We are interested in fundamental aspects of transcription by RNA polymerase II, the role of chromatin structure in the regulation of transcription, and chromatin assembly and dynamics. These phenomena are remarkable and fascinating molecular processes. Transcription by RNA polymerase II. In the area of transcription, we have been studying the RNA polymerase II core promoter. The best-known core promoter motif is the TATA box. Yet, the TATA box is present in only about 10 to 15% of human genes. We therefore focussed on the transcription of TATA-less genes, and discovered two new core promoter motifs – the DPE and the MTE. Both the DPE and MTE are conserved from Drosophila to humans, and are located downstream of the transcription start site. Although it is generally presumed that core promoters function via a common mechanism to direct the initiation of transcription, we found that there are sharp differences in the properties of TATA-dependent versus DPE-dependent core promoters. For example, the coregulatory protein NC2 (negative cofactor 2; also known as Dr1-Drap1) is not only a repressor of TATA-dependent transcription (as known previously), but also an activator of DPE-dependent transcription (as we discovered). We also found that some transcriptional enhancers function specifically with core promoters that contain either a DPE or a TATA box. We are currently analyzing DPE-specific enhancers with the aim of learning more about how the DPE specificity is achieved. These studies should also shed new light on enhancer function in general. Enhancers were discovered over 20 years ago, but the molecular mechanism of their action remains largely a mystery. Our analysis of DPE-specific enhancers should provide extra ‘leverage’ (e.g., by enabling us to distinguish between specific and nonspecific effects) that will be useful in the study of enhancer function. Thus, our current research on the basic transcription process is focussed on the discovery and analysis of new core promoter motifs as well as the molecular basis of enhancer–core promoter specificity. Notably, the core promoter is not a generic element – rather, there are many different types of core promoters whose distinct functions are dictated by the presence or absence of sequence motifs such as the TATA, Inr, MTE, and DPE. Chromatin structure and transcriptional regulation. Chromatin has existed for several hundred million years, and therefore, DNA-utilizing factors have evolved to function optimally with chromatin rather than with plain (‘naked’) DNA. In the area of chromatin and transcription, we have used a primarily biochemical approach to study the transcriptional properties of chromatin templates. This work contributed to the current model that sequence-specific transcription factors function largely to counteract chromatin-mediated repression (antirepression hypothesis). We have found that the use of chromatin templates in vitro much more accurately recreates transcriptional phenomena as seen in vivo, relative to that seen with naked DNA templates in vitro. For instance, by using chromatin templates in vitro, we were able to achieve estrogen- and antiestrogen-regulated transcription by the human estrogen receptor. The success of these experiments was a direct consequence of the use of chromatin rather than naked DNA templates. More recently, we have been able to perform chromatin transcription reactions with a completely purified set of chromatin assembly factors and transcription factors. We anticipate that this defined chromatin transcription system will allow the analysis of a broad range of chromatin-based transcriptional phenomena in the future. Chromatin assembly and dynamics. A third area of focus in the lab is chromatin assembly and dynamics. Chromatin assembly has been a largely underappreciated phenomenon. Yet, it is important to consider that the replication and repair of our chromosomes requires both DNA synthesis and chromatin assembly. Thus, chromatin assembly is critically important for cell growth as well as for the maintenance of the integrity of the genome. Our studies of chromatin assembly began with the development of a crude extract from Drosophila embryos, termed the S-190, that is competent for ATP-dependent chromatin assembly. The S-190 extract was indeed very useful and effective, but we ultimately sought to assemble chromatin with purified factors rather than with a crude extract. We therefore embarked on the fractionation and purification of the essential components in the S-190 that catalyze chromatin assembly. This work eventually led to the purification and cloning of the minimal set of factors that can mediate the ATP-dependent assembly of periodic nucleosome arrays. Today, we are now able to assemble tens of milligrams of chromatin with purified recombinant factors. Our studies of chromatin assembly are largely focussed on the mechanism of the process. For instance, we have found that the ATP-utilizing motor protein, ACF, functions processively in the assembly of chromatin. We have also found a second ATP-utilizing protein, CHD1, that participates in chromatin assembly. ACF and CHD1 also function as chromatin remodeling enzymes – that is, they catalyze the movement of nucleosomes in an ATP-dependent manner. Chromatin assembly and remodeling are essential for the replication, organization, repair, and utilization of our chromosomes, and the analysis of these processes continues to be an exciting and stimulating journey. Selected papers on transcription by RNA polymerase II Butler, J. E. F., and Kadonaga, J. T. (2001). Enhancer-promoter specificity mediated by DPE or TATA core promoter motifs. Genes Dev. 15, 2515-2519. Butler, J. E. F., and Kadonaga, J. T. (2002). The RNA polymerase II core promoter: a key component in the regulation of gene expression. Genes Dev. 16, 2583-2592. Kadonaga, J. T. (2004). Regulation of RNA polymerase II transcription by sequence-specific DNA-binding factors. Cell 116, 247-257. Lim, C. Y., Santoso, B., Boulay, T., Dong, E., Ohler, U., and Kadonaga, J. T. (2004). The MTE, a new core promoter element for transcription by RNA polymerase II. Genes Dev. 18, 1606-1617. Juven-Gershon, T., Cheng, S., and Kadonaga, J. T. (2006). Rational design of a super core promoter that enhances gene expression. Nature Methods 3, 917-922. Selected papers on chromatin dynamics Fyodorov, D. V., and Kadonaga, J. T. (2002). Dynamics of ATP-dependent chromatin assembly by ACF. Nature 418, 897-900. Alexiadis, V., and Kadonaga, J. T. (2002). Strand pairing by Rad54 and Rad51 is enhanced by chromatin. Genes Dev. 16, 2767-2771. Fyodorov, D. V., Blower, M. D., Karpen, G. H., and Kadonaga, J. T. (2004). Acf1 confers unique activities to ACF/CHRAC and promotes the formation rather than disruption of chromatin in vivo. Genes Dev. 18, 170-183. Lusser, A., Urwin, D. L., and Kadonaga, J. T. (2005). Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly. Nat. Struct. Mol. Biol. 12, 160-166. Santoso, B., and Kadonaga, J. T. (2006). Reconstitution of chromatin transcription with purified components reveals a chromatin-specific repressive activity of p300. Nat. Struct. Mol. Biol. 13, 131-139. Jim Kadonaga was an undergraduate in Chemistry at MIT, where he received the American Institute of Chemists Certificate as well as the Alpha Chi Sigma Prize. He then carried out his graduate studies in the Department of Chemistry at Harvard University, where he was a DuPont Fellow. Jim was a postdoctoral associate at UC Berkeley as a Fellow of the Miller Institute for Basic Research in Science. He joined the faculty at UCSD in 1988, and is presently a Professor in the Section of Molecular Biology. |
|
