Journal: Carcinogenesis

Article Title: Estrogen-mediated epigenetic repression of the imprinted gene cyclin-dependent kinase inhibitor 1C in breast cancer cells

doi: 10.1093/carcin/bgr017

Figure Lengend Snippet: 11p15.5 epigenetic and genetic states and expression of CDKN1C in breast cancer cells. (A) The DNA methylation status of the 11p15.5 ICR differentially methylated domain was analyzed by high-resolution bisulfite PCR pyrosequencing in primary tumors (n = 306), breast cancer cell lines (n = 52) and normal breast tissues (n = 19). Methylation levels of individual CpG sites (circles) are shown in a color gradient ranging from 0% (white) to 100% (blue). Left, diagram of CpG sites interrogated by the pyrosequencing assay. Right, results presented in rows of columns. (B) Multiple linear regression analysis of impact of 11p15.5 ICR methylation and genetic status on CDKN1C expression in breast cancer cells. The combined 11p15.5 ICR methylation, copy number and CDKN1C mRNA levels in 16 breast cancer cell lines were assessed by bisulfite PCR pyrosequencing, DNA copy number qPCR and qRT–PCR, respectively. DNA copy number of 11p15.5 ICR was quantified by absolute standard curve of cloned PCR products as described in supplementary Figure S4 (available at Carcinogenesis Online). Relative CDKN1C ΔCt expression values were obtained by normalizing to the 36B4 reference gene. (C) The effect of E2 on CDKN1C expression in breast cancer cells with 11p15.5 ICR hypermethylation. MDA-MB-453 and T47D cells were stimulated with 10 nM E2 or vehicle for 12 h. CDKN1C mRNA levels were assessed as described in Figure 1. Results are the average of two independent experiments. Columns, mean (n = 6); bars, SD.

Article Snippet: Approximately 300 ng of genomic DNA was bisulfite modified with EZ DNA Methylation Kit (Zymo Research, Irivine, CA) according to the manufacturer’s protocol.

Techniques: Expressing, DNA Methylation Assay, Methylation, Pyrosequencing Assay, Quantitative RT-PCR, Clone Assay

Journal: Carcinogenesis

Article Title: Estrogen-mediated epigenetic repression of the imprinted gene cyclin-dependent kinase inhibitor 1C in breast cancer cells

doi: 10.1093/carcin/bgr017

Figure Lengend Snippet: Potential mechanisms causing repression of CDKN1C in breast cancer cells. (A) Proposed model for epigenetic repression of CDKN1C through coordinated loop formation with the 11p15.5 ICR. CTCF binding to the ICR and CDKN1C locus and forms a long-range intrachromosomal loop via dimerization of CTCF. Ligand-bound ERα complex (orange and blue sphere) may mediate silencing through the formation of a secondary loop that serves both to sequester upstream tissue-specific enhancers and to recruit PRC2 and HDAC1 to the 11p15.5 ICR. CTCF serves as a scaffold to secure the PRC2 complex that methylates H3K27, leading to the formation of a repressive chromatin state at the CDKN1C locus. (B) Proposed regulatory mechanism of CDKN1C-AS. The formation of a double-stranded RNA may negatively regulate stability, transport and/or translation of the sense CDKN1C transcript. (C) Summary of three potential mechanisms causing CDKN1C repression in breast cancer cells. DNA methylation status of 11p15.5 ICR is indicated by large oval: white (unmethylated), black (methylated). Upper left, in the normal imprinted domain unmethylated 11p15.5 ICR on the paternal allele (♂) functions as a silencer and a promoter for KCNQ1OT1 transcription, repressing CDKN1C expression. The methylated maternal allele (♀) cannot function as a silencer or a promoter for KCNQ1OT1, thus permitting expression of CDKN1C. Upper right, the CDKN1C-AS transcript represses CDKN1C in trans, potentially through a double-stranded RNA mechanism. Under certain cellular conditions, this may be induced by estrogen-mediated upregulation of CDKN1C-AS. Lower left, DNA hypomethylation resulting from genetic loss of the methylated 11p15.5 ICR allele leads to aberrant domain silencer activity mediated by unrestricted CTCF binding and KCNQ1OT1 transcription, repressing CDKN1C expression. Lower right, estrogen induces KCNQ1OT1 transcription and CTCF recruitment to mediate ICR silencer activity, which in turn direct epigenetic repression of the CDKN1C locus.

