post - translational modifications of the n - terminal tails of core histones , including methylation , acetylation , ubiquitylation and phosphorylation , influence chromatin configuration , which can modulate accessibility of transcription factors and transcriptional activity of nearby genes 1 .
four lysine residues in the n - terminal tail of histone h3 , k4 , k9 , k27 and k36 , are primary targets of specific histone methyltransferases and demethylases .
methylated h3k27 is abundant , and three different statues ( mono- , di- or tri - methylation ) exist .
for example , based on quantitative mass spectrometry 50% of h3k27 in embryonic stem ( es ) cells is dimethylated , 15% trimethylated and 15% monomethylated 2 .
di- and tri - methylation are characteristic of pcg ( polycomb group ) target genes and are associated with transcriptional repression 3 .
prc1 catalyzes the monoubiquitylation of histone h2a and prc2 contributes to the methylation of h3k27 4 .
prc2 is composed of several proteins , including eed , suz12 , rbap46/48 and either ezh1 or ezh2 .
ezh1 and 2 are characterized by a set domain , which functions as a histone - lysine n - methyltransferases 5 - 8 .
while prc2 complexes containing ezh2 are primarily responsible for h3k27 methylation , prc2 complexes containing ezh1 can also catalyze h3k27 methylation 9 , or compact chromatin through mechanisms other than histone methylation .
experiments with cell - specific ezh2 knockout mice have demonstrated that ezh2 influences proliferation and differentiation of stem cells and progenitor cells in three distinct cell types , adipocytes , keratinocytes , and neurons 11 - 13 .
experimental evidence from several systems suggests that h3k27 methylation is mainly achieved by ezh2 and to a lesser extent by ezh1 .
the critical importance of ezh2 is reflected by the fact that ezh2-null mice die in utero , while ezh1-null mice have no overt defects , possibly due to compensatory functions of ezh2 .
however , ezh1 can also compensate to some extent for ezh2 as loss of both ezh1 and 2 evoke more severe consequences than loss of ezh2 alone 14 .
in addition , contribution to h3k27 monomethylation by g9a , a well - known h3k9 methyltransferase , was observed in vitro and in vivo 15 , 16 .
although no changes in h3k27 di- and tri - methylation were observed between wild type and g9a es cells , the extent of monomethylation was significantly decreased in g9a   es cells .
it is possible that g9a introduces a monomethyl group on h3k27 in the absence of ezh2 and that this is sufficient the binding by embryonic ectoderm development ( eed ) , a component of the prc2 complex , which contains the methyl - lysine histone - binding domain wd40 .
jmjd3 ( kdm6b , [ jumonji - domain containing protein 3 ] ) and utx ( kdm6a [ ubiquitously transcribed tetratricopeptide repeat gene on the x chromosome ] ) are demethylases acting on h3k27 ( figure 1b ) .
they remove the gene - inactivating h3k27 di - methyl and tri - methyl marks and thereby presumably contribute to the maintenance of gene expression .
abberant of utx and jmjd3 levels have been associated with cancer and defective differentiation programs 17 .
excessive ezh2 concentrations have been reported as a marker of aggressive breast cancer 18 , 19 and associated with invasion and cancer progression .
chinnaiyan and colleagues tested ezh2 protein levels in 280 breast cancer patients using high - density tissue microarray 20 .
ezh2 levels were elevated in patients with invasive breast carcinoma relative to normal or atypical hyperplasia .
notably , increased expression was already observed in ductal carcinoma in situ ( dcis ) , a precursor of invasive carcinoma 21 .
elevated ezh2 concentration has been associated with increased tumor cell proliferation in various cancers including breast , prostate and melanoma 22 .
the two - layered regulatory loop includes transcriptional regulation of the ezh2 gene and ensuing consequences of associated h3k27me3 ( figure 2 ) .
e2f , a target of the retinoblastoma protein ( prb ) , plays a critical role in regulating cell cycle progression through activating genes that control entry into the s phase and genes associated with dna replication 23 . upon phosphorylation of prb ,
activated e2f binds to promoter regions of ezh2 and eed , and controls their expression .
however , helin and colleagues confirmed that the interrupt of ezh2 and eed expression was not enough to increase expression of negative regulators of cell cycle including p14arf , but positive regulators of cell proliferation , cyclind1 ( ccnd1 ) , cycline1 ( ccne1 ) , cyclina2 ( ccna2 ) and cyclinb1 ( ccnb1 ) , significantly decreased in ezh2 lacking cells .
they concluded that the prc complex , including ezh2 , is required for the activation or maintenance of the activated state of certain genes in proliferating cells through h3k27 methylation 24 .
