Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer

The mechanism of DNA hypermethylation-associated tumor suppressor gene silencing in cancer remains incompletely understood. Here, we show by chromatin immunoprecipitation that for three genes (P16, MLH1, and the O(6)-methylguanine-DNA methyltransferase gene, MGMT), histone H3 Lys-9 methylation directly correlates and histone H3 Lys-9 acetylation inversely correlates with DNA methylation in three neoplastic cell lines. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) resulted in moderately increased Lys-9 acetylation at silenced loci with no effect on Lys-9 methylation and minimal effects on gene expression. By contrast, treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5Aza-dC) rapidly reduced Lys-9 methylation at silenced loci and resulted in reactivation for all three genes. Combined treatment with 5Aza-dC and TSA was synergistic in reactivating gene expression through simultaneous effects on Lys-9 methylation and acetylation, which resulted in a robust increase in the ratio of Lys-9 acetylated and methylated histones at loci showing dense DNA methylation. By contrast to Lys-9, histone H3 Lys-4 methylation inversely correlated with promoter DNA methylation, was not affected by TSA, and was increased moderately at silenced loci by 5Aza-dC. Our results suggest that reduced H3 Lys-4 methylation and increased H3 Lys-9 methylation play a critical role in the maintenance of promoter DNA methylation-associated gene silencing in colorectal cancer.     

DNA methylation-independent reversion of gemcitabine resistance by hydralazine in cervical cancer cells

Background

Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells.

Methodology/Principal Findings

The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity.

Conclusions/Significance

Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.     

Effect of trichostatin A and 5-Aza-2��-deoxycytidine on transgene reactivation and epigenetic modification in transgenic pig fibroblast cells

Transgenic technology has greatly facilitated our understanding of gene function, producing pharmaceutical proteins, and generating models for the study of human diseases. However, epigenetic silencing is still the most major limitation. In this study, we employed DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC) and histone deacetylase inhibitor Trichostatin A (TSA) to study the reactivation of silenced green fluorescent protein (GFP) transgene driven by the cytomegalovirus (CMV) promoter in three fibroblast cell lines from transgenic pigs (tPFs). Analysis showed that porcine fetal fibroblasts (PFF) treated with 0.5 μM 5-Aza-dC for 48 h or 0.25 μM TSA for 24 h had no significantly relevant deaths and no considerably morphological changes. We observed that transgene underwent progressive silencing in a long time course of culture in vitro, and this was correlated with DNA hypermethylation and hypoacetylation of specific histone H3 lysines in the CMV promoter region. Moreover, silenced transgene could be reactivated with 5-Aza-dC or/and TSA treatment by reversing the CMV promoter status of histone hypoacetylation and DNA hypermethylation, and the combination treatment with both agents resulted in a synergistic activation of the transgene, suggesting a cross talk between histone acetylation and DNA methylation. Furthermore, the combination treatment once per 10 days could maintain transgene expression in a high level for more than 60 days by sustaining DNA hypomethylation and histone hyperacetylation. In conclusion, our results suggest that methyltransferase inhibitor 5-Aza-dC and histone deacetylase inhibitor TSA can reactivate silenced transgene and maintain transgene expression by induction of DNA hypomethylation and histone hyperacetylation in the promoter region.     

Epigenetic dysregulation in mesenchymal stem cell aging and spontaneous differentiation

Background

Mesenchymal stem cells (MSCs) hold great promise for the treatment of difficult diseases. As MSCs represent a rare cell population, ex vivo expansion of MSCs is indispensable to obtain sufficient amounts of cells for therapies and tissue engineering. However, spontaneous differentiation and aging of MSCs occur during expansion and the molecular mechanisms involved have been poorly understood.

Methodology/Principal Findings

Human MSCs in early and late passages were examined for their expression of genes involved in osteogenesis to determine their spontaneous differentiation towards osteoblasts in vitro, and of genes involved in self-renewal and proliferation for multipotent differentiation potential. In parallel, promoter DNA methylation and hostone H3 acetylation levels were determined. We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP. Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly. Notably, the altered expression of these genes were closely associated with epigenetic dysregulation of histone H3 acetylation in K9 and K14, but not with methylation of CpG islands in the promoter regions of most of these genes. bFGF promoted MSC proliferation and suppressed its spontaneous osteogenic differentiation, with corresponding changes in histone H3 acetylation in TERT, Oct4, Sox2, Runx2 and ALP genes.

Conclusions/Significance

Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.     

Parent-of-origin specific histone acetylation and reactivation of a key imprinted gene locus in Prader-Willi syndrome

To examine the chromatin basis of imprinting in chromosome 15q11-q13, we have investigated the status of histone acetylation of the SNURF-SNRPN locus, which is a key imprinted gene locus in Prader-Willi syndrome (PWS). Chromatin immunoprecipitation (ChIP) studies revealed that the unmethylated CpG island of the active, paternally derived allele of SNURF-SNRPN was associated with acetylated histones, whereas the methylated maternally derived, inactive allele was specifically hypoacetylated. The body of the SNURF-SNRPN gene was associated with acetylated histones on both alleles. Furthermore, treatment of PWS cells with the DNA methyltransferase inhibitor 5-azadeoxycytidine (5-aza-dC) induced demethylation of the SNURF-SNRPN CpG island and restoration of gene expression on the maternal allele. The reactivation was associated with increased H4 acetylation but not with H3 acetylation at the SNURF-SNRPN CpG island. These findings indicate that (1) a significant role for histone deacetylation in gene silencing is associated with imprinting in 15q11-q13 and (2) silenced genes in PWS can be reactivated by drug treatment.     

The essential role of histone H3 Lys9 di-methylation and MeCP2 binding in MGMT silencing with poor DNA methylation of the promoter CpG island

Silencing of the O (6)-methylguanine-DNA methyltransferase (MGMT) gene, a key to DNA repair, is involved in carcinogenesis. Recent studies have focused on DNA hypermethylation of the promoter CpG island. However, cases showing silencing with DNA hypomethylation certainly exist, and the mechanism involved is not elucidated. To clarify this mechanism, we examined the dynamics of DNA methylation, histone acetylation, histone methylation, and binding of methyl-CpG binding proteins at the MGMT promoter region using four MGMT negative cell lines with various extents of DNA methylation. Histone H3K9 di-methylation (H3me2K9), not tri-methylation, and MeCP2 binding were commonly seen in all MGMT negative cell lines regardless of DNA methylation status. 5Aza-dC, but not TSA, restored gene expression, accompanied by a decrease in H3me2K9 and MeCP2 binding. In SaOS2 cells with the most hypomethylated CpG island, 5Aza-dC decreased H3me2K9 and MeCP2 binding with no effect on DNA methylation or histone acetylation. H3me2K9 and DNA methylation were restricted to in and around the island, indicating that epigenetic modification at the promoter CpG island is critical. We conclude that H3me2K9 and MeCP2 binding are common and more essential for MGMT silencing than DNA hypermethylation or histone deacetylation. The epigenetic mechanism leading to silent heterochromatin at the promoter CpG island may be the same in different types of cancer irrespective of the extent of DNA methylation.     

An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs

Background

Small RNAs generated by RNA polymerase IV (Pol IV) are the most abundant class of small RNAs in flowering plants. In Arabidopsis thaliana Pol IV-dependent short interfering (p4-si)RNAs are imprinted and accumulate specifically from maternal chromosomes in the developing seeds. Imprinted expression of protein-coding genes is controlled by differential DNA or histone methylation placed in gametes. To identify epigenetic factors required for maternal-specific expression of p4-siRNAs we analyzed the effect of a series of candidate mutations, including those required for genomic imprinting of protein-coding genes, on uniparental expression of a representative p4-siRNA locus.

Results

Paternal alleles of imprinted genes are marked by DNA or histone methylation placed by DNA METHYLTRANSFERASE 1 or the Polycomb Repressive Complex 2. Here we demonstrate that repression of paternal p4-siRNA expression at locus 08002 is not controlled by either of these mechanisms. Similarly, loss of several chromatin modification enzymes, including a histone acetyltransferase, a histone methyltransferase, and two nucleosome remodeling proteins, does not affect maternal expression of locus 08002. Maternal alleles of imprinted genes are hypomethylated by DEMETER DNA glycosylase, yet expression of p4-siRNAs occurs irrespective of demethylation by DEMETER or related glycosylases.

Conclusions

Differential DNA methylation and other chromatin modifications associated with epigenetic silencing are not required for maternal-specific expression of p4-siRNAs at locus 08002. These data indicate that there is an as yet unknown epigenetic mechanism causing maternal-specific p4-siRNA expression that is distinct from the well-characterized mechanisms associated with DNA methylation or the Polycomb Repressive Complex 2.     

Curcumin Modulates DNA Methylation in Colorectal Cancer Cells

Aim

Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells.

Materials and Methods

To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2′-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR.

Results

As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines.

Conclusions

Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical.