Yanming Wang Assistant Professor of Biochemistry and Molecular Biology   332 South Frear Lab University Park, PA 16802 Telephone: (814) 865-3775 E-mail:yuw12@psu.edu B.A. in Biochemistry, Shandong University, China Ph.D. in Molecular, Cellular, and Development Biology, Iowa State University Postdoctoral fellow, The Rockefeller University Wang Lab Web Site Member of Center for Eukaryotic Gene Regulation

Epigenetic Histone modifications in Cell Differentiation and Cancer

In our bodies, proliferating stem cells produce two types of daughter cells: one type keeps the stem cell identity, while the other type replenishes the pool of terminal differentiated somatic cells (Fig.1).  Somatic cells express a distinct set of genes compared with their precursor stem cells.  Further, the homeostasis of stem cell proliferation and differentiation keeps the balance of the number of dead and live cells.  Over proliferation of stem cells or the failure of cell differentiation can produce large amount undifferentiated cells leading to cancers.

Figure 1. The balance of stem cell differentiation and division keeps a brake on tumorigenesis

DNA in eukaryotic cell nucleus is organized with histones to form chromatin.  Nucleosome, the basic structural unit of chromatin, consists of 146bp of DNA wrapped around an octamer of core histones, including two of each histone H3, H4, H2A, and H2B (Fig.2).  Since histones are separated into daughter DNA strands during the S phase of the cell cycle, post-translational modifications of histones can serve as epigenetic information for the heritable changes of gene expression.

Figure 2. Histones are major carriers of epigenetic information.

Histones can by post-translationally modified by acetylation, ADP-ribosylation, methylation, phosphorylation, ubiquitination, and probably even more to be coming.  Histone modifications impact on the structure of chromatin as well as the association of histone- and DNA-binding proteins (Fig.3). Epigenetic histone modifications play important roles in cell differentiation as well as in maintaining the stem cell identity.  The study of enzymes and binding proteins that regulate covalent histone modifications in chromatin biology will open up exciting possibility for new biomedicine in cancer treatment.

Figure 3. Selected enzymes that modify histones are shown.

We are interested in the epigenetic role of histone modifications in cell differentiation and cancer.  Our overall objective is to understand how histone modifications regulate cell growth, proliferation, differentiation, and how deregulated histone modifications can lead to misfortunes like cancers.  Our specific goals are to 1) understand the establishment of histone modification patterns and chromatin structure determined by primary DNA sequences; 2) understand the role of histone Arg methylation in cell differentiation and cancer.

Project 1: Map of Histone Methylation in Chromatin Structure and Function in Drosophila

Based on the previous results from us and others (see below for references), we hypothesize that histone methylation defines a “functional genome” by marking chromatin domains, such as condensed chromatin (e.g., H3K9^Me and H4K20^Me), decondensed chromatin (e.g., H3K4^Me, see Fig. 4), silenced genes (e.g., H3K27^Me), and active genes (e.g., H4R3^Me).  To test this, we aim to 1) map H3K4^Me on several representative genes as well as on the interband DNA sequences on the tip (bands 1-5) of the X chromosome; 2) carry out bioinformatics analysis of decondensed/interband DNA sequences to search for the boundaries between interbands and bands as well as shared DNA sequences between interbands.  We favor the view that conserved DNA elements may be shared by different interbands; 3) experimentally dissect the functional elements of interbands in transfected cells and transgenic flies by P-element mediated transformation; 4)explore the rigidity and plasticity of genomic distribution of histone methylation by comparing H3K4^Me on ecdysone and heat-shock inducible gene promoters before and after activation in S2 cells.

Figure 4. Histone H3 K4 methylation marks decondensed chromatin, the interbands of Drosophila polytene chromosomes.

