Methylation-mediated silencing of TMS1/ASC is accompanied by histone hypoacetylation and CpG island-localized changes in chromatin architecture.

Aberrant methylation of CpG-dense islands in the promoter regions of genes is an acquired epigenetic alteration associated with the silencing of tumor suppressor genes in human cancers. In a screen for endogenous targets of methylation-mediated gene silencing, we identified a novel CpG island-associated gene, TMS1, which is aberrantly methylated and silenced in response to the ectopic expression of DNA methyltransferase-1. TMS1 functions in the regulation of apoptosis and is frequently methylated and silenced in human breast cancers. In this study, we characterized the methylation pattern and chromatin architecture of the TMS1 locus in normal fibroblasts and determined the changes associated with its progressive methylation. In normal fibroblasts expressing TMS1, the CpG island is defined by an unmethylated domain that is separated from densely methylated flanking DNA by distinct 5' and 3' boundaries. Analysis of the nucleoprotein architecture of the locus in intact nuclei revealed three DNase I-hypersensitive sites that map within the CpG island. Strikingly, two of these sites coincided with the 5'- and 3'-methylation boundaries. Methylation of the TMS1 CpG island was accompanied by loss of hypersensitive site formation, hypoacetylation of histones H3 and H4, and gene silencing. This altered chromatin structure was confined to the CpG island and occurred without significant changes in methylation, histone acetylation, or hypersensitive site formation at a fourth DNase I-hypersensitive site 2 kb downstream of the TMS1 CpG island. The data indicate that there are sites of protein binding and/or structural transitions that define the boundaries of the unmethylated CpG island in normal cells and that aberrant methylation overcomes these boundaries to direct a local change in chromatin structure, resulting in gene silencing.     

Regional methylation of the 5' end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.

In mammalians, demethylation of specific promoter regions often correlates with gene activation; inversely, dense methylation of CpG islands leads to gene silencing, probably mediated by methyl-CpG binding proteins. In cell lines and cancers, inhibition of tissue-specific genes and tumor suppressor genes expression seems to be related to such hypermethylation. The 5' end of the breast cancer predisposition gene BRCA1 is embedded in a large CpG island of approximately 2.7 kb in length. In human sporadic breast cancers, the down-regulation of BRCA1 does not seem to be related to BRCA1 gene alterations. Southern blot analysis and the bisulfite sequencing method indicate that the BRCA1 CpG island is regionally methylated in all human tissues analyzed and unmethylated in the gametes, suggesting a role for DNA methylation in the control of gene expression. We have therefore investigated the potential role of methyl-CpG binding proteins in the regulation of BRCA1 gene expression. In vitro, partial methylation of constructs containing this region strongly inhibits gene expression in the presence of MeCP2 protein. Moreover, in the five human cell lines analyzed, chemically induced hypomethylation is associated with BRCA1 gene activation. These data suggest that methyl-CpG binding proteins might be associated with the control of BRCA1 gene expression and that methyl-DNA binding proteins may participate in the regulation of gene expression in mammalian cells.     

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, CK8, CK18, EGFR, MUC1, and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥ 40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.Key words: BRCA1, methylation, epigenetics, triple negative, breast cancer, retionblastoma tumor suppressor gene, pRb, p16     

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, EGFR, MUC1 and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.     

BRCA1 promoter methylation in peripheral blood DNA was identified in sporadic breast cancer and controls

Objective Epigenetics, particularly DNA methylation, has recently been shown to be important in breast cancer initiation. We investigated the clinical and prognostic importance of whole blood breast cancer early onset gene 1 (BRCA1) DNA methylation in sporadic breast cancer. Methods Genomic DNA was extracted from the peripheral blood cells (PBCs) of 902 breast cancer patients at diagnosis, with no BRCA1 mutation, and 990 control women. DNA methylation was measured by quantitative analysis of methylated alleles (QAMA) to estimate the extent of methylation of 2 CpG sites in the promoter region of BRCA1 oncosuppressor. Results BRCA1 promoter methylation rate in PBCs was 47.1% with a 95% confidence interval [46.1; 48.1] in breast cancer patients, and 45.9% with a 95% confidence interval [45.0; 46.8] in controls. We found a trend toward BRCA1 promoter hypermethylation in PBCs of sporadic breast cancer patients compared with controls. Association between methylation and clinicopathological features was evaluated using statistical tests. BRCA1 promoter methylation in PBCs increased significantly in breast cancer patients compared with controls, for age over 70 years (p = 0.022), in post-menopausal status (p = 0.013), for a body mass index (BMI) <20 (p = 0.0095) or a waist-to-hip ratio (WHR) ≤76.8 (p = 0.0027). We also found an association of increased BRCA1 promoter methylation in PBCs with ACA/ACA genotype for the SNP Thr594Thr in ESR (estrogen receptor gene), known to be associated with breast cancer risk (p = 0.092), reflecting the reduced presence of this genotype in this breast cancer case-control study. Conclusion Analysis of site-specific DNA methylation in PBCs by QAMA provides quantitative DNA methylation values that may serve as important prognostic indicators.     

