Comparative anatomy of chromosomal domains with imprinted and non-imprinted allele-specific DNA methylation.

Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM.

Activation of type III interferon genes by pathogenic bacteria in infected epithelial cells and mouse placenta.

Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-λ) genes, whose products play important roles in epithelial innate immunity against viruses. Here, we studied the expression of IFN-λ genes in cultured human epithelial cells infected with different pathogenic bacteria and in the mouse placenta infected with Listeria monocytogenes. We first showed that in intestinal LoVo cells, induction of IFN-λ genes by L. monocytogenes required bacterial entry and increased further during the bacterial intracellular phase of infection. Other Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, also induced IFN-λ genes when internalized by LoVo cells. In contrast, Gram-negative bacteria Salmonella enterica serovar Typhimurium, Shigella flexneri and Chlamydia trachomatis did not substantially induce IFN-λ. We also found that IFN-λ genes were up-regulated in A549 lung epithelial cells infected with Mycobacterium tuberculosis and in HepG2 hepatocytes and BeWo trophoblastic cells infected with L. monocytogenes. In a humanized mouse line permissive to fetoplacental listeriosis, IFN-λ2/λ3 mRNA levels were enhanced in placentas infected with L. monocytogenes. In addition, the feto-placental tissue was responsive to IFN-λ2. Together, these results suggest that IFN-λ may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues.

CHRNA5 as negative regulator of nicotine signaling in normal and cancer bronchial cells: effects on motility, migration and p63 expression.

Genome-wide association studies have linked lung cancer risk with a region of chromosome 15q25.1 containing CHRNA3, CHRNA5 and CHRNB4 encoding α3, α5 and ß4 subunits of nicotinic acetylcholine receptors (nAChR), respectively. One of the strongest associations was observed for a non-silent single-nucleotide polymorphism at codon 398 in CHRNA5. Here, we have used pharmacological (antagonists) or genetic (RNA interference) interventions to modulate the activity of CHRNA5 in non-transformed bronchial cells and in lung cancer cell lines. In both cell types, silencing CHRNA5 or inhibiting receptors containing nAChR α5 with α-conotoxin MII exerted a nicotine-like effect, with increased motility and invasiveness in vitro and increasing calcium influx. The effects on motility were enhanced by addition of nicotine but blocked by inhibiting CHRNA7, which encodes the homopentameric receptor α7 subunit. Silencing CHRNA5 also decreased the expression of cell adhesion molecules P120 and ZO-1 in lung cancer cells as well as the expression of DeltaNp63α in squamous cell carcinoma cell lines. These results demonstrate a role for CHRNA5 in modulating adhesion and motility in bronchial cells, as well as in regulating p63, a potential oncogene in squamous cell carcinoma.

Epigenetic mechanisms in hepatocellular carcinoma: how environmental factors influence the epigenome.

Epigenetic mechanisms maintain heritable changes in gene expression and chromatin organization over many cell generations. Importantly, deregulated epigenetic mechanisms play a key role in a wide range of human malignancies, including liver cancer. Hepatocellular carcinoma (HCC), which originates from the hepatocytes, is by far the most common liver cancer, with rates and aetiology that show considerable geographic variation. Various environmental agents and lifestyles known to be risk factors for HCC (such as infection by hepatitis B virus (HBV) and hepatitis C virus (HCV), chronic alcohol intake, and aflatoxins) are suspected to promote its development by eliciting epigenetic changes, however the precise gene targets and underlying mechanisms have not been elucidated. Many recent studies have exploited conceptual and technological advances in epigenetics and epigenomics to investigate the role of epigenetic events induced by environmental factors in HCC tumors and non-tumor precancerous (cirrhotic) lesions. These studies have identified a large number of genes and pathways that are targeted by epigenetic deregulation (changes in DNA methylation, histone modifications and RNA-mediated gene silencing) during the development and progression of HCC. Frequent identification of aberrant epigenetic changes in specific genes in cirrhotic tissue is consistent with the notion that epigenetic deregulation of selected genes in pre-malignant lesions precedes and promotes the development of HCC. In addition, several lines of evidence argue that some environmental factors (such as HBV virus) may abrogate cellular defense systems, induce silencing of host genes and promote HCC development via an "epigenetic strategy". Finally, profiling studies reveal that HCC tumors and pre-cancerous lesions may exhibit epigenetic signatures associated with specific risk factors and tumor progression stage. Together, recent evidence underscores the importance of aberrant epigenetic events induced by environmental factors in liver cancer and highlights potential targets for biomarker discovery and future preventive and therapeutic strategies.

Aberrant DNA methylation distinguishes hepatocellular carcinoma associated with HBV and HCV infection and alcohol intake.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most frequent human cancers and a major cause of cancer-related death worldwide. The major risk factors for developing HCC are infection by hepatitis B virus (HBV) and hepatitis C virus (HCV), chronic alcoholism, and aflatoxins; however, critical gene targets remain largely unknown. Herein, we sought to establish DNA methylation patterns in HCC and corresponding cirrhotic tissues and to identify DNA methylation changes associated with major risk factors. METHODS: We have established assays for quantitative analysis of DNA methylation levels in a panel of seven cancer-associated genes and repetitive elements, and combined these assays with a series of HCC tumors, associated with major risk factors, collected from two different geographical areas. RESULTS: We found a high frequency of aberrant hypermethylation of specific genes (RASSF1A, GSTP1, CHRNA3, and DOK1) in HCC tumors as compared to control cirrhotic or normal liver tissues, suggesting that aberrant hypermethylation exhibits non-random and tumor-specific patterns in HCC. Importantly, our analysis revealed an association between alcohol intake and the hypomethylation of MGMT and between hypermethylation of GSTP1 and HBV infection, indicating that hypermethylation of the genes analyzed in HCC tumors exhibits remarkably distinct patterns depending on associated risk factors. CONCLUSIONS: This study identifies aberrant DNA methylation of specific cellular genes in HCC and the major risk factors associated with these changes, providing information that could be exploited for biomarker discovery in clinics and molecular epidemiology.

DNA methylation of hepatitis B virus (HBV) genome associated with the development of hepatocellular carcinoma and occult HBV infection.

Recent studies have identified the presence of methylation in the hepatitis B virus (HBV) genome and have suggested that it may be an important mechanism regulating transcription and replication of the HBV virus. However, it remains unclear whether this phenomenon is associated with occult hepatitis. Here, we performed a detailed analysis of DNA methylation in the HBV genome in liver samples of patients at different stages of hepatocarcinoma development and in in vitro infected hepatocytes and found discrete CpG sites in the HBV genome that are recurrently hypermethylated in cancer but not in chronic hepatitis tissue.

Quantitative detection of DNA methylation states in minute amounts of DNA from body fluids.

Quantitative and reliable estimation of DNA methylation levels for multiple genomic regions pose a major challenge where starting DNA is available in very low quantity. Here we review major advances in the development of techniques for quantitative detection of DNA methylation in minute amount of DNA and describe a detailed protocol for quantitative Methylation Analysis of Minute DNA amounts after whole Bisulfitome Amplification (qMAMBA), a combination of techniques that allows quantitative and sensitive detection of DNA methylation at multiple CpG sites and for multiple gene assays. Recently we successfully used this technique to quantitatively detect DNA methylation for a set of cancer-related genes in lung cancer patient plasma samples [18]. This method involves genome-wide amplification of bisulfite-modified DNA template followed by quantitative methylation detection using pyrosequencing. This allows a precise assessment of DNA methylation at CpG sites and could be adapted for high-throughput settings. It can also be applied in conjunction with studies involving single-cells or laser capture microdissected samples. Thus, this method should facilitate DNA methylation studies aiming to discover epigenetic biomarkers, and should prove particularly valuable in profiling a large sample series of body fluids from molecular epidemiology studies.

Aberrant DNA methylation links cancer susceptibility locus 15q25.1 to apoptotic regulation and lung cancer.

