Analysis of candidate colitis genes in the Gdac1 locus of mice deficient in glutathione peroxidase-1 and -2.

BACKGROUND: Mice that are deficient for glutathione peroxidases 1 and 2 (GPX) show large variations in the penetrance and severity of colitis in C57BL/6J and 129S1/SvImJ backgrounds. We mapped a locus contributing to this difference to distal chromosome 2 (∼119-133 mbp) and named it glutathione peroxidase-deficiency-associated colitis 1 (Gdac1). The aim of this study was to identify the best gene candidates within the Gdac1 locus contributing to the murine colitis phenotype. METHOD/PRINCIPAL FINDINGS: We refined the boundaries of Gdac1 to 118-125 mbp (95% confidence interval) by increasing sample size and marker density across the interval. The narrowed region contains 128 well-annotated protein coding genes but it excludes Fermt1, a human inflammatory bowel disease candidate that was within the original boundaries of Gdac1. The locus we identified may be the Cdcs3 locus mapped by others studying IL10-knockout mice. Using in silico analysis of the 128 genes, based on published colon expression data, the relevance of pathways to colitis, gene mutations, presence of non-synonymous-single-nucleotide polymorphisms (nsSNPs) and whether the nsSNPs are predicted to have an impact on protein function or expression, we excluded 42 genes. Based on a similar analysis, twenty-five genes from the remaining 86 genes were analyzed for expression-quantitative-trait loci, and another 15 genes were excluded. CONCLUSION/SIGNIFICANCE: Among the remaining 10 genes, we identified Pla2g4f and Duox2 as the most likely colitis gene candidates, because GPX metabolizes PLA2G4F and DUOX2 products. Pla2g4f is a phospholipase A2 that has three potentially significant nsSNP variants and showed expression differences across mouse strains. PLA2G4F produces arachidonic acid, which is a substrate for lipoxygenases and, in turn, for GPXs. DUOX2 produces H(2)O(2) and may control microbial populations. DUOX-1 and -2 control microbial populations in mammalian lung and in the gut of several insects and zebrafish. Dysbiosis is a phenotype that differentiates 129S1/SvImJ from C57BL/6J and may be due to strain differences in DUOX2 activity.

Effects of endocrine disruptors on imprinted gene expression in the mouse embryo.

Environmental endocrine disruptors (EDs) are synthetic chemicals that resemble natural hormones and are known to cause epigenetic perturbations. EDs have profound effects on development and fertility. Imprinted genes had been identified as susceptible loci to environmental insults by EDs because they are functionally haploid, and because the imprints undergo epigenetic resetting between generations. To screen for possible epigenetic perturbations caused by EDs at imprinted loci, we treated pregnant mice daily between 8.5 and 12.5 days post coitum (dpc) with di-(2-ethylhexyl)-phthalate (DEHP), bisphenol A (BPA), vinclozolin (VZ), or control oil vehicle. After isolating RNA from the placenta, yolk sac, amnion, head, body, heart, liver, lung, stomach, and intestines of 13.5 dpc embryos we measured the allele-specific expression of 38 imprinted transcripts using multiplex single nucleotide primer extension (SNuPE) assays. In this representative data set we identified only a small number of transcripts that exhibited a substantial relaxation of imprinted expression with statistical significance: Slc22a18 with 10% relaxation in the embryo after BPA treatment; Rtl1as with 11 and 16% relaxation in the lung and placenta, respectively after BPA treatment; and Rtl1 with 12% relaxation in the yolk sac after DEHP treatment. Additionally, the standard deviation of allele-specificity increased in various organs after ED treatment for several transcripts including Igf2r, Rasgrf1, Usp29, Slc38a4, and Xist. Our data suggest that the maintenance of strongly biased monoallelic expression of imprinted genes is generally insensitive to EDs in the 13.5 dpc embryo and extra-embryonic organs, but is not immune to those effects.

MIRA-SNuPE, a quantitative, multiplex method for measuring allele-specific DNA methylation.

