H3K9 Deacetylation Research Articles

Chromatin determinants impart camptothecin sensitivity

Camptothecin‐induced locking of topoisomerase 1 on DNA generates a physical barrier to replication fork progression and creates topological stress. By allowing replisome rotation, absence of the Tof1/Csm3 complex promotes the conversion of impending topological stress to DNA catenation and causes camptothecin hypersensitivity. Through synthetic viability screening, we discovered that histone H4 K16 deacetylation drives the sensitivity of yeast cells to camptothecin and that inactivation of this pathway by mutating H4 K16 or the genes SIR1‐4 suppresses much of the hypersensitivity of tof1∆ strains towards this agent. We show that disruption of rDNA or telomeric silencing does not mediate camptothecin resistance but that disruption of Sir1‐dependent chromatin domains is sufficient to suppress camptothecin sensitivity in wild‐type and tof1∆ cells. We suggest that topoisomerase 1 inhibition in proximity of these domains causes topological stress that leads to DNA hypercatenation, especially in the absence of the Tof1/Csm3 complex. Finally, we provide evidence of the evolutionarily conservation of this mechanism.

HOPX hypermethylation promotes metastasis via activating SNAIL transcription in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is characterized by a high rate of local invasion and early distant metastasis. Increasing evidence indicates that epigenetic abnormalities play important roles in NPC development. However, the epigenetic mechanisms underlying NPC metastasis remain unclear. Here we investigate aberrantly methylated transcription factors in NPC tissues, and we identify the HOP homeobox HOPX as the most significantly hypermethylated gene. Consistently, we find that HOXP expression is downregulated in NPC tissues and NPC cell lines. Restoring HOPX expression suppresses metastasis and enhances chemosensitivity of NPC cells. These effects are mediated by HOPX-mediated epigenetic silencing of SNAIL transcription through the enhancement of histone H3K9 deacetylation in the SNAIL promoter. Moreover, we find that patients with high methylation levels of HOPX exhibit poor clinical outcomes in both the training and validation cohorts. In summary, HOPX acts as a tumour suppressor via the epigenetic regulation of SNAIL transcription, which provides a novel prognostic biomarker for NPC metastasis and therapeutic target for NPC treatment.

Histone H3 Lysine 9 Acetylation Obstructs ATM Activation and Promotes Ionizing Radiation Sensitivity in Normal Stem Cells.

SummaryDynamic spatiotemporal modification of chromatin around DNA damage is vital for efficient DNA repair. Normal stem cells exhibit an attenuated DNA damage response (DDR), inefficient DNA repair, and high radiosensitivity. The impact of unique chromatin characteristics of stem cells in DDR regulation is not yet recognized. We demonstrate that murine embryonic stem cells (ES) display constitutively elevated acetylation of histone H3 lysine 9 (H3K9ac) and low H3K9 tri-methylation (H3K9me3). DNA damage-induced local deacetylation of H3K9 was abrogated in ES along with the subsequent H3K9me3. Depletion of H3K9ac in ES by suppression of monocytic leukemia zinc finger protein (MOZ) acetyltransferase improved ATM activation, DNA repair, diminished irradiation-induced apoptosis, and enhanced clonogenic survival. Simultaneous suppression of the H3K9 methyltransferase Suv39h1 abrogated the radioprotective effect of MOZ inhibition, suggesting that high H3K9ac promoted by MOZ in ES cells obstructs local upregulation of H3K9me3 and contributes to muted DDR and increased radiosensitivity.

SirT1 activator represses the transcription of TNF‑α in THP‑1 cells of a sepsis model via deacetylation of H4K16.

Sepsis is a systemic inflammatory response resulting from the excessive production of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α. Sirtuin 1 (SirT1) actively deacetylates histone proteins, and facilitates chromatin compaction and gene silencing. In the present study, a cell model of sepsis, comprising lipopolysaccharide (LPS)-tolerant THP-1 cells, was used to investigate whether the SirT1 activator, resveratrol, repressed the transcription of TNF-α. Chromatin immunoprecipitation and real-time PCR were used to determine the transcription of the TNF-α promoter. The result revealed that the binding of SirT1 to the TNF-α promoter was decreased by LPS stimulation in normal cells. However, in LPS-tolerant cells, nuclear protein levels of SirT1 remained elevated, and LPS stimulation had no significant effect on the binding of SirT1 to the TNF-α promoter. However, the activity of SirT1 was increased and binding of ace-H4K16 to the TNF-α promoter was decreased with resveratrol treatment in the tolerant cells. It was concluded that resveratrol stimulated sirtuin activity in LPS-tolerant THP-1 cells, and repressed TNF-α transcription through the deacetylation of H4K16, without affecting the methylation of H3K9. Resveratrol offers potential as an infective candidate to alleviate inflammation in patients with sepsis.

Molecular Mechanism of DNA Topoisomerase I-Dependent rDNA Silencing: Sir2p Recruitment at Ribosomal Genes

Saccharomyces cerevisiae sir2Δ or top1Δ mutants exhibit similar phenotypes involving ribosomal DNA, including (i) loss of transcriptional silencing, resulting in non-coding RNA hyperproduction from cryptic RNA polymerase II promoters; (ii) alterations in recombination; and (iii) a general increase in histone acetylation. Given the distinct enzymatic activities of Sir2 and Top1 proteins, a histone deacetylase and a DNA topoisomerase, respectively, we investigated whether genetic and/or physical interactions between the two proteins could explain the shared ribosomal RNA genes (rDNA) phenotypes. We employed an approach of complementing top1Δ cells with yeast, human, truncated, and chimeric yeast/human TOP1 constructs and of assessing the extent of non-coding RNA silencing and histone H4K16 deacetylation. Our findings demonstrate that residues 115–125 within the yeast Top1p N-terminal domain are required for the complementation of the top1∆ rDNA phenotypes. In chromatin immunoprecipitation and co-immunoprecipitation experiments, we further demonstrate the physical interaction between Top1p and Sir2p. Our genetic and biochemical studies support a model whereby Top1p recruits Sir2p to the rDNA and clarifies a structural role of DNA topoisomerase I in the epigenetic regulation of rDNA, independent of its known catalytic activity.

Control of Leaf Senescence by an MeOH-Jasmonates Cascade that Is Epigenetically Regulated by OsSRT1 in Rice

Although considerable progress has been made in identifying the genes regulating accumulation of hormones that are involved in leaf senescence, only a few studies have focused on natural variations in jasmonates content and much less on the underlying genetic basis. Moreover, the epigenetic regulation of jasmonate-mediated leaf senescence remains largely unknown. In this study, we carried out metabolic profiling of a worldwide collection of rice accessions and demonstrated that there are substantial variations in jasmonate levels among these accessions. A subsequent metabolite-based genome-wide association study identified candidates for two major quantitative genes (QTGs), OsPME1 and OsTSD2, affecting the content of jasmonates. Further investigations using a series of relevant mutants and transgenic lines revealed the MeOH-jasmonate cascade plays an important role in regulating leaf senescence. Moreover, we showed that OsSRT1, one of the two Sir2 (silent information regulator 2) homologs in rice, negatively regulates leaf senescence by repressing expression of the biosynthetic genes of this metabolic cascade and at least particially through histone H3K9 deacetylation of OsPME1. Taken together, our results indicate that the MeOH-jasmonates cascade and its epigenetic regulation are crucial for controlling leaf senescence process in rice.

Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation.

BackgroundUltraviolet-B (UVB) exposure attributes to the formation of age-related nuclear cataract (ARNC), which is mediated with DNA damage. DNA damage, an important factor for pathogenesis of ARNC, is induced by UVB, and is generally resolved by the nucleotide excision repair (NER) repair mechanism. Cockayne syndrome complementation group B (CSB) protein coded by ERCC6 is a vital component for NER. However, we found no association between selected ERCC6 polymorphisms and ARNC. In this study, we investigated whether UVB exposure could alter ERCC6 expression and the process could involve epigenetic changes of DNA methylation and/or histone acetylation of ERCC6 in the lens epithelial cells (LECs). We also assessed the involvement of those coordinated changes in lens tissue from ARNC patients.ResultsmRNA and protein expression of ERCC6 in lens tissue (LECs) were lower in ARNCs than those in the controls. This reduction corresponded to methylation of a CpG site at the ERCC6 promoter and histone modifications (methylation and acetylation) nearby this site. UVB-treated human lens epithelium B3 (HLE-B3) and 239T cell presented (1) increased apoptosis, suggesting reduced UV-damage repair, (2) hypermethylation of the CpG site located at position -441 (relative to transcription start site) within the binding region for transcriptional factor Sp1 in the ERCC6 promoter, (3) the enhancement of histone H3K9 deacetylation, (4) induction in DNA methyltransferases 3b (DNMT3b) and histone deacetylase1 (HDAC1) associated to the CpG site of ERCC6 by CHIP assay.ConclusionsThese findings suggest an orchestrated mechanism triggered by UVB radiation where the concurrent association of specific hypermethylation CpG site, H3K9 deacetylation of ERCC6, and repression of ERCC6 gene expression. Taken together, with the similar changes in the lens tissue from ARNC patients, our data unveiled a possible mechanism of epigenetic modification of DNA repair gene in the pathogenesis of ARNC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0229-y) contains supplementary material, which is available to authorized users.

A crucial role of SUMOylation in modulating Sirt6 deacetylation of H3 at lysine 56 and its tumor suppressive activity.

Sirt6 is a histone deacetylase with NAD(+)-dependent activity. Sirt6 has been shown as a tumor suppressor partially via inhibiting the expression of c-Myc target genes and ribosome biogenesis. However, how to regulate Sirt6 activity is largely unknown. In this study, we identify that Sirt6 can be modified by small ubiquitin-like modifier. Sirt6 SUMOylation deficiency specifically decreases its deacetylation of H3K56 but not H3K9 in vivo. Mechanistically, we find that SUMOylation deficiency decreases Sirt6 binding with c-Myc, decreasing Sirt6 occupancy on the locus of c-Myc target genes. Therefore, Sirt6 SUMOylation deficiency reduces its deacetylation of H3k56 and its repression of c-Myc target genes. Moreover, Sirt6 SUMOylation deficiency reduces its suppression of cell proliferation and tumorigenesis. Thus, these results reveal that SUMOylation has an important role in regulation of Sirt6 deacetylation on H3K56, as well as its tumor suppressive activity.

1,4-Dihydropyridines Active on the SIRT1/AMPK Pathway Ameliorate Skin Repair and Mitochondrial Function and Exhibit Inhibition of Proliferation in Cancer Cells.

Modulators of sirtuins are considered promising therapeutic targets for the treatment of cancer, cardiovascular, metabolic, inflammatory, and neurodegenerative diseases. Here we prepared new 1,4-dihydropyridines (DHPs) bearing changes at the C2/C6, C3/C5, C4, or N1 position. Tested with the SIRTainty procedure, some of them displayed increased SIRT1 activation with respect to the prototype 3a, high NO release in HaCat cells, and ameliorated skin repair in a mouse model of wound healing. In C2C12 myoblasts, two of them improved mitochondrial density and functions. All the effects were reverted by coadministration of compound C (9), an AMPK inhibitor, or of EX-527 (10), a SIRT1 inhibitor, highlighting the involvement of the SIRT1/AMPK pathway in the action of DHPs. Finally, tested in a panel of cancer cells, the water-soluble form of 3a, compound 8, displayed antiproliferative effects in the range of 8-35 μM and increased H4K16 deacetylation, suggesting a possible role for SIRT1 activators in cancer therapy.

Global mapping of the regulatory interactions of histone residues

Histone residues can serve as platforms for specific regulatory function. Here we constructed a map of regulatory associations between histone residues and a wide spectrum of chromatin regulation factors based on gene expression changes by histone point mutations in Saccharomyces cerevisiae. Detailed analyses of this map revealed novel associations. Regarding the modulation of H3K4 and K36 methylation by Set1, Set2, or Jhd2, we proposed a role for H4K91 acetylation in early Pol II elongation, and for H4K16 deacetylation in late elongation and crosstalk with H3K4 demethylation for gene silencing. The association of H3K56 with nucleosome positioning suggested that this lysine residue and its acetylation might contribute to nucleosome mobility for transcription activation. Further insights into chromatin regulation are expected from this approach.

Arsenic Trioxide Reduces Global Histone H4 Acetylation at Lysine 16 through Direct Binding to Histone Acetyltransferase hMOF in Human Cells.

Histone post-translational modification heritably regulates gene expression involved in most cellular biological processes. Experimental studies suggest that alteration of histone modifications affects gene expression by changing chromatin structure, causing various cellular responses to environmental influences. Arsenic (As), a naturally occurring element and environmental pollutant, is an established human carcinogen. Recently, increasing evidence suggests that As-mediated epigenetic mechanisms may be involved in its toxicity and carcinogenicity, but how this occurs is still unclear. Here we present evidence that suggests As-induced global histone H4K16 acetylation (H4K16ac) partly due to the direct physical interaction between As and histone acetyltransferase (HAT) hMOF (human male absent on first) protein, leading to the loss of hMOF HAT activity. Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells. However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation. Using As-immobilized agarose, we confirmed that As binds directly to hMOF, and that this interaction was competitively inhibited by free As2O3. Also, the direct interaction of As and C2CH zinc finger peptide was verified by MAIDI-TOF mass and UV absorption. In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure. These data suggest a theoretical basis for elucidating the mechanism of As toxicity.

MPP8 and SIRT1 crosstalk in E-cadherin gene silencing and epithelial-mesenchymal transition.

As a critical developmental process, epithelial-mesenchymal transition (EMT) involves complex transcriptional reprogramming and has been closely linked to malignant progression. Although various epigenetic modifications, such as histone deacetylation and H3K9 methylation, have been implicated in this process, how they are coordinated remains elusive. We recently revealed that MPP8 couples H3K9 methylation and DNA methylation for E-cadherin gene silencing and promotes tumor cell migration, invasion, and EMT. Here, we show that MPP8 cooperates with the class III HDAC SIRT1 in this process through their physical interaction. SIRT1 antagonizes PCAF-catalyzed MPP8-K439 acetylation to protect MPP8 from ubiquitin-proteasome-mediated proteolysis. Conversely, MPP8 recruits SIRT1 for H4K16 deacetylation after binding to methyl-H3K9 on target promoters. Consequently, disabling either MPP8 methyl-H3K9 binding or SIRT1 interaction de-represses E-cadherin and reduces EMT phenotypes, as does knockdown of MPP8 or SIRT1 in prostate cancer cells. These results illustrate how SIRT1 and MPP8 reciprocally promote each other's function and coordinate epithelial gene silencing and EMT.

Suppression of silent information regulator 1 activity in noncancerous tissues of hepatocellular carcinoma: Possible association with non-B non-C hepatitis pathogenesis.

Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase. In mice, mSirt1 deficiency causes the onset of fatty liver via regulation of the hepatic nutrient metabolism pathway. In this study, we demonstrate SIRT1 expression, activity and NAD+ regulation using noncancerous liver tissue specimens from hepatocellular carcinoma patients with non-B non-C (NBNC) hepatitis. SIRT1 expression levels were higher in NBNC patients than in healthy donors, while SIRT1 histone H3K9 deacetylation activity was suppressed in NBNC patients. In the liver of hepatitis patients, decreased NAD+ amounts and its regulatory enzyme nicotinamide phosphoribosyltransferase expression levels were observed, and this led to inhibition of SIRT1 activity. SIRT1 expression was associated with HIF1 protein accumulation in both the NBNC liver and liver cancer cell lines. These results may indicate that the NBNC hepatitis liver is exposed to hypoxic conditions. In HepG2 cells, hypoxia induced inflammatory chemokines, such as CXCL10 and MCP-1. These inductions were suppressed in rich NAD+ condition, and by SIRT1 activator treatment. In conclusion, hepatic SIRT1 activity was repressed in NBNC patients, and normalization of NAD+ amounts and activation of SIRT1 could improve the inflammatory condition in the liver of NBNC hepatitis patients.

Dual Roles of Histone H3 Lysine 9 Acetylation in Human Embryonic Stem Cell Pluripotency and Neural Differentiation

Early neurodevelopment requires cell fate commitment from pluripotent stem cells to restricted neural lineages, which involves the epigenetic regulation of chromatin structure and lineage-specific gene transcription. However, it remains unclear how histone H3 lysine 9 acetylation (H3K9Ac), an epigenetic mark representing transcriptionally active chromatin, is involved in the neural commitment from pluripotent embryonic stem cells (ESCs). In this study, we demonstrate that H3K9Ac gradually declines during the first 4 days of in vitro neural differentiation of human ESCs (hESCs) and then increases during days 4-8. Consistent with this finding, the H3K9Ac enrichment at several pluripotency genes was decreased, and H3K9Ac occupancies at the loci of neurodevelopmental genes increased during hESC neural commitment. Inhibiting H3K9 deacetylation on days 0-4 by histone deacetylase inhibitors (HDACis) promoted hESC pluripotency and suppressed its neural differentiation. Conversely, HDACi-elicited up-regulation of H3K9 acetylation on days 4-8 enhanced neural differentiation and activated multiple neurodevelopmental genes. Mechanistically, HDACis promote pluripotency gene transcription to support hESC self-renewal through suppressing HDAC3 activity. During hESC neural commitment, HDACis relieve the inhibitory activities of HDAC1/5/8 and thereby promote early neurodevelopmental gene expression by interfering with gene-specific histone acetylation patterns. Furthermore, p300 is primarily identified as the major histone acetyltransferase involved in both hESC pluripotency and neural differentiation. Our results indicate that epigenetic modification plays pivotal roles during the early neural specification of hESCs. The histone acetylation, which is regulated by distinct HDAC members at different neurodevelopmental stages, plays dual roles in hESC pluripotency maintenance and neural differentiation.

BRMS1L suppresses breast cancer metastasis by inducing epigenetic silence of FZD10.

BRMS1L (breast cancer metastasis suppressor 1 like, BRMS1-like) is a component of Sin3A-histone deacetylase (HDAC) co-repressor complex that suppresses target gene transcription. Here we show that reduced BRMS1L in breast cancer tissues is associated with metastasis and poor patient survival. Functionally, BRMS1L inhibits breast cancer cells migration and invasion by inhibiting epithelial-mesenchymal transition. These effects are mediated by epigenetic silencing of FZD10, a receptor for Wnt signalling, through HDAC1 recruitment and histone H3K9 deacetylation at the promoter. Consequently, BRMS1L-induced FZD10 silencing inhibits aberrant activation of WNT3-FZD10-β-catenin signalling. Furthermore, BRMS1L is a target of miR-106b and miR-106b upregulation leads to BRMS1L reduction in breast cancer cells. RNA interference-mediated silencing of BRMS1L expression promotes metastasis of breast cancer xenografts in immunocompromised mice, whereas ectopic BRMS1L expression inhibits metastasis. Therefore, BRMS1L provides an epigenetic regulation of Wnt signalling in breast cancer cells and acts as a breast cancer metastasis suppressor.

Histone deacetylase 3 promotes pancreatic cancer cell proliferation, invasion and increases drug-resistance through histone modification of P27, P53 and Bax

Pancreatic cancer is one of the most aggressive solid malignancies with a dismal survival rate. Recent studies have shown that high expression levels of histone deacetylase3 (HDAC3) correlate with malignant phenotype. However, the expression patterns and biological role of HDAC3 in pancreatic cancer remain unclear. In this study, our data showed that a higher level of HDAC3 protein expression was found in pancreatic cancer as compared to paired paracancerous tissues. Consistently, higher expression level of HDAC3 was found in all of the eight pancreatic cancer cell lines relative to human pancreatic ductal epithelial cells (HPDE). In addition, further function analysis revealed that HDAC3 can function as oncogenic protein, which could promote pancreatic cancer cell proliferation, migration and invasion, and may increase drug resistance. Moreover, the functional involvement of HDAC3 was partially correlated with post-induction repression of P53, P27 and Bax gene transcription, acting via H3K9 deacetylation. Taken together, our data suggest that HDAC3 participates in the pathogenesis and progression of pancreatic cancer through histone modification, which might be a pivotal epigenetic target against this devastating disease.

Studying SIRT6 regulation using H3K56 based substrate and small molecules

SIRT6 is a modulator of chromatin structure having an important role in healthy ageing, and there is a crucial need to find specific modulators for it. Therefore, the activity of SIRT6 should be studied using a variety of methods. We examined the capability of SIRT6 to deacetylate a set of five fluorogenic substrates based on p53 and histone H3 sequences. The substrate designed around H3K56 deacetylation site exhibited the best signal-to-background ratio and was chosen for further studies. Nicotinamide is a known inhibitor for sirtuins, and it was found to be less potent inhibitor for SIRT6 than it is for SIRT1. In addition, we studied 15 other small molecule sirtuin modulators using the H3K56 based substrate. EX-527, quercetin and three pseudopeptidic compounds were found to be the most potent SIRT6 inhibitors, exhibiting over 50% deacetylation inhibition. These findings describe the first modulators of SIRT6 activity at the physiologically important H3K56 deacetylation site.

Regulation of the Histone Deacetylase Hst3 by Cyclin-dependent Kinases and the Ubiquitin Ligase SCFCdc4

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) is a modification of new H3 molecules deposited throughout the genome during S-phase. H3K56ac is removed by the sirtuins Hst3 and Hst4 at later stages of the cell cycle. Previous studies indicated that regulated degradation of Hst3 plays an important role in the genome-wide waves of H3K56 acetylation and deacetylation that occur during each cell cycle. However, little is known regarding the mechanism of cell cycle-regulated Hst3 degradation. Here, we demonstrate that Hst3 instability in vivo is dependent upon the ubiquitin ligase SCF(Cdc4) and that Hst3 is phosphorylated at two Cdk1 sites, threonine 380 and threonine 384. This creates a diphosphorylated degron that is necessary for Hst3 polyubiquitylation by SCF(Cdc4). Mutation of the Hst3 diphospho-degron does not completely stabilize Hst3 in vivo, but it nonetheless results in a significant fitness defect that is particularly severe in mutant cells treated with the alkylating agent methyl methanesulfonate. Unexpectedly, we show that Hst3 can be degraded between G2 and anaphase, a window of the cell cycle where Hst3 normally mediates genome-wide deacetylation of H3K56. Our results suggest an intricate coordination between Hst3 synthesis, genome-wide H3K56 deacetylation by Hst3, and cell cycle-regulated degradation of Hst3 by cyclin-dependent kinases and SCF(Cdc4).

GATA-dependent regulatory switches establish atrioventricular canal specificity during heart development.

The embryonic vertebrate heart tube develops an atrioventricular canal that divides the atrial and ventricular chambers, forms atrioventricular conduction tissue and organizes valve development. Here we assess the transcriptional mechanism underlying this localized differentiation process. We show that atrioventricular canal-specific enhancers are GATA-binding site-dependent and act as switches that repress gene activity in the chambers. We find that atrioventricular canal-specific gene loci are enriched in H3K27ac, a marker of active enhancers, in atrioventricular canal tissue and depleted in H3K27ac in chamber tissue. In the atrioventricular canal, Gata4 activates the enhancers in synergy with Bmp2/Smad signalling, leading to H3K27 acetylation. In contrast, in chambers, Gata4 cooperates with pan-cardiac Hdac1 and Hdac2 and chamber-specific Hey1 and Hey2, leading to H3K27 deacetylation and repression. We conclude that atrioventricular canal-specific enhancers are platforms integrating cardiac transcription factors, broadly active histone modification enzymes and localized co-factors to drive atrioventricular canal-specific gene activity.

Calcium-sensing receptor silencing in colorectal cancer is associated with promoter hypermethylation and loss of acetylation on histone 3.

The calcium-sensing receptor (CaSR) is suggested to mediate the antiproliferative effects of calcium in colon. However, in colorectal cancer (CRC) the expression of the CaSR is silenced and the underlying mechanisms leading to its loss are poorly understood. We investigated whether loss of the CaSR expression in colorectal tumors is caused by DNA hypermethylation and imbalance of transcriptionally permissive/repressive histone alterations. We observed significantly lower CaSR mRNA expression (n = 65, p < 0.001) in colorectal tumors compared with the adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of the CaSR protein also. The CaSR promoter was methylated to a greater extent in tumors compared with adjacent mucosa as determined by bisulfite sequencing (n = 20, p < 0.01) and by pyrosequencing (n = 45, p < 0.001), and methylation correlated inversely with mRNA expression (n = 20, ρ = −0.310, p < 0.05 and n = 45, ρ = −0.588, p < 0.001). Treatments with 5-aza-2′-deoxycytidine (DAC), a DNA methyltransferase inhibitor and/or with two different histone deacetylase inhibitors, trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) restored the expression of CaSR in colon cancer cells. Restored CaSR expression in Coga1A and HT29 cells was functional. Inhibition of lysine-specific demethylase 1 (LSD1) to prevent demethylation of mono- and dimethylated H3K4, increased CaSR expression only marginally. Our data show that hypermethylation of the CaSR promoter and H3K9 deacetylation, but not H3K4me2 demethylation are important factors that cause silencing of the CaSR in colorectal cancer.