A diverse range of biological phenomena, including many human diseases, can be traced to the proper or improper expression of a gene. New insights into the mechanisms of gene expression will have a broad impact upon science and society.
The objective of our work is to understand the regulation of RNA polymerase II transcription. We study the transcription process as well as the chromatin template to illuminate the mechanisms by which genes are regulated in animals.
The RNA Polymerase II Core Promoter
In the area of transcription, we have focused on the RNA polymerase II core promoter. The core promoter is the stretch of DNA that directs transcription initiation, and it typically comprises sequences from about -40 to +40 nt relative to the +1 transcription start site. The core promoter is the gateway to transcription – it is the site at which the activating signals ultimately converge.
Core promoters are diverse in terms of their structure and activity. The transcriptional activity of core promoters is driven by DNA sequence motifs such as the TATA box, initiator (Inr), TCT motif, downstream core promoter element (DPE), motif ten element (MTE), and others (Fig. 1). Any particular core promoter will have one or more core promoter motifs. There are, however, no universal core promoter elements. In addition, there are core promoter motifs that have yet to be discovered.
We study core promoters at three levels: DNA sequence motifs, transcription factors, and biological networks.
(1). DNA sequence motifs. We have identified and characterized new core promoter elements, such as the DPE and MTE, and articulated the nature and properties of the TCT motif. We are continuing to search for new core promoter elements as well as to gain a better understanding of the functions of the known elements.
(2). Transcription factors. We have found that TATA-less transcription with TCT-dependent core promoters does not require TBP (TATA box-binding protein), but is instead dependent upon TRF2 (TBP-related factor 2). We also discovered that Caudal protein (a DNA-binding transcription factor that is a key regulator of the Hox genes) is a DPE-specific activator. This finding is a specific example of the concept that sometranscriptional enhancers are specific for core promoters with DPE or TATA motifs (Fig. 2). More generally, we seek to understand the different sets of transcription factors that function at different types of core promoters.
(3). Biological networks. We found that some core promoter motifs have specific functions in biological networks – the DPE with the Hox gene network and the TCT motif with the ribosomal protein gene network (Fig. 3). Notably, both the DPE and TCT motifs function with TRF2 rather than TBP. Thus, these findings have also led us to investigate the evolution of TRF2 (Fig. 4). It is important to understand the function of the core promoter in a broader biological context.
Our current studies include the identification and analysis of new core promoter motifs and structures, particularly in humans. We are also investigating the distinct mechanisms of transcription from different types of core promoters as well as the basis of enhancer-core promoter specificity. The core promoter continues to be a rich source of new discoveries.
Chromatin Assembly and Dynamics
Chromatin assembly is important for processes, such as DNA replication, transcription, and repair, that involve the disruption and reformation of nucleosomes. 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. We then undertook the biochemical fractionation of the essential components in the S-190 extract. 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
With purified and defined factors, we have been studying the mechanism of chromatin assembly. This work led to the discovery of the prenucleosome, which is a stable conformational isomer of the nucleosome that associates with approximately 80 bp DNA (Fig. 5). Prenucleosomes are formed rapidly upon the deposition of histones onto DNA, and can be converted into canonical nucleosomes by an ATP-driven chromatin assembly factor such as ACF. Moreover, p300 acetylates histone H3K56 in prenucleosomes but not in nucleosomes.
Multiple independent lines of evidence suggest that prenucleosomes or prenucleosome-like particles are at active chromatin such as enhancers and promoters (Fig. 6). First, there is a strong correlation between psoralen-crosslinked species observed with prenucleosomes assembled in vitro and the active yeast PHO5 promoter in vivo. Second, chromatin particles with prenucleosome-sized DNA and core histones are observed at the upstream region of active promoters in mouse cells. Third, prenucleosomes, but not nucleosomes, can be acetylated at H3K56 by p300, and H3K56 acetylation occurs at active promoters and enhancers in yeast, Drosophila, and humans. It is possible that the transcriptional machinery is optimized to function in conjunction with a prenucleosome. More generally, prenucleosomes or prenucleosome-like particles might be generated at dynamic chromatin.
Our studies of chromatin dynamics took an unexpected turn in the course our studies of the HARP protein (also known as SMARCAL1). We were 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). We thus embarked on the analysis of HARP with the notion that it might alter chromatin structure; however, HARP did 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 DNA, such as single DNA strands that are bound by RPA, the major single-stranded DNA-binding protein in the nucleus (Fig. 7). In addition, we found a second annealing helicase termed annealing helicase 2 (AH2, also known as ZRanB3). Unlike HARP, which forms a stable complex with RPA, AH2 does not bind to RPA. In these studies, we have identified a new DNA-modifying activity that acts in opposition to the DNA-unwinding activities of helicases and polymerases.
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 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 California Division, 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. Jim also served as the Chair of the Graduate Program in Biological Sciences from 2008 to 2014. 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.