Biochem - Martinez, Ernest

My research interest is in the characterization of the factors and molecular mechanisms involved in the regulation of chromatin structure and gene-selective transcription by RNA polymerase II, with emphasis on the control of mammalian cell proliferation and differentiation. Research in the lab currently focuses on 3 interrelated projects

(i) Analysis of the structure, function, and regulation of human multi protein transcription coregulator complexes including GCN5/PCAF acetylase complexes (such as STAGA).

(ii) Investigation of the molecular mechanisms involved in the control of gene expression and chromatin/epigenetic modification by the MYC oncoproteins as they relate to MYC-dependent cell proliferation of “normal” (e.g. stem or progenitor) cells and cancer cells.

(iii) Analysis of the basal transcription machinery required for specific transcription of genes with different core promoter structure including the role of chromatin, TAFs, and novel cofactors in core promoter selectivity.

A combination of cellular, molecular biological and biochemical techniques are used, including the reconstitution of transcription regulatory processes in vitro with purified native and recombinant factors and chromatin-assembled physiological target genes.

I. Structure, function, and regulation of human GCN5 acetyltransferase complexes.

In eukaryotes, genomic DNA is packaged by histones into nucleosomes, the basic repeating units of chromatin that further fold into higher order chromatin structures and generally inhibit sequence-specific protein-DNA interactions. Eukaryotic cells have evolved two major enzymatic mechanisms to modify chromatin structure (and to alleviate its repressive nature): (i) ATP-dependent nucleosome remodeling by protein complexes that use the energy of ATP hydrolysis to alter the association of core histones with DNA and (ii) covalent modifications of core histones, including acetylation by histone acetyltransferases (HATs), that regulate core histone interactions with either DNA, adjacent nucleosomes and/or other regulatory proteins. GCN5 is the prototypical HAT with transcription regulatory functions and exists as part of several different multi-protein complexes in eukaryotic cells. Two main types of GCN5 complexes have been identified in yeast: the so-called ADA and SAGA complexes. In metazoans, GCN5 homologues are also incorporated into several distinct complexes, which include SAGA-type (e.g. drosophila SAGA and mammalian STAGA and PCAF complexes) and ATAC-type complexes. The functions of these and other potential GCN5 complexes remain to be determined in mammalian cells. Mammals have two distinct GCN5 homologues (PCAF and GCN5). GCN5 is ubiquitously expressed and required for early embryogenesis, whereas PCAF is dispensable and expressed in a more tissue-restricted manner. Human GCN5 and PCAF assemble in similar SAGA-type complexes: the STAGA (SPT3-TAF9-GCN5-Acetylase) complex and the PCAF complex, respectively. Our studies have identified most the subunits of human STAGA, which include an ataxia telangiectasia mutated (ATM)-related protein TRRAP that was originally identified through its interaction with MYC and E2F1 and contributes to cellular transformation by MYC in vitro. Accordingly, We have recently shown that STAGA functions as a transcription coactivator for MYC in human cells and are interested in further characterizing the detailed molecular mechanisms involved (see point II. below). STAGA contains many other subunits including proteins involved in regulation of transcription, protein ubiquitination, processing of mRNA and repair of UV-damaged DNA (Martinez et al., 2001; Wang et al. unpublished), suggesting the interesting possibility that STAGA couples chromatin modification to various processes in gene expression and genome maintenance. We are interested in elucidating the function of the various components of STAGA and in characterizing other GCN5/PCAF complexes in human cells.

II. Control of genetic/epigenetic information and cell proliferation by the MYC oncoprotein.

Tight control of c-myc gene expression is critical for normal development and cell proliferation in response to growth stimuli and for self-renewal and differentiation of stem cells. Mutations that affect the expression levels and/or the amino acid sequence of the MYC oncoprotein are among the most commonly found in human cancers. MYC is a DNA/chromatin-binding transcription factor that interacts with a large fraction of the genome and regulates gene expression and chromatin structure to influence many biological processes including cell division, growth, pluripotency, differentiation, and apoptosis. MYC over-production or its unscheduled expression can be devastating and depending on the cell type and context (e.g. in conjunction with other genetic alterations) may lead to either cell death or immortalization and cell transformation. Experiments in mice have demonstrated that MYC overexpression induces a variety of tumors. We are interested in identifying and characterizing the cofactors and molecular mechanisms that mediate MYC functions in normal and cancer cells at the gene/chromatin level. We have recently shown an important function of the p300/CBP coactivator-HAT and the STAGA and Mediator complexes in MYC-dependent transcription activation of the human telomerase reverse transcriptase (hTERT) gene, a critical target of MYC immortalizing and transforming functions and are further characterizing the mechanisms involved (Fig. 1). Our laboratory has also shown that MYC is acetylated in mammalian cells at various lysine residues by the cofactors-HATs it recruits - i.e., by p300/CBP and by GCN5/STAGA (Zhang et al., 2005; Faiola et al., 2005). We are further investigating how acetylation affects MYC functions.

