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Shilatifard Lab
 Ali Shilatifard, Ph.D.
Investigator
ash@stowers.org
Shilatifard Lab Research Website

     Regulation of gene transcription by RNA polymerase II is critical for development and differentiation, and its misregulation contributes to pathogenesis of many cancers, including leukemia. The overall goal of our laboratory is to define the molecular mechanisms underlying leukemogenesis and identify potential targets for therapy through detailed studies of proteins and protein complexes that regulate chromatin modifications, transcription initiation, and transcription elongation.

     For example, homeotic (HOX) proteins are transcriptional regulators essential for normal hematopoiesis, and their misregulation is associated with hematological malignancies. Similarly, the mixed lineage leukemia (MLL) protein normally positively regulates multiple HOX genes, and several chromosomal rearrangements and translocations that make MLL chimeric proteins cause various forms of leukemia. Such malignancies presumably arise through changes in the hematopoiesis program, which result from misregulation of HOX genes by the MLL fusion proteins.

     Much of our understanding of the mechanisms — by which MLL, its target genes such as the HOX gene family, and its chimeras function — arises from studies on their close homologues in model organisms, such as yeast and Drosophila. Therefore, our laboratory takes full advantage of the powers of genetic, biochemistry, and cell biology in yeast, Drosophila, and mammalian systems to decipher the role of these factors during development and how their misregulation results in the pathogenesis of hematological malignancies.

Studies in yeast S. cerevisiae
     Although the MLL gene was cloned some twenty years ago, the first molecular function for an MLL homolog was defined by our laboratory when we demonstrated that the yeast Set1 (the MLL homolog) is a component of a large complex, COMPASS, that methylates histones in the early transcribed region of genes. Subsequent work by us and others demonstrated that MLL is part of a homologous complex in higher organisms and the components of its complex behave similarly from yeast to human.

     To better define the molecular machinery required for proper histone methylation by COMPASS in yeast and MLL complex in human, we devised a global functional proteomic screen, which we call Global Proteomic analysis in S. cerevisiae (GPS). In GPS, we test extracts of each of the non-essential yeast gene deletion mutants in different mating types (~15,000 strains) for defects in modifications of histones by Western blotting. Employing an antibody specific to histone H3 methylated on its fourth lysine, GPS revealed that ubiquitination of lysine 123 of histone H2B by Rad6 (the E2 conjugating enzyme) is required for histone methylation by COMPASS. We have taken advantage of GPS and have been able to put together a molecular pathway of factors required for proper histone methylation by COMPASS. This include a role for Bre1 as Rad6’s E3 ligase and the elongation factor, the Paf1 complex, and the Bur1/Bur2 kinase for proper regulation H2B monoubiquitination.

     We are employing GPS in yeast to better define the molecular machinery required for COMPASS function and have also applied this screen to other posttranslational modifications of histones such as H3K36 methylation, H3K79 methylation and H3K56 acetylation. There is no doubt that the application of GPS will be extremely informative in defining such molecular pathways.

Studies in Drosophila Melanogaster
     One fusion partner of MLL in acute myelogenous leukemia (AML) is the ELL protein. We showed that human ELL functions as a transcription elongation factor. We have identified the Drosophila homolog of ELL and demonstrated it to be essential for development. Drosophila ELL associates with elongating RNA polymerase II in vivo on chromosomes and is a positive regulator of Notch signaling pathway. This has suggested to us that human ELL might also participate in the same process.

     Following the identification of three ELL related proteins in humans, we showed that they all share a conserved C terminal domain which is not required for transcription elongation properties of ELLs. We have shown that in Drosophila, ELL’s C-terminal domain is essential for development and the equivalent region of human ELL is critical for hematopoietic immortalization. Therefore, defining the molecular role of ELL’s C-terminal domain in Drosophila development is a major focus of our laboratory.

