Biochemistry Department

Current Research in the Martinez Lab

Our lab’s 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 epigenetic coregulator complexes that link cell signaling and histone/chromatin modifications to regulation of the basal transcription machinery. Such human complexes discovered by us include the two distinct GCN5/PCAF acetylase complexes STAGA and ATAC.

(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-induced proliferation of “normal” cells (e.g., stem and progenitor cells) and cancer cells.

(iii)  Analysis of the basal transcription machinery and fundamental mechanisms controlling specific transcription by RNA polymerase II of genes with different core promoter structures, including the role of chromatin, TAFs, and novel cofactors (TICs) in core promoter-selective gene regulation.

A combination of cellular, molecular biological, biochemical, proteomics and genomics approaches are used, including the reconstitution of gene-specific transcription regulatory processes in vitro with purified native and/or recombinant transcription factors and chromatin components.

I. Structure, function, and regulation of mammalian GCN5/PCAF acetyltransferase complexes.
In eukaryotes, genomic DNA is packaged by histones into nucleosomes, the basic repeating units of chromatin, which further fold into higher order chromatin structures that often restrict sequence-specific protein-DNA interactions and hinder DNA-based enzymatic processes such as transcription, replication and repair. Specific epigenetic modifications on histones and DNA control the structure of chromatin into transcriptionally active (euchromatin) or repressive (heterochromatin) states. Eukaryotes have evolved two major enzymatic mechanisms to modify chromatin structure: (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 (or DNA), 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 (Fig.1).  Two main types of GCN5 complexes have been identified in yeast: the so-called ADA and SAGA complexes. In metazoans, GCN5 homologues (which include PCAF in mammals) are also incorporated into several distinct complexes, including SAGA-type complexes (e.g., mammalian STAGA) and metazoan-specific complexes called ATAC.

Figure 001 Image

Fig.1: Eukaryotic GCN5/PCAF complexes.
The identified protein components of the distinct types of complexes currently known in yeast (y), Drosophila (d) and humans (h), and the total estimated size of the complexes are indicated. Subunits indicated in parentheses are variably or less stably associated (e.g. salt concentration-dependent) in the respective complexes. Shaded blocks are groups of homologs specific of each type of complex. The GCN5-ADA3-STAF36/Sgf29 module is conserved in all complexes. HCF-1 is an abundant nuclear cofactor and promiscuous adaptor for several different coactivators; it was not detectable by LC-MS/MS in highly purified STAGA or ATAC, and thus may only be weakly associated (adapted from Wang et al., 2008).

Besides the HAT/coactivator roles of GCN5/PCAF and their ability to also modify (acetylate) many other proteins, including cancer-associated gene regulatory proteins (p53, MYC) and regulators of metabolic processes (PGC1 coactivators), the functions of the different GCN5/PCAF complexes remain poorly understood. The two mammalian homologues GCN5 and PCAF are differentially expressed during development. GCN5 is ubiquitously expressed and required for early embryogenesis, whereas PCAF is dispensable and expressed in a more tissue-restricted manner. However, human GCN5 and PCAF assemble into otherwise (so far) indistinguishable complexes of either SAGA-type or ATAC-type composition. Why should GCN5/PCAF HATs assemble into such large complexes? What are the functions of the many GCN5/PCAF-associated factors? Why do metazoans need several distinct GCN5/PCAF complexes? Are GCN5/PCAF complexes deregulated during neoplastic, metabolic and/or other disease processes? These and other questions related to the molecular, cellular and physiological activities of the GCN5/PCAF complexes remain to be answered. 

II. Cofactors and molecular mechanisms that regulate the functions of the MYC oncoprotein.
Tight control of c-myc gene expression and proper regulation of the activity of its product the MYC oncoprotein are critical for normal development and cell proliferation in response to growth stimuli and for self-renewal and differentiation of stem cells. Alterations that affect the expression levels and/or activity of the MYC oncoprotein are among the most commonly found in human cancers. For instance, MYC expression is deregulated in ~100% Bukitt lymphoma and in 40-70% of all other human cancers. MYC is a DNA/chromatin-binding transcription factor that interacts with a large fraction of the genome, influences epigenetic marks on chromatin, and regulates gene expression to influence many biological processes including cell division, growth, differentiation, and apoptosis. In addition, MYC is one of four factors sufficient to reprogram adult somatic cells into pluripotent embryonic-like stem cells. MYC over-production or its unscheduled expression or activity 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 deregulation induces a variety of tumors and its de-activation often leads to tumor regression. We are interested in identifying and characterizing the cofactors and molecular mechanisms that mediate MYC functions in normal and cancer cells at the genetic and epigenetic levels. We hypothesize that the oncogenic activities of deregulated MYC might result (in part) from its unrestrained or altered activities in both gene transcription and modification of chromatin structure and epigenetic information, the latter affecting other DNA proccesses besides transcription.

