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David S. GilmourProfessor of Molecular and Cell Biology
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Transcriptional Regulation of the hsp70 Heat Shock Gene in Drosophila
Gene expression requires RNA polymerase II (Pol II) to associate with one end of a gene, the promoter, and accurately transcribe sequence information in the DNA into RNA. At the opposite end of the gene, Pol II disengages from the DNA by a process called termination. Termination releases the RNA from the DNA allowing the RNA to function in protein synthesis.Our lab is interested in understanding processes that control transcription elongation and termination. These processes are important to the well being of animals. For example, replication of HIV depends on the action of a protein from the virus called Tat, which alters the Pol II so that Pol II can transcribe from one end of the viral genome to the other end (Price 2000). Work on estrogen responsive genes suggests that defects in another regulator of elongation called NELF could contribute to aberrant proliferation of breast epithelial cells (Aiyar et al. 2004). Expression of the proto-oncogenes, c-fos and c-myc, appear to be regulated by blocks to elongation that occur shortly downstream from the start of each gene (Krumm et al. 1992; Strobl and Eick 1992; Plet et al. 1995; Pinaud and Mirkovitch 1998).
We are using the hsp70 heat shock gene of Drosophila as a model for investigating mechanisms by which cells control elongation. This gene is rapidly induced in response to heat shock. Under normal growth conditions when the gene is not expressed, Pol II initiates transcription but stops elongation in the region 20 to 40 nucleotides downstream. This block to elongation sets up the gene so that it can be rapidly induced in response to heat shock (Wang et al. 2005). We have identified key components of this block to elongation. Two proteins, NELF and DSIF have been shown to slow the elongation process (Wada et al. 1998; Yamaguchi et al. 1999; Renner et al. 2001). Depletion of either protein from in vitro transcription reactions or ablation of one subunit of NELF from salivary glands with RNAi impairs the ability of Pol II to accumulate in the promoter proximal region of hsp70 (Wu et al. 2003; Wu et al. 2005). Recent work done in collaboration with the Henderson and Gergen labs indicates the NELF is involved in repressing HIV transcription (Zhang et al. 2007) and regulating transcription of genes involved in Drosophila development (Wang et al. 2007). Moreover, our unpublished data indicates that a NELF-mediated block to elongation occurs on over 1000 genes in Drosophila.
Results from biochemical studies suggest a mechanism in which one
subunit of NELF associates with the nascent transcript emerging from
the Pol II (Yamaguchi
et al. 2002). We speculate that the ensemble of NELF and DSIF
restrain the movement of the transcript from the Pol II at the hsp70
gene by forming a rigid body. Importantly, NELF and DSIF alone
appear to slow but not stop elongation so the establishment of a stable
block to elongation probably involves other cellular components such as
chromatin structure (Brown
et al. 1996; Corey
et al. 2003). Transcriptional activation seems to involve an
activator-dependent process that causes NELF to dissociate from the Pol
II complex (Wu
et al. 2003; Wu
et al. 2005). DSIF remains associated with the Pol II
and stimulates elongation (Yamada et al. 2006).
Another protein that controls elongation in eukaryotic cells is
Pcf11. This protein was first identified as part of a complex
involved in processing the 3’ ends of mRNA (Amrani
et al. 1997). Mutations in this protein prevent Pol II from
terminating transcription in the correct location (Birse
et al. 1998; Sadowski
et al. 2003). We believe we have determined the mechanism of
termination. Pcf11 is able to form a bridge between the CTD of Pol II
and the nascent transcript (Zhang
et
al. 2005; Zhang
and Gilmour 2006). The CTD is an unusual part
of the Pol II that
projects from the surface of the Pol II molecule (Meinhart
and Cramer 2004; Noble
et al. 2005). The CTD had been implicated in termination but
the mechanism was not known (McCracken
et al. 1997). We propose that force generated by
conformational changes in the CTD are transmitted to the nascent
transcript by Pcf11, and the resulting force on the nascent transcript
pulls the transcript out of the elongation complex. Signals at
the end of the gene such as the polyadenylation signal and pause
signals are likely to trigger this reaction (Proudfoot
et al. 2002; Luo
and Bentley 2004).
Since Pcf11 is also found in the body of the gene (Licatalosi
et al. 2002; Kim
et al. 2004; Zhang
and Gilmour 2006), we speculate that Pcf11 has
the
potential to cause Pol II to prematurely termination before reaching
the end of the gene. Our recent results from the analysis of HIV
transcription support this hypothesis (Zhang
et al. 2007). Several transcription regulators, including
Tat and NF-kappaB, activate transcription by promoting elongation (Cullen
1990; Garber
and Jones 1999; Price
2000; Barboric
et al. 2001). We posit that these regulators are modifying
the Pol II at the beginning of the gene so it resists the action of
Pcf11.
Our research is now concentrating on understanding the functions of
NELF, DSIF, and Pcf11 in cells and elucidating the biochemical basis
for how these proteins control gene expression.
Publications relevant to current work:
Gilchrist, D.A., S. Nechaev, C. Lee, S.K.B. Ghosh, J.B. Collins, L. Li, D.S. Gilmour, K. Adelman. 2008. NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly. Genes and Development 15:1921-1933.Li, P., H. Yao, Z. Zhang, M. Li, Y. Luo, P.R. Thompson, D.S. Gilmour, Y. Wang. 2008. Regulation of p53 target gene expression by peptidylarginine deiminase 4. Molecular and Cellular Biology 28:4745-4758.
Lee, C., X. Li, A. Hechmer, M. Eisen, M.D. Biggin, B.J. Venters, C. Jiang, J. Li, B.F. Pugh, D.S. Gilmour. 2008. NELF and GAGA factor are linked to promoter-proximal pausing at many genes in Drosophila. Molecular and Cellular Biology 28:3290-3300.
Mavrich, T.N., C. Jiang, I.P. Ioshikhes, X. Li, B.J. Venters, S.J. Zanton, L.P. Tomsho, J. Qi, R.L. Glaser, S.C. Schuster, D.S. Gilmour, I. Albert, B.F. Pugh. 2008. Nucleosome organization in the Drosophila genome. Nature 453:358-362.
Klatt, A., Z. Zhang, P. Kalantart, P.A. Hankey, D.S. Gilmour, A.J. Henderson. 2008. The receptor tyrosine kinase RON represses HIV-1 transcription by targeting RNA polymerase processivity. The Journal of Immunology 180:1670-1677.
Gilmour, D.S. and R. Fan. 2007. Derailing the locomotive: Transcription termination. Journal of Biological Chemistry 283:661-664.
Zhang, Z., A. Klatt, A.J. Henderson, and D.S. Gilmour. 2007. Transcription termination factor Pcf11 limits the processivity of Pol II on an HIV provirus to repress gene expression. Genes and Development 21:1609-1614.
Wang, X., C. Lee, D.S. Gilmour, and J.P. Gergen. 2007. Transcription elongation controls cell fate specification in the Drosophila embryo. Genes and Development 21:1031-1036.
Zhang, Z., A. Klatt, D.S. Gilmour, and A.J. Henderson. 2007. Negative elongation factor NELF represses human immunodeficiency virus transcription by pausing the RNA polymerase II complex. Journal of Biological Chemistry 282:16981-16988.
Zhang, Z. and D.S. Gilmour. 2006. Pcf11 is a transcription termination factor in Drosophila that dismantles the elongation complex by bridging the CTD of RNA polymerase II to the nascent transcript. Molecular Cell 21:65-74.
Zhang, Z., J. Fu, and D.S. Gilmour. 2005. CTD-dependent dismantling of an RNA Polymerase II elongation complex by the pre-mRNA 3' end processing factor, Pcf11. Genes and Development 19:1572-1580.
Wang, Y.V., H. Tang, D.S. Gilmour. 2005. Identification in vivo of different rate-limiting steps associated with transcriptional activators in the presence and absence of a GAGA element. Molecular and Cellular Biology, 25:3543-3552.
Wu, C-H, C. Lee, R. Fan, M.J. Smith, Y. Yamaguchi, H. Handa, and D.S. Gilmour. 2005. Molecular Characterization of Drosophila NELF. Nucleic Acids Research 33:1268-1279.
Zhang, Z., C.-H. Wu, and D.S. Gilmour. 2004. Analysis of Pol II elongation complexes by native gel electrophoresis: evidence for a novel CTD-mediated termination mechanism. Journal of Biological Chemistry 279:23223-23228.
Wu, C.-H., Y. Yamaguchi, L.R. Benjamin, M. Horvat-Gordon, J. Washinski, E. Enerly, J. Larsson, A. Lambertsson, H. Handa, and D. Gilmour. 2003. NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila. Genes and Development 17:1402-1414.
Tang, H., Y. Liu, L. Madabusi, and D.S. Gilmour. 2000. Promoter proximal pausing on the hsp70 promoter in Drosophila depends upon the upstream regulator. Molecular and Cellular Biology 20:2569-2580.
Benjamin, L.R., and D.S. Gilmour. (1998) Nucleosomes are not necessary for promoter-proximal pausing in vitro on the Drosophila hsp70 promoter. Nucleic Acids Research 26:1051-1055.
Weber, J.A., D. Taxman, Q. Lu, and D.S. Gilmour. (1997) The molecular architecture of the hsp70 promoter after deleting the TATA box or the upstream regulatory region. Molecular and Cellular Biology 17:3799-3808.
Li, B., J.A. Weber, Y. Chen, A.L. Greenleaf, and D.S. Gilmour. 1996. Analyses of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract. Molecular and Cellular Biology 16:5433-5443.
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