Non-canonical functions of EZH2 in cancer

Mutations in chromatin modifying genes frequently occur in many kinds of cancer. Most mechanistic studies focus on their canonical functions, while therapeutic approaches target their enzymatic activity. Recent studies, however, demonstrate that non-canonical functions of chromatin modifiers may be equally important and therapeutically actionable in different types of cancer. One epigenetic regulator that demonstrates such a dual role in cancer is the histone methyltransferase EZH2. EZH2 is a core component of the polycomb repressive complex 2 (PRC2), which plays a crucial role in cell identity, differentiation, proliferation, stemness and plasticity. While much of the regulatory functions and oncogenic activity of EZH2 have been attributed to its canonical, enzymatic activity of methylating lysine 27 on histone 3 (H3K27me3), a repressive chromatin mark, recent studies suggest that non-canonical functions that are independent of H3K27me3 also contribute towards the oncogenic activity of EZH2. Contrary to PRC2’s canonical repressive activity, mediated by H3K27me3, outside of the complex EZH2 can directly interact with transcription factors and oncogenes to activate gene expression. A more focused investigation into these non-canonical interactions of EZH2 and other epigenetic/chromatin regulators may uncover new and more effective therapeutic strategies. Here, we summarize major findings on the non-canonical functions of EZH2 and how they are related to different aspects of carcinogenesis

Cells exhibiting strong p16INK4a promoter activation in vivo display features of senescence

The activation of cellular senescence throughout the lifespan promotes tumor suppression, whereas the persistence of senescent cells contributes to aspects of aging. This theory has been limited, however, by an inability to identify and isolate individual senescent cells within an intact organism. Toward that end, we generated a murine reporter strain by “knocking-in” a fluorochrome, tandem-dimer Tomato (tdTom), into exon 1α of the p16 INK4a locus. We used this allele (p16 tdTom ) for the enumeration, isolation, and characterization of individual p16 INK4a -expressing cells (tdTom + ). The half-life of the knocked-in transcript was shorter than that of the endogenous p16 INK4a mRNA, and therefore reporter expression better correlated with p16 INK4a promoter activation than p16 INK4a transcript abundance. The frequency of tdTom + cells increased with serial passage in cultured murine embryo fibroblasts from p16 tdTom/+ mice. In adult mice, tdTom + cells could be readily detected at low frequency in many tissues, and the frequency of these cells increased with aging. Using an in vivo model of peritoneal inflammation, we compared the phenotype of cells with or without activation of p16 INK4a and found that tdTom + macrophages exhibited some features of senescence, including reduced proliferation, senescence-associated β-galactosidase (SA-β-gal) activation, and increased mRNA expression of a subset of transcripts encoding factors involved in SA-secretory phenotype (SASP). These results indicate that cells harboring activation of the p16 INK4a promoter accumulate with aging and inflammation in vivo, and display characteristics of senescence

An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation

B-cell lymphoma and melanoma harbor recurrent mutations in the gene encoding the EZH2 histone methyltransferase, but the carcinogenic role of these mutations is unclear. Here we describe a mouse model in which the most common somatic EZH2 gain-of-function mutation (Y646F in human, Y641F in the mouse) can be conditionally expressed. Expression of Ezh2Y641F in mouse B-cells or melanocytes caused high-penetrance lymphoma or melanoma, respectively. Bcl2 overexpression or p53 loss, but not c-Myc overexpression, further accelerated lymphoma progression, and expression of mutant B-Raf but not mutant N-Ras further accelerated melanoma progression. Although expression of Ezh2Y641F increased abundance of global H3K27 trimethylation (H3K27me3), it also caused a widespread redistribution of this repressive mark, including a loss of H3K27me3 associated with increased transcription at many loci. These results suggest that Ezh2Y641F induces lymphoma and melanoma through a vast reorganization of chromatin structure inducing both repression and activation of polycomb-regulated loci

Ataxin1L Is a Regulator of HSC Function Highlighting the Utility of Cross-Tissue Comparisons for Gene Discovery

<div><p>Hematopoietic stem cells (HSCs) are rare quiescent cells that continuously replenish the cellular components of the peripheral blood. Observing that the ataxia-associated gene <i>Ataxin-1-like</i> (<i>Atxn1L</i>) was highly expressed in HSCs, we examined its role in HSC function through <i>in vitro</i> and <i>in vivo</i> assays. Mice lacking Atxn1L had greater numbers of HSCs that regenerated the blood more quickly than their wild-type counterparts. Molecular analyses indicated <i>Atxn1L</i> null HSCs had gene expression changes that regulate a program consistent with their higher level of proliferation, suggesting that <i>Atxn1L</i> is a novel regulator of HSC quiescence. To determine if additional brain-associated genes were candidates for hematologic regulation, we examined genes encoding proteins from autism- and ataxia-associated protein–protein interaction networks for their representation in hematopoietic cell populations. The interactomes were found to be highly enriched for proteins encoded by genes specifically expressed in HSCs relative to their differentiated progeny. Our data suggest a heretofore unappreciated similarity between regulatory modules in the brain and HSCs, offering a new strategy for novel gene discovery in both systems.</p> </div

<i>Atxn1L^−/−</i> mice have enhanced HSC function.

<p>A. Schematic of the experimental design. Equal numbers of bone marrow (BM) cells from WT (competitors) and <i>Atxn1L</i>^−/− or WT (donor) cells were transplanted into lethally irradiated recipients. For secondary transplants, HSCs were purified from primary recipients and transplanted into new recipients along with fresh whole BM competitor cells. B. Competitive whole BM transplants comparing the engraftment ability of WT vs <i>Atxn1L^−/−</i> cells. White and grey bars indicate peripheral blood contribution at 4 and 16 weeks post transplant. Red bars indicate donor cell contribution to bone marrow after 16 weeks. C. Peripheral blood chimerism after purified HSC transplantation at the indicated weeks. Twenty purified HSCs from WT and <i>Atxn1L^−/−</i>mice were transplanted along with 250,000 WT competitor BM cells. D. Analysis of the proportion of donor-derived HSCs obtained from transplant recipients as pooled from 5 mice. HSCs were defined as SP^KSL+CD150+ cells. E. Peripheral blood chimerism after secondary transplants from HSC-transplanted mice from (D) at the indicated weeks (n = 5). F. Limiting dilution competitive repopulation assay with the indicated numbers of WT and <i>Atxn1L^−/−</i> BM cells. The table shows the number of mice tested in each group and the number of mice that were engrafted with donor cells (contribution to blood>0.1%). G. The graph shows the percentage of mice that contain less than 0.1% multi-lineage engraftment 12 weeks post transplant. The HSC frequency was calculated using the L-Calc software according to Poisson statistics (two-tailed t-test; p = 0.016). (* <i>P<0.05</i>, ** <i>P<0.01</i>). All bone marrow transplantation experiments were repeated at least twice with similar results. All graphs display the mean plus standard error.</p