Robert Tjian

介绍 Robert Tjian



HHMI INVESTIGATORS







The first person who have cloned the Eukaryotic transcription factor



Biomedical Scientists, Ranked by Total Citations

(Based on papers published and cited 1990-June 1997)

paper Total Citations

43 Robert Tjian HHMI, UC Berkeley 88 7870

the 1994 California Scientist of the Year.



Mechanisms of Gene Regulation in Animal Cells

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Robert Tjian, Ph.D.

Investigator,

University of California, Berkeley



Biography...





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Summary: Robert Tjian is interested in the biochemistry of gene regulation in metazoans. In particular, what is the nature of the molecular machinery that controls the turning up and down of gene expression in animal cells, and how does disruption of this highly regulated process lead to various disease states?



Our main research interest involves the means by which the genetic information stored in DNA molecules is retrieved in a controlled and orderly fashion during the biochemical process called transcription, which leads to the production of specific proteins in animal cells. We take a biochemical approach to the problem of gene control and have devised various means of isolating the individual components of the cell responsible for transcription and of reconstructing this complex reaction in the test tube. In this way, we can study how specific genes are turned on and off during cell growth and development of eukaryotic organisms. The mechanisms that govern the switching on and off of genes are of fundamental importance in understanding the normal metabolic processes that maintain and perpetuate living cells, as well as in deciphering the basis of disease and other cellular or genetic disorders.



Proteins That Regulate Gene Expression

Among the most important families of proteins in the nucleus are the sequence-specific transcription factors that bind to DNA at selected sites and regulate the expression of genes in a tissue-specific and developmentally regulated fashion. A significant technical advance was the development of biochemical methods that allow the purification of these rare and fragile transcription factors. Through the use of specific DNA-affinity chromatography procedures developed in this laboratory, it is now possible to isolate, in pure form, minute quantities of transcription proteins. This in turn allows us to isolate the genes that encode these regulatory proteins.



The ability to identify and manipulate this biologically important class of genes provides a powerful approach toward understanding their structure and function. These sequence-specific transcription factors not only regulate the temporal cascade of gene expression during development of multicellular organisms but also are often good candidates for genes that lead to cancer. For example, many of these transcription factors turn out to be either oncogenes or tumor-suppressor genes.



How Promoter-Specific Regulators Trigger Transcription

A fundamental question that was unanswered until recently is the mode of action by which sequence-specific DNA-binding proteins direct transcription. To address this critical issue, our group fractionated and isolated the multiple components necessary to reconstitute transcription in the test tube. In the process of dissecting the general transcriptional apparatus, we discovered previously undetected components that serve as the functional bridge between upstream regulatory proteins, such as the human transcription factor SP1, and the initiation complex that contains RNA polymerase. These novel factors we have called coactivators appear to be part of the missing link that directs promoter-selective transcription in animal cells and is likely to represent a diverse and essential class of regulatory proteins.



There has been significant progress in the purification, cloning, and characterization of transcriptional coactivators. The first class of coactivators we identified consist of the TATA-binding protein (TBP)-associated factors (TAFs), which make up the basal transcription complex called TFIID. Biochemical characterization of these factors revealed that transcriptional regulators can bind selective TAFII subunits of the TFIID complex. We have also successfully completed the assembly of transcriptionally active TBP-TAF complexes reconstituted from the recombinant subunits of TFIID. The specificity and function of individual TAFs mediating transcriptional activation remains to be determined, however, and we continue to make progress toward this goal. At the same time, we have begun to search for additional classes of coactivators.