Our lab has identified coregulators (such as p300/CBP, STAGA and Mediator) that mediate the chromatin-modification and transcription stimulatory functions of MYC on specific target genes and shown that both MYC and its partner MAX are reversibly acetylated in mammalian cells by some of these coactivator-HATs. How differential MYC/MAX acetylation by these different cofactors affect MYC functions is an area of ongoing investigation in our lab. We have also begun to decipher the detailed molecular mechanisms by which MYC activates transcription of specific genes in cancer cells and identified the direct interplay of STAGA and Mediator coregulator complexes in MYC-dependent activation of the human telomerase reverse transcriptase (hTERT) gene in human cervical carcinoma cells  (Fig.2).

Fiqure 2 Image

Fig. 2: Model for the interplay of various coregulator complexes during MYC-dependent transcription activation of the human TERT promoter (adapted from Liu et al., 2008). MYC coregulators (p300, STAGA, and Mediator) are colored in blue. The basal transcription machinery is depicted in yellow and a nucleosome in schematized in green (DNA is the grey line). Bold dashed lines indicate protein-protein interactions necessary for MYC-dependent recruitment of Mediator and activation of TERT transcription in human cervical cancer cells. Light dashed lines indicate possible additional interactions of Mediator with other components of the transcription machinery, which are insufficient for Mediator recruitment. The thin arrows pointing to “Ac” indicate acetylation by GCN5 and p300 of histones and MYC/MAX. The thick blue arrow indicates a transcription activating function of Mediator that is distinct from TFIID and RNA polymerase II recruitment to the core promoter.

III. Core promoter-selective transcription by RNA polymerase II
RNA polymerase II is the enzyme that transcribes all protein-coding genes and many non-coding regions of the genome. It is now well established that specific transcription initiation by RNA polymerase II requires accessory “general/basal transcription initiation factors” (GTFs) that recognize the core promoter region of genes and is modulated by sequence-specific DNA-binding “regulators” that bind to promoter-proximal or distal regulatory and enhancer regions. The regulators, with the help of “coregulators”, ultimately modulate the assembly and/or function of the basal transcription machinery at the core promoter. How this is accomplished is only partially understood and is core promoter structure/context-dependent. Thus, a complete understanding of gene regulation at the transcriptional level requires the characterization of the core promoter-selective functions of the basal transcription machinery. This is investigated at the molecular level in our lab by reconstituting specific transcription in vitro with purified factors.

The general/basal transcription machinery comprises RNA polymerase II (Pol II) and six GTFs, 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 genes. 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 GTFs 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 (and other downstream promoter elements such as the DPE) 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 control transcription of almost half (40-46%) of human and yeast genes (Fig. 3).

Figure 3 Image

Fig. 3:  Frequencies of different core promoter types in human and yeast gene promoters. (A) Human gene promoters (10,271 total) from DBTSS were searched by scanning a 110 nt window within the -80 to +80 region relative to TSS (+1) for the existence of TATA (532 different sequences) and INR elements. TATA only, genes with a TATA box but no INR in the -80 to +80 region; TATA+INR group includes both genes/promoters having TATA and INR at a fixed orientation and distance from each other (TATA box 15 to 30 nt upstream of the INR, 211 genes, 2.1%) as well as genes with a TATA and INR in any orientation and spacing within the -80 to +80 region (1358 promoters, 13.2%); INR only, genes with an INR element but no TATA box; None, genes with neither a TATA box nor an INR element in the -80 to +80 region. (B) S. cerevisiae promoters (6165 genes) were searched for the presence of at least one TATA element (TATAWAWR) in the -150 to +1 region and/or one INR element in the -25 to +25 region and grouped into different promoter categories as in (A). Adapted from Yang et al., 2007.

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 our 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:

An unsolved question is: how is TFIID/TBP (and other GTFs) stably and functionally recruited to the large class of TATA-less promoters? 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 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 TATA-INR synergy and INR-directed transcription from TAF-dependent TATA-less promoters. We postulate that these TIC cofactors might actively convey the core promoter/gene-selective regulatory functions of enhancers.

In conclusion, because of the broad conservation of the Initiator (INR) element and the preponderance of Initiator-containing and TATA-less promoters in eukaryotes, the identification and characterization of novel TATA-less promoter-specific basal (co)factors (TICs) and cognate Initiator-dependent transcription mechanisms is critical to our understanding of the general and most fundamental mechanisms of eukaryotic gene regulation.

Acknowledgements and Disclaimer

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 (Projects I and 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.

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