ezh2 expression is also controlled by hypoxia through hif response elements ( hre ) in the ezh2 gene promoter 25 . in hypoxic microenvironment ,
lastly , mek / erk / elk pathway can also leads to ezh2 overexpression in erbb2-overexpressing breast cancer cell lines 26 .
a set of genes associated with cell proliferation and invasion is regulated by ezh2 . among them
rad51 , runx3 , and cdkn1c ( p57 ) regulate cell proliferation , and foxc1 and cdh1 ( e - cadherin ) have been linked to metastasis .
the runt - related ( runx ) family is associated with normal development and neoplasia and runx3 is an established tumor suppressor gene in gastric cancer 46 .
inactivation of runx3 expression through dna hypermethylation has been reported in various cancers , including those of prostate , lung and pancreas .
notably , upon reducing ezh2 expression in the mcf-7 breast cancer cell line , runx3 expression was recovered .
it has been suggested that runx3 down - regulation is controlled by h3k27me3 through ezh2 and histone deacetylation through hdac1 . even though dna methylation affects repression of runx3 , h3k27me3 by ezh2 has a critical role in down - regulation of runx3 .
runx3 reduction leads to the decrease of the cyclin - dependent kinase inhibitor p21expression , which results in the induction of cell proliferation in breast cancer 28 .
foxc1 , a member of the forkhead box transcription factor family , plays an important role in differentiation . increased ezh2 influences transcriptional repression of foxc1 through the accumulation of h3k27me3 and diminished acetylation of h3/h4 in mda - mb-231 cell lines .
elevated foxc1 concentrations lead to reduced migration and invasion in vitro and in vivo 29 .
the cyclin - dependent kinase ( cdk ) inhibitor , cdkn1c ( p57 ) encodes the tumor suppressor p57 protein .
reduced cdkn1c expression in various breast cancer cell lines was associated with ezh2 overexpression and increased h3k27me3 30 . upon treatment with the histone methylation inhibitor 3-deazaneplanocin a ( dznep ) , recovery of cdkn1c expression was observed suggesting a direct link between h3k27me3 and transcriptional suppression .
the transmembrane receptor e - cadherin maintains epithelial cellular adhesion and integrity and it has been proposed that its down - regulation leads to enhanced invasion in cancer . in ezh2-overexpressing breast epithelial cells , reduced cdh1 ( e - cadherin ) expression correlated with h3k27me3 levels in the promoter .
however , elevated expression of ezh2 was not sufficient to increase enzymatic activity for h3k27me3 at the cdh1 promoter , which also required hdac activity .
increased hdac activity leads to the removal of acetyl groups from h3k27 , a prerequisite for ezh2-controlled histone methylation .
increased ezh2 induces reduction of cdh1 expression , and it leads to metastasis and invasion in breast cancer as well as pancreatic cancer and ovarian cancer 31 - 33 .
ezh2 also appears to regulate breast tumor initiating cells ( btics ) through suppressing rad51 expression .
pho and gaga motifs , which are targets for prc2 complexes , are located in the rad51 promoter and h3k27me3 coincides with these sites .
reduction of rad51 affects chromosome breaks , deletion and translocation as well as increase of centrosome number and genome instability through activated raf / perk/  - catenin signaling 25 .
mutations in brca1 are a cause of basal like breast carcinomas that are er , pr and her2-neu negative ( triple negative ) and increased ezh2 concentrations were found mainly in basal type breast carcinomas 34 .
germline brca1 mutations predispose women to develop breast cancer , and patients have a poor prognosis , in part because brca1 's role in dna repair and genomic stability 35 .
even though the majority of basal breast cancers do not carry brca1 germline mutations , reduced concentration of brca1 protein and mrna levels were reported in these patients 36 .
however , the mechanisms causing decreased brca1 expression in basal like carcinomas remains to be established .
ezh2 is frequently overexpressed in er - negative breast cancer cell lines , and a reduction of ezh2 results in suppressed cell proliferation .
biological effects of ezh2 knockdowns were observed in vivo and in vitro system using xenograft transplantations . reduced tumor growth was confirmed in xenograft mouse models injected with ezh2 knockdown cells 37 .
ezh2 overexpression inhibits brca1 phosphorylation ( ser1423 ) and thereby promotes an increase of cdc25c , an essential player for g2/m checkpoint control .
once cdc25c is activated , it induces the activity of the cdc2/cyclinb1 complex . in summary , elevated ezh2 concentrations in er - negative breast cancer cell lines confer increased cell proliferation in part possibly through the inhibition of brca1 phosphorylation 37 .
