Erasmus MC : Chromatin regulation in development and disease.

Groupleader: C. Peter Verrijzer

Address:
Peter Verrijzer
Dept. of Biochemistry, room Ee642
Erasmus University Medical Center
Dr Molewaterplein 50
3015 GE Rotterdam
The Netherlands
Phone: +31-107043461
Email: c.verrijzer@erasmusmc.nl

Research goal:
To understand the mechanism of gene expression control during development and disease.

Mechanisms of Eukaryotic transcription control.
We are interested in how the expression of the eukaryotic genome is regulated. In particular, we focus on the role of chromatin regulation in development and disease. Over the last decade or so, it has become clear that chromatin structure forms an integral part of the mechanisms by which gene transcription is controlled in eukaryotic cells. Our studies focus on three related topics: (1) The role of SWI/SNF-class ATP-dependent chromatin remodeling complexes in transcription regulation during development and disease. (2) Transcription control by protein (de)ubiquitylation. (3) Mechanism of gene silencing by Polycomb group proteins. For many of our studies, we use Drosophila as a model organism because it allows an integrated combination of biochemistry, proteomics and developmental genetics. In addition, we investigate the mechanism of tumor suppression by hSNF5, a core subunit of human SWI/SNF remodeling complexes. For these studies we use human tumor cell lines and mouse models.

Chromatin remodelers in disease.
The multi-subunit SWI/SNF ATP-dependent chromatin remodeling complexes are highly conserved molecular motors that play crucial roles in diverse cellular processes, including expression and duplication of the genome. Human SNF5/INI1 is a universal SWI/SNF subunit and tumor-suppressor lost in malignant rhabdoid tumors (MRTs), rare but highly aggressive pediatric cancers. We found that re-expression of hSNF5 in MRT cells caused an accumulation in G0/G1, cellular senescence and apoptosis. Cellular senescence is largely the result of direct transcriptional activation of the tumor-suppressor p16INK4a by hSNF5 (Oruetxebarria et al. 2004). Loss of hSNF5 function in MRT cells promotes chromosomal instability by compromised mitosis (Vries et al., 2005). hSNF5 activates the mitotic checkpoint through the p16INK4a-cyclinD/CDK4-pRb-E2F pathway, revealing a convergence of tumor suppressor pathways. We propose that inactivation of hSNF5 causes both the selective growth advantage and the genetic instability necessary for tumor initiation and progression (figure 1).

We will continue to dissect the cellular effector circuitries of hSNF5-mediated tumor suppression. Special attention will be on cancer-relevant cellular processes such as apoptosis, chromosomal instability or metastasis. We have already established assays to study the role of hSNF5 in cellular senescence, apoptosis, chromosomal stability and cell migration and invasion. Combined with genome-wide gene expression profiling, shRNA knock down analysis of candidate effectors, and studies of hSNF5 conditional KO mice, these assays will be used to address the role of hSNF5 in tumorigenesis. We anticipate that these investigations will help us to gain insight in the corruption of multiple cellular pathways due to loss of the hSNF5 tumor suppressor in MRTs. Because cancer due to loss of hSNF5 is so extremely aggressive, insight in the pathways involved might be relevant to understanding other forms of cancer.

Chromatin remodelers in development.
We will continue to study the function of the SWI/SNF remodelers during Drosophila development by combining genetic and proteomic screens. We identified unique SWI/SNF subcomplexes in the fruit fly Drosophila melanogaster, which appeared to control distinct transcriptional programs (Mohrmann et al., 2004). Indeed, our recent results indicated that the two major SWI/SNF-class remodelers in Drosophila, named BAP and PBAP (Polybromo and Brahma associated proteins), perform distinct functions in the cell (figure 2).

Our goal now is to obtain a comprehensive and detailed overview of the biochemical pathways regulated by distinct SWI/SNF subunits. Our main objectives are to: (1) Identify the role of individual BAP/PBAP subunits on basic cellular processes including cell cycle, cell growth, metabolism and hormone regulation. (2) To determine the transcriptional circuitries controlled by individual BAP/PBAP subunits that are responsible for cellular phenotypes by genome-wide expression profiling and ChIP on chip studies (3) To identify BAP/PBAP interacting partners using proteomic screens. (4) The in vivo validation of candidate partners or pathways utilizing genetic assays. Finally, we will study the developmental functions of BAP170, a novel SWI/SNF subunit we recently discovered, in flies as well as mice.

Transcription regulation by protein (de)ubiquitylation.
Despite the fact that histones were the first proteins found to be ubiquityated, many questions remain concerning the physiological consequences of mono-ubiquitination of histones H2A and H2B. We purified the essential Drosophila ubiquitin protease USP7 as a heteromeric complex with guanosine 5’-monophosphate synthetase (GMPS) (van der Knaap et al., 2005). We found that USP7/GMPS contributes to epigenetic gene silencing by Polycomb and deubiquitylates histone H2B (figure 3).