Article Snippet: Approximately 300 ng of genomic DNA was bisulfite modified with EZ DNA Methylation Kit (Zymo Research, Irivine, CA) according to the manufacturer’s protocol.

Techniques: Binding Assay, DNA Methylation Assay, Methylation, Expressing, Activity Assay

Journal: Gut

Article Title: Glutathione peroxidase 7 has potential tumor suppressor functions that are silenced by location-specific methylation in oesophageal adenocarcinoma

doi: 10.1136/gutjnl-2013-304612

Figure Lengend Snippet: A) A schematic chart shows GPX7 genomic structure. GPX7 has 3 exons as shown in black boxes. A CpG island with dense CpG sites is present around the transcription start site (TSS); each vertical bar represents one CpG site. Pyrosequencing assays that covered CpG sites from −162 to +138, relative to TSS were designed (Supplementary Table 1). B) Summary of pyrosequencing results in normal oesophageal squamous epithelia (NS), normal gastric mucosa (NG), Barrett’s oesophagus (BO) and oesophageal adenocarcinoma (OAC) (additional details in Table 1). Hypermethylation of CpG sites from +13 to +64 (dash-line square) was only seen in OAC. C) Comparison of the average DNA methylation levels (CpG sites from +13 to +64) in NS, NG, BO, and OAC samples. D) Spearman correlation analysis demonstrated inverse correlation between methylation levels (+13 to +64) and GPX7 mRNA expression fold (r=−0.37, p=0.001).

Article Snippet: DNA bisulfite treatment and pyrosequencing analysis The DNA was bisulfite modified using an EZ DNA Methylation Gold Kit (Zymo Research, Orange, California, USA) according to the manufacturer’s protocol.

Techniques: DNA Methylation Assay, Methylation, Expressing

Journal: Gut

Article Title: Glutathione peroxidase 7 has potential tumor suppressor functions that are silenced by location-specific methylation in oesophageal adenocarcinoma

doi: 10.1136/gutjnl-2013-304612

Figure Lengend Snippet: GPX7 DNA copy number, methylation level, and mRNA expression in oesophageal cell lines and primary tumors

Article Snippet: DNA bisulfite treatment and pyrosequencing analysis The DNA was bisulfite modified using an EZ DNA Methylation Gold Kit (Zymo Research, Orange, California, USA) according to the manufacturer’s protocol.

Techniques: Methylation, Expressing, DNA Methylation Assay

Journal: Epigenetics

Article Title: Estrogen signaling, through estrogen receptor β, regulates DNA methylation and its machinery in male germ line in adult rats

doi: 10.1080/15592294.2017.1309489

Figure Lengend Snippet: Effect of PPT and DPN treatment on DNA methylation. (A) Decrease in global 5mC content in spermatozoa after DPN treatment. All values are expressed as means ± SEM. ** represents P < 0.01. (B) Methylation mapping of 15 CpG sites and total methylation of H19 DMR by pyrosequencing after PPT and DPN treatments. ‘a’ indicates P < 0.05, ‘b’ indicates P < 0.01, and ‘c’ indicates P < 0.001. n = 8.

Article Snippet: Methylation analysis of spermatozoa DNA by bisulfite pyrosequencing Sperm genomic DNA was subjected to bisulphite modification using MethylCode Bisulfite Conversion Kit (Invitrogen) as per manufacturer's protocol.

Techniques: DNA Methylation Assay, Methylation

Journal: Epigenetics

Article Title: Estrogen signaling, through estrogen receptor β, regulates DNA methylation and its machinery in male germ line in adult rats

doi: 10.1080/15592294.2017.1309489

Figure Lengend Snippet: DNA methylation analysis of LINE-1 promoter by pyrosequencing after PPT and DPN treatments. Data represented as mean ± SEM n = 8. ** P < 0.01 ; *** P < 0.001.

Article Snippet: Methylation analysis of spermatozoa DNA by bisulfite pyrosequencing Sperm genomic DNA was subjected to bisulphite modification using MethylCode Bisulfite Conversion Kit (Invitrogen) as per manufacturer's protocol.

Techniques: DNA Methylation Assay, Methylation