Transcriptional regulation and chromatin dynamics
We are interested in fundamental aspects of transcription by RNA polymerase II and chromatin dynamics. The analysis of these remarkable and fascinating molecular processes has led to many new and unexpected discoveries.
Transcription by RNA Polymerase II. In the area of transcription, we have focused on the RNA polymerase II core promoter. The core promoter is the stretch of DNA, which typically ranges from −40 to +40 relative to the +1 start site, that directs the initiation of transcription. Although it was often presumed that core promoters function via a common mechanism, it is now apparent that there is considerable diversity in core promoter structure and function.
We study core promoter motifs at three levels: DNA sequence motifs, protein factors, and networks. First, core promoters contain DNA sequence motifs such as the TATA box, Inr, MTE, DPE, and TCT elements. Note, however, that there are no universal core promoter motifs. Second, there are the protein factors that function specifically with the DNA sequence elements. Third, there are gene networks that are regulated by particular core promoter elements.
Depending on the presence or absence of specific core promoter elements, each core promoter has its own distinct properties. The best-known core promoter motif is the TATA box; however, the TATA box is present in only about 10 to 15% of human genes. In our studies of TATA-less promoters, we discovered two new core promoter motifs – the DPE and the MTE. Both the DPE and MTE are downstream of the transcription start site and are conserved from Drosophila to humans. It is interesting to note, for example, that the promoters of nearly all of the Drosophila homeotic (Hox) genes contain a DPE motif and lack a TATA box. [The promoters lacking a DPE motif are those associated with the evolutionarily most recent genes, Ubx and Abd-A. Hence, all of the more ancient Hox genes have TATA-less, DPE-containing core promoters.] Moreover, Caudal, a sequence-specific DNA-binding protein that is a master regulator of the Hox genes, is a DPE-specific activator. Thus, enhancer-core promoter specificity can be used in the regulation of gene networks.
We also study the TCT motif, which encompasses the transcription start site of nearly all ribosomal protein gene promoters in Drosophila and mammals. The TCT element resembles the initiator (Inr), but is not recognized by the canonical TFIID complex (which binds to the Inr) and cannot function in lieu of an Inr. Thus, the TCT motif is a novel transcriptional element that is distinct from the Inr. Strikingly, a single T-to-A nucleotide substitution converts the TCT element into a functionally active Inr. In other words, the difference between the TCT element and the Inr is a single key nucleotide.
The TCT-based RNA polymerase II transcription system is dedicated to the synthesis of ribosomal proteins and is complementary to the RNA polymerase I and RNA polymerase III transcription systems that are dedicated to the synthesis of rRNAs and tRNAs. We are further investigating the regulation of ribosomal protein gene transcription via the TCT motif. This work should lead to new insights into the control of ribosome biogenesis and cell growth.
The core promoter continues to be a rich source of new discoveries, which is consistent with the strategic position of the core promoter in the transcription process. In our analysis of the TCT motif, we saw that rare and highly specialized core promoter elements can be of immense biological importance. There are likely to be additional rare and undiscovered core promoter elements. Thus, our ongoing studies include the discovery and analysis of new core promoter motifs. We are also interested in the molecular basis of enhancer–core promoter specificity. 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, DPE, and TCT elements.
Chromatin Structure and Dynamics. Chromatin assembly is important for processes, such as DNA replication, transcription, and repair, that involve the disruption and reformation of chromatin.
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 useful and effective, but we ultimately sought to identify the specific factors that mediate chromatin assembly. 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 core set of factors that can mediate the ATP-dependent assembly of periodic nucleosome arrays. Now, we are able to assemble chromatin with purified recombinant factors [ACF or CHD1, NAP1, core histones, histone H1 (optional), DNA, and ATP].
We are currently examining the mechanism of the chromatin assembly process. In the assembly of extended nucleosome arrays on relaxed DNA under physiological conditions, ACF is required for the formation of nucleosomes in a process that requires the hydrolysis of ATP. We also found a second ATP-utilizing protein, CHD1, that functions to assemble nucleosomes. We are currently studying the mechanism of the assembly process. This work has led to a model in which histones are rapidly deposited onto the DNA to give non-nucleosomal histone-DNA complexes ("prenucleosomes"), which are converted into canonical nucleosomes in a processive manner by a motor protein such as ACF. We are interested in the function of different histone chaperones and motor proteins in this process as well as the mechanism by which ACF converts prenucleosomes to canonical nucleosomes.
Annealing Helicases: ATP-driven DNA-Rewinding Motor Proteins. Our studies of chromatin dynamics took an unexpected turn in the course our studies of the HARP protein. We were initially interested in HARP because it 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 found to be responsible for a rare human disorder known as Schimke immuno-osseous dysplasia (SIOD), which usually leads to death in early childhood. We thus 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. We found, however, that HARP does not exhibit chromatin remodeling activity. Instead, we discovered that HARP possesses a novel annealing (reverse) helicase activity. In other words, HARP is a motorized molecular zipper that rewinds complementary single strands of DNA that are stably bound by RPA (replication protein A, the major single-stranded DNA binding protein in the eukaryotic nucleus). Hence, we have identified a new DNA-modifying activity that opposes the DNA-unwinding activities of helicases and polymerases. In addition, we have found a second annealing helicase, which we term AH2, for annealing helicase 2. Unlike HARP, which forms a stable complex with RPA, AH2 does not bind to RPA. Intriguingly, AH2 has an HNH motif, which is a protein sequence that is typically found in nucleases. Thus far, however, we have not detected a nuclease activity in AH2, but it is possible that there are specific conditions in which a latent nuclease activity in AH2 becomes activated. Our current efforts are directed toward the identification and characterization of additional annealing helicases as well as the illumination of the biological processes in which HARP is involved in the cell.
Jim Kadonaga was an undergraduate in Chemistry at MIT, where he received the Alpha Chi Sigma Prize as well as the American Institute of Chemists Certificate. He then carried out his graduate studies with Jeremy R. Knowles in the Department of Chemistry at Harvard University, where he was a DuPont Fellow. Jim was a postdoctoral associate with Robert Tjian at UC Berkeley as a Fellow of the Miller Institute for Basic Research in Science, American Cancer Society, and Lucille P. Markey Charitable Trust. He joined the faculty at UCSD in 1988 and served as the Chair of the Section of Molecular Biology from 2003 to 2007. In 2012, he received the UCSD Chancellor's Associates Award for Excellence in Research in Science and Engineering. Jim is presently a Distinguished Professor in the Section of Molecular Biology, and serves as the Chair of the Graduate Program in Biological Sciences.