James T. Kadonaga

Distinguished Professor
Section of Molecular Biology, UCSD

e-mail: jkadonaga@ucsd.edu

We are interested in fundamental aspects of transcription by RNA polymerase II and chromatin dynamics. The study of these remarkable and fascinating molecular processes has led to exciting adventures in science.

    Transcription by RNA polymerase II. In the area of transcription, we have focussed on the RNA polymerase II core promoter. The core promoter is the stretch of DNA (which typically encompasses -40 to +40 relative to the +1 start site) that directs the initiation of transcription. Although it is often presumed that core promoters function via a common mechanism to direct the initiation of transcription, it is now apparent that there is considerable diversity in core promoter structure and function.

    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 investigated 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. We have found that there are sharp differences in the properties of TATA-dependent versus DPE-dependent core promoters. For example, Caudal, which is a sequence-specific DNA-binding protein that is a master regulator of the homeotic (Hox) genes, is a DPE-specific activator. Thus, enhancer-core promoter specificity can be used to create gene regulatory networks.

    Our current research in transcription 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 dynamics. Chromatin has existed for at least several hundred million years; 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.

    In the area of chromatin dynamics, we have devoted considerable effort toward the analysis of chromatin assembly. 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 [ACF, NAP1, core histones, histone H1 (optional), DNA, and ATP].

    We are currently examining the mechanism of the chromatin assembly process. For instance, we have found that the ATP-utilizing motor protein, ACF, functions processively in the assembly of chromatin. We also found a second ATP-utilizing protein, CHD1, that participates in chromatin assembly. In addition, ACF and CHD1 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.

    Our studies of chromatin dynamics took an interesting turn in the course of our analysis of the HARP protein. HARP is in the same family of ATPases (the SNF2 family) as the chromatin assembly proteins ISWI (the ATPase subunit of ACF) and CHD1. In addition, mutations in HARP were known to be responsible for a rare human disorder known as Schimke immuno-osseous dysplasia (SIOD), which usually leads to death in early childhood. We therefore embarked on the analysis of HARP with the notion that it may catalyze chromatin remodeling in conjunction with a specific biochemical process, such as DNA repair. Instead, we discovered that HARP possesses a novel reverse (annealing) helicase activity--that is, HARP is a motorized molecular zipper that rewinds separated complementary strands of DNA. In this manner, we have identified a new DNA-modifying enzyme that opposes the DNA-unwinding activities of helicases and polymerases. Moreover, it is likely that the SIOD is caused by a deficiency of annealing helicase activity. Our adventures in science continue as we seek to identify additional annealing helicases as well as to understand the biological processes in which HARP is involved in the cell.

Kadonaga, J. T. (2004). Regulation of RNA polymerase II transcription by sequence-specific DNA-binding factors. Cell 116, 247-257.

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.

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.

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.

Juven-Gershon, T., Hsu, J.-Y., Theisen, J. W., and Kadonaga, J. T. (2008). The RNA polymerase II core promoter-- the gateway to transcription. Curr. Opin. Cell Biol. 20, 253-259

Hsu, J.-Y., Juven-Gershon, T., Marr, M. T., 2nd, Wright, K. J., Tjian, R., and Kadonaga, J. T. (2008). TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription. Genes Dev. 22, 2353-2358.

Juven-Gershon, T., Hsu, J.-Y., and Kadonaga, J. T. (2008). Caudal, a key developmental regulator, is a DPE-specific transcription factor. Genes Dev. 22, 2823-2830.

Yusufzai, T., and Kadonaga, J. T. (2008). HARP is an ATP-driven annealing helicase. Science 322, 748-750.

    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 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.