Maternal exposure to a Western-style diet causes differences in intestinal microbiota composition and gene expression of suckling mouse pups.

SCOPE: The long-lasting consequences of nutritional programming during the early phase of life have become increasingly evident. The effects of maternal nutrition on the developing intestine are still underexplored. METHODS AND RESULTS: In this study, we observed (1) altered microbiota composition of the colonic luminal content, and (2) differential gene expression in the intestinal wall in 2-week-old mouse pups born from dams exposed to a Western-style (WS) diet during the perinatal period. A sexually dimorphic effect was found for the differentially expressed genes in the offspring of WS diet-exposed dams but no differences between male and female pups were found for the microbiota composition. Integrative analysis of the microbiota and gene expression data revealed that the maternal WS diet independently affected gene expression and microbiota composition. However, the abundance of bacterial families not affected by the WS diet (Bacteroidaceae, Porphyromonadaceae, and Lachnospiraceae) correlated with the expression of genes playing a key role in intestinal development and functioning (e.g. Pitx2 and Ace2). CONCLUSION: Our data reveal that maternal consumption of a WS diet during the perinatal period alters both gene expression and microbiota composition in the intestinal tract of 2-week-old offspring.

The peroxisome proliferator-activated receptors under epigenetic control in placental metabolism and fetal development.

The placental metabolism can adapt to the environment throughout pregnancy to both the demands of the fetus and the signals from the mother. Such adaption processes include epigenetic mechanisms, which alter gene expression and may influence the offspring's health. These mechanisms are linked to the diversity of prenatal environmental exposures, including maternal under- or overnutrition or gestational diabetes. The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that contribute to the developmental plasticity of the placenta by regulating lipid and glucose metabolism pathways, including lipogenesis, steroidogenesis, glucose transporters, and placental signaling pathways, thus representing a link between energy metabolism and reproduction. Among the PPAR isoforms, PPARγ appears to be the main modulator of mammalian placentation. Certain fatty acids and lipid-derived moieties are the natural activating PPAR ligands. By controlling the amounts of maternal nutrients that go across to the fetus, the PPARs play an important regulatory role in placenta metabolism, thereby adapting to the maternal nutritional status. As demonstrated in animal studies, maternal nutrition during gestation can exert long-term influences on the PPAR methylation pattern in offspring organs. This review underlines the current state of knowledge on the relationship between environmental factors and the epigenetic regulation of the PPARs in placenta metabolism and offspring development.

DNA Methylation and Expression Patterns of Selected Genes in First-Trimester Placental Tissue from Pregnancies with Small-for-Gestational-Age Infants at Birth.

Variations in DNA methylation levels in the placenta are thought to influence gene expression and are associated with complications of pregnancy, like fetal growth restriction (FGR). The most important cause for FGR is placental dysfunction. Here, we examined whether changes in DNA methylation, followed by gene expression changes, are mechanistically involved in the etiology of FGR. In this retrospective case-control study, we examined the association between small-for-gestational-age (SGA) children and both DNA methylation and gene expression levels of the genes WNT2, IGF2/H19, SERPINA3, HERVWE1, and PPARG in first-trimester placental tissue. We also examined the repetitive element LINE-1. These candidate genes have been reported in the literature to be associated with SGA. We used first-trimester placental tissue from chorionic villus biopsies. A total of 35 SGA children (with a birth weight below the 10th percentile) were matched to 70 controls based on their gestational age. DNA methylation levels were analyzed by pyrosequencing and mRNA levels were analyzed by real-time PCR. None of the average DNA methylation levels, measured for each gene, showed a significant difference between SGA placental tissue compared to control tissue. However, hypermethylation of WNT2 was detected on two CpG positions in SGA. This was not associated with changes in gene expression. Apart from two CpG positions of the WNT2 gene, in early placenta samples, no evident changes in DNA methylation or expression were found. This indicates that the already reported changes in term placenta are not present in the early placenta, and therefore must arise after the first trimester.

Sexually dimorphic characteristics of the small intestine and colon of prepubescent C57BL/6 mice.