Nicotinic acetylcholine receptor (nAChR) genes form a highly conserved gene cluster at the lung cancer susceptibility locus 15q25.1. In this study, we show that the CHRNalpha3 gene encoding the nAChRalpha3 subunit is a frequent target of aberrant DNA hypermethylation and silencing in lung cancer, whereas the adjacent CHRNbeta4 and CHRNalpha5 genes exhibit moderate and no methylation, respectively. Treatment of cancer cells exhibiting CHRNalpha3 hypermethylation with DNA methylation inhibitors caused demethylation of the CHRNalpha3 promoter and gene reactivation. Restoring CHRNalpha3 levels through ectopic expression induced apoptotic cell death. Small hairpin RNA-mediated depletion of nAChRalpha3 in CHRNalpha3-expressing lung cancer cells elicited a dramatic Ca(2+) influx response in the presence of nicotine, followed by activation of the Akt survival pathway. CHRNalpha3-depleted cells were resistant to apoptosis-inducing agents, underscoring the importance of epigenetic silencing of the CHRNalpha3 gene in human cancer. In defining a mechanism of epigenetic control of nAChR expression in nonneuronal tissues, our findings offer a functional link between susceptibility locus 15q25.1 and lung cancer, and suggest nAChRs to be theranostic targets for cancer detection and chemoprevention.

Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids.

Cell-free circulating DNA isolated from the plasma of individuals with cancer has been shown to harbor cancer-associated changes in DNA methylation, and thus it represents an attractive target for biomarker discovery. However, the reliable detection of DNA methylation changes in body fluids has proven to be technically challenging. Here we describe a novel combination of methods that allows quantitative and sensitive detection of DNA methylation in minute amounts of DNA present in body fluids (quantitative Methylation Analysis of Minute DNA amounts after whole Bisulfitome Amplification, qMAMBA). This method involves genome-wide amplification of bisulphite-modified DNA template followed by quantitative methylation detection using pyrosequencing and allows analysis of multiple genes from a small amount of starting DNA. To validate our method we used qMAMBA assays for four genes and LINE1 repetitive sequences combined with plasma DNA samples as a model system. qMAMBA offered high efficacy in the analysis of methylation levels and patterns in plasma samples with extremely small amounts of DNA and low concentrations of methylated alleles. Therefore, qMAMBA will facilitate methylation studies aiming to discover epigenetic biomarkers, and should prove particularly valuable in profiling a large sample series of body fluids from molecular epidemiology studies as well as in tracking disease in early diagnostics.

Quantitative analysis of DNA methylation profiles in lung cancer identifies aberrant DNA methylation of specific genes and its association with gender and cancer risk factors.

The global increase in lung cancer burden, together with its poor survival and resistance to classical chemotherapy, underscores the need for identification of critical molecular events involved in lung carcinogenesis. Here, we have applied quantitative profiling of DNA methylation states in a panel of five cancer-associated genes (CDH1, CDKN2A, GSTP1, MTHFR, and RASSF1A) to a large case-control study of lung cancer. Our analyses revealed a high frequency of aberrant hypermethylation of MTHFR, RASSF1A, and CDKN2A in lung tumors as compared with control blood samples, whereas no significant increase in methylation levels of GSTP1 and CDH1 was observed, consistent with the notion that aberrant DNA methylation occurs in a tumor-specific and gene-specific manner. Importantly, we found that tobacco smoking, sex, and alcohol intake had a strong influence on the methylation levels of distinct genes (RASSF1A and MTHFR), whereas folate intake, age, and histologic subtype had no significant influence on methylation states. We observed a strong association between MTHFR hypermethylation in lung cancer and tobacco smoking, whereas methylation levels of CDH1, CDKN2A, GSTP1, and RASSF1A were not associated with smoking, indicating that tobacco smoke targets specific genes for hypermethylation. We also found that methylation levels in RASSF1A, but not the other genes under study, were influenced by sex, with males showing higher levels of methylation. Together, this study identifies aberrant DNA methylation patterns in lung cancer and thus exemplifies the mechanism by which environmental factors may interact with key genes involved in tumor suppression and contribute to lung cancer.

Estrogen and androgen repression of two female specific lacrimal lipocalins in hamster: Pituitary independent and sex hormone receptor mediated action.

Sexual dimorphism in lacrimal gland (LG) gene expression is believed to be due to direct inductive effects of androgens mediated by androgen receptors (AR) but hypophysectomy dramatically curtails these inductive effects. Since, functional estrogen receptors (ER) could not be detected in LG, estrogen effects on LG are believed to be indirectly mediated by changes in levels of pituitary hormones. We found that two lipocalins expressed in female hamster LG display an unusual and marked repression by both androgens and estrogens, which could be detected both at the level of transcripts and proteins. Here, we investigate whether these repressions, (i) require presence of pituitary and (ii) are mediated by androgen and estrogen receptors. Pituitary-ablation but not gonadectomy reduced LG weights in hamster. However, both pituitary-ablation and gonadectomy induced abundant expression of the LG lipocalins, which were markedly repressed by androgen or estrogen treatment. AR- and ER-antagonists prevented these repressions and only ER-alpha- but not ER-beta-specific agonist could mimic the estrogenic repression. AR transcript and protein and ER-alpha transcript were also detected in hamster LG. Thus, pituitary factors are neither essential for the expression of these LG lipocalins nor for their estrogenic or androgenic repressions and these repressions are very likely mediated by functional ER and AR present in LG.

Purification, cloning and regulation of a novel acid-lipase-like protein of hamster expressed in lacrimal glands and tears during lactation.

We report a novel 48-kDa tear acid-lipase-like protein (TALLP), which is markedly induced in lacrimal glands (LG) and secreted in tears of hamster dams during lactation. TALLP is undetectable in LG and tears of normal hamsters, but is also induced after gonadectomy in both sexes and this is prevented by androgen, estrogen or thyroid hormone treatment. These observations and the obliteration of TALLP upon cessation of lactation suggest that endogenous estrogens (in females) and androgens (in males) completely repress TALLP expression. Purified TALLP is monomeric, contains approximately 18% N-glycosylation and several pI isoforms. TALLP expression was tissue-specific and immunolocalized in LG acinar cells. The cDNA deduced amino-acid sequence of TALLP precursor (398 residue, containing a 19 residues signal-peptide) showed only 43-48% identity with all known mammalian acid-lipases, including even those of other rodents, suggesting that TALLP is a prototype of a new category, within the acid-lipase family. Surprisingly, although the catalytic triad residues and other sequence features important for lipolytic activity are conserved in TALLP, it has no detectable lipase activity. However, TALLP binds the polarity sensitive hydrophobic probe, 1-aminoanthracene (K(d)=12 microM). TALLP might have a unique substrate-specificity or a lipid-binding/carrier function in tears of hamster dams. This is the first report of an acid-lipase-like protein secreted in tears of any species. Since TALLP lacks the usual lipase activity, it can be an excellent model to understand better what other structural features in acid-lipases influence their catalytic activity.

Marked sexual dimorphism of lacrimal gland peroxidase in hamster: repression by androgens and estrogens.

Peroxidase secreted in tears by the lacrimal glands is a marker of secretory activity of these glands and is believed to have an antimicrobial function. We report for the first time a marked sex difference in lacrimal gland (LG) peroxidase in hamsters ( approximately 3.4-fold higher activity in females), which is due to an unusual repression by physiological levels of androgens in males. LG peroxidase activity was markedly induced in a time-dependent manner after gonadectomy in males and also females ( approximately 8- and 2-fold, respectively) and was strongly repressed by androgen treatment in a dose- and time-dependent manner. Estrogen treatment of gonadectomized hamsters could also repress LG peroxidase but not below female levels. These repressions by androgens and estrogens were significantly prevented upon co-treatment with their respective receptor antagonists. Western blotting showed that differences in LG peroxidase specific activity, in different sex hormonal states and treatments were due to changes in the levels of peroxidase protein in LG. A tear peroxidase with a clear sex difference suggests that it might also have other novel function(s) in hamster tears.