R. Alan Harris

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.

Functional annotation of the human brain methylome identifies tissue-specific epigenetic variation across brain and blood

BackgroundDynamic changes to the epigenome play a critical role in establishing and maintaining cellular phenotype during differentiation, but little is known about the normal methylomic differences that occur between functionally distinct areas of the brain. We characterized intra- and inter-individual methylomic variation across whole blood and multiple regions of the brain from multiple donors.ResultsDistinct tissue-specific patterns of DNA methylation were identified, with a highly significant over-representation of tissue-specific differentially methylated regions (TS-DMRs) observed at intragenic CpG islands and low CG density promoters. A large proportion of TS-DMRs were located near genes that are differentially expressed across brain regions. TS-DMRs were significantly enriched near genes involved in functional pathways related to neurodevelopment and neuronal differentiation, including BDNF, BMP4, CACNA1A, CACA1AF, EOMES, NGFR, NUMBL, PCDH9, SLIT1, SLITRK1 and SHANK3. Although between-tissue variation in DNA methylation was found to greatly exceed between-individual differences within any one tissue, we found that some inter-individual variation was reflected across brain and blood, indicating that peripheral tissues may have some utility in epidemiological studies of complex neurobiological phenotypes.ConclusionsThis study reinforces the importance of DNA methylation in regulating cellular phenotype across tissues, and highlights genomic patterns of epigenetic variation across functionally distinct regions of the brain, providing a resource for the epigenetics and neuroscience research communities.

Season of Conception in Rural Gambia Affects DNA Methylation at Putative Human Metastable Epialleles

Throughout most of the mammalian genome, genetically regulated developmental programming establishes diverse yet predictable epigenetic states across differentiated cells and tissues. At metastable epialleles (MEs), conversely, epigenotype is established stochastically in the early embryo then maintained in differentiated lineages, resulting in dramatic and systemic interindividual variation in epigenetic regulation. In the mouse, maternal nutrition affects this process, with permanent phenotypic consequences for the offspring. MEs have not previously been identified in humans. Here, using an innovative 2-tissue parallel epigenomic screen, we identified putative MEs in the human genome. In autopsy samples, we showed that DNA methylation at these loci is highly correlated across tissues representing all 3 embryonic germ layer lineages. Monozygotic twin pairs exhibited substantial discordance in DNA methylation at these loci, suggesting that their epigenetic state is established stochastically. We then tested for persistent epigenetic effects of periconceptional nutrition in rural Gambians, who experience dramatic seasonal fluctuations in nutritional status. DNA methylation at MEs was elevated in individuals conceived during the nutritionally challenged rainy season, providing the first evidence of a permanent, systemic effect of periconceptional environment on human epigenotype. At MEs, epigenetic regulation in internal organs and tissues varies among individuals and can be deduced from peripheral blood DNA. MEs should therefore facilitate an improved understanding of the role of interindividual epigenetic variation in human disease.

High-fat maternal diet during pregnancy persistently alters the offspring microbiome in a primate model

The intestinal microbiome is a unique ecosystem and an essential mediator of metabolism and obesity in mammals. However, studies investigating the impact of the diet on the establishment of the gut microbiome early in life are generally lacking, and most notably so in primate models. Here we report that a high-fat maternal or postnatal diet, but not obesity per se, structures the offspring’s intestinal microbiome in Macaca fuscata (Japanese macaque). The resultant microbial dysbiosis is only partially corrected by a low-fat, control diet after weaning. Unexpectedly, early exposure to a high-fat diet diminished the abundance of non-pathogenic Campylobacter in the juvenile gut, suggesting a potential role for dietary fat in shaping commensal microbial communities in primates. Our data challenge the concept of an obesity-causing gut microbiome, and rather provide evidence for a contribution of the maternal diet in establishing the microbiota, which in turn affects intestinal maintenance of metabolic health.

Gibbon genome and the fast karyotype evolution of small apes.