III. Role of chromatin structure, TAFs, and novel cofactors in core promoter selectivity.

The class II basal transcription machinery comprises RNA polymerase II (Pol II) and accessory “general” or basal transcription factors, i.e., TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, that are ubiquitous and interact with the core promoter region surrounding the transcription start site(s) of most class II genes (Fig.1). Until relatively recently it was thought that the core promoters of most eukaryotic genes function in a similar manner by merely serving as “landing pads” for the basal transcription machinery to specify the transcription initiation site. The general model has been that TFIID, through its TATA-binding protein (TBP) subunit binds first to the TATA box (or to a TATA-like sequence) thought to be present in the core promoter of most genes and then nucleates the assembly (i.e., recruitment) of the other general/basal transcription factors and Pol II into a stable complex that allows specific transcription initiation. Whether this is indeed the general mechanism for transcription initiation by Pol II at most eukaryotic genes remains unclear, but has been increasingly questioned. It has been known for quite some time that optimal induction of gene-selective transcription by distal enhancers often requires specific core promoter DNA sequences and that core promoter elements like the TATA box and/or the initiator (INR) element are not present in all genes. In fact, in collaboration with the labs of F. Sladek and T. Jiang at UCR, we have found that the consensus INR element (YYANWYY) is more frequent than the TATA box in the promoters of human and yeast genes. We have shown that the vast majority of human core promoters (~76%) not only lack the consensus TATA box sequence but also many other TATA-like AT-rich sequences (i.e., 532 different 8-mer DNA sequences) that form minor grooves compatible with the DNA-binding surface of the TBP molecular saddle. Our results have further indicated that the metazoan INR consensus sequence (YYANWYY) is also conserved in yeast and suggested that the INR element may influence transcription of almost half (40-46%) of the human and yeast genes. Our genome-wide computational and Gene Ontology analyses have revealed unexpected similarities in the frequency of specific core promoter types in yeast and humans and in the biological processes associated with the corresponding genes and have suggested that the process of transcription initiation and start site selection might be remarkably conserved in all eukaryotes (Yang et al., 2007). These and earlier results (Martinez et al. 1994, 1995, 1998) have challenged the idea that transcription initiation by Pol II is always dominated by the first recognition of the TATA box by TBP/TFIID and support the notion that specific transcription initiation at most eukaryotic genes (from yeast to human) is likely to rely on alternative pathways/mechanisms often involving the function of the INR element and other cofactors essential for INR function and TATA-independent transcription (i.e., TICs, see below). The lists of human and yeast genes/promoters having and lacking TATA and/or INR elements (from Yang et al., 2007) are available hereafter:

List of all human core promoter types with and without TATA and/or INR: Click here to see list

An unsolved question is: how is TFIID/TBP stably and functionally recruited to TATA-less promoters or to core promoters that are "masked" by nucleosomes or otherwise inaccessible? We (and others) have shown that TBP-associated factors (TAFs) can functionally interact with specific core promoter sequences (e.g., INR and other downstream promoter sequences) and are essential for basal transcription from TATA-less promoters. However, TAF-promoter interactions are not sufficient for functional TFIID recruitment to TAF/INR-dependent TATA-less promoters but additional TAF- and INR-dependent Cofactors (TICs) are required (Martinez et al., 1998). We are interested in identifying these TIC cofactors and the molecular mechanisms involved in INR-directed transcription from TAF-dependent TATA-less promoters.

Our research has been funded by grants from the University of California Cancer Research Committee (Project I.), the NIH Cancer Institute (Project II.) and NSF (Project III.). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of any of the above Granting Agencies.

Liu, X., Vorontchikhina, M., Wang, Y.-L., Faiola, F. and Martinez, E. (2007) STAGA recruits Mediator to the MYC oncoprotein to stimulate transcription and cell proliferation. Mol. Cell. Biol. In Press.

Faiola, F., Wu, Y.-T., Pan, S., Zhang, K., Farina, A. and Martinez, E. (2007) Max is acetylated by p300 at several nuclear localization residues. Biochem. J., 403, 397-407.

Yang, C., Bolotin, E., Jiang, T., Sladek, F. and Martinez, E. (2007) Prevalence of the Initiator over the TATA box in human and yeast genes and identification of DNA motifs enriched in human TATA-less core promoters. Gene, 389, 52-65. (doi:10.1016/j.gene.2006.09.029)

Faiola, F., Liu, X., Lo, S.-Y., Pan, S., Zhang, K., Lymar, E., Farina, A. and Martinez , E. (2005) Dual regulation of c-Myc by p300 via acetylation-dependent control of Myc protein turnover and coactivation of Myc-induced transcription. Mol. Cell. Biol., 25, 10220-10234.

