ChIP analysis of the histone modifications at the D4Z4 repeats in facioscapulohumeral muscular dystrophy (FSHD)

Genomic DNA must exist in a particular chromatin configuration and modification of this structure is essential for the correct control of gene expression. There are several human genetic disorders that are caused by misregulation of epigenetic gene control. Facioscapulohumeral muscular dystrophy (FSHD) is a disease that may be caused by alterations in chromatin structure. FSHD is the third most common form of muscular dystrophy. The majority of FSHD cases show contraction of the D4Z4 repeats on the 4q35 chromosome (FSHD1). However, a small number of FSHD cases show no contraction at this region (FSHD2), but share epigenetic changes at the D4Z4 region with the FSHD1 patients. In 2009, Zeng et al. reported a specific loss of H3K9me3 histone modification at the D4Z4 repeats in FSHD patients. The main focus of this study was to verify the published data by Zeng et al (2009) and further investigate the histone modification changes at the D4Z4 array. Chromatin immunoprecipitation (ChIP) coupled with real-time quantitative PCR (qPCR) was employed to investigate the histone modifications within the D4Z4 array. The results obtained were in agreement with the previously published data on the reduction of H3K9me3 histone modification at the D4Z4 repeats in FSHD patients. However, contradictory to the previous data, the reduction of this histone modification was also observed on other genomic regions. A global reduction of H3K27me3 was also observed in FSHD patients

ChIP analysis of the histone modifications at the D4Z4 repeats in facioscapulohumeral muscular dystrophy (FSHD)

Genomic DNA must exist in a particular chromatin configuration and modification of this structure is essential for the correct control of gene expression. There are several human genetic disorders that are caused by misregulation of epigenetic gene control. Facioscapulohumeral muscular dystrophy (FSHD) is a disease that may be caused by alterations in chromatin structure. FSHD is the third most common form of muscular dystrophy. The majority of FSHD cases show contraction of the D4Z4 repeats on the 4q35 chromosome (FSHD1). However, a small number of FSHD cases show no contraction at this region (FSHD2), but share epigenetic changes at the D4Z4 region with the FSHD1 patients. In 2009, Zeng et al. reported a specific loss of H3K9me3 histone modification at the D4Z4 repeats in FSHD patients. The main focus of this study was to verify the published data by Zeng et al (2009) and further investigate the histone modification changes at the D4Z4 array. Chromatin immunoprecipitation (ChIP) coupled with real-time quantitative PCR (qPCR) was employed to investigate the histone modifications within the D4Z4 array. The results obtained were in agreement with the previously published data on the reduction of H3K9me3 histone modification at the D4Z4 repeats in FSHD patients. However, contradictory to the previous data, the reduction of this histone modification was also observed on other genomic regions. A global reduction of H3K27me3 was also observed in FSHD patients

Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer

<p>Hypomethylation of LINE-1 repeats in cancer has been proposed as the main mechanism behind their activation; this assumption, however, was based on findings from early studies that were biased toward young and transpositionally active elements. Here, we investigate the relationship between methylation of 2 intergenic, transpositionally inactive LINE-1 elements and expression of the LINE-1 chimeric transcript (LCT) 13 and LCT14 driven by their antisense promoters (L1-ASP). Our data from DNA modification, expression, and 5′RACE analyses suggest that colorectal cancer methylation in the regions analyzed is not always associated with LCT repression. Consistent with this, in HCT116 colorectal cancer cells lacking DNA methyltransferases DNMT1 or DNMT3B, LCT13 expression decreases, while cells lacking both DNMTs or treated with the DNMT inhibitor 5-azacytidine (5-aza) show no change in LCT13 expression. Interestingly, levels of the H4K20me3 histone modification are inversely associated with LCT13 and LCT14 expression. Moreover, at these LINE-1s, H4K20me3 levels rather than DNA methylation seem to be good predictor of their sensitivity to 5-aza treatment. Therefore, by studying individual LINE-1 promoters we have shown that in some cases these promoters can be active without losing methylation; in addition, we provide evidence that other factors (e.g., H4K20me3 levels) play prominent roles in their regulation.</p

miR-21 expression in primary colorectal tissue samples, calculated relatively to that of the control miRNA RNU44, was studied in primary tumour and matched adjacent normal mucosa (n = 45; A); in Dukes' A (n = 9), B (n = 23) and C (n = 13) stages (B); in lymph node negative (No; n = 32) versus lymph node positive (Yes, n = 13) cases (C); and in tumours displaying lower (pT1 & pT2; n = 11) versus higher (pT3 & pT4; n = 34) depth of invasion (D).

<p>Column bar graphs indicate the median and whiskers demonstrate the IQR of miR-21 expression. Note that significant increase in miR-21 expression is observed in tumours, and this increases further with worsening stage, lymphnode involvement and depth of invasion. Statistical significance was calculated using the Wilcoxon signed-rank test in A, the Kruskall-Wallis test in B, and the Mann-Whitney U test in C and D.</p

Inflammation and MiR-21 Pathways Functionally Interact to Downregulate PDCD4 in Colorectal Cancer

<div><p>Inflammation plays a direct role in colorectal cancer (CRC) progression; however the molecular mechanisms responsible for this effect are unclear. The inflammation induced cyclooxygenase 2 (COX-2) enzyme required for the production of Prostaglandin E2 (PGE₂), can promote colorectal cancer by decreasing expression of the tumour suppressor gene Programmed Cell Death 4 (PDCD4). As PDCD4 is also a direct target of the oncogene microRNA-21 (miR-21) we investigated the relationship between the COX-2 and miR-21 pathways in colorectal cancer progression. Gene expression profile in tumour and paired normal mucosa from 45 CRC patients demonstrated that up-regulation of COX-2 and miR-21 in tumour tissue correlates with worse Dukes' stage. In vitro studies in colonic adenocarcinoma cells revealed that treatment with the selective COX-2 inhibitor NS398 significantly decreased miR-21 levels (p = 0.0067) and increased PDCD4 protein levels (p<0.001), whilst treatment with PGE₂ up-regulated miR-21 expression (p = 0.019) and down-regulated PDCD4 protein (p<0.05). These findings indicate that miR-21 is a component of the COX-2 inflammation pathway and that this pathway promotes worsening of disease stage in colorectal cancer by inducing accumulation of PGE₂ and increasing expression of miR-21 with consequent downregulation of the tumour suppressor gene PDCD4.</p></div

Proposed model of how the inflammation pathway can promote colorectal cancer progression by linking inflammation to downregulation of PDCD4 via miR-21 induction mediated by COX-2 driven accumulation of PGE₂.

<p>Persistent inflammation at the tumour site drives increase in COX-2 expression leading to accumulation of PGE₂. This in turn leads to the increase in expression of miR-21 and subsequent downregulation of PDCD4 protein levels facilitating progressive increase to more invasive and metastatic forms of colorectal cancer (from Dukes' A to B, C and D). Our experiments indicate that this pathway can be blocked by COX-2 inhibitors (NS398 and Aspirin) by preventing accumulation of PGE₂ as well as by miR-21 inhibitors. ↑ Activation, Inhibition. See text for further details.</p

miR-21 and <i>PDCD4</i> expression in HCA-7 cells treated with the COX-2 inhibitor NS398.

<p>Following 72 hours treatment significant decrease in miR-21 levels is seen in NS398 treated cells (<b>A</b>). <i>PDCD4</i> mRNA levels are not affected by NS398 treatment (<b>B</b>). Western blot analysis of PDCD4 (<b>C</b> top panel for a representative blot) reveals a significant increase in protein levels in NS398 treated cells as compared to untreated or vehicle alone (DMSO) treated cells, when quantified relatively to beta actin protein (BACT) as a loading control (<b>C</b> bottom panel). The column bar graph indicates the mean and the whiskers demonstrate the standard error of the mean (SEM). Statistical significance was calculated using the unpaired t-test. Experiments were repeated three times and analysed in triplicate. Untreated = cells cultured in media; DMSO = cells cultured in media supplemented with 0.1% DMSO vehicle; NS398 = cells cultured in media supplemented with NS398 prepared in DMSO to a final concentration of 100 µM NS398 and 0.1% DMSO.</p

miR-21 and <i>PDCD4</i> expression in HCA-7 cells treated with miR-21 inhibitor.

<p>Following 72 hours from transfection significant decrease in miR-21 levels are seen only in cells transfected with miR-21 inhibitor (<b>A</b>). <i>PDCD4</i> mRNA levels were not affected by inhibition of miR-21 (<b>B</b>). Western blot analysis of PDCD4 (<b>C</b> top panel for a representative blot) reveals a significant increase in protein levels in cells transfected with the inhibitor as compared to untreated or scramble control treated cells, when quantified relatively to beta actin protein (BACT) as a loading control (<b>C</b> bottom panel). The column bar graph indicates the mean and the whiskers demonstrate the standard error of the mean (SEM). Statistical significance was calculated using the unpaired t-test. Experiments were repeated three times and analysed in duplicate. Untreated = cells culture in media; scramble = cells transfected with scramble RNA inhibitors; miR-21 inhibitor = cells treated with the miR-21 RNA inhibitor.</p

Expression of <i>PDCD4</i> and <i>COX-2</i> mRNA in primary colorectal tissue samples calculated relatively to that of the control gene <i>GAPDH</i>.

<p>Combined relative expression of PDCD4 and COX-2 was analysed in primary tumour versus matched adjacent normal mucosa (n = 45; A and C respectively), and in normal (N) and tumour (T) tissues from Dukes' A (n = 9), B (n = 23) and C (n = 13) stages (B and D respectively). The column bar graph indicates the median and the whiskers demonstrate the IQR of mRNA expression. Statistical significance was calculated using the Wilcoxon signed-rank test in A and C; the Kruskall-Wallis test in B and D. Note that <i>PDCD4</i> is downregulated in tumour versus normal, but no differences are seen in the remaining expression in tumours of different Dukes' stages. By contrast <i>COX-2</i> levels increase in tumour and with disease stage.</p

Relative expression of miR-21 with common clinical-pathological features of colorectal cancer.

<p>Relative expression of miR-21 with common clinical-pathological features of colorectal cancer.</p