Identification of TBP-Related Factors

Eukaryotic cells were originally thought to contain a single TBP that directs transcription by all cellular RNA polymerases. However, we recently identified two additional TBP-related factors, TRF-1 and TRF-2. Like TBP, the TRF molecules can interact with the basal transcription factors TFIIA and TFIIB, and TRF-1 has also been shown to direct RNA polymerase II transcription in vitro. The TRFs can also form a complex containing multiple associated subunits reminiscent of the TBP-TAF complexes. Antibody staining of embryos and polytene chromosomes reveals cell-type-specific expression and gene-selective properties consistent with the notion that the TRFs may direct transcription of specific subsets of genes in metazoans. These findings suggest TRFs are homologs of TBP that direct tissue- and gene-specific transcription. The discovery of multiple TFIID-like complexes suggests that, in animal cells, the molecular machinery regulating gene expression has become highly elaborated to accommodate the large number of genes active during development and differentiation.



Discovery and Characterization of a Cell-Type-Specific TAF

In addition to multiple TRFs, we have also discovered tissue- or cell-type-specific TAFs. Our recent in vitro biochemical and in vivo genetic analyses reveal that TAFII105 is selectively expressed in the granulosa cells of the mouse ovary. Deletion of this gene by homologous recombination results in female mice that are viable but infertile due to a defect in the development of follicle cells that are required to produce normal oocytes. By using a "gene chip" approach, we were able to identify a specific set of genes that are unable to be transcribed in the absence of TAFII105, thus leading to a defect in ovarian development. Our findings not only establish TAFII105 as a novel cell-type-specific component of the mammalian transcription machinery but may also help reveal the molecular basis of certain types of human infertility.



Isolation of CRSP and ARC, Multisubunit Transcriptional Cofactors

Previous studies showed that the interaction of the glutamine-rich activation domains of SP1 with the TAFII subunits of TFIID is important for transcriptional activation in vitro. These early studies used a partially purified transcription system, however, and did not address the question of whether other cofactors may be necessary to fully potentiate activation by SP1. To identify additional potential cofactors, we developed several types of in vitro transcription assays to detect coactivators. These studies identified two new human cofactor complexes, CRSP (cofactor required for SP1) and ARC (activator-recruited cofactor), that together with the TAFIIs help mediate transcriptional activation. Although these complexes share several common subunits, their structural and functional relationships remained unknown. We recently found that affinity-purified ARC consists of two distinct multisubunit complexes; a larger complex, denoted ARC-L, and a smaller coactivator, CRSP. Importantly, reconstituted in vitro transcription with biochemically separated ARC-L and CRSP reveals differential cofactor functions. The ARC-L complex is transcriptionally inactive; the CRSP complex is highly active. Structural determination by electron microscopy (EM) and three-dimensional reconstruction indicate that ARC-L and CRSP are substantially different in size and shape. Moreover, EM analysis of independently derived CRSP complexes reveals distinct conformations induced by different activators. These results suggest that CRSP may potentiate transcription via specific activator-induced conformational changes.



Integrating Transcription and Chromatin Transactions

The diversity of transcriptional cofactors that mediate gene activation or repression by promoter-specific proteins includes specialized protein complexes that are thought to help RNA polymerase navigate through chromatin. To examine how specificity is achieved, we have reconstructed an integrated transcription reaction with chromatin templates that recapitulates the tight promoter control and ligand dependence of nuclear receptor transactivation observed in vivo. This transcription system unmasked an activity in human nuclear fractions that strongly potentiates ligand-dependent transcription. Using this integrated chromatin-dependent transcription reaction, we have purified PBAF, a multisubunit cofactor that directs ligand-dependent transactivation by nuclear hormone receptors. By contrast, a highly related cofactor, human SWI/SNF, and the ISWI-containing chromatin-remodeling complex ACF both failed to potentiate transcription. We also found that transcriptional activation mediated by nuclear hormone receptors requires TAFs as well as the multisubunit cofactor CRSP. Thus, our studies demonstrate functional selectivity among highly related complexes involved in gene regulation and help define a more complete set of cofactors required to activate transcription.



A grant from the National Institutes of Health provided partial support for the work on SP1, CRSP, ARC and other human enhancer and basal transcription factors.