BACKGROUND: There is increasing appreciation for sexually dimorphic effects, but the molecular mechanisms underlying these effects are only partially understood. In the present study, we explored transcriptomics and epigenetic differences in the small intestine and colon of prepubescent male and female mice. In addition, the microbiota composition of the colonic luminal content has been examined. METHODS: At postnatal day 14, male and female C57BL/6 mice were sacrificed and the small intestine, colon and content of luminal colon were isolated. Gene expression of both segments of the intestine was analysed by microarray analysis. DNA methylation of the promoter regions of selected sexually dimorphic genes was examined by pyrosequencing. Composition of the microbiota was explored by deep sequencing. RESULTS: Sexually dimorphic genes were observed in both segments of the intestine of 2-week-old mouse pups, with a stronger effect in the small intestine. Amongst the total of 349 genes displaying a sexually dimorphic effect in the small intestine and/or colon, several candidates exhibited a previously established function in the intestine (i.e. Nts, Nucb2, Alox5ap and Retnlγ). In addition, differential expression of genes linked to intestinal bowel disease (i.e. Ccr3, Ccl11 and Tnfr) and colorectal cancer development (i.e. Wt1 and Mmp25) was observed between males and females. Amongst the genes displaying significant sexually dimorphic expression, nine genes were histone-modifying enzymes, suggesting that epigenetic mechanisms might be a potential underlying regulatory mechanism. However, our results reveal no significant changes in DNA methylation of analysed CpGs within the selected differentially expressed genes. With respect to the bacterial community composition in the colon, a dominant effect of litter origin was found but no significant sex effect was detected. However, a sex effect on the dominance of specific taxa was observed. CONCLUSIONS: This study reveals molecular dissimilarities between males and females in the small intestine and colon of prepubescent mice, which might underlie differences in physiological functioning and in disease predisposition in the two sexes.

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia.

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2-94.7% high-grade CIN and in 59.3-100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.

Overproduction of a rice aldo-keto reductase increases oxidative and heat stress tolerance by malondialdehyde and methylglyoxal detoxification.

The accumulation of toxic compounds generated by the interaction between reactive oxygen species and polyunsaturated fatty acids of membrane lipids can significantly damage plant cells. A plethora of enzymes act on these reactive carbonyls, reducing their toxicity. Based on the chromosomal localization and on their homology with other stress-induced aldo-keto reductases (AKRs) we have selected three rice AKR genes. The transcription level of OsAKR1 was greatly induced by abscisic acid and various stress treatments; the other two AKR genes tested were moderately stress-inducible. The OsAKR1 recombinant protein exhibited a high nicotinamide adenine dinucleotide phosphate-dependent catalytic activity to reduce toxic aldehydes including glycolysis-derived methylglyoxal (MG) and lipid peroxidation-originated malondialdehyde (MDA). The function of this enzyme in MG detoxification was demonstrated in vivo in E. coli and in transgenic plants overproducing the OsAKR1 protein. Heterologous synthesis of the OsAKR1 enzyme in transgenic tobacco plants resulted in increased tolerance against oxidative stress generated by methylviologen (MV) and improved resistance to high temperature. In these plants lower levels of MDA were detected both following MV and heat treatment due to the activity of the OsAKR1 enzyme. The transgenic tobaccos also exhibited higher AKR activity and accumulated less MG in their leaves than the wild type plants; both in the presence and absence of heat stress. These results support the positive role of OsAKR1 in abiotic stress-related reactive aldehyde detoxification pathways and its use for improvement of stress tolerance in plants.

Gene promoter methylation patterns throughout the process of cervical carcinogenesis.

OBJECTIVES: To determine methylation status of nine genes, previously described to be frequently methylated in cervical cancer, in squamous intraepithelial lesions (SIL). METHODS: QMSP was performed in normal cervix, low-grade (L)SIL, high-grade (H)SIL, adenocarcinomas and squamous cell cervical cancers, and in corresponding cervical scrapings. RESULTS: Only CCNA1 was never methylated in normal cervices and rarely in LSILs. All other genes showed methylation in normal cervices, with CALCA, SPARC and RAR-beta(2) at high levels. Methylation frequency of 6 genes (DAPK, APC, TFPI2, SPARC, CCNA1 and CADM1) increased with severity of the underlying cervical lesion. DAPK showed the highest increase in methylation frequency between LSIL and HSIL (10% vs. 40%, p<0.05), while CCNA1 and TFPI2 were most prominently methylated in cervical cancers compared to HSILs (25% vs. 52%, p<0.05, 30% vs. 58%, p<0.05). CADM1 methylation in cervical cancers was related to depth of invasion (p<0.05) and lymph vascular space involvement (p<0.01), suggesting a role in invasive potential of cervical cancers. Methylation ratios in scrapings reflected methylation status of the underlying lesions (p<0.05). CONCLUSION: Methylation of previously reported cervical cancer specific genes frequently occurs in normal epithelium. However, frequency of methylation increases during cervical carcinogenesis, with CCNA1 and DAPK as the best markers to distinguish normal/LSIL from HSIL/cancer lesions.

Methylation markers for CCNA1 and C13ORF18 are strongly associated with high-grade cervical intraepithelial neoplasia and cervical cancer in cervical scrapings.