Zhang, K., Faiola, F. and Martinez, E. (2005) Six lysine residues on c-Myc are direct substrates for acetylation by p300. Biochem. Biophys. Res. Commun., 336, 274-280.

Farina, A., Faiola, F. and Martinez , E. (2004) Reconstitution of an E box-binding Myc:Max complex with recombinant full-length proteins expressed in Escherichia coli. Protein Express. Purif., 34, 215-222.

Liu, X., Tesfai, J., Evrard, Y.A., Dent, S.Y.R., and Martinez, E. (2003) c-Myc transformation domain recruits the human STAGA complex and requires TRRAP and GCN5 acetylase activity for transcription activation. J. Biol. Chem., 278, 20405-20412.

Martinez , E. (2002) Multi-protein complexes in eukaryotic gene transcription. Plant Mol. Biol., 50, 925-947 .

Martinez, E., Palhan, V., Tjernberg, A., Lymar, E.S., Gamper, A.M., Kundu, T.K., Chait, B.T. and Roeder, R.G. (2001) Human STAGA complex is a chromatin-acetylating transcription coactivator that interacts with pre-mRNA splicing and DNA damage-binding factors in vivo. Mol. Cell. Biol., 21, 6782-6795.

Teichman, M., Wang, Z., Martinez, E., Tjernberg, A., Zhang, D., Vollmer, F., Chait, B.T. and Roeder, R.G. (1999) Human TATA-binding protein-related factor-2 (hTRF2) stably associates with hTFIIA in HeLa cells. Proc. Natl. Acad. Sci. USA, 96, 13720-13725.

Gu, W., Malik, S., Ito, M., Yuan, C.-X., Fondell, J.D., Zhang, X.L., Martinez , E., Qin, J. and Roeder, R.G. (1999) A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation. Mol. Cell, 3, 97-108.

Martinez , E., Kundu, T.K., Fu, J. and Roeder, R.G. (1998) A human SPT3-TAFII31-GCN5-L acetylase complex distinct from TFIID. J. Biol. Chem., 273, 23781-23785.

Martinez , E., Ge, H., Tao, Y., Yuan, C.-X., Palhan, V. and Roeder, R.G. (1998) Novel cofactors and TFIIA mediate functional core promoter selectivity by the human TAFII150-containing TFIID complex. Mol. Cell. Biol., 18, 6571-6583.

Tao, Y., Guermah, M., Martinez , E., Oelgeschläger, T., Hasegawa, S., Takada, R., Yamamoto, T., Horikoshi, M. and Roeder, R.G. (1997) Specific interactions and potential functions of human TAFII100. J. Biol. Chem., 272, 6714-6721.

Ge, H., Martinez , E., Chiang, C.-M. and Roeder, R.G. (1996) Activator-Dependent Transcription by Mammalian RNA Polymerase II: In Vitro Reconstitution with General Transcription Factors and Cofactors. In Methods in Enzymology (Edited by Sankar Adhya), Academic Press Inc.,Vol.274, 57-71.

Martinez, E., Zhou, Q., L'Etoile, N., Oelgeschläger, T., Berk, A.J. and Roeder, R.G. (1995) Core promoter-specific function of a mutant transcription factor TFIID defective in TATA box-binding. Proc. Natl. Acad. Sci. USA, 92, 11864-11868.

Martinez, E., Chiang, C.-M., Ge, H. and Roeder, R.G. (1994) TATA-binding protein-associated factor(s) in TFIID function through the initiator to direct basal transcription from a TATA-less class II promoter. EMBO J., 13, 3115-3126.

Martinez, E., Lagna, G. and Roeder, R.G. (1994) Overlapping transcription by RNA polymerases II and III of the Xenopus TFIIIA gene in somatic cells. J. Biol. Chem., 269, 25692-25698.

Martinez, E., Dusserre, Y., Wahli, W. and Mermod, N. (1991) Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor. Mol. Cell. Biol., 11, 2937-2945.

Wahli, W. and Martinez, E. (1991) Superfamily of steroid nuclear receptors : positive and negative regulators of gene expression. FASEB J., 5, 2243-2249.

Martinez, E. and Wahli, W. (1991) Characterization of hormone response elements. In Nuclear Hormone Receptors (Edited by M. G. Parker), Academic Press Limited, New York, London, pp 125-153.

Martinez, E., Givel, F. and Wahli, W. (1991) A common ancestor DNA motif for invertebrate and vertebrate hormone response elements. EMBO J., 10, 263-268.

Martinez, E. and Wahli, W. (1989) Cooperative binding of estrogen receptor to imperfect estrogen-responsive DNA elements correlates with their synergistic hormone-dependent enhancer activity. EMBO J., 8, 3781-3791.

Martinez, E., Givel, F. and Wahli, W. (1987) The estrogen-responsive element as an inducible enhancer : DNA sequence requirements and conversion to a glucocorticoid-responsive element. EMBO J., 6, 3719-3727.