- Pasteur Institute, Paris, France, Research Fellow in Genetic Engineering, 1977.
- University of Geneva, Switzerland, Research Fellow and Research Associate in Molecular Biology, 1972-1976.
- Harvard University, Cambridge, MA, U.S., PhD in Biophysics, 1973.
- McGill University, Montreal, BSc in Physics, 1967.
MY RESEARCH OVERVIEW (GO TO SCIENTIFIC OVERVIEW)
From the time when I first learned as a graduate student that there was a relatively new field called “molecular biology,” I have been fascinated with understanding the mechanisms that turn genes on or off. It is, after all, the specific set of genes that is turned on in a cell or organism that defines what exactly that cell is, how it will respond to its environment, and whether it is normal or diseased. The research in the Greenblatt lab has changed over time from a focus on how genes are regulated in bacteria to how genes are regulated in yeast and, most recently, to how genes are regulated in human cells. Of the 20,000 or so human genes, over 1600 encode proteins whose job it is to regulate human gene expression by interacting with DNA. It has become apparent, however, that these proteins often interact among themselves or with other proteins to cooperatively control gene expression. As part of the Proteomics Research Center, we are using mass spectrometry to determine how these proteins interact with each other, and we are using advanced sequencing technologies to understand how these proteins interact with DNA or RNA. What these proteins regulate is RNA polymerase II, the enzyme that transcribes protein-coding genes, and we are also studying how modifications to RNA polymerase II control its activity. For me, the vast interaction networks that control human gene expression and are now coming into view are truly the “secrets of life.” To the extent that we are deciphering these “secrets,” it has only been possible because of our collaborations with many talented colleagues in the Donnelly Centre.
SCIENTIFIC RESEARCH OVERVIEW
Research in the Greenblatt lab is now focused entirely on the regulation of human gene expression. Our current research projects mainly concern either the ~1600 human DNA-binding proteins or how modifications of the RNA polymerase II CTD impact gene expression:
1. C2H2 zinc finger transcription factors
In collaboration with the Emili lab, we have been using affinity purification and mass spectrometry to identify protein-protein interactions for bacterial, yeast, and human proteins, and our current focus is the ~1600 human proteins that putatively bind DNA. Because so little is known about them, we began with the >700 C2H2 zinc finger proteins, and our most notable finding has been their remarkable propensity to interact with each other to form heterodimers. Our working hypothesis is that their extensive interaction network involving >1000 protein-protein interactions has a central role in organizing DNA topology in the nucleus. This collaborative project with the Hughes lab has already shown that most, or perhaps almost all, of the C2H2 proteins do indeed recognize specific sequence motifs in human cells. We would now like to know whether most of these proteins regulate the transcription of protein-coding or other genes and what mechanisms they use to regulate transcription. We will use ChIP-seq to analyze their effects of various aspects of transcription by RNA polymerase II and ChIA-PET to determine whether their regulatory mechanisms involve DNA looping.
ChIP-seq data for a large number of C2H2 proteins has indicated that ~20% of the C2H2 proteins preferentially target DNA encoding alternatively spliced exons. As a consequence, we are now collaborating with the Blencowe lab to determine whether many of the C2H2 proteins have a major role in regulating alternative splicing. We would like to determine whether C2H2 proteins regulate splicing by provoking chromatin modifications that alter the kinetics of elongation by RNA polymerase II, by interacting with RNA as well as with DNA, or by recruiting RNA-binding proteins to influence splicing, and whether these regulatory mechanisms, whatever they are, involve DNA looping mediated by heterodimerizing C2H2 proteins.
2. Transcriptional basis of neurodegenerative disease
In another project we have discovered a new modification of the RNA polymerase II CTD, a symmetrical dimethylation of R1810. We found that R1810me2s is recognized by the tudor domain of SMN, a protein that is mutated in spinal muscular atrophy (SMA). R1810me2s and SMN are components of a pathway in which the helicase Senataxin resolves R-loops to facilitate termination by RNA polymerase II. We are now investigating whether two RNA-binding proteins, FUS and TDP-43, which are sometimes mutated in amyotrophic lateral sclerosis (ALS), are also components of this pathway, as well as the effects of defects in this pathway on gene expression, genome stability, and alternative splicing. Defects in this pathway may be major contributors to neurodegenerative disease.
3. Transcriptional basis of tumorigenesis
We are also characterizing three proteins, RPRD1A, RPRD1B, and RPRD2, that interact with the RNA polymerase II CTD, most strongly when it is phosphorylated on S2. RPRD1B is an oncoprotein over-expressed in the vast majority of human tumours. We found that RPRD1A and RPRD1B have important roles in recruiting the CTD S5 phosphatase RPAP2 during the transition from initiation to elongation. Our current focus is on determining to what extent the RPRD proteins are important for recruiting the nucleosome assembly factor SPT6 in order to suppress cryptic initiation of transcription. Which of these mechanistic aspects of RPRD protein function is related to tumorigenesis by RPRD1B remains to be seen.
- Human-chromatin-related protein interactions identify a demethylase complex required for chromosome segregation. Marcon E, Ni Z, Pu S, Turinsky AL, Trimble SS, Olsen JB, Silverman-Gavrila R, Silverman-Gavrila L, Phanse S, Guo H, Zhong G, Guo X, Young P, Bailey S, Roudeva D, Zhao D, Hewel J, Li J, Gräslund S, Paduch M, Kossiakoff AA, Lupien M, Emili A, Wodak SJ, Greenblatt J. Cell Rep. 2014 Jul 10;8(1):297-310.
- RPRD1A and RPRD1B are human RNA polymerase II C-terminal domain scaffolds for Ser5 dephosphorylation. Ni Z, Xu C, Guo X, Hunter GO, Kuznetsova OV, Tempel W, Marcon E, Zhong G, Guo H, Kuo WH, Li J, Young P, Olsen JB, Wan C, Loppnau P, El Bakkouri M, Senisterra GA, He H, Huang H, Sidhu SS, Emili A, Murphy S, Mosley AL, Arrowsmith CH, Min J, Greenblatt JF. Nat Struct Mol Biol. 2014 Aug;21(8):686-95.
- Interaction landscape of membrane-protein complexes in Saccharomyces cerevisiae. Babu M, Vlasblom J, Pu S, Guo X, Graham C, Bean BD, Burston HE, Vizeacoumar FJ, Snider J, Phanse S, Fong V, Tam YY, Davey M, Hnatshak O, Bajaj N, Chandran S, Punna T, Christopolous C, Wong V, Yu A, Zhong G, Li J, Stagljar I, Conibear E, Wodak SJ, Emili A, Greenblatt JF. Nature. 2012 Sep 27;489(7417):585-9.