PURPOSE: Recently, we reported 13 possible cervical cancer-specific methylated biomarkers identified by pharmacologic unmasking microarray in combination with large-genome computational screening. The aim of the present study was to perform an in-depth analysis of the methylation patterns of these 13 candidate genes in cervical neoplasia and to determine their diagnostic relevance. EXPERIMENTAL DESIGN AND RESULTS: Five of the 13 gene promoters (C13ORF18, CCNA1, TFPI2, C1ORF166, and NPTX1) were found to be more frequently methylated in frozen cervical cancer compared with normal cervix specimens. Quantitative methylation analysis for these five markers revealed that both CCNA1 and C13ORF18 were methylated in 68 of 97 cervical scrapings from cervical cancer patients and in only 5 and 3 scrapings, respectively, from 103 healthy controls (P < 0.0005). In cervical scrapings from patients referred with an abnormal Pap smear, CCNA1 and C13ORF18 were methylated in 2 of 43 and 0 of 43 CIN 0 (no cervical intraepithelial neoplasia) and in 1 of 41 and 0 of 41 CIN I, respectively. Furthermore, 8 of 43 CIN II, 22 of 43 CIN III, and 3 of 3 microinvasive cancer patients were positive for both markers. Although sensitivity for CIN II or higher (for both markers 37%) was low, specificity (96% and 100%, respectively) and positive predictive value (92% and 100%, respectively) were high. CONCLUSION: Methylation of CCNA1 and C13ORF18 in cervical scrapings is strongly associated with CIN II or higher-grade lesions. Therefore, these markers might be used for direct referral to gynecologists for patients with a methylation-positive scraping.

Genome-wide promoter analysis uncovers portions of the cancer methylome.

DNA methylation has a role in mediating epigenetic silencing of CpG island genes in cancer and other diseases. Identification of all gene promoters methylated in cancer cells "the cancer methylome" would greatly advance our understanding of gene regulatory networks in tumorigenesis. We previously described a new method of identifying methylated tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of re-expression on microarray analysis. In this study, we modified and greatly improved the selection of candidates based on new promoter structure algorithm and microarray data generated from 20 cancer cell lines of 5 major cancer types. We identified a set of 200 candidate genes that cluster throughout the genome of which 25 were previously reported as harboring cancer-specific promoter methylation. The remaining 175 genes were tested for promoter methylation by bisulfite sequencing or methylation-specific PCR (MSP). Eighty-two of 175 (47%) genes were found to be methylated in cell lines, and 53 of these 82 genes (65%) were methylated in primary tumor tissues. From these 53 genes, cancer-specific methylation was identified in 28 genes (28 of 53; 53%). Furthermore, we tested 8 of the 28 newly identified cancer-specific methylated genes with quantitative MSP in a panel of 300 primary tumors representing 13 types of cancer. We found cancer-specific methylation of at least one gene with high frequency in all cancer types. Identification of a large number of genes with cancer-specific methylation provides new targets for diagnostic and therapeutic intervention, and opens fertile avenues for basic research in tumor biology.

Dicot and monocot plants differ in retinoblastoma-related protein subfamilies.

The present study supports the view that the retinoblastoma functions are shared by two distinct retinoblastoma-related (RBR) protein subfamilies in the monocot cereal species, whereas dicot plants have only a single RBR protein. Genes encoding RBR proteins were identified and characterized in alfalfa (Medicago sativa), rice (Oryza sativa), and wheat (Triticum aestivum). The alfalfa MsRBR gene encodes a new member of the dicot RBR proteins (subfamily A). A comparison was made of two rice genes, OsRBR1 (subfamily B) and OsRBR2 (subfamily C), which exhibit differences in exon-intron organization and share only 52% amino acid sequence identity. The plant RBR proteins can be categorized into three distinct subfamilies, in which the similarity between members is greater than the similarity to other RBR proteins from the same species. Comparison of the transcript levels in various tissues revealed that the expression of the OsRBR1 gene was high in embryos or cultured cells and gradually decreased from the basal region to the tip of the leaves. The OsRBR2 gene displayed more transcripts in differentiated tissues, such as leaves and roots. In contrast, the mRNA level of the MsRBR gene did not differ significantly in either mature leaves or cultured cells. The results of yeast two-hybrid pairwise interaction assays demonstrated differences between the rice RBR variants in the interactions with the phosphatase 2A B'' regulatory subunit and an unknown protein. The in silico and functional data presented in this work highlight considerable differences between dicot and monocot species in the retinoblastoma regulatory pathways and permit an improved classification of RBR proteins in higher plants.