brca1 is a nuclear protein involved in numerous activities , including dna repair and estrogen receptor modulation .
it has been proposed that akt-1 ( ser473 ) , but not akt-2 and 3 , are phosphorylated and activated by ezh2 in the ezh2 overexpressing breast cancer cell line cal51 , and nuclear location of brca1 protein is suppressed in these cells by a yet to be determined mechanism ( figure 3 ) .
this in turn results in aberrant mitoses with extra centrosomes , and genomic instability 38 .
elevated ezh2 concentrations were observed in mouse mammary epithelium from which the brca1 gene had been deleted . as a result gene expression related to differentiation was suppressed and excessive cell proliferation was promoted .
it was confirmed that brca1-deficient cells are more sensitive to dznep , a potential treatment , when compared with brca1-proficient cells 39 .
genome - wide approaches have been applied to identify genomic localizations and target genes of prc complexes in human and mouse es cells .
this system has been explored using a combination of chromatin immunoprecipitation ( chip ) , chip on chip and microarray analyses 40 - 43 .
transcription factors related with developmental regulation , including homeobox protein ( hox ) , gata binding protein ( gata ) , and runt related transcription factor ( runx ) are regulated by the prc2 complex in human es cells .
these include genes encoding the transcription factor families fox , sox , gata and tbx 40 . moreover , enriched h3k27me3 was found in proximal promoter regions of these genes , which were also derepressed in suz12- and eed - null cells 40 , 42 supporting the notion that their suppression is dependent on the prc complex .
chip - seq analyses with antibodies against ezh2 , ring1b , h3k4me3 and h3k27me3 have been performed to identify the conservation of chromatin states in orthologous genomic loci in human and mouse es cells 44 .
bivalent promoters , which carry h3k4me3 and h3k27me3 marks , are highly conserved between mouse and human es cells .
in addition to prc2 , prc1 binding was identified in the above genes . in general , h3k27me3 retention was most pronounced in regions that were occupied by both prc1 and 2 , while poor retention was detected on sequences occupied by prc2 only .
these findings suggest that prc2 catalyzes and regulates h3k27 methylation but is not sufficient to maintain the methylation status of respective target genes .
genome - wide approaches have been used to predict the importance of the prc2 complex in the regulation of developmental transcription through h3k27 methylation .
however , it is not clear what concentration of ezh2 is required to regulate h3k27 methylation , and whether excess ezh2 has other functions besides its methyltransferase activity .
the suggestion that excess ezh2 , and thereby altered histone modifications , leads to a changed epigenetic genomic landscape , and thereby contributes to cancer progression , is mainly based on correlative studies and cell culture investigations .
although biochemical studies are capable of linking elevated ezh2 concentrations with altered genetic programs in cancer , clear genetic evidence is scarce . it will be necessary to conduct decisive genetic experiments in mice to link ezh2 to the h3k27 methylation status and subsequent genetic programs in relevant breast cancer models , such as mice carrying mutant brca1 45 .
it will be necessary to explore breast cancer initiation and progression in relevant mouse models in which h3k27 methylation is disrupted . by ablating the ezh1 and ezh2 genes in mammary epithelium
the mammary genome will lack h3k27me3 marks and upon deletion of the utx and jmjd3 genes a persistent h3k27me3 should be expected .
such experiments , although labor intensive and time - consuming , shall provide definitive answers on the role of h3k27 methylation in development and the etiology of cancer .
 histone modifications are thought to control the regulation of genetic programs in normal physiology and cancer . 
 methylation ( mono- , di- , and tri - methylation ) on histone h3 lysine ( k ) 27 induces transcriptional repression , and thereby participates in controlling gene expression patterns . 
 enhancer of zeste ( ezh ) 2 , a methyltransferase and component of the polycomb repressive complex 2 ( prc2 ) , plays an essential role in the epigenetic maintenance of the h3k27me3 repressive chromatin mark . 
 abnormal ezh2 expression has been associated with various cancers including breast cancer . here , we discuss the contribution of ezh2 and the prc2 complex in controlling the h3k27 methylation status and subsequent consequences on genomic instability and the cell cycle in breast cancer cells . 
 we also discuss distinct molecular mechanisms used by ezh2 to suppress brca1 functions . 
Transcriptional regulation modulated by the H3K27 methylation status
EZH2 and human breast cancer
Dynamic signaling regulated by EZH2
A BRCA1-EZH2 connection
Genome-wide approaches to explore PRC
Moving forward using mouse genetics