5-methyl-C (5mC) and 5-hydroxymethyl-C (5hmC) are epigenetic marks with well known and putative roles in gene regulation, respectively. These two DNA covalent modifications cannot be distinguished by bisulfite sequencing or restriction digestion, the standard methods of 5mC detection. The methylated CpG island recovery assay (MIRA), however, specifically detects 5mC but not 5hmC. We further developed MIRA for the analysis of allele-specific CpG methylation at differentially methylated regions (DMRs) of imprinted genes. MIRA specifically distinguished between the parental alleles by capturing the paternally methylated H19/Igf2 DMR and maternally methylated KvDMR1 in mouse embryo fibroblasts (MEFs) carrying paternal and maternal duplication of mouse distal Chr7, respectively. MIRA in combination with multiplex single nucleotide primer extension (SNuPE) assays specifically captured the methylated parental allele from normal cells at a set of maternally and paternally methylated DMRs. The assay correctly recognized aberrant biallelic methylation in a case of loss-of imprinting. The MIRA-SNuPE assays revealed that placenta exhibited less DNA methylation bias at DMRs compared to yolk sac, amnion, brain, heart, kidney, liver and muscle. This method should be useful for the analysis of allele-specific methylation events related to genomic imprinting, X chromosome inactivation and for verifying and screening haplotype-associated methylation differences in the human population.

Coordinated allele-specific histone acetylation at the differentially methylated regions of imprinted genes.

Genomic imprinting is an epigenetic inheritance system characterized by parental allele-specific gene expression. Allele-specific DNA methylation and chromatin composition are two epigenetic modification systems that control imprinted gene expression. To get a general assessment of histone lysine acetylation at imprinted genes we measured allele-specific acetylation of a wide range of lysine residues, H3K4, H3K18, H3K27, H3K36, H3K79, H3K64, H4K5, H4K8, H4K12, H2AK5, H2BK12, H2BK16 and H2BK46 at 11 differentially methylated regions (DMRs) in reciprocal mouse crosses using multiplex chromatin immunoprecipitation SNuPE assays. Histone acetylation marks generally distinguished the methylation-free alleles from methylated alleles at DMRs in mouse embryo fibroblasts and embryos. Acetylated lysines that are typically found at transcription start sites exhibited stronger allelic bias than acetylated histone residues in general. Maternally methylated DMRs, that usually overlap with promoters exhibited higher levels of acetylation and a 10% stronger allele-specific bias than paternally methylated DMRs that reside in intergenic regions. Along the H19/Igf2 imprinted domain, allele-specific acetylation at each lysine residue depended on functional CTCF binding sites in the imprinting control region. Our results suggest that many different histone acetyltransferase and histone deacetylase enzymes must act in concert in setting up and maintaining reciprocal parental allelic histone acetylation at DMRs.

Allele-specific H3K79 Di- versus trimethylation distinguishes opposite parental alleles at imprinted regions.

Imprinted gene expression corresponds to parental allele-specific DNA CpG methylation and chromatin composition. Histone tail covalent modifications have been extensively studied, but it is not known whether modifications in the histone globular domains can also discriminate between the parental alleles. Using multiplex chromatin immunoprecipitation-single nucleotide primer extension (ChIP-SNuPE) assays, we measured the allele-specific enrichment of H3K79 methylation and H4K91 acetylation along the H19/Igf2 imprinted domain. Whereas H3K79me1, H3K79me2, and H4K91ac displayed a paternal-specific enrichment at the paternally expressed Igf2 locus, H3K79me3 was paternally biased at the maternally expressed H19 locus, including the paternally methylated imprinting control region (ICR). We found that these allele-specific differences depended on CTCF binding in the maternal ICR allele. We analyzed an additional 11 differentially methylated regions (DMRs) and found that, in general, H3K79me3 was associated with the CpG-methylated alleles, whereas H3K79me1, H3K79me2, and H4K91ac enrichment was specific to the unmethylated alleles. Our data suggest that allele-specific differences in the globular histone domains may constitute a layer of the "histone code" at imprinted genes.

A risk variant in an miR-125b binding site in BMPR1B is associated with breast cancer pathogenesis.

