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James T. Kadonaga


Transcriptional Regulation and Chromatin Dynamics

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 decipher the mechanisms by which genes are regulated in animals.

Development of Sequence-specific DNA Affinity Chromatography & the Purification and Cloning of Transcription Factor Sp1

Prior to my arrival at UCSD, I was involved in early studies of eukaryotic sequence-specific DNA-binding transcriptional factors as a postdoctoral researcher in the laboratory of Robert Tjian at UC Berkeley. In this work, I developed sequence-specific DNA affinity chromatography for the purification of transcription factor Sp1 (Fig. 1). The purification of Sp1 enabled me to isolate its corresponding cDNA and to characterize its binding to DNA via three zinc finger motifs (Kadonaga et al., 1987). Sequence-specific DNA affinity chromatography has since been used by many investigators to purify numerous factors that function in a wide range of biological processes. The remainder of the work below was carried out in my lab at UCSD.

Sequence-Specific DNA Affinity Chromatography

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, as it is the site at which the signals that activate transcription ultimately converge.

Core promoters are diverse in terms of their structure and function. 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. 2). 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.

Core Promoters

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. We also seek to gain a better understanding of the known elements.

(2). Transcription factors. In Drosophila, 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 some transcriptional enhancers are specific for core promoters with DPE or TATA motifs (Fig. 3). More generally, we seek to understand the different sets of transcription factors that function at different types of core promoters.

A Role for Core Promoters in Enhancer Specificity

(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. 4). Notably, in Drosophila, 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. 5). It is important to understand the function of the core promoter in a broader biological context.

Core Promoter Motifs in Biological Networks Evolution of TRF2

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.

Early Studies of the Role of Chromatin in the Regulation of Transcription

We carried out early experiments on the role of chromatin structure in the regulation of transcription by RNA polymerase II. These studies were initiated in the late 1980s – this was a time when many people in the transcription field felt that chromatin was unimportant. We were primarily motivated to perform this work because chromatin is the natural state of DNA in the eukaryotic nucleus. We found that sequence-specific DNA-binding transcription factors act primarily to counteract chromatin-mediated repression – we termed this phenomenon "antirepression" (Laybourn and Kadonaga, 1991, 1992; Paranjape et al., 1994). This paradigm was in contrast to the prevailing belief that sequence-specific factors increase the rate of the transcription reaction in a manner that is independent of chromatin structure. These studies of chromatin and transcription led to the model for transcriptional activation that is shown in Fig. 6 (Pazin et al., 1994). In this project, we were also able to achieve ligand-regulated transcription in vitro by the estrogen receptor by using chromatin templates (Kraus and Kadonaga, 1998). Today, it is generally accepted that sequence-specific activators counteract chromatin-mediated repression of basal (unactivated) transcription, and chromatin is extensively studied as

Model for steps leading to the activation of transcription

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 and purification of the essential components in the S-190 extract. This work included the discovery, purification, and cloning of ACF (ATP-utilizing chromatin assembly and remodeling factor; Ito et al., 1997, 1999) and the discovery that Asf1 is a chromatin assembly factor that serves as a chaperone for newly synthesized histones H3 and H4 (Tyler et al., 1999). The analysis of the mechanism of the ATP-dependent assembly of periodic nucleosome arrays led to the model shown in Fig. 7.

Chromatin assembly

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. 8). 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.

Prenucleosomes summary slide

Multiple independent lines of evidence suggest that prenucleosomes or prenucleosome-like particles are at active chromatin such as promoters (Fig. 9). 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 are observed at active chromatin. In other words, prenucleosomes are generated in dynamic chromatin at which there is active disassembly and reassembly of nucleosomes.

Prenucleosomes at active promoters

Annealing Helicases

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. 10). 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. More generally, in these studies, we have identified a DNA structure-modifying activity that acts in opposition to the DNA-unwinding activities of helicases and polymerases.

