Professor of MCD Biology
B.A. UC Berkeley
Ph.D. UC San Francisco
Postdoctorate, Harvard Medical School
Chromatin and Transcription
Eukaryotes store their DNA in the nucleus as a protein-DNA complex called chromatin. The basic repeating unit of chromatin is the nucleosome, which consists of 160 basepairs of DNA wrapped around an octamer of histones. The spatial arrangement of nucleosomes on a gene, i.e. its chromatin structure, influences its transcription by modulating the ability of regulatory proteins to access specific DNA sequences and by modulating therate at which RNA polymerase travels down genes. We are interested in understanding the role chromatin plays in gene expression and the mechanisms by which chromatin structure is manipulated to regulate transcription.
We study this problem in the yeast, Saccharomyces cerevisiae, using biochemistry and genetics. We focus on two proteins, Spt4 and Spt5, that form a complex and appear to modulate transcription by interacting with chromatin. Genetic analyses of Spt4 and Spt5 have led us to propose that they help remove nucleosomes from the path of transcribing RNA polymerase II and then reassemble nucleosomes after polymerase has moved on down the template. Thus we believe that the Spt4-Spt5 complex both facilitates transcription by removing a nucleosomal barrier to transcript elongation and also suppresses inappropriate transcription by reassembling nucleosomes behind transcribing polymerases.
Consistent with our model, we have found that the Spt4-Spt5 complex associates with RNA polymerase II, and that it is recruited to transcribed regions of genes in cells. The human homologs of Spt4 and Spt5 also play a role in transcription elongation and furthermore, may play an important role in regulating HIV transcription.
To further elucidate Spt4-Spt5 complex function, we affinity purifyied Spt5 from wildtype and mutant yeast strains to identify proteins that interact with Spt5 under different transcription states. Among the proteins that copurify with Spt5 are the members of the Paf1 complex, Spt6, FACT and Chd1. All of these proteins are implicated in transcription elongation and chromatin structure. Of particular interest is the Paf1 complex, which is required for several specific histone methylation events.
Our identification of proteins that copurify with Spt5 also suggest intriguing links to pre-mRNA processing. For example, the proteins responsible for adding the cap structure to the 5' end of mRNAs co-purified with Spt5. This result suggests that Spt5 may also play a role in pre-mRNA processing in addition to transcription elongation. Consistent with this idea, we have found that spt5 mutations cause splicing defects. In a collaboration with Manny Ares, we have used splicing-sensitive DNA microarrays to monitor splicing, on a genome-wide basis, in more than 100 different mutants defective for all aspects of gene expression. Surprisingly, we observe that mutations affecting proteins in a diverse set of processes alter splicing. This suggests that splicing is coupled to a diverse set of processes, including transcription elongation.
Xiao Y, Yang YH, Burckin TA, Shiue L, Hartzog GA, Segal MR.
Analysis of a splice array experiment elucidates roles of chromatin elongation factor spt4-5 in splicing. PLoS Comput Biol. 2005 Sep;1(4):e39.
Burckin T, Nagel R, Mandel-Gutfreund Y, Shiue L, Clark TA, Chong JL, Chang TH, Squazzo S, Hartzog G, Ares M Jr. Exploring functional relationships between components of the gene expression machinery. Nat Struct Mol Biol. 2005 Feb;12(2):175-82.
Simic R., Lindstrom D.L., Tran H.G., Roinick K.L., Costa P.J., Johnson A.D., Hartzog G.A. and Arndt K.M. The Saccharomyces cerevisiae chromatin-modifying protein Chd1 localizes to the transcribed reg, ions of genes and interacts with transcription elongation factors. EMBO J. 22(8): 1846-1856 (2003).
Lindstrom D.L., Squazzo S., Mustser N., Burckin T., Wachter K., Emigh C., McCleery J., Yates J. and Hartzog G.A. The Spt4-Spt5 complex associates with multiple protein complexes invovled in transcription elongation and pre-mRNA processing. Molecular and Cellular Biology 23(4): 1368-78 (2003).
Hartzog, G.A. Recent advances in transcription elongation. Current Opinions in Genetics and Development 13(2): 119-126 (2003).
Squazzo, S.L., Costa, P.J., Lindstrom, D.L., Kumer, K.E., Simic, R., Jennings, J.L., Link, A.J., Arndt, K.M., and Hartzog, G.A. The Paf1 complex physically and functionally associates with transcription elongation factors in vivo. EMBO J 21(7): 1764-74 (2002).
Lindstrom, L. and Hartzog, G. Genetic interactions of Spt4-Spt5 and TFIIS with the RNA Polymerase II CTD and CTD modifying enzymes in Saccharomyces cerevisiae. Genetics 159: 487-497 (2001).
Murray, S., Udupa, R., Yao, S., Hartzog, G. and Prelich, G. Phosphorylation of the RNA Polymerase II carboxy-terminal domain by the Bur1 cyclin-dependent kinase. Molecular and Cellular Biology 21(13): 4089-96 (2001).
Wada, T., Takagai, T., Yamaguchi, Y., Ferdous, A., Imai, T., Hirose, S., Sugimoto, S., Yano, K., Hartzog, G.A., Winston, F., Buratowski, S. and Handa, H. DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes & Development 12: 343-356 (1998).
Hartzog, G.A., Wada, T., Handa, H. and Winston, F. Evidence that Spt4, Spt5 and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. Genes & Development
12: 357-369 (1998).