DNA methylation down-regulates CDX1 gene expression in colorectal cancer cell lines

CDX1 is a homeobox protein that inhibits proliferation of intestinal epithelial cells and regulates intestine-specific genes involved in differentiation. CDX1 expression is developmentally and spatially regulated, and its expression is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines. However, very little is known about the molecular mechanism underlying the regulation of CDX1 gene expression. In this study, we characterized the CDX1 gene structure and identified that its gene promoter contained a typical CpG island with a CpG observed/expected ratio of 0.80, suggesting that the CDX1 gene is a target of aberrant methylation. Alterations of DNA methylation in the CDX1 gene promoter were investigated in a series of colorectal cancer cell lines. Combined Bisulfite Restriction Analysis (COBRA) and bisulfite sequencing analysis revealed that the CDX1 promoter is methylated in CDX1 non-expressing colorectal cancer cell lines but not in human normal colon tissue and T84 cells, which express CDX1. Treatment with 5'-aza-2'-deoxycytidine (5-azaC), a DNA methyltransferase inhibitor, induced CDX1 expression in the colorectal cancer cell lines. Furthermore, de novo methylation was determined by establishing stably transfected clones of the CDX1 promoter in SW480 cells and demethylation by 5-azaC-activated reporter gene expression. These results indicate that aberrant methylation of the CpG island in the CDX1 promoter is one of the mechanisms that mediate CDX1 down-regulation in colorectal cancer cell lines.     

Treatment of tumor cells with histone deacetylase inhibitors results in altered recruitment of methyl-CpG binding proteins to a methylated CpG island

When human cancer cells with silencing of the CDH1 gene associated with CpG island methylation and histone deacetylation were treated with histone deacetylase inhibitors, alteration in recruitment of methyl-CpG binding proteins (MBPs) to the methylated CDH1-CpG island was observed, as well as altered histone acetylation status. This change was independent of the histone deacetylase inhibitor used. These results suggest that histone hyperacetylation provides a more open chromatin structure conformation for the recruitment of additional MBPs.     

Extensive methylation of a part of the CpG island located 3.0-4.5 kbp upstream to the chicken alpha-globin gene cluster may contribute to silencing the globin genes in non-erythroid cells

Here, we show that in the chicken genome, the domain of alpha-globin genes is preceded by a CpG island of which the downstream part ( approximately 0.65 kbp) is heavily methylated in lymphoid cells; it is either non-methylated or undermethylated in erythroid cells. Recombinant plasmids were constructed with the corresponding DNA fragment (called "uCpG") placed upstream to a reporter CAT gene expressed from the promoter of the alpha(D) chicken globin gene. Selective methylation of CpG dinucleotides within the uCpG fragment suppressed fivefold the expression of the CAT gene, when neither this gene itself nor the alpha(D) promoter were methylated. Methylation of CpG dinucleotides within the alpha(D) gene promoter did not modify the suppression effect exerted by methylated uCpG. We interpret these results within the frame of the hypothesis postulating, that methylation of the upstream CpG island of the chicken alpha-globin gene domain may play an essential role in silencing the alpha-globin genes in non-erythroid cells.     

Clinical significance of promoter hypermethylation of RASSF1A, RARbeta2, BRCA1 and HOXA5 in breast cancers of Indian patients

Promoter hypermethylation of genes is implicated in the pathogenesis of many cancers, including breast cancer. Herein, we analyzed the promoter methylation status of a panel of critical growth regulatory genes, RASSF1A, RARbeta2, BRCA1 and HOXA5, in 54 breast cancers and 5 distant normal breast tissues of Indian patients. The methylation data were correlated with clinicopathological characteristics and hormone receptor status to determine the impact of methylation in breast carcinogenesis. Promoter hypermethylation of RASSF1A was observed in 39/54 (72%), HOXA5 in 36/54 (67%), BRCA1 in 15/54 (28%) and RARbeta2 in 8/54 (15%) breast cancers. Our most significant findings were the association of RASSF1A methylation with nodal metastasis (p=0.05); and RARbeta2 methylation with age (all tumors in patients in the older age group were methylated, p=0.04). Further, the interactions between DNA methylation and hormone receptor biology in breast cancer cells are beginning to be clearly understood. In this context the association of HOXA5 methylation with loss of ERalpha (p=0.009) is noteworthy.     

The ERCC1 N118N polymorphism does not change cellular ERCC1 protein expression or platinum sensitivity

A hallmark of aberrant DNA methylation-associated silencing is reversibility. However, long-term stability of reactivated promoters has not been explored. To examine this issue, spontaneous reactivant clones were isolated from mouse embryonal carcinoma cells bearing aberrantly silenced Aprt alleles and re-silencing frequencies were determined as long as three months after reactivation occurred. Despite continuous selection for expression of the reactivated Aprt alleles, exceptionally high spontaneous re-silencing frequencies were observed. A DNA methylation analysis demonstrated retention of sporadic methylation of CpG sites in a protected region of the Aprt promoter in many reactivant alleles suggesting a role for these methylated sites in the re-silencing process. In contrast, a chromatin immunoprecipitation (ChIP) analysis for methyl-H3K4, acetyl-H3K9, and dimethyl-H3K9 levels failed to reveal a specific histone modification that could explain high frequency re-silencing. These results demonstrate that aberrantly silenced and reactivated promoters retain a persistent memory of having undergone the silencing process and suggest the failure to eliminate all CpG methylation as a potential contributing mechanism.