Laboratory of Biochemistry and Molecular Biology
Robert G. Roeder
Arnold and Mabel Beckman Professor
The processes of animal cell growth, differentiation and infection by viruses result from the differential expression of specific genes, controlled primarily at the level of transcription. Our broad objectives are to understand the specific regulatory events that control these processes, as well as more fundamental aspects of transcription activation and repression mechanisms. To this end, our specific objectives are to determine the nature and mechanism of action of both the general transcription initiation factors that are commonly used by all genes and the gene- and cell type-specific factors that directly regulate target genes in response to various growth, developmental and viral stimuli. Our experimental strategy has been (1) to reconstitute cell-free systems in which cloned genes, as free DNA or after assembly into chromatin, are accurately transcribed by general factors and regulated by gene-specific factors, (2) to dissect biochemically these systems and to purify and clone the individual factors and (3) to study the structure, function and regulation of these factors by a combination of biochemical and genetic (e.g., transgenic and knockout mice) analyses.
Since the ubiquitous general transcription factors represent the ultimate targets of the various gene-specific factors, their characterization is essential. The transcription of protein-coding genes involves RNA polymerase II and a number of common factors (TFIIA, -B, -D, -E, -F and -H) that form functional preinitiation complexes (on promoters) via an ordered assembly pathway. The multisubunit TFIID plays a key role in this process by virtue of its ability to bind directly to core promoter elements (notably the TATA box and initiator elements). Similarly, the transcription of small structural RNA genes (5S RNA, tRNA) by RNA polymerase III involves the assembly of common factors (TFIIIC and TFIIIB) into highly stable complexes via an ordered pathway. Most of the general factors (classes II and III) have been purified and individual subunits cloned for further structural and functional studies.
Some of the general factors have been shown to be direct targets for specific regulatory factors, while more recent studies have implicated a number of general and gene-specific cofactors in the function of gene-specific activators. The general cofactors include both negative cofactors (NC2, TAFs) that repress (basal) promoter activity in the absence of activators and positive cofactors (PC1, PC2, PC3 and PC4) that, in the presence of activators, reverse the action of negative cofactors and effect a net increase in overall promoter activity. Other positive cofactors that may function as activator-specific or core promoter-specific coactivators include the TAF subunits (around 14) associated with the small TATA-binding protein (TBP) in natural TFIID, novel polypeptides (SRBs, MEDs) in an RNA polymerase II interacting SMCC/Mediator complex, a B-cell-specific coactivator (OCA-B) that interacts with upstream DNA-binding factors (OCT-1, OCT-2) to activate lymphoid-specific promoters at late stages of B-cell differentiation, an OCA-B related factor (OCA-S) important for OCT-1-dependent activation of a histone gene in S-phase of the cell cycle and a large complex of coactivators (TRAPs) that interact with the thyroid hormone receptor (and other nuclear receptors) in a ligand-dependent way. The human SMCC/Mediator complex has recently been shown to be equivalent to the earlier-described TRAP complex and, through subunit specific interactions, to mediate the function of a variety of activators (including several nuclear receptors, the tumor suppressor p53, and the herpes virus activator VP16). These various classes of cofactors, as well as others (p300/CBP, STAGA) implicated in chromatin template modifications, offer important new mechanisms for gene regulation.
The specific genes and regulatory factors currently under analysis include viral genes (adenovirus, herpesvirus and HIV-1) activated by viral-coded (E1A, VP16 and Tat) and cellular (NFkB, SP1 and USF) factors, histone genes activated during the cell cycle (via OCT-1 and OCA-S), lymphoid-specific (immunoglobulin, other) genes activated during B-cell differentiation (via OCT-1, OCA-B, NFkB and other factors), genes activated during development and homeostatic responses by nuclear hormone receptors (thyroid hormone receptor, retinoic acid receptor, peroxisome proliferation-activated receptor, estrogen receptor and androgen receptor) in conjunction with the TRAP and other coactivators, cellular protooncogenes (cyclin D) activated by growth stimuli and genes activated or repressed by tumor suppressors (p53). Our studies are also directed toward the mechanisms by which these factors in turn are regulated in the various biological responses.