Project 2: Histone Arg Methylation in Cell Differentiation and Carcinogenesis in Mammals

Multiple residues on histones H3 and H4 are subject of methylation, which are catalyzed by the protein Arg methyltransferase (PRMT) family of enzymes.  There are over 7 members in the PRMT protein family in the human genome.  Among these, PRMT1 methylates histone H4 and many other cellular proteins, while CARM1 (also called PRMT4) methylates histone H3.  The methylation of histone by PRMT1 and CARM1 plays a role in transcriptional activation mediated by p53 and nuclear hormone receptors.  Recently, PRMT5 was reported to methylate both histone H3 and H4 to repress gene expression.  Therefore, histone Arg methylation mediated by different PRMTs can have opposite effect on transcription.  Since p53 and nuclear hormone receptors are important gate keepers for gene expression in normal cells to prevent tumorigenesis, to study the role of histone Arg methylation during transcription is important to understand the type of cancers in which p53 and nuclear hormone pathways are involved.

Although PRMTs are known for a long period of time, enzymes that remove methyl groups from Arg had not been know.  Importantly, our recent work has identified a new pathway to keep the steady state balance of histone Arg methylation.  We found that PAD4 can decrease histone Arg methylation by a newly characterized demethylimination activity (Fig. 5).  This new role of PAD4 suggests that histone Arg methylation is not a one way street, but a two way road to keep the yin-yang balance of methylation. 

Figure 5. The multiple ways of protein Arg modifications: methylation, citrullination, and demethylimination.

Currently, we aim to understand the biological function of PAD4 by exploring following directions: 1) to understand the regulation of PAD4 enzymatic activity by other interacting protein partners in human cells; 2) to analyze the role of PAD4 in antagonizing PRMTs during transcription mediated by nuclear hormone receptors and p53; 3) to dissect the involvement of PAD4 during mouse embryonic stem cell differentiation induced by retinoic acid; lastly, 4) we are dissecting the role of PAD4 in development using the mouse model system.

Representative Publications:

• Wang, Y., Wysocka J., Sayegh, J., Lee, Y.H., Perlin J.R., Leonelli, L., Sonbuchner, L.S., McDonald, C.H., Cook, R.G., Dou, Y., Roeder, R.G., Clarke, S., Stallcup, M.R., Allis, C.D., Coonrod, S.A. (2004) Human PAD4 regulates histone arginine methylation levels via demethylimination. Science 306, 279-83.  Suggested reading for project 1
• Fischle, W.*, Wang, Y.*, Jacobs, S.A.*, Kim, Y., Allis, C.D., and S. Khorasanizadeh (2003) Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by polycomb and HP1 chromodomains. Gen. & Dev. 17: 1870-81. (* indicating authors of equal contribution) Suggested reading for project 1.
• Zhang, W, Wang, Y., Long J., Girton, J., Johansen, J., and Johansen, K.M. (2003) A developmentally regulated splice-variant from the complex lola locus encoding multiple different zinc-finger domain proteins interacts with the chromosomal kinase JIL-1. Biol. Chem. 278(13):11696-704
• Nishioka, K., Rice, J.C., Sarma, K., Erdjument-Bromage, H., Werner, J., Wang, Y., Chuikov, S., Valenzuela., P., Tempst, P., Steward, R., Lis, J.T., Allis, C.D., and Reinberg, D. (2002) PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin. Mol. Cell. 9(6): 1201-13. Suggested reading for project 1.
• Wang, Y., Zhang, W., Jin, Y., Johansen, J., and Johansen, K.M. (2001) The JIL-1 tandem kinase mediates histone H3 phosphorylation and is required for maintenance of chromatin structure in Drosophila. Cell 105: 433-43. Suggested reading for project 1.
• Jin, Y. *, Wang, Y. *, Johansen, J. and Johansen, K.M. (2000) JIL-1, a chromosomal kinase implicated in regulation of chromatin structure, associates with the MSL dosage compensation complex. J. Cell Biol. 149: 1005-10. (* indicating authors of equal contribution)

Reviews and Book Chapters