Transcriptional Regulation and Chromatin Dynamics
We are interested in fundamental aspects of transcription by RNA polymerase II as well as chromatin assembly and dynamics. The analysis of these remarkable molecular processes has been a fascinating scientific journey.
Transcription by RNA Polymerase II. In the area of transcription, we have focused on the RNA polymerase II core promoter. The core promoter is often referred to as the 'gateway to transcription', as the consolidated output of the signals that control the activation of genes must pass through this 'gateway'. The core promoter is defined to be the stretch of DNA that directs transcription initiation, and it typically comprises sequences from about -40 to +40 nt relative to the +1 start site. It is now clearly evident that there is considerable diversity in the structure and activity of core promoters. There are a variety of DNA sequence elements, termed core promoter motifs (or elements), that can potentially be present in any given core promoter. These core promoter motifs include the TATA box, Initiator (Inr), TCT motif, downstream core promoter element (DPE), motif ten element (MTE), and others. There are no universal core promoter elements.
We study core promoter motifs at three levels: DNA sequence motifs, protein factors, and networks. First, there are the core promoter sequence motifs such as the TATA box, Inr, MTE, DPE, and TCT elements. 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. Moreover, we have found a new and distinct transcription system that is dedicated to the transcription of TCT-dependent core promoters. Much of our future efforts will be directed toward the identification and characterization of the factors in this new transcription system.
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. Together, these three specialized transcription systems yield most of the proteins and RNAs that are required for translation.
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 a rare and highly specialized core promoter element 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. Furthermore, for practical applications, we have developed synthetic "Super Core Promoters" that can be used for high levels of stable gene expression in metazoans. Hence, in conclusion, 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 Assembly 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 as the motor protein; NAP1 or other histone chaperone; core histones; histone H1 (optional); DNA; and ATP]. 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.
Our analysis of the mechanism of chromatin assembly led to the discovery of the prenucleosome, which is a novel non-nucleosomal histone-DNA particle that is an intermediate in the pathway leading to the formation of nucleosomes. In a rapid process (< 15 sec) that does not require a motor protein, core histones are deposited by a chaperone (such as NAP1) onto the DNA to give prenucleosomes. Then, in a slower step (~10 min), prenucleosomes are converted by an ATP-dependent motor protein (such as ACF or Chd1) into periodic nucleosome arrays. Unlike canonical nucleosomes, prenucleosomes do not supercoil DNA. Purified prenucleosomes contain all four core histones and can be converted into periodic nucleosome arrays by either ACF or Chd1 motor proteins. By atomic force microscopy, prenucleosomes are indistinguishable from nucleosomes. Thus, the existence of prenucleosomes resolves a nearly 40-year-old paradox in chromatin assembly – which is that nucleosome-like particles (as observed by electron microscopy) are rapidly formed within seconds at DNA replication forks, but canonical nucleosomes (as assessed by nuclease digestion assays) require about 10 minutes to be assembled. Now, we know that the rapidly-formed nucleosome-like particles at DNA replication forks are probably prenucleosomes. This conclusion is consistent with the rapid formation of prenucleosomes and their nucleosome-like appearance. The process that was previously known as "chromatin maturation" is likely to include the conversion of prenucleosomes ("immature" or "nascent" chromatin) into "mature" chromatin consisting of canonical nucleosomes.
Through the analysis of chromatin-remodeling-defective versions of Chd1, we further found that ATP-dependent chromatin assembly is functionally distinct from ATP-dependent chromatin remodeling. In other words, chromatin remodeling activity is not required for the ATP-dependent formation of nucleosomes. However, when nucleosomes are formed with chromatin-remodeling-defective Chd1 proteins, the resulting chromatin consists of randomly-distributed nucleosomes rather than periodic nucleosome arrays. Thus, both chromatin assembly and remodeling activities are required for the formation of regularly-spaced arrays of nucleosomes.
We are currently studying the structure and function of prenucleosomes as well as the process by which prenucleosomes are converted into canonical nucleosomes. In this work, we will examine the biochemical and biological characteristics of prenucleosomes. It is possible, for instance, that prenucleosomes are formed whenever chromatin disruption and reassembly occurs. We will also investigate the functions of specific core histone subregions as well as different histone chaperones in the formation and maturation of prenucleosomes. These studies should provide new insights into the disruption and reassembly of nucleosomes that occurs during transcription, replication, and DNA repair.
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-like 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 might 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, such as single DNA strands that are bound by RPA, the major single-stranded DNA-binding protein in the 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. 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. Since 2008, Jim has been serving as the Chair of the Graduate Program in Biological Sciences. In 2012, he received the UCSD Chancellor's Associates Award for Excellence in Research in Science and Engineering. Jim is presently the Amylin Endowed Chair and a Distinguished Professor in the Section of Molecular Biology.