Gibbons are small arboreal apes that display an accelerated rate of evolutionary chromosomal rearrangement and occupy a key node in the primate phylogeny between Old World monkeys and great apes. Here we present the assembly and analysis of a northern white-cheeked gibbon (Nomascus leucogenys) genome. We describe the propensity for a gibbon-specific retrotransposon (LAVA) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage. We further show that the gibbon genera (Nomascus, Hylobates, Hoolock and Symphalangus) experienced a near-instantaneous radiation ∼5 million years ago, coincident with major geographical changes in southeast Asia that caused cycles of habitat compression and expansion. Finally, we identify signatures of positive selection in genes important for forelimb development (TBX5) and connective tissues (COL1A1) that may have been involved in the adaptation of gibbons to their arboreal habitat.

GASZ Is Essential for Male Meiosis and Suppression of Retrotransposon Expression in the Male Germline

Nuage are amorphous ultrastructural granules in the cytoplasm of male germ cells as divergent as Drosophila, Xenopus, and Homo sapiens. Most nuage are cytoplasmic ribonucleoprotein structures implicated in diverse RNA metabolism including the regulation of PIWI-interacting RNA (piRNA) synthesis by the PIWI family (i.e., MILI, MIWI2, and MIWI). MILI is prominent in embryonic and early post-natal germ cells in nuage also called germinal granules that are often associated with mitochondria and called intermitochondrial cement. We find that GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) co-localizes with MILI in intermitochondrial cement. Knockout of Gasz in mice results in a dramatic downregulation of MILI, and phenocopies the zygotene–pachytene spermatocyte block and male sterility defect observed in MILI null mice. In Gasz null testes, we observe increased hypomethylation and expression of retrotransposons similar to MILI null testes. We also find global shifts in the small RNAome, including down-regulation of repeat-associated, known, and novel piRNAs. These studies provide the first evidence for an essential structural role for GASZ in male fertility and epigenetic and post-transcriptional silencing of retrotransposons by stabilizing MILI in nuage.

Maternal methyl-donor supplementation induces prolonged murine offspring colitis susceptibility in association with mucosal epigenetic and microbiomic changes

Developmental epigenetic changes, such as DNA methylation, have been recognized as potential pathogenic factors in inflammatory bowel diseases, the hallmark of which is an exaggerated immune response against luminal microbes. A methyl-donor (MD) diet can modify DNA methylation at select murine genomic loci during early development. The components of the MDs are routinely incorporated into prenatal human supplements. Therefore, we studied the effects of maternal MD supplementation on offspring colitis susceptibility and colonic mucosal DNA methylation and gene expression changes in mice as a model. Additionally, we investigated the offspring mucosal microbiomic response to the maternal dietary supplementation. Colitis was induced by dextran sulfate sodium. Colonic mucosa from offspring of MD-supplemented mothers following reversal to control diet at weaning was interrogated by methylation-specific microarrays and pyrosequencing at postnatal days 30 (P30) and P90. Transcriptomic changes were analyzed by microarray profiling and real-time reverse transcription polymerase chain reaction. The mucosal microbiome was studied by high throughput pyrosequencing of 16S rRNA. Maternal MD supplementation induced a striking susceptibility to colitis in offspring. This phenotype was associated with colonic mucosal DNA methylation and expression changes. Metagenomic analyses did not reveal consistent bacteriomic differences between P30 and P90, but showed a prolonged effect of the diet on the offspring mucosal microbiome. In conclusion, maternal MD supplementation increases offspring colitis susceptibility that associates with persistent epigenetic and prolonged microbiomic changes. These findings underscore that epigenomic reprogramming relevant to mammalian colitis can occur during early development in response to maternal dietary modifications.

A maternal high-fat diet modulates fetal SIRT1 histone and protein deacetylase activity in nonhuman primates

In nonhuman primates, we previously demonstrated that a maternal high‐fat diet (MHFD) induces fetal nonalcoholic fatty liver disease (NