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Michael Hampsey
Regulation of gene expression; yeast geneticsResearch Fields
Research Description My laboratory studies the regulation of gene expression in eukaryotic organisms. We are especially interested in transcription of protein-encoding genes by RNA polymerase II (Pol II). The experimental organism used in our studies is the yeast Saccharomyces cerevisiae , which enables us to use a powerful combination of classical genetics, molecular biology and modern biochemistry to address fundamental questions in medical biochemistry. Our current efforts are focused on two related issues: 1) Coupling of Pol II transcription with pre-mRNA processing and nuclear export; and 2) The role of gene loops that juxtapose promoter-terminator regions in Pol II transcription. A remarkable feature of coupled Pol II transcription and mRNA processing is the extent to which these two processes are conserved among eukaryotic organisms. Gene loops also appear to be a general feature of Pol II transcription in eukaryotes and might be involved in “memory” of recent transcriptional activity and translocation of mRNA from the nucleus to the cytoplasm.
1) Coupling of 3' end processing to Pol II transcription. Nascent mRNA undergoes modifications that include 5' capping, splicing, 3' endonucleolytic cleavage/polyadenylation, mRNP formation and nuclear export. These events occur co-transcriptionally and involve the recruitment and exchange of processing factors to the C-terminal domain (CTD) of the Rpb1 subunit of Pol II. The CTD, a reiterated heptad repeat (YSPTSPS) is phosphorylated and dephosphorylated at Ser2 and Ser5 (and probably Ser7) during the transcription cycle. We discovered that the Ssu72 protein is a CTD Ser5-P phosphatase and an integral component of the CPF 3' end processing complex. We are now focused on determining (i) how Ssu72-mediated Ser5-P dephosphorylation affects Pol II progression through the transcription cycle; (ii) how Ssu72 is regulated by the transcriptional, processing and nuclear export machineries; and (iii) the relationship of Ssu72 to other CTD phosphatases, including Fcp1 and Rtr1. We are collaborating on this project with Professor Claire Moore, Tufts Medical School . This work is supported by NIH grant RO1 GM068887.
2) Role of “gene loops” in transcription. Although Ssu72 is a component of the CPF 3' end processing complex, we first identified this protein based on genetic and physical interactions with TFIIB, a transcription initiation factor. As such, Ssu72 defined an unexpected link between the Pol II initiation and termination machineries. These observations led us to investigate potential physical interactions between the promoter and terminator regions. Our results revealed that gene loops are a general feature of Pol II transcription and underlie “memory” of recent transcriptional activity, defined as the very rapid transcriptional response following a cycle of activation and repression. We also identified several components of the nuclear pore complex as suppressors of looping-defective mutants, suggesting that gene loops and transcriptional memory are functionally integrated with mRNA export to the cytoplasm. Gene loops are unlikely to be a feature unique to yeast, as r ecent studies in the Proudfoot laboratory (Oxford) have also identified gene loops in mammalian cells, including promoter-terminator juxtaposition at the breast cancer BRCA1 gene, and between the 5' and 3' LTRs of the HIV provirus. This work is supported by NIH grant RO1 GM39484. Selected Publications: KRISHNAMURTHY, S., M. A. GHAZY, C. MOORE and M. HAMPSEY (2009) Functional interaction of the Ess1 prolyl isomerase with components of the RNA polymerase II initiation and termination machineries. Mol. Cell. Biol. 29:2925-2934. SINGH, B.N., A. ANSARI and M. HAMPSEY (2009) Detection of transient short-range chromatin interactions by 3C in yeast. Methods 47:000-000 [in press] GHAZY, M., X. HE, B.N. SINGH, M. HAMPSEY and C. MOORE (2009) The essential N-terminus of the Pta1 scaffold protein is required for snoRNA transcription termination and Ssu72 function but is dispensable for pre-mRNA 3'-end processing. Mol. Cell. Biol. 29: 2296-2307 . SINGH, B. N. and M. HAMPSEY (2007) A transcription-independent role for TFIIB in gene looping. Mol. Cell 27:806-816. REYES-REYES, M. and M. HAMPSEY (2007) Role for the Ssu72 C-terminal domain phosphatase in RNA polymerase II transcription elongation. Mol. Cell. Biol. 27:926-936. ANSARI, A. and M. HAMPSEY (2005) A role for the CPF 3'-end processing machinery in RNAP II-dependent gene looping. Genes & Dev. 19:2969-2978. KRISHNAMURTHY, S., X. HE, M. REYES-REYES, C. MOORE AND M. HAMPSEY (2004) Ssu72 is a RNA polymerase II CTD phosphatase. Mol. Cell 14:387-394. CHEN, B.-S. and M. HAMPSEY (2004) Functional Interaction Between TFIIB and the Rpb2 Subunit of RNA polymerase II: implications for the mechanism of transcription start site selection. Mol. Cell. Biol . 24: 3983-3991. WOYCHIK, N. A. and M. HAMPSEY (2002) The RNA polymerase II machinery: structure illuminates function. Cell 108:453-463.
Hampsey Laboratory:
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