MicroRNAs regulate diverse cellular processes and play an integral role in cancer pathogenesis. Genomic variation within miRNA target sites may therefore be important sources for genetic differences in cancer risk. To investigate this possibility, we mapped HapMap single nucleotide polymorphisms (SNP) to putative miRNA recognition sites within genes dysregulated in estrogen receptor-stratified breast tumors and used local linkage disequilibrium patterns to identify high-ranking SNPs in the Cancer Genetic Markers of Susceptibility (CGEMS) breast cancer genome-wide association study for further testing. Two SNPs, rs1970801 and rs11097457, scoring in the top 100 from the CGEMS study, were in strong linkage disequilibrium with rs1434536, an SNP that resides within a miR-125b target site in the 3' untranslated region of the bone morphogenic receptor type 1B (BMPR1B) gene encoding a transmembrane serine/threonine kinase. We validated the CGEMS association findings for rs1970801 in an independent cohort of admixture-corrected cases identified from families with multiple case histories. Subsequent association testing of rs1434536 for these cases and CGEMS controls with imputed genotypes supported the association. Furthermore, luciferase reporter assays and overexpression of miR-125b-mimics combined with quantitative reverse transcription-PCR showed that BMPR1B transcript is a direct target of miR-125b and that miR-125b differentially regulates the C and T alleles of rs1434536. These results suggest that allele-specific regulation of BMPR1B by miR-125b explains the observed disease risk. Our approach is general and can help identify and explain the mechanisms behind disease association for alleles that affect miRNA regulation.

Meta-analysis of breast cancer microarray studies in conjunction with conserved cis-elements suggest patterns for coordinate regulation.

BACKGROUND: Gene expression measurements from breast cancer (BrCa) tumors are established clinical predictive tools to identify tumor subtypes, identify patients showing poor/good prognosis, and identify patients likely to have disease recurrence. However, diverse breast cancer datasets in conjunction with diagnostic clinical arrays show little overlap in the sets of genes identified. One approach to identify a set of consistently dysregulated candidate genes in these tumors is to employ meta-analysis of multiple independent microarray datasets. This allows one to compare expression data from a diverse collection of breast tumor array datasets generated on either cDNA or oligonucleotide arrays. RESULTS: We gathered expression data from 9 published microarray studies examining estrogen receptor positive (ER+) and estrogen receptor negative (ER-) BrCa tumor cases from the Oncomine database. We performed a meta-analysis and identified genes that were universally up or down regulated with respect to ER+ versus ER- tumor status. We surveyed both the proximal promoter and 3' untranslated regions (3'UTR) of our top-ranking genes in each expression group to test whether common sequence elements may contribute to the observed expression patterns. Utilizing a combination of known transcription factor binding sites (TFBS), evolutionarily conserved mammalian promoter and 3'UTR motifs, and microRNA (miRNA) seed sequences, we identified numerous motifs that were disproportionately represented between the two gene classes suggesting a common regulatory network for the observed gene expression patterns. CONCLUSION: Some of the genes we identified distinguish key transcripts previously seen in array studies, while others are newly defined. Many of the genes identified as overexpressed in ER- tumors were previously identified as expression markers for neoplastic transformation in multiple human cancers. Moreover, our motif analysis identified a collection of specific cis-acting target sites which may collectively play a role in the differential gene expression patterns observed in ER+ versus ER- breast cancer tumors. Importantly, the gene sets and associated DNA motifs provide a starting point with which to explore the mechanistic basis for the observed expression patterns in breast tumors.

Polymorphism in the androgen receptor and mammographic density in women taking and not taking estrogen and progestin therapy.

There is some evidence that women with a higher number of CAG repeat lengths on the androgen receptor (AR) gene have increased breast cancer risk. We evaluated the association between AR-CAG repeat length and mammographic density, a strong breast cancer risk factor, in 404 African-American and Caucasian breast cancer patients. In postmenopausal estrogen progestin therapy users, carriers of the less active AR-CAG had statistically significantly higher mean percentage of density (41.4%) than carriers of the more active AR-CAG (25.7%; P = 0.04). Our results raise the question of whether the number of AR-CAG repeats predicts breast cancer risk in estrogen progestin therapy users.