-selective functions (SPTs) involving interactions with the TATA-binding protein (TBP). In particular, yeast SPT3 has been shown to interact directly with TBP. Here we report the molecular cloning of a cDNA encoding a human homologue of yeast SPT3. Amino acid sequence comparisons between human SPT3 (hSPT3) and its counterparts in different yeast species reveal three highly conserved domains, with the most conserved 92-amino acid N-terminal domain being 25% identical with human TAFII18. Despite the significant sequence similarity with TAFII18, native hSPT3 is not a bona fide TAFII because it is not associated in vivoeither with human TBP/TFIID or with a TFIID-related TBP-free TAFII complex. However, we present evidence that hSPT3 is associated in vivo with TAFII31 and the recently described longer form of human GCN5 (hGCN5-L) in a novel human complex that has histone acetyltransferase activity. We propose that the human SPT3-TAFII31-GCN5-L acetyltransferase (STAGA) complex is a likely homologue of the yeast SAGA complex.

TATA-binding protein-associated factor(s) in TFIID function through the initiator to direct basal transcription from a TATA-less class II promoter.

The RNA polymerase II (Pol II) basal transcription factor TFIID is composed of the TATA box‐binding protein (TBP) and several TBP‐associated factors (TAFs). TBP is required for Pol II transcription from TATA‐containing and TATA‐less promoters. TATA‐less promoters of mRNA‐encoding genes often contain an initiator element at the transcription start site that is sufficient to direct accurate Pol II transcription. Here we address the mechanisms of functional TBP recruitment to the TATA‐less initiator‐dependent promoter of the mouse terminal deoxynucleotidyl transferase (TdT) gene. We show that the natural TATA‐less TdT initiator region is sufficient to promote low levels of specific transcription in vitro and to direct the assembly of a stable preinitiation complex. In contrast to what is observed for several other promoters lacking a consensus TATA element, the TATA‐binding activity of TBP is not required for the functional recruitment of TFIID to the natural TATA‐less TdT and beta‐polymerase promoters. Moreover, a comparison of TBP and highly purified epitope‐tagged TFIID reveals that one or several TAFs function independently of distal regulatory elements to mediate initiator‐directed (basal) transcription from the natural TATA‐less TdT core promoter in crude nuclear extracts. Furthermore, by using a transcription system reconstituted with purified components, we present the first evidence for a basal transcription function of TAFs through the TdT initiator element. Altogether, our results suggest an alternative pathway for TFIID recruitment to initiator‐dependent TATA‐less class II promoters in which TAF(s) recruit TBP by interacting either directly or indirectly with the initiator region.

Cooperative binding of estrogen receptor to imperfect estrogen-responsive DNA elements correlates with their synergistic hormone-dependent enhancer activity.

The Xenopus vitellogenin (vit) gene B1 estrogen‐inducible enhancer is formed by two closely adjacent 13 bp imperfect palindromic estrogen‐responsive elements (EREs), i.e. ERE‐2 and ERE‐1, having one and two base substitutions respectively, when compared to the perfect palindromic consensus ERE (GGTCANNNTGACC). Gene transfer experiments indicate that these degenerated elements, on their own, have a low or no regulatory capacity at all, but in vivo act together synergistically to confer high receptor‐ and hormone‐dependent transcription activation to the heterologous HSV thymidine kinase promoter. Thus, the DNA region upstream of the vitB1 gene comprising these two imperfect EREs separated by 7 bp, was called the vitB1 estrogen‐responsive unit (vitB1 ERU). Using in vitro protein‐DNA interaction techniques, we demonstrate that estrogen receptor dimers bind cooperatively to the imperfect EREs of the vitB1 ERU. Binding of a first receptor dimer to the more conserved ERE‐2 increases approximately 4‐ to 8‐fold the binding affinity of the receptor to the adjacent less conserved ERE‐1. Thus, we suggest that the observed synergistic estrogen‐dependent transcription activation conferred by the pair of hormone‐responsive DNA elements of the vit B1 ERU is the result of cooperative binding of two estrogen receptor dimers to these two adjacent imperfect EREs.

Human ATAC Is a GCN5/PCAF-containing Acetylase Complex with a Novel NC2-like Histone Fold Module That Interacts with the TATA-binding Protein

Eukaryotic GCN5 acetyltransferases influence diverse biological processes by acetylating histones and non-histone proteins and regulating chromatin and gene-specific transcription as part of multiprotein complexes. In lower eukaryotes and invertebrates, these complexes include the yeast ADA complex that is still incompletely understood; the SAGA (Spt-Ada-Gcn5 acetylase) complexes from yeast to Drosophila that are mostly coactivators; and the ATAC (Ada Two-A containing) complex, only known in Drosophila and still poorly characterized. In contrast, vertebrate organisms, express two paralogous GCN5-like acetyltransferases (GCN5 and PCAF), which have been found so far only in SAGA-type complexes referred to hereafter as the STAGA (SPT3-TAF9-GCN5/PCAF acetylase) complexes. We now report the purification and characterization of vertebrate (human) ATAC-type complexes and identify novel components of STAGA. We show that human ATAC complexes incorporate in addition to GCN5 or PCAF (GCN5/PCAF), other epigenetic coregulators (ADA2-A, ADA3, STAF36, and WDR5), cofactors of chromatin assembly/remodeling and DNA replication machineries (POLE3/CHRAC17 and POLE4), the stress- and TGFβ-activated protein kinase (TAK1/MAP3K7) and MAP3-kinase regulator (MBIP), additional cofactors of unknown function, and a novel YEATS2-NC2β histone fold module that interacts with the TATA-binding protein (TBP) and negatively regulates transcription when recruited to a promoter. We further identify the p38 kinase-interacting protein (p38IP/FAM48A) as a novel component of STAGA with distant similarity to yeast Spt20. These results suggest that vertebrate ATAC-type and STAGA-type complexes link specific extracellular signals to modification of chromatin structure and regulation of the basal transcription machinery.

Dual Regulation of c-Myc by p300 via Acetylation-Dependent Control of Myc Protein Turnover and Coactivation of Myc-Induced Transcription

ABSTRACT The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases (HATs) GCN5 and Tip60. Here, we report a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. We show that p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1 to 110 and acetylates Myc in a TAD-dependent manner in vivo at several lysine residues located between the TAD and DNA-binding domain. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. Whereas p300 and CBP can stabilize Myc independently of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene, and p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. Our results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation.