Gene MAGEA1 belongs to a group of human germline-specific genes that rely on DNA methylation for repression in somatic tissues. Many of these genes, termed cancer-germline (CG) genes, become demethylated and activated in a wide variety of tumors, where they encode tumor-specific antigens. The process leading to DNA demethylation of CG genes in tumors remains unclear. Previous data suggested that histone acetylation might be involved. Here, we investigated the relative contribution of DNA methylation and histone acetylation in the epigenetic regulation of gene MAGEA1. We show that MAGEA1 DNA hypomethylation in expressing melanoma cells is indeed correlated with local increases in histone H3 acetylation (H3ac). However, when MAGEA1-negative cells were exposed to a histone deacetylase inhibitor (TSA), we observed only short-term activation of the gene and detected no demethylation of its promoter. As a more sensitive assay, we used a cell clone harboring a methylated MAGEA1/hph construct, which confers resistance to hygromycin upon stable re-activation. TSA induced only transient de-repression of the transgene, and did not lead to the emergence of hygromycin-resistant cells. In striking contrast, transient depletion of DNA-methyltransferase-1 in the reporter cell clone gave rise to a hygromycin-resistant population, in which the re-activated MAGEA1/hph transgene displayed not only marked DNA hypomethylation, but also significant reversal of histone marks, including gains in H3ac and H3K4me2, and losses of H3K9me2. Collectively, our results indicate that DNA methylation has a dominant role in the epigenetic hierarchy governing MAGEA1 expression.

DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. DNA methylation is considered an important mechanism for silencing of retroelements in the mammalian genome. However, the methylation of human endogenous retroviruses (HERVs) is not well investigated. The aim of this study was to investigate the transcriptional potential of HERV-Fc1 proviral 5′LTR in more detail, and examined the specific influence of CpG methylation on this LTR in number of cell lines. Specifically, the role of demethylating chemicals e.g. 5-aza-2′ deoxycytidine and Trichostatin-A, in inducing or reactivating expression of HERV-Fc1 specific sequences and the mechanisms were investigated. In our present study, 5-aza-dC is shown to be a powerful inducer of HERV-Fc1, and at the same time it strongly inhibits methylation of DNA. Treatment with this demethylating agent 5-aza-dC, results in significantly increased levels of HERV-Fc1 expression in cells previously not expressing HERV-Fc1, or with a very low expression level. The extent of expression of HERV-Fc1 RNAs precisely correlates with the apparent extent of demethylation of the related DNA sequences. In conclusion, the results suggest that inhibition of DNA methylation/histone deacetylase can interfere with gene silencing mechanisms affecting HERV-Fc1 expression in human cells.

Histone deacetylase (HDAC) inhibitors have been shown to induce cell cycle arrest and apoptosis in cancer cells. However, the mechanisms of HDAC inhibitor induced apoptosis are incompletely understood. In this study, depsipeptide, a novel HDAC inhibitor, was shown to be able to induce significant apoptotic cell death in human lung cancer cells. Further study showed that Bim, a BH3-only proapoptotic protein, was significantly upregulated by depsipeptide in cancer cells, and Bim's function in depsipeptide-induced apoptosis was confirmed by knockdown of Bim with RNAi. In addition, we found that depsipeptide-induced expression of Bim was directly dependent on acetylation of forkhead box class O1 (FoxO1) that is catalyzed by cyclic adenosine monophosphate-responsive element-binding protein-binding protein, and indirectly induced by a decreased four-and-a-half LIM-domain protein 2. Moreover, our results demonstrated that FoxO1 acetylation is required for the depsipeptide-induced activation of Bim and apoptosis, using transfection with a plasmid containing FoxO1 mutated at lysine sites and a luciferase reporter assay. These data show for the first time that an HDAC inhibitor induces apoptosis through the FoxO1 acetylation-Bim pathway.

Among the human genome, p53 is one of the first tumor suppressor genes to be discovered. It has a wide range of functions covering cell cycle control, apoptosis, genome integrity maintenance, metabolism, fertility, cellular reprogramming and autophagy. Although different possible underlying mechanisms for p53 regulation have been proposed for decades, none of them is conclusive. While much literature focuses on the importance of individual post-translational modifications, further explorations indicate a new layer of p53 coordination through the interplay of the modifications, which builds up a complex 'network'. This review focuses on the necessity, characteristics and mechanisms of the crosstalk among post-translational modifications and its effects on the precise and selective behavior of p53.

Histone deacetylase inhibitor (HDACi) has been shown to demethylate the mammalian genome, which further strengthens the concept that DNA methylation and histone modifications interact in regulation of gene expression. Here, we report that an HDAC inhibitor, depsipeptide, exhibited significant demethylating activity on the promoters of several genes, including p16, SALL3, and GATA4 in human lung cancer cell lines H719 and H23, colon cancer cell line HT-29, and pancreatic cancer cell line PANC1. Although expression of DNA methyltransferase 1 (DNMT1) was not affected by depsipeptide, a decrease in binding of DNMT1 to the promoter of these genes played a dominant role in depsipeptide-induced demethylation and reactivation. Depsipeptide also suppressed expression of histone methyltransferases G9A and SUV39H1, which in turn resulted in a decrease of di- and trimethylated H3K9 around these genes' promoter. Furthermore, both loading of heterochromatin-associated protein 1 (HP1α and HP1β) to methylated H3K9 and binding of DNMT1 to these genes' promoter were significantly reduced in depsipeptide-treated cells. Similar DNA demethylation was induced by another HDAC inhibitor, apicidin, but not by trichostatin A. Our data describe a novel mechanism of HDACi-mediated DNA demethylation via suppression of histone methyltransferases and reduced recruitment of HP1 and DNMT1 to the genes' promoter.

Deregulated expression of E2F1 not only promotes S-phase entry but also induces apoptosis. Although it has been well documented that E2F1 is able to induce p53-dependent apoptosis via raising ARF activity, the mechanism by which E2F induces p53-independent apoptosis remains unclear. Here we report that E2F1 can directly bind to and activate the promoter of Smac/DIABLO, a mitochondrial proapoptotic gene, through the E2F1-binding sites BS2 (−542 ∼ −535 bp) and BS3 (−200 ∼ −193 bp). BS2 and BS3 appear to be utilized in combination rather than singly by E2F1 in activation of Smac/DIABLO. Activation of BS2 and BS3 are E2F1-specific, since neither E2F2 nor E2F3 is able to activate BS2 or BS3. Using the H1299 ER-E2F1 cell line where E2F1 activity can be conditionally induced, E2F1 has been shown to upregulate the Smac/DIABLO expression at both mRNA and protein levels upon 4-hydroxytamoxifen treatment, resulting in an enhanced mitochondria-mediated apoptosis. Reversely, reducing the Smac/DIABLO expression by RNA interference significantly diminishes apoptosis induced by E2F1. These results may suggest a novel mechanism by which E2F1 promotes p53-independent apoptosis through directly regulating its downstream mitochondrial apoptosis-inducing factors, such as Smac/DIABLO.

Generally, histone deacetylase (HDAC) inhibitor-induced p21Waf1/Cip1 expression is thought to be p53 independent. Here we found that an inhibitor of HDAC, depsipeptide (FR901228), but not trichostatin A (TSA), induces p21Waf1/Cip1 expression through both p53 and Sp1/Sp3 pathways in A549 cells (which retain wild-type p53). This is demonstrated by measuring relative luciferase activities of p21 promoter constructs with p53 or Sp1 binding site mutagenesis and was further confirmed by transfection of wild-type p53 into H1299 cells (p53 null). That p53 was acetylated after depsipeptide treatment was tested by sequential immunoprecipitation/Western immunoblot analysis with anti-acetylated lysines and anti-p53 antibodies. The acetylated p53 has a longer half-life due to a significant decrease in p53 ubiquitination. Further study using site-specific antiacetyllysine antibodies and transfection of mutated p53 vectors (K319/K320/K321R mutated and K373R/K382R mutations) into H1299 cells revealed that depsipeptide specifically induces p53 acetylation at K373/K382, but not at K320. As assayed by coimmunoprecipitation, the K373/K382 acetylation is accompanied by a recruitment of p300, but neither CREB-binding protein (CBP) nor p300/CBP-associated factor (PCAF), to the p53 C terminus. Furthermore, activity associated with the binding of the acetylated p53 at K373/K382 to the p21 promoter as well as p21Waf1/Cip1 expression is significantly increased after depsipeptide treatment, as tested by chromatin immunoprecipitations and Western blotting, respectively. In addition, p53 acetylation at K373/K382 is confirmed to be required for recruitment of p300 to the p21 promoter, and the depsipeptide-induced p53 acetylation at K373/K382 is unlikely to be dependent on p53 phosphorylation at Ser15, Ser20, and Ser392 sites. Our data suggest that p53 acetylation at K373/K382 plays an important role in depsipeptide-induced p21Waf1/Cip1 expression.

DNA methylation in the promoter of certain genes is associated with transcriptional silencing. Methylation affects gene expression directly by interfering with transcription factor binding and/or indirectly by recruiting histone deacetylases through methyl-DNA-binding proteins. In this study, we demonstrate that the human lung cancer cell line H719 lacks p53-dependent and -independent p21Cip1 expression. p53 response to treatment with gamma irradiation or etoposide is lost due to a mutation at codon 242 of p53 (C→W). Treatment with depsipeptide, an inhibitor of histone deacetylase, was unable to induce p53-independent p21Cip1 expression because the promoter of p21Cip1 in these cells is hypermethylated. By analyzing luciferase activity of transfected p21Cip1 promoter vectors, we demonstrate that depsipeptide functions on Sp1-binding sites to induce p21Cip1 expression. We hypothesize that hypermethylation may interfere with Sp1/Sp3 binding. By using an electrophoretic mobility shift assay, we show that, although methylation within the consensus Sp1-binding site did not reduce Sp1/Sp3 binding, methylation outside of the consensus Sp1 element induced a significant decrease in Sp1/Sp3 binding. Depsipeptide induced p21Cip1 expression was reconstituted when cells were pretreated with 5-aza-2′-deoxycytidine. Our data suggest, for the first time, that hypermethylation around the consensus Sp1-binding sites may directly reduce Sp1/Sp3 binding, therefore leading to a reduced p21Cip1 expression in response to depsipeptide treatment.

Abstract

Epigenetic changes, including DNA methylation, are a common finding in cancer. In lung cancers methylation of cytosine residues may affect tumor initiation and progression in several ways, including the silencing of tumor suppressor genes through promoter methylation and by providing the targets for adduct formation of polycyclic aromatic hydrocarbons present in combustion products of cigarette smoke. Although the importance of aberrant DNA methylation is well established, the extent of DNA methylation in lung cancers has never been determined. Restriction landmark genomic scanning (RLGS) is a highly reproducible two-dimensional gel electrophoresis that allows the determination of the methylation status of up to 2000 promoter sequences in a single gel. We selected 1184 CpG islands for RLGS analysis and determined their methylation status in 16 primary non-small cell lung cancers. Some tumors did not show methylation whereas others showed up to 5.3% methylation in all CpG islands of the profile. Cloning of 21 methylated loci identified 11 genes and 6 ESTs. We demonstrate that methylation is part of the silencing process of BMP3B in primary tumors and lung cancer cell lines.

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.

Chromosome 8q24 is the most commonly amplified region across multiple cancer types, and the typical length of the amplification suggests that it may target additional genes to MYC. To explore the roles of the genes most frequently included in 8q24 amplifications, we analyzed the relation between copy number alterations and gene expression in three sets of endometrial cancers (N = 252); and in glioblastoma, ovarian, and breast cancers profiled by TCGA. Among the genes neighbouring MYC, expression of the bromodomain-containing gene ATAD2 was the most associated with amplification. Bromodomain-containing genes have been implicated as mediators of MYC transcriptional function, and indeed ATAD2 expression was more closely associated with expression of genes known to be upregulated by MYC than was MYC itself. Amplifications of 8q24, expression of genes downstream from MYC, and overexpression of ATAD2 predicted poor outcome and increased from primary to metastatic lesions. Knockdown of ATAD2 and MYC in seven endometrial and 21 breast cancer cell lines demonstrated that cell lines that were dependent on MYC also depended upon ATAD2. These same cell lines were also the most sensitive to the histone deacetylase (HDAC) inhibitor Trichostatin-A, consistent with prior studies identifying bromodomain-containing proteins as targets of inhibition by HDAC inhibitors. Our data indicate high ATAD2 expression is a marker of aggressive endometrial cancers, and suggest specific inhibitors of ATAD2 may have therapeutic utility in these and other MYC-dependent cancers.

Adiponectin, an abundant adipose tissue-derived protein, exerts protective effect against cardiovascular disease. Adiponectin receptors (AdipoR1 and AdipoR2) mediate the beneficial effects of adiponectin on the cardiovascular system. However, the alteration of AdipoRs in cardiac remodeling is not fully elucidated. Here, we investigated the effect of angiotensin II (AngII) on cardiac AdipoRs expression and explored the possible molecular mechanism. AngII infusion into rats induced cardiac hypertrophy, reduced AdipoR1 but not AdipoR2 expression, and attenuated the phosphorylations of adenosine monophosphate-activated protein kinase and acetyl coenzyme A carboxylase, and those effects were all reversed by losartan, an AngII type 1 (AT1) receptor blocker. AngII reduced expression of AdipoR1 mRNA and protein in cultured neonatal rat cardiomyocytes, which was abolished by losartan, but not by PD123319, an AT2 receptor antagonist. The antioxidants including reactive oxygen species (ROS) scavenger NAC, NADPH oxidase inhibitor apocynin, Nox2 inhibitor peptide gp91 ds-tat, and mitochondrial electron transport chain complex I inhibitor rotenone attenuated AngII-induced production of ROS and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. AngII-reduced AdipoR1 expression was reversed by pretreatment with NAC, apocynin, gp91 ds-tat, rotenone, and an ERK1/2 inhibitor PD98059. Chromatin immunoprecipitation assay demonstrated that AngII provoked the recruitment of c-Myc onto the promoter region of AdipoR1, which was attenuated by PD98059. Moreover, AngII-induced DNA binding activity of c-Myc was inhibited by losartan, NAC, apocynin, gp91 ds-tat, rotenone, and PD98059. c-Myc small interfering RNA abolished the inhibitory effect of AngII on AdipoR1 expression. Our results suggest that AngII inhibits cardiac AdipoR1 expression in vivo and in vitro and AT1 receptor/ROS/ERK1/2/c-Myc pathway is required for the downregulation of AdipoR1 induced by AngII.

Differential positioning of the histone variant H2A.Z in a p53 dependent manner was shown to regulate p21 transcription. Whether H2A.Z is involved in p21 activity in the absence of p53 is not known. The p21 gene is repressed in estrogen receptor (ER) negative cell lines that are p53−/− and hormone independent for their growth. Here we demonstrate that class I and II pan Histone deacetylase inhibitors (HDACi) induce p21 transcription and reduce cell proliferation of MDA-MB231, an ERα-negative mammary tumor cell line, in a H2A.Z dependent manner. H2A.Z is associated with the transcription start site (TSS) of the repressed p21 gene. Depleting H2A.Z did not lead to transcription of p21 but annihilated the stimulating effect of HDACi on this gene. Acetylation of H2A.Z but not of H3K9 at the p21 promoter correlated with p21 activation. We further show that HDACi treatment reduced the presence of the p400 chromatin remodeler at the p21 TSS. We propose a model in which association of p400 negatively affects p21 transcription by interfering with acetylation of H2A.Z.

We previously identified actin filament-associated protein 1-like 1 (AFAP1L1) as a metastasis-predicting marker from the gene-expression profiles of 65 spindle cell sarcomas, and demonstrated the up-regulation of AFAP1L1 expression to be an independent risk factor for distant metastasis in multivariate analyses. Little is known, however, about how the expression of AFAP1L1 is regulated. Luciferase reporter assays showed tandem binding motives of a specificity protein (Sp) located at −85 to −75 relative to the transcriptional start site to be essential to the promoter activity. Overexpression of Sp1 and Sp3 proteins transactivated the proximal AFAP1L1 promoter construct, and electrophoretic mobility shift assays showed that both Sp1 and Sp3 were able to bind to this region in vitro. Chromatin immunoprecipitation experiments, however, revealed that Sp3 is the major factor binding to the proximal promoter region of the AFAP1L1 gene in AFAP1L1- positive cells. Treatment with mithramycin A, an inhibitor of proteins binding to GC-rich regions, prevented Sp3 from binding to the proximal promoter region of AFAP1L1 and decreased its expression in a dose-dependent manner. Finally, knocking down Sp3 using small inhibitory RNA duplex (siRNA) reduced AFAP1L1 expression significantly, which was partially restored by expressing siRNA-resistant Sp3. These findings indicate a novel role for Sp3 in sarcomas as a driver for expression of the metastasis-related gene AFAP1L1.

Histone methylation regulates normal stem cell fate decisions through a coordinated interplay between histone methyltransferases and demethylases at lineage specific genes. Malignant transformation is associated with aberrant accumulation of repressive histone modifications, such as polycomb mediated histone 3 lysine 27 (H3K27me3) resulting in a histone methylation mediated block to differentiation. The relevance, however, of histone demethylases in cancer remains less clear. We report that JMJD3, a H3K27me3 demethylase, is induced during differentiation of glioblastoma stem cells (GSCs), where it promotes a differentiation-like phenotype via chromatin dependent (INK4A/ARF locus activation) and chromatin independent (nuclear p53 protein stabilization) mechanisms. Our findings indicate that deregulation of JMJD3 may contribute to gliomagenesis via inhibition of the p53 pathway resulting in a block to terminal differentiation.

The endoplasmic reticulum (ER) has a unique, network-like morphology. The ER structures are composed of tubules, cisternae, and three-way junctions. This morphology is highly conserved among eukaryotes, but the molecular mechanism that maintains ER morphology has not yet been elucidated. In addition, certain Brassicaceae plants develop a unique ER-derived organelle called the ER body. This organelle accumulates large amounts of PYK10, a β-glucosidase, but its physiological functions are still obscure. We aimed to identify a novel factor required for maintaining the morphology of the ER, including ER bodies, and employed a forward-genetic approach using transgenic Arabidopsis thaliana (GFP-h) with fluorescently-labeled ER. We isolated and investigated a mutant (designated endoplasmic reticulum morphology3, ermo3) with huge aggregates and abnormal punctate structures of ER. ERMO3 encodes a GDSL-lipase/esterase family protein, also known as MVP1. Here, we showed that, although ERMO3/MVP1/GOLD36 was expressed ubiquitously, the morphological defects of ermo3 were specifically seen in a certain type of cells where ER bodies developed. Coimmunoprecipitation analysis combined with mass spectrometry revealed that ERMO3/MVP1/GOLD36 interacts with the PYK10 complex, a huge protein complex that is thought to be important for ER body-related defense systems. We also found that the depletion of transcription factor NAI1, a master regulator for ER body formation, suppressed the formation of ER-aggregates in ermo3 cells, suggesting that NAI1 expression plays an important role in the abnormal aggregation of ER. Our results suggest that ERMO3/MVP1/GOLD36 is required for preventing ER and other organelles from abnormal aggregation and for maintaining proper ER morphology in a coordinated manner with NAI1.

L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21cip1 gene, mRNA and protein in cancer cells but not p27kip1; (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21cip1 gene but not p27kip1 detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.

The retinoblastoma protein (pRb) and the related proteins Rb2/p130 and 107 represent the “pocket protein” family of cell cycle regulators. A key function of these proteins is the cell cycle dependent modulation of E2F-regulated genes. The biological activity of these proteins is controlled by acetylation and phosphorylation in a cell cycle dependent manner. In this study we attempted to investigate the interdependence of acetylation and phosphorylation of Rb2/p130 in vitro. After having identified the acetyltransferase p300 among several acetyltransferases to be associated with Rb2/p130 during S-phase in NIH3T3 cells in vivo, we used this enzyme and the CDK4 protein kinase for in vitro modification of a variety of full length Rb2/p130 and truncated versions with mutations in the acetylatable lysine residues 1079, 128 and 130. Mutation of these residues results in the complete loss of Rb2/p130 acetylation. Replacement of lysines by arginines strongly inhibits phosphorylation of Rb2/p130 by CDK4; the inhibitory effect of replacement by glutamines is less pronounced. Preacetylation of Rb2/p130 strongly enhances CDK4-catalyzed phosphorylation, whereas deacetylation completely abolishes in vitro phosphorylation. In contrast, phosphorylation completely inhibits acetylation of Rb2/p130 by p300. These results suggest a mutual interdependence of modifications in a way that acetylation primes Rb2/p130 for phosphorylation and only dephosphorylated Rb2/p130 can be subject to acetylation. Human papillomavirus 16-E7 protein, which increases acetylation of Rb2/p130 by p300 strongly reduces phosphorylation of this protein by CDK4. This suggests that the balance between phosphorylation and acetylation of Rb2/p130 is essential for its biological function in cell cycle control.

G-quadruplex structures, formed from guanine rich sequences, have previously been shown to be involved in various physiological processes including cancer-related gene expression. Furthermore, G-quadruplexes have been found in several oncogene promoter regions, and have been shown to play a role in the regulation of gene expression. The mutagenic properties of oxidative stress on DNA have been widely studied, as has the association with carcinogenesis. Guanine is the most susceptible nucleotide to oxidation, and as such, G-rich sequences that form G-quadruplexes can be viewed as potential “hot-spots” for DNA oxidation. We propose that oxidation may destabilise the G-quadruplex structure, leading to its unfolding into the duplex structure, affecting gene expression. This would imply a possible mechanism by which oxidation may impact on oncogene expression. This work investigates the effect of oxidation on two biologically relevant G-quadruplex structures through 500 ns molecular dynamics simulations on those found in the promoter regions of the c-Kit and c-Myc oncogenes. The results show oxidation having a detrimental effect on stability of the structure, substantially destabilising the c-Kit quadruplex, and with a more attenuated effect on the c-Myc quadruplex. Results are suggestive of a novel route for oxidation-mediated oncogenesis and may have wider implications for genome stability.

Mapping DNase I hypersensitive sites (DHSs) within nuclear chromatin is a traditional and powerful method of identifying genetic regulatory elements. DHSs have been mapped by capturing the ends of long DNase I-cut fragments (>100,000 bp), or 100–1200 bp DNase I-double cleavage fragments (also called double-hit fragments). But next generation sequencing requires a DNA library containing DNA fragments of 100–500 bp. Therefore, we used short DNA fragments released by DNase I digestion to generate DNA libraries for next generation sequencing. The short segments are 100–300 bp and can be directly cloned and used for high-throughput sequencing. We identified 83,897 DHSs in 2,343,479 tags across the human genome. Our results indicate that the DHSs identified by this DHS assay are consistent with those identified by longer fragments in previous studies. We also found: (1) the distribution of DHSs in promoter and other gene regions of similarly expressed genes differs among different chromosomes; (2) silenced genes had a more open chromatin structure than previously thought; (3) DHSs in 3′untranslated regions (3′UTRs) are negatively correlated with level of gene expression.

Histone deacetylase (HDAC) proteins have a role in promoting neuronal survival in vitro, but the mechanism underlying this function has not been identified. Here we provide evidence that components of the neuronal microenvironment, including non-neuronal cells and defined culture media, can mitigate midbrain neuronal cell death induced by HDAC inhibitor treatment. Using microarrays we further identified gene expression changes taking place in non-neuronal cells as a result of HDAC inhibition. This analysis demonstrated that HDAC inhibitor treatment results in the down-regulation of immunity related signaling factors, in particular the Toll-like receptors (TLR). TLR signaling is active in cultured midbrain cells, yet blocking TLR receptors is not sufficient to cause neuronal cell death. In contrast, selective activation of this pathway using TLR ligands can modestly block the effects of HDAC inhibition. Furthermore, we observed that the negative effects of HDAC inhibitor treatment on neuronal survival could be more substantially blocked by the cytokine Interleukin-6 (IL-6), which is a major downstream target of TLR signaling. These data suggest that HDACs function to promote neuronal survival by activating a TLR and IL-6 dependent pathway.

During epidermal cell differentiation, keratin 14 (K14) expression is down-regulated, p53 expression varies, and the expression of the p53 target genes, p21 and 14-3-3σ, increases. These trends suggest that the relative transcriptional activity of p53 is increased during epidermal cell differentiation. To determine the relationship between K14 and p53, we constructed K14 promoters of various sizes and found that wild-type p53 could repress the promoter activity of all of the K14 promoter constructs in H1299 cells. K14-p160 contains an SP1 binding site mutation that prevents p53 from repressing K14 expression. Using a DNA affinity precipitation assay, we confirmed that p53 forms a complex with SP1 at the SP1 binding site between nucleotides -48 and -43 on the K14 promoter. Thus, our data indicate that p53 acts as a co-repressor to down-regulate K14 expression by binding to SP1. Next, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal cell differentiation model to examine the inhibition of K14 expression caused by increased p53 activity. Human ovarian teratocarcinoma C9 cells were treated with TPA to induce differentiation. Over-expression of the dominant negative p53 mutant ΔTAp53, which inhibits p53 activity, prevented the TPA-induced K14 down-regulation in C9 cells. Furthermore, treatment of normal primary human foreskin keratinocytes (PHFK) with the p53 inhibitor pifithrin-α (PFT-α) showed that the inhibition of p53 activity relieves K14 repression during epidermal cell differentiation. Finally, we found that TPA induces the phosphorylation of p53 at residue 378, which enhances the affinity of p53 to bind to Sp1 and repress K14 expression.

Human PDHA2 is a testis-specific gene that codes for the E1α subunit of Pyruvate Dehydrogenase Complex (PDC), a crucial enzyme system in cell energy metabolism. Since activation of the PDHA2 gene in somatic cells could be a new therapeutic approach for PDC deficiency, we aimed to identify the regulatory mechanisms underlying the human PDHA2 gene expression. Functional deletion studies revealed that the −122 to −6 promoter region is indispensable for basal expression of this TATA-less promoter, and suggested a role of an epigenetic program in the control of PDHA2 gene expression. Indeed, treatment of SH-SY5Y cells with the hypomethylating agent 5-Aza-2′-deoxycytidine (DAC) promoted the reactivation of the PDHA2 gene, by inducing the recruitment of the RNA polymerase II to the proximal promoter region and the consequent increase in PDHA2 mRNA levels. Bisulfite sequencing analysis revealed that DAC treatment induced a significant demethylation of the CpG island II (nucleotides +197 to +460) in PDHA2 coding region, while the promoter region remained highly methylated. Taken together with our previous results that show an in vivo correlation between PDHA2 expression and the demethylation of the CpG island II in testis germ cells, the present results show that internal methylation of the PDHA2 gene plays a part in its repression in somatic cells. In conclusion, our data support the novel finding that methylation of the PDHA2 coding region can inhibit gene transcription. This represents a key mechanism for absence of PDHA2 expression in somatic cells and a target for PDC therapy.

Short-chain fatty acids (SCFAs), especially butyrate, affect cell differentiation, proliferation, and motility. Butyrate also induces cell cycle arrest and apoptosis through its inhibition of histone deacetylases (HDACs). In addition, butyrate is a potent inducer of histone hyper-acetylation in cells. Therefore, this SCFA provides an excellent in vitro model for studying the epigenomic regulation of gene expression induced by histone acetylation. In this study, we analyzed the differential in vitro expression of genes induced by butyrate in bovine epithelial cells by using deep RNA-sequencing technology (RNA-seq). The number of sequences read, ranging from 57,303,693 to 78,933,744, were generated per sample. Approximately 11,408 genes were significantly impacted by butyrate, with a false discovery rate (FDR) <0.05. The predominant cellular processes affected by butyrate included cell morphological changes, cell cycle arrest, and apoptosis. Our results provided insight into the transcriptome alterations induced by butyrate, which will undoubtedly facilitate our understanding of the molecular mechanisms underlying butyrate-induced epigenomic regulation in bovine cells.

5-aza-2′-deoxycytidine (5-aza-CdR) is used extensively as a demethylating agent and acts in concert with histone deacetylase inhibitors (HDACI) to induce apoptosis or inhibition of cell proliferation in human cancer cells. Whether the action of 5-aza-CdR in this synergistic effect results from demethylation by this agent is not yet clear. In this study we found that inhibition of cell proliferation was not observed when cells with knockdown of DNA methyltransferase 1 (DNMT1), or double knock down of DNMT1-DNMT3A or DNMT1-DNMT3B were treated with HDACI, implying that the demethylating function of 5-aza-CdR may be not involved in this synergistic effect. Further study showed that there was a causal relationship between 5-aza-CdR induced DNA damage and the amount of [3H]-5-aza-CdR incorporated in DNA. However, incorporated [3H]-5-aza-CdR gradually decreased when cells were incubated in [3H]-5-aza-CdR free medium, indicating that 5-aza-CdR, which is an abnormal base, may be excluded by the cell repair system. It was of interest that HDACI significantly postponed the removal of the incorporated [3H]-5-aza-CdR from DNA. Moreover, HDAC inhibitor showed selective synergy with nucleoside analog-induced DNA damage to inhibit cell proliferation, but showed no such effect with other DNA damage stresses such as γ-ray and UV, etoposide or cisplatin. This study demonstrates that HDACI synergistically inhibits cell proliferation with nucleoside analogs by suppressing removal of incorporated harmful nucleotide analogs from DNA.