Model of Annealing Helicases

NDF, a Nucleosome Destabilizing Factor that Facilitates Transcription through Nucleosomes

Our studies of prenucleosomes led us to examine factors that mediate the destabilization or disassembly of conventional nucleosomes. To this end, we devised a nucleosome destabilization assay that was based on the ability of p300 to acetylate histone H3K56 in a partially unwrapped nucleosome. By using this assay, we identified a nucleosome destabilizing activity in Drosophila embryo extracts, and then purified this activity by conventional chromatography. This work led to the identification of a factor that we term NDF, for nucleosome destabilizing factor. NDF is present in most animals, and perhaps in plants, but it does not appear to have a homolog in Saccharomyces cerevisiae.

NDF has a conserved PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner, and facilitates transcription by RNA polymerase II through nucleosomes in a purified and defined transcription system as well as in cell nuclei (Fig. 11). Upon transcriptional induction, NDF is recruited to the transcribed regions of thousands of genes and co-localizes with a subset of H3K36me3-enriched regions (Fig. 12). Notably, the recruitment of NDF to gene bodies is accompanied by an increase in the transcript levels of many of the NDF-enriched genes. In addition, the global loss of NDF results in a decrease in the RNA levels of many genes. In humans, NDF is present at high levels in all tested tissue types, is essential in stem cells, and is frequently overexpressed in breast cancer. These findings indicate that NDF is a nucleosome destabilizing factor that is recruited to gene bodies during transcriptional activation and facilitates RNA polymerase II transcription through nucleosomes.

The properties of NDF indicate that it has a key role in the transcription of chromatin by RNA polymerase II in animals. The further analysis of NDF should reveal additional factors and mechanisms that orchestrate the procession of RNA polymerase II through active genes.

NDF, a nucleosome destabilizing factor NDF is recuited to gene bodies upon transcriptional induction

Select Publications

  • Fei, J., Ishii, H., Hoeksema, M. A., Meitinger, F., Kassavetis, G. A., Glass, C. K., Ren, B., and Kadonaga, J. T. (2018). NDF, a nucleosome destabilizing factor that facilitates transcription through nucleosomes. Genes Dev. 32, 682-694. PMCID: PMC6004073
  • Khuong, M.T., Fei, J., Cruz-Becerra, G., and Kadonaga, J.T. (2017). A simple and versatile system for the ATP-dependent assembly of chromatin. J. Biol. Chem. 292, 19478-19490. PMCID: PMC5702684
  • Vo ngoc, L., Wang, Y.-L., Kassavetis, G.A., and Kadonaga, J.T. (2017). The punctilious RNA polymerase II core promoter. Genes Dev. 31, 1289-1301. PMCID: PMC5580651
  • Vo ngoc, L., Cassidy, C.J., Huang, C.Y., Duttke, S.H.C., and Kadonaga, J.T. (2017). The human initiator is a distinct and abundant element that is precisely positioned in focused core promoters. Genes Dev. 31, 6-11. PMCID: PMC5287114
  • Fei, J., Torigoe, S.E., Brown, C.R., Khuong, M.T., Kassavetis, G.A., Boeger, H., and Kadonaga, J.T. (2015). The prenucleosome, a stable conformational isomer of the nucleosome. Genes Dev.  29, 2563-2575. PMCID: PMC4699385
  • Khuong, M.T., Fei, J., Ishii, H., and Kadonaga, J.T. (2015). Prenucleosomes and active chromatin. Cold Spring Harbor Symp. Quant. Biol.  80, in press. doi: 10.1101/sqb.2015.80.027300
  • Duttke, S.H.C., Lacadie, S.A., Ibrahim, M.M., Glass, C.K., Corcoran, D.L., Benner, C., Heinz, S., Kadonaga, J.T.‡, and Ohler, U.‡ (2015). Human promoters are intrinsically directional. Mol. Cell  57, 674-684. ‡Co-corresponding authors. PMCID: PMC4336624
  • Ishii, H., Kadonaga, J. T. ‡, and Ren, B. ‡ (2015). MPE-seq, a new method for the genome-wide analysis of chromatin structure. Proc. Natl. Acad. Sci. USA   112, E3457-E3465. ‡Co-corresponding authors. PMCID: PMC4500276
  • Duttke, S. H. C., Doolittle, R. F., Wang, Y.-L., and Kadonaga, J. T. (2014). TRF2 and the evolution of the bilateria. Genes Dev.  28, 2071-2076. PMCID: PMC4180970
  • Wang, Y.-L., Duttke, S. H. C., Chen, K., Johnston, J., Kassavetis, G. A., Zeitlinger, J., and Kadonaga, J. T. (2014). TRF2, but not TBP, mediates the transcription of ribosomal protein genes. Genes Dev.  28, 1550-1555. PMCID: PMC4102762
  • Torigoe, S. E., Patel, A., Khuong, M. T., Bowman, G. D., and Kadonaga, J. T. (2013). ATP-dependent chromatin assembly is functionally distinct from chromatin remodeling. eLife  2, e00863. PMCID: PMC3748710
  • Kadonaga, J. T. (2012). Perspectives on the RNA polymerase II core promoter. WIREs Dev. Biol.  1, 40-51. PMCID: PMC3695423
  • Torigoe, S. E, Urwin, D. L., Ishii, H., Smith, D. E., and Kadonaga, J. T. (2011). Identification of a rapidly formed non-nucleosomal histone-DNA intermediate that is converted into chromatin by ACF. Mol. Cell  43, 638-648. PMCID: PMC3160715
  • Yusufzai, T., and Kadonaga, J. T. (2010). Annealing helicase 2 (AH2), a DNA-rewinding motor with an HNH motif. Proc. Natl. Acad. Sci. USA  107, 20970-20973. PMCID: PMC3000258
  • Parry, T. J., Theisen, J. W. M., Hsu, J.-Y., Wang, Y.-L., Corcoran, D. L., Eustice, M., Ohler, U., and Kadonaga, J. T. (2010). The TCT motif, a key component of an RNA polymerase II transcription system for the translational machinery. Genes Dev.  24, 2013-2018. PMCID: PMC2939363
  • Juven-Gershon, T., and Kadonaga, J. T. (2010). Regulation of gene expression via the core promoter and the basal transcriptional machinery. Dev. Biol. 339, 225-229. PMCID: PMC2830304
  • Yusufzai, T., Kong, X., Yokomori, K., and Kadonaga, J. T. (2009). The annealing helicase HARP is recruited to DNA repair sites via an interaction with RPA. Genes Dev.  23, 2400-2404. PMCID: PMC2764493
  • Rattner, B. P., Yusufzai, T., and Kadonaga, J. T. (2009). HMGN proteins act in opposition to ATP-dependent chromatin remodeling factors to restrict nucleosome mobility. Mol. Cell  34, 620-626. PMCID: PMC2709789
  • Yusufzai, T., and Kadonaga, J. T. (2008). HARP is an ATP-driven annealing helicase. Science  322, 748-750. PMCID: PMC2587503
  • 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. PMCID: PMC2569877
  • 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. PMCID: PMC2532932
  • Santoso, B., and Kadonaga, J. T. (2006). Reconstitution of chromatin transcription with purified components reveals a chromatin-specific repressive activity of p300. Nature Struct. Mol. Biol. 13, 131-139.
  • Lusser, A., Urwin, D. L., and Kadonaga, J. T. (2005). Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly. Nature Struct. Mol. Biol. 12, 160-166.
  • 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.
  • Kadonaga, J. T. (2004). Regulation of RNA polymerase II transcription by sequence-specific DNA-binding factors. Cell 116, 247-257.
  • 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.