     We have also taken advantage of RNAi technology in Drosophila to reduce the levels of factors required for proper histone modifications to define their role in a living organism. We have shown that the components of the Rad6/Bre1, the Paf1 complex, and other factors are required for histone methylation. Furthermore, we have recently identified that the trithorax group gene in Drosophila, called little imaginal discs, encodes a histone trimethyl H3K4 demethylase. We are planning to follow through with our Drosophila studies to better define the molecular machinery involved in histone methylation and how misregulation of their activities result in cellular immortalization.

Studies in Mammalian Model Systems
     Chromosomal rearrangements resulting in alteration of gene expression are a major cause of hematological malignancies. Our goal is to advance the understanding of the biochemical and molecular mechanisms of rearrangement-based leukemia, and to provide insights into how translocations affect cellular division by altering gene expression. Using mammalian model systems such as tissue culture and mouse genetics, we plan to explore the regulation of gene expression via the MLL gene and its translocation partners found in human leukemia. We are currently defining the molecular composition of the MLL complexes and how translocations alter its biochemical function and integrity, resulting in leukemic pathogenesis. We are also planning to define the mechanism of targeting of the MLL complex and its histone methyltransferase activity to chromatin to determine its normal cellular functions and its mistargeting and disregulation in leukemogenesis.

Overall summary of research plans
     The ongoing experiments in our laboratory will take advantage of our biochemical and cell biological expertise in the mammalian systems to identify the molecular mechanism of gene expression regulation by MLL-complexes and their chimeras found in translocations in leukemia. Since the molecular regulation of gene expression via MLL and its related complexes seem to be highly conserved among eukaryotic organisms, our expertise in yeast and Drosophila puts us in a unique position to identify the basic components of this machinery in mammals. Our GPS biochemical screen in yeast was very successful in identifying the molecular mechanism of histone methylation and transcriptional regulation via COMPASS. We identified a wide range of genes involved in the regulation of the enzymatic activity of COMPASS. We plan to test the role of these gene products in the regulation of the enzymatic activity of the MLL complex in the mammalian system and to define how translocation of MLL can result in pathogenesis of acute leukemia in the hope of identifying potential targets for therapy.

Selected publications

Lee JS, Smith E, Shilatifard A. The language of histone crosstalk. Cell. 2010;142(5):682-5. Abstract

H. M. Herz and A. Shilatifard The JARID2-PRC2 duality. Genes Dev 2010;24(9):857-861. Abstract

Herz HM, Madden LD, Chen Z, Bolduc C, Buff E, Gupta R, Davuluri R, Shilatifard A, Hariharan IK, Bergmann A. The H3K27me3 demethylase dUTX is a suppressor of notch- and Rb-dependent tumors in Drosophila. Mol Cell Biol. 2010;30(10):2485-97. Abstract.

Lin C, Smith ER, Takahashi H, Lai KC, Martin-Brown S, Florens L, Washburn MP, Conaway JW, Conaway RC, Shilatifard A. AFF4, a Component of the ELL/P-TEFb Elongation Complex and a Shared Subunit of MLL Chimeras, Can Link Transcription Elongation to Leukemia.  Mol Cell. 2010;37(3):429-437.  Abstract

Mohan M, Herz HM, Takahashi YH, Lin C, Lai KC, Zhang Y, Washburn MP, Florens L, and Shilatifard A. Linking H3K79 trimethylation to Wnt signaling through a novel Dot1-containing complex (DotCom). Genes Dev. 2010;24(6):574-89. Abstract

Herz HM, Nakanishi S, Shilatifard A. The Curious Case of Bivalent Marks [published ahead of print August 14, 2009]. Dev Cell. 2009.

Nakanishi S, Lee JS, Gardner KE, Gardner JM, Takahashi YH, Chandrasekharan MB, Sun ZW, Osley MA, Strahl BD, Jaspersen SL, Shilatifard A. Histone H2BK123 monoubiquitination is the critical determinant for H3K4 and H3K79 trimethylation by COMPASS and Dot1. J Cell Biol. 2009;186:371-377. Abstract

Schulze JM, Jackson J, Nakanishi S, Gardner JM, Hentrich T, Haug J, Johnston M, Jaspersen SL, Kobor MS, Shilatifard A. Linking Cell Cycle to Histone Modifications: SBF and H2B Monoubiquitination Machinery and Cell-Cycle Regulation of H3K79 Dimethylation [published ahead of print August 12 2009]. Mol Cell. 2009. Abstract

Trievel RC, Shilatifard A. WDR 5, a complexed protein. Nat Struct Mol Biol. 2009;16:678-680. Abstract

Smith E, Shilatifard A. Developmental biology. Histone cross-talk in stem cells. Science. 2009;323:221-222. Abstract

Kim J, Guermah M, McGinty RK, Lee JS, Tang Z, Milne TA, Shilatifard A, Muir TW, Roeder RG. RAD 6-Mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells. Cell. 2009;137:459-471. Abstract

Dang W, Steffen KK, Perry R, Dorsey JA, Johnson FB, Shilatifard A, Kaeberlein M, Kennedy BK, Berger SL. Histone H4 lysine 16 acetylation regulates cellular lifespan. Nature. 2009;459:802-807. Abstract

Berger SL, Kouzarides T, Shiekhattar R, Shilatifard A. An operational definition of epigenetics. Genes Dev. 2009;23:781-783. Abstract

Nakanishi S, Sanderson BW, Delventhal KM, Bradford WD, Staehling-Hampton K, Shilatifard A. A comprehensive library of histone mutants identifies nucleosomal residues required for H3K4 methylation. Nat Struct Mol Biol. 2008;15:881-888. Abstract

Shilatifard A. Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation. Curr Opin Cell Biol. 2008;20:341-348. Abstract

Allis CD, Berger SL, Cote J, Dent S, Jenuwien T, Kouzarides T, Pillus L, Reinberg D, Shi Y, Shiekhattar R, Shilatifard A, Workman J, Zhang Y. New Nomenclature for Chromatin-Modifying Enzymes. Cell. 2007;131:633-636. Abstract

Bhaumik SR, Smith E, Shilatifard A. Covalent modifications of histones during development and disease pathogenesis. Nat Struct Mol Biol. 2007;14:1008-1016. Abstract

Eissenberg JC, Lee MG, Schneider J, Ilvarsonn A, Shiekhattar R, Shilatifard A. The trithorax-group gene in Drosophila little imaginal discs encodes a trimethylated histone H3 Lys4 demethylase. Nat Struct Mol Biol. 2007a;14:344-346. Abstract

Lee JS, Shukla A, Schneider J, Swanson SK , Washburn MP, Florens L, Bhaumik SR, Shilatifard A. Histone crosstalk between H2B monoubiquitination and H3 methylation mediated by COMPASS. Cell. 2007;131:1084-1096. Abstract

Lee MG, Norman J, Shilatifard A, Shiekhattar R. Physical and functional association of a trimethyl H3K4 demethylase and Ring6a/MBLR, a polycomb-like protein. Cell. 2007b;128:877-887. Abstract

Smith E, Shilatifard A. The A, B, Gs of silencing. Genes Dev. 2007;21:1141-1144. Abstract

Krogan NJ, Cagney G, Yu H, Zhong G, Guo X, Ignatchenko A, Li J, Pu S, Datta N, Tikuisis AP, Punna T, Peregrin-Alvarez JM, Shales M, Zhang X, Davey M, Robinson MD, Paccanaro A, Bray JE, Sheung A, Beattie B, Richards DP, Canadien V, Lalev A, Mena F, Wong P, Starostine A, Canete MM, Vlasblom J, Wu S, Orsi C, Collins SR, Chandran S, Haw R, Rilstone JJ, Gandi K, Thompson NJ, Musso G, St Onge P, Ghanny S, Lam MH, Butland G, Altaf-Ul AM, Kanaya S, Shilatifard A, O'Shea E, Weissman JS, Ingles CJ, Hughes TR, Parkinson J, Gerstein M, Wodak SJ, Emili A, Greenblatt JF. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature. 2006;440:637-643. Abstract

Pavri R, Zhu B, Li G, Trojer P, Mandal S, Shilatifard A, Reinberg D. Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II. Cell. 2006;125:703-717. Abstract

Shilatifard A. Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression. Annu Rev Biochem. 2006;75:243-269. Abstract

Steward MM, Lee JS, O'Donovan A, Wyatt M, Bernstein BE, Shilatifard A. Molecular regulation of H3K4 trimethylation by ASH 2L, a shared subunit of MLL complexes. Nature Struct Mol Biol. 2006;13:852-854. Abstract

Baillat D, Hakimi MA, Naar AM, Shilatifard A, Cooch N, Shiekhattar R. Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II. Cell. 2005;123:265-276. Abstract

Emre NC , Ingvarsdottir K, Wyce A, Wood A, Krogan NJ , Henry KW, Li K, Marmorstein R, Greenblatt JF, Shilatifard A, Berger SL. Maintenance of low histone ubiquitylation by Ubp10 correlates with telomere-proximal Sir2 association and gene silencing. Mol Cell. 2005;17:585-594. Abstract

Schneider J, Wood A, Lee JS, Schuster R, Dueker J, Maguire C, Swanson SK, Florens L, Washburn MP, Shilatifard A. Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression. Mol Cell. 2005;19:849-856. Abstract

Wood A, Schneider J, Dover J, Johnston M, Shilatifard A. The Bur1/Bur2 complex is required for histone H2B monoubiquitination by Rad6/Bre1 and histone methylation by COMPASS. Mol Cell. 2005a;20:589-599. Abstract

Wood A, Shilatifard A. Guided by COMPASS on a journey through chromosome segregation. Nat Struct Mol Biol. 2005;12:839-840. Abstract

Krogan NJ, Dover J, Wood A, Schneider J, Heidt J, Boateng MA, Dean K, Ryan OW, Golshani A, Johnston M, Greenblatt JF, Shilatifard A. The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation. Mol Cell. 2003a;11:721-729. Abstract

Shilatifard A, Conaway RC, Conaway JW. The RNA polymerase II elongation complex. Annu Rev Biochem. 2003;72:693-715. Abstract

Wood A, Krogan NJ, Dover J, Schneider J, Heidt J, Boateng MA, Dean K, Golshani A, Zhang Y, Greenblatt JF, Johnston M, Shilatifard A. Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Mol Cell. 2003a;11:267-274. Abstract

Dover J, Schneider J, Tawiah-Boateng MA, Wood A, Dean K, Johnston M, Shilatifard A. Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6. J Biol Chem. 2002;277:28368-28371. Abstract

Eissenberg JC, Ma J, Gerber MA, Christensen A, Kennison JA, Shilatifard A. dELL is an essential RNA polymerase II elongation factor with a general role in development. Proc Natl Acad Sci U S A. 2002;99:9894-9899. Abstract

Miller T, Krogan NJ , Dover J, Erdjument-Bromage H, Tempst P, Johnston M, Greenblatt JF, Shilatifard A. COMPASS: a complex of proteins associated with a trithorax-related SET domain protein. Proc Natl Acad Sci U S A. 2001;98:12902-12907. Abstract

DiMartino JF, Miller T, Ayton PM, Landewe T, Hess JL, Cleary ML, Shilatifard A. A carboxy-terminal domain of ELL is required and sufficient for immortalization of myeloid progenitors by MLL-ELL. Blood. 2000;96:3887-3893. Abstract

Shilatifard A, Duan DR , Haque D, Florence C, Schubach WH, Conaway JW, Conaway RC. ELL2, a new member of an ELL family of RNA polymerase II elongation factors. Proc Natl Acad Sci U S A. 1997b;94:3639-3643. Abstract

Shilatifard A, Lane WS, Jackson KW, Conaway RC, Conaway JW. An RNA polymerase II elongation factor encoded by the human ELL gene. Science. 1996;271:1873-1876. Abstract