It has become increasingly clear that the pathogenesis of many forms of cancer have a significant epigenetic component. Many cancer types are characterized by gene inactivation, and genes can be silenced through epigenetic mechanisms such as DNA methylation and/or the acquisition of repressive histone 3 modifications such as the methylation of lysines 27 or 9. Methylation of H3K27 is catalyzed by the Polycomb protein Ezh2, the catalytic component of Polycomb Repressive Complex 2 (PRC2). Recently, a specific mutation in the catalytic site of Ezh2 that hyperactivates its H3K27me3 methyltransferase activity while eliminating its ability to monomethylate H3K27 been detected with high frequency in one specific subtype of cancer: the germinal center (GC) B cell subtype of diffuse large B cell lymphoma (GCB-DLBCL). This hyperactive Ezh2 mutation is not present in any other types of lymphomas examined, suggesting that it may be uniquely relevant for GCB- DLBCL lymphomagenesis. Ezh2 transcription is repressed in naove B cells, but it is upregulated in GC B cells. We hypothesize that Ezh2 expression is normally increased at this stage to repress a specific subset of genes that must be downregulated to allow the differentiation of a naove B cell into a GC B cell, and of a GC B cell into a post-GC B cell. Once Ezh2 methylates H3K27, H3K27me3 can be recognized by the Polycomb Repressive Complex 1 (PRC1). The catalytic component of this repressive PRC1 complex is Ring1b, which monoubiquitinates H2A on lysine 119 (uH2A), leading to further repression. However, H2AK119 can be ubiquitinylated by other complexes also. Furthermore, unlike Drosophila PRC1, the basic 4 components in mammalian PRC1 have many alternative paralogs, resulting in a variety of different PRC1 complexes with different affinities for H3K27me3. Very little is known about the biological consequences of PRC1 complexes having different compositions, although it is known that the 4 mammalian paralogs of the histone binding subunit have varying affinities for H3K27me3. We hypothesize that a direct downstream consequence of more H3K27me3 in a subset of GCB-DLBCL may be that there will be more PCR1 recruitment and/or recruitment of different PRC1 complexes. This could result in more uH2A and/or more DNA methylation on some genes. Consequently, these genes are likely to display the most dysregulation. This project will characterize the Polycomb-mediated epigenetic changes that take place in normal human B cells as they differentiate into GC B cells, then into post-GC memory B cells. We will perform ChIP-seq to identify PRC1 components binding to H3K27me3, and compare this to the expression profile and DNA methylation profile to determine which genes are normally repressed in a Polycomb-mediated manner. We will analyze a large set of GC-DLBCL, will determine the epigenetic profile of lymphomas that bear or do not bear the Ezh2 mutation, and will relate this to their gene expression profile. These studies will provide insight into the dysregulation of genes in GCB- DLBCL, and will also provide novel information on the normal epigenetic mechanisms by which genes are repressed in a Polycomb-dependent manner during the late steps of B lymphocyte differentiation.
Diffuse large B cell lymphoma (DLBCL) is an aggressive disease that accounts for 40% of all non- Hodgkins lymphomas, and the GCB subset has been described to have a unique mutation in the histone methyltransferase, Ezh2. Finding genes that are specifically misregulated due to the Ezh2 mutation, and understanding the downstream consequences of bearing this mutant Ezh2, will be a major step forward in pinpointing key molecular targets that may be involved in the pathogenesis of GCB-DLBCL. The novel information we will gain regarding the various mechanisms of Polycomb- mediated repression in normal germinal center B cells, the cell type that undergoes the malignant transformation to GCB-DLBCL, will be essential to understand the misregulation that occurs in lymphomagenesis.