  • Alexiadis, V., and Kadonaga, J. T. (2002). Strand pairing by Rad54 and Rad51 is enhanced by chromatin. Genes Dev. 16, 2767-2771.
  • 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.
  • Fyodorov, D. V., and Kadonaga, J. T. (2002). Dynamics of ATP-dependent chromatin assembly by ACF. Nature 418, 897-900.
  • 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.
  • Willy, P. J., Kobayashi, R., and Kadonaga, J. T. (2000). A basal transcription factor that activates or represses transcription. Science 290, 982-984.
  • Tyler, J. K., Adams, C. R., Chen, S.-R., Kobayashi, R., Kamakaka, R. T., and Kadonaga, J. T. (1999). The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402, 555-560.
  • Ito, T., Levenstein, M. E., Fyodorov, D. V., Kutach, A. K., Kobayashi, R., and Kadonaga, J. T. (1999). ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly. Genes Dev. 13, 1529-1539.
  • Blackwood, E. M., and Kadonaga, J. T. (1998). Going the distance: a current view of enhancer action. Science 281, 60-63.
  • Kraus, W. L., and Kadonaga, J. T. (1998). p300 and estrogen receptor cooperatively activate transcription via differential enhancement of initiation and reinitiation. Genes Dev. 12, 331-342.
  • Burke, T. W., and Kadonaga, J. T. (1997). The downstream core promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila. Genes Dev. 11, 3020-3031.
  • Ito, T., Bulger, M., Pazin, M. J., Kobayashi, R., and Kadonaga, J. T. (1997). ACF, an ISWI-containing and ATP-utilizing chromatin assembly and remodeling factor. Cell 90, 145-155.
  • Pazin, M. J., and Kadonaga, J. T. (1997). What's up and down with histone deacetylation and transcription? Cell 89, 325-328.
  • Pazin, M. J., Bhargava, P., Geiduschek, E. P., and Kadonaga, J. T. (1997). Nucleosome mobility and the maintenance of nucleosome positioning. Science 276, 809-812.
  • Pazin, M. J., and Kadonaga, J. T. (1997). SWI2/SNF2 and related proteins: ATP-driven motors that disrupt protein-DNA interactions? Cell 88, 737-740.
  • Burke, T. W., and Kadonaga, J. T. (1996). Drosophila TFIID binds to a conserved downstream basal promoter element that is present in many TATA-box-deficient promoters. Genes Dev. 10, 711-724.
  • Pazin, M. J., Kamakaka, R. T., and Kadonaga, J. T. (1994). ATP-dependent nucleosome reconfiguration and transcriptional activation from preassembled chromatin templates. Science 266, 2007-2011.
  • Paranjape, S. M., Kamakaka, R. T., and Kadonaga, J. T. (1994). Role of chromatin structure in the regulation of transcription by RNA polymerase II. Annu. Rev. Biochem. 63, 265-297.
  • Kamakaka, R. T., Bulger, M., and Kadonaga, J. T. (1993). Potentiation of RNA polymerase II transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly. Genes Dev. 7, 1779-1795.
  • Tyree, C. M., George, C. P., Lira-DeVito, L. M., Wampler, S. L., Dahmus, M. E., Zawel, L., and Kadonaga, J. T. (1993). Identification of minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II. Genes Dev. 7, 1254-1265.
  • Laybourn, P. J., and Kadonaga, J. T. (1992). Threshold phenomena and long-distance activation of transcription by RNA polymerase II. Science 257, 1682-1685.
  • Laybourn, P. J., and Kadonaga, J. T. (1991). Role of nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase II. Science 254, 238-245.
  • Croston, G. E., Kerrigan, L. A., Lira, L., Marshak, D. R., and Kadonaga, J. T. (1991). Sequence-specific antirepression of histone H1-mediated inhibition of basal RNA polymerase II transcription. Science 251, 643-649.
  • Kamakaka, R. T., Tyree, C. M., and Kadonaga, J. T. (1991). Accurate and efficient RNA polymerase II transcription with a soluble nuclear fraction derived from Drosophila embryos. Proc. Natl. Acad. Sci. USA 88, 1024-1028.
  • Kadonaga, J. T., Courey, A. J., Ladika, J., and Tjian, R. (1988). Distinct regions of Sp1 modulate DNA binding and transcriptional activation. Science 242, 1566-1570.
  • Kadonaga, J. T., Carner, K. R., Masiarz, F. R., and Tjian, R. (1987). Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 51, 1079-1090.
  • Kadonaga, J. T., and Tjian, R. (1986). Affinity purification of sequence-specific DNA binding proteins. Proc. Natl. Acad. Sci. USA 83, 5889-5893.


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 UCSD faculty in 1988 and was one of 15 scientists to be named as a Presidential Faculty Fellow by President George H.W. Bush in 1992. In 1994, he was elected to Fellow of the American Association for the Advancement of Science, and in 1995, he was elected to Fellow of the American Academy of Microbiology. He 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. In 2017, he was elected to the American Academy of Arts & Sciences. Jim is presently the Amylin Endowed Chair and a Distinguished Professor in the Section of Molecular Biology. Profile in J. Cell Biol.