Strikingly, association with GMPS was essential for H2B deubiquitylation. Our findings implicated a biosynthetic enzyme in chromatin control via ubiquitin regulation. An important future direction in the lab is to study the role of protein (de)ubiquitylation in transcriptional control. We plan to follow up on our earlier findings and study the functions of USP7/GMPS as well as other ubiquitin proteases and ligases. Special focus will be on the mechanism by which GMPS can regulate USP7 activity and the role of protein (de)ubiquitylation in Polycomb silencing. We found that USP7/GMPS functions not only at homeotic loci but also in silencing at pericentric heterochromatin and ecdysone-regulated genes. In addition, we identified several new protein targets for USP7/GMPS. Thus, the aim of this project is to map the protein and gene network controlled by USP7/GMPS, using an integrated proteomic and genetic approaches. Finally, we have developed genetic screens to study the regulatory networks involving protein (de)ubiquitylation in Drosophila. A first screen already revealed several antagonizing ubiquitin proteases and ligases, which are subject of our future investigations. We aim to identify their protein targets, mechanism of recruitment and their critical sets of developmentally controlled target genes.

Mechanism of gene silencing by Polycomb group proteins.
Research over the last decade has emphasized the critical role of covalent chromatin modifications in epigenetic gene regulation. However, how specialized DNA sequence elements can bring a linked gene under epigenetic control has remained unclear. We identified a Polycomb-binding element (PBE), a small conserved sequence element downstream of Pleiohomeotic (PHO) binding sites (Mohd-Sarip et al., 2005). Importantly, the PBE is required for PcG silencing in vivo (figure 4).

PHO sites and their juxtaposed PBEs function as an integrated DNA platform for the synergistic assembly of a PC repressive complex. We suggest that the formation of a PcG repressive nucleoprotein complex that we called silenceosome is governed by the same molecular principles that direct enhanceosome assembly. In our future studies we will continue to pursue the issue of how specific DNA sequences bring target genes under epigenetic Polycomb group control, using a combination of in vivo genetics and in vitro biochemical and imaging studies. Furthermore, we will investigate the role of protein ubiquitylation in Polycom group silencing.

Selected publications.

Mohd-Sarip, A., Cléard, F., Mishra, R.K., Karch, F., and Verrijzer, C.P. (2005). Synergistic Recognition of an Epigenetic DNA Element by Pleiohomeotic and a Polycomb Core Complex. Genes Dev. 19: 1755-1760.

Vries, R.G.J., Bezrookove, V., Zuijderduijn, L.M.P., Kheradmand Kia, S., Houweling, A, Oruetxebarria, I, Raap, A.K., and Verrijzer, C.P. (2005)

Cancer–associated mutations in chromatin-remodeler hSNF5 promote chromosomal instability by compromising the mitotic checkpoint. Genes Dev. 19: 665-670.

Van der Knaap, J.A., Kumar, B.R.P., Moshkin, Y.M., Langenberg, K., Krijgsveld, J., Heck, A.J., Karch, F., and Verrijzer, C.P. (2005). GMP Synthetase Stimulates Histone H2B Deubiquitylation by the Epigenetic Silencer USP7. Mol. Cell 17: 695-707.

Mohrmann, L., and Verrijzer, C.P. (2005). Composition and functional specificity of SWI2/SNF2 class chromatin remodeling complexes. Biochim. Biophys. Acta 1681: 59-73.

Mohrmann, L., Langenberg, K., Krijgsveld, J., Kal, A.J., Heck, A.J.R., and Verrijzer, C.P. (2004). Differential targeting of two distinct SWI/SNF-related Drosophila chromatin-remodeling complexes. Mol. Cell. Biol. 24: 3077-3088.

Oruetxebarria, I. Venturini, F., Kekarainen, T., Houweling, A., Zuijderduijn, L.M.P, Mohd-Sarip, A., Hoeben, R., and Verrijzer, C.P. (2004).

p16INK4a is required for hSNF5 chromatin-remodeler induced cellular senescence in malignant rhabdoid tumor cells. J. Biol. Chem. 279: 3807-3816.

Moshkin, Y.U., Armstrong J.A., Maeda, R.K., Tamkun, J.W., Verrijzer, C.P., Kennison, J.A. and Karch, F. (2002). Histone chaperone ASF1 cooperates with the Brahma chromatin-remodeling machinery. Genes Dev. 16: 2621-2626.

Mohd-Sarip, A., Venturini, F., Chalkley, G.E. and Verrijzer, C. P. (2002). Pleiohomeotic can link Polycomb to DNA and mediate transcriptional repression Mol. Cell. Biol. 22: 7473-7483.

Mahmoudi, M., Katsani, K.R. and Verrijzer, C.P. (2002). GAGA can mediate enhancer function in trans by linking two separate DNA molecules. EMBO J. 21: 1775-1781.

Katsani, K.R., Arredondo, J.J., Kal A.J. and Verrijzer, C.P. (2001). A homeotic mutation in the trithorax SET domain impedes histone binding. Genes Dev. 15: 2197-2202.

Mahmoudi, T. and Verrijzer, C.P. (2001). Chromatin silencing and activation by Polycomb and trithorax group proteins. Oncogene 20, 3055-3066.

Kal, A.J., Mahmoudi, T., Zak, N.B. and Verrijzer, C.P. (2000). The Drosophila Brahma complex is an essential coactivator for the trithorax group protein Zeste. Genes Dev. 14: 1058-1071.