Richard George Antonius Bernardus Sewalt

The polycomb group protein EZH2 is involved in progression of prostate cancer

Prostate cancer is a leading cause of cancer-related death in males and is second only to lung cancer. Although effective surgical and radiation treatments exist for clinically localized prostate cancer, metastatic prostate cancer remains essentially incurable. Here we show, through gene expression profiling, that the polycomb group protein enhancer of zeste homolog 2 (EZH2) is overexpressed in hormone-refractory, metastatic prostate cancer. Small interfering RNA (siRNA) duplexes targeted against EZH2 reduce the amounts of EZH2 protein present in prostate cells and also inhibit cell proliferation in vitro. Ectopic expression of EZH2 in prostate cells induces transcriptional repression of a specific cohort of genes. Gene silencing mediated by EZH2 requires the SET domain and is attenuated by inhibiting histone deacetylase activity. Amounts of both EZH2 messenger RNA and EZH2 protein are increased in metastatic prostate cancer; in addition, clinically localized prostate cancers that express higher concentrations of EZH2 show a poorer prognosis. Thus, dysregulated expression of EZH2 may be involved in the progression of prostate cancer, as well as being a marker that distinguishes indolent prostate cancer from those at risk of lethal progression.

EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells

The Polycomb Group Protein EZH2 is a transcriptional repressor involved in controlling cellular memory and has been linked to aggressive prostate cancer. Here we investigate the functional role of EZH2 in cancer cell invasion and breast cancer progression. EZH2 transcript and protein were consistently elevated in invasive breast carcinoma compared with normal breast epithelia. Tissue microarray analysis, which included 917 samples from 280 patients, demonstrated that EZH2 protein levels were strongly associated with breast cancer aggressiveness. Overexpression of EZH2 in immortalized human mammary epithelial cell lines promotes anchorage-independent growth and cell invasion. EZH2-mediated cell invasion required an intact SET domain and histone deacetylase activity. This study provides compelling evidence for a functional link between dysregulated cellular memory, transcriptional repression, and neoplastic transformation.

Characterization of Interactions between the Mammalian Polycomb-Group Proteins Enx1/EZH2 and EED Suggests the Existence of Different Mammalian Polycomb-Group Protein Complexes

ABSTRACT In Drosophila melanogaster, thePolycomb-group (PcG) andtrithorax-group (trxG) genes have been identified as repressors and activators, respectively, of gene expression. Both groups of genes are required for the stable transmission of gene expression patterns to progeny cells throughout development. Several lines of evidence suggest a functional interaction between the PcG and trxG proteins. For example, genetic evidence indicates that the enhancer of zeste [E(z)] gene can be considered both a PcG and a trxGgene. To better understand the molecular interactions in which the E(z) protein is involved, we performed a two-hybrid screen with Enx1/EZH2, a mammalian homolog of E(z), as the target. We report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog ofeed, a murine PcG gene which has extensive homology with the Drosophila PcG gene extra sex combs(esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. Our findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins.

C-Terminal Binding Protein Is a Transcriptional Repressor That Interacts with a Specific Class of Vertebrate Polycomb Proteins

ABSTRACT Polycomb (Pc) is part of a Pc group (PcG) protein complex that is involved in repression of gene activity during Drosophilaand vertebrate development. To identify proteins that interact with vertebrate Pc homologs, we performed two-hybrid screens withXenopus Pc (XPc) and human Pc 2 (HPC2). We find that the C-terminal binding protein (CtBP) interacts with XPc and HPC2, that CtBP and HPC2 coimmunoprecipitate, and that CtBP and HPC2 partially colocalize in large PcG domains in interphase nuclei. CtBP is a protein with unknown function that binds to a conserved 6-amino-acid motif in the C terminus of the adenovirus E1A protein. Also, theDrosophila CtBP homolog interacts, through this conserved amino acid motif, with several segmentation proteins that act as repressors. Similarly, we find that CtBP binds with HPC2 and XPc through the conserved 6-amino-acid motif. Importantly, CtBP does not interact with another vertebrate Pc homolog, M33, which lacks this amino acid motif, indicating specificity among vertebrate Pc homologs. Finally, we show that CtBP is a transcriptional repressor. The results are discussed in terms of a model that brings together PcG-mediated repression and repression systems that require corepressors such as CtBP.

Poorly Differentiated Breast Carcinoma is Associated with Increased Expression of the Human Polycomb Group EZH2 Gene

Polycomb group (PcG) genes contribute to the maintenance of cell identity, cell cycle regulation, and oncogenesis. We describe the expression of five PcG genes (BMI-1, RING1, HPC1, HPC2, and EZH2) innormal breast tissues, invasive breast carcinomas, and their precursors. Members of the HPC-HPH/PRC1 PcG complex, including BMI-1, RING1, HPC1, and HPC2, were detected in normal resting and cycling breast cells. The EED-EZH/PRC2 PcG complex protein EZH2 was only found in rare cycling cells, whereas normal resting breast cells were negative for EZH2. PcG gene expression patterns in ductal hyperplasia (DH), well-differentiated ductal carcinoma in situ (DCIS), and well-differentiated invasive carcinomas closely resembled the pattern in healthy cells. However, poorly differentiated DCIS and invasive carcinomas frequently expressed EZH2 in combination with HPC-HPH/PRC1 proteins. Most BMI-1/EZH2 double-positive cells in poorly differentiated DCIS were resting. Poorly differentiated invasive carcinoma displayed an enhanced rate of cell division within BMI-1/EZH2 double-positive cells. We propose that the enhanced expression of EZH2 in BMI-1(+) cells contributes to the loss of cell identity in poorly differentiated breast carcinomas, and that increased EZH2 expression precedes high frequencies of proliferation. These observations suggest that deregulated expression of EZH2 is associated with loss of differentiation and development of poorly differentiated breast cancer in humans.

Unique polycomb gene expression pattern in Hodgkin's lymphoma and Hodgkin's lymphoma-derived cell lines

Human Polycomb-group (PcG) genes play a crucial role in the regulation of embryonic development and regulation of the cell cycle and hematopoiesis. PcG genes encode proteins that form two distinct PcG complexes, involved in maintenance of cell identity and gene silencing patterns. We recently showed that expression of the BMI-1 and EZH2 PcG genes is separated during normal B-cell development in germinal centers, whereas Hodgkin/Reed-Sternberg (H/RS) cells co-express BMI-1 and EZH2. In the current study, we used immunohistochemistry and immunofluorescence to determine whether the binding partners of these PcG proteins are also present in H/RS cells and H/RS-derived cell lines. PcG expression profiles were analyzed in combination with expression of the cell cycle inhibitor p16INK4a, because experimental model systems indicate that p16 is a downstream target of Bmi-1. We found that H/RS cells and HL-derived cell lines co-express all core proteins of the two known PcG complexes, including BMI-1, MEL-18, RING1, HPH1, HPC1, and -2, EED, EZH2, YY1, and the HPC2 binding partner, CtBP. Expression of HPC1 has not been found in normal mature B cells and other malignant lymphomas of B-cell origin, suggesting that the PcG expression profile of H/RS is unique. In contrast to Bmi-1 transgenic mice where p16INK4a is down-regulated, 27 of 52 BMI-1POS cases of HL revealed strong nuclear expression of p16INK4a. We propose that abnormal expression of BMI-1 and its binding partners in H/RS cells contributes to development of HL. However, abnormal expression of BMI-1 in HL is not necessarily associated with down-regulation of p16INK4a.

Interference with the Expression of a Novel Human Polycomb Protein, hPc2, Results in Cellular Transformation and Apoptosis

Polycomb (Pc) is involved in the stable and heritable repression of homeotic gene activity during Drosophila development. Here, we report the identification of a novel human Pc homolog, hPc2. This gene is more closely related to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1). However, the hPc2 and CBX2/hPc1 proteins colocalize in interphase nuclei of human U-2 OS osteosarcoma cells, suggesting that the proteins are part of a common protein complex. To study the functions of the novel human Pc homolog, we generated a mutant protein, delta hPc2, which lacks an evolutionarily conserved C-terminal domain. This C-terminal domain is important for hPc2 function, since the delta hPc2 mutant protein which lacks the C-terminal domain is unable to repress gene activity. Expression of the delta hPc2 protein, but not of the wild-type hPc2 protein, results in cellular transformation of mammalian cell lines as judged by phenotypic changes, altered marker gene expression, and anchorage-independent growth. Specifically in delta hPc2-transformed cells, the expression of the c-myc proto-oncogene is strongly enhanced and serum deprivation results in apoptosis. In contrast, overexpression of the wild-type hPc2 protein results in decreased c-myc expression. Our data suggest that hPc2 is a repressor of proto-oncogene activity and that interference with hPc2 function can lead to derepression of proto-oncogene transcription and subsequently to cellular transformation.

Identification of anti-repressor elements that confer high and stable protein production in mammalian cells

The expression of transgenic proteins is often low and unstable over time, a problem that may be due to integration of the transgene in repressed chromatin. We developed a screening technology to identify genetic elements that efficiently counteract chromatin-associated repression. When these elements were used to flank a transgene, we observed a substantial increase in the number of mammalian cell colonies that expressed the transgenic protein. Expression of the shielded transgene was, in a copy number–dependent fashion, substantially higher than the expression of unprotected transgenes. Also, protein production remained stable over an extended time period. The DNA elements are small, not exceeding 2,100 base pairs (bp), and they are highly conserved between human and mouse, at both the functional and sequence levels. Our results demonstrate the existence of a class of genetic elements that can readily be applied to more efficient transgenic protein production in mammalian cells.

Selective Interactions between Vertebrate Polycomb Homologs and the SUV39H1 Histone Lysine Methyltransferase Suggest that Histone H3-K9 Methylation Contributes to Chromosomal Targeting of Polycomb Group Proteins

ABSTRACT Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures.

Transcriptional Repression Mediated by Polycomb Group Proteins and Other Chromatin-associated Repressors Is Selectively Blocked by Insulators

Polycomb group (PcG) proteins repress gene activity over a considerable distance, possibly by spreading along the chromatin fiber. Insulators or boundary elements, genetic elements within the chromatin, may serve to terminate the repressing action of PcG proteins. We studied the ability of insulators to block the action of chromatin-associated repressors such as PcG proteins, HP1, and MeCP2. We found that the Drosophila special chromatin structure insulator completely blocks transcriptional repression mediated by all of the repressors we tested. The Drosophilagypsy insulator was able to block the repression mediated by the PcG proteins Su(z)2 and RING1, as well as mHP1, but not the repression mediated by MeCP2 and the PcG protein HPC2. The 5′-located DNase I-hypersensitive site in the chicken β-globin locus displayed a limited ability to block repression, and a matrix or scaffold attachment region element was entirely unable to block repression mediated by any repressor tested. Our results indicate that insulators can block repression mediated by PcG proteins and other chromatin-associated repressors, but with a high level of selectivity. This high degree of specificity may provide a useful assay to define and characterize distinct classes of insulators.