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.
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.
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 p16
INK4a, 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
p16
INK4a
is down-regulated, 27 of 52 BMI-1
POS cases of HL revealed strong nuclear expression of p16
INK4a. 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
p16
INK4a
.
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.
We have identified a MAR/SAR recognition signature (MRS) which is common to a large group of matrix and scaffold attachment regions. The MRS is composed of two degenerate sequences (AATAAYAA and ...AWWRTAANNWWGNNNC) within close proximity. Analysis of <300 kb of genomic sequence from a variety of eukaryotic organisms shows that the MRS faithfully predicts 80% of MARs and SARs. In each case where we find a MRS, the corresponding DNA region binds specifically to the nuclear scaffold. Although all MRSs are associated with a SAR, not all known SARs and MARs contain a MRS, suggesting that at least two classes exist, one containing a MRS, the other not. Evidence is presented that the two sequence elements of the bipartite MRS occupy a position on the nucleosome near the dyad axis, together creating a putative protein binding site. The identification of a MAR- and SAR-associated DNA element is an important step forward towards understanding the molecular mechanisms of these elements. It will allow: (i) analysis of the genomic location of SARs, e.g. in relationship to genes, based on sequence information alone, rather than on the basis of an elaborate biochemical assay; (ii) identification and analysis of proteins that specifically bind to the MRS.
Polycomb group (PcG) genes encode two chromatin‐binding protein complexes, the PRC1 and the PRC2 PcG complexes, which are essential for the maintenance of cell identity and play a role in ...oncogenesis. PcG complexes were recently identified as novel regulators of hematopoiesis, and appear to be expressed in a non‐overlapping pattern in resting and mature follicular B cells. Using highly specific antisera in combination with immunohistochemistry and triple immunofluorescence, we investigated the expression pattern of nine human PcG genes in germinal center (GC) B cells and highly purified germinal center B cell subpopulations. PcG proteins were detected in characteristic binding patterns that were not necessarily related to mutually exclusive expression of the two PcG complexes. We conclude that the two PcG complexes are expressed throughout GC development, and that the fine composition of each complex is determined by the differentiation status of the cell. In addition, a subset of dividing cells with a centrocyte CD marker profile was identified that co‐expresses core components of the PRC1 and PRC2 complex. We propose that these cells reflect a transitional stage between resting and dividing follicular B lymphocytes, and that they possibly represent the healthy precursors of nodal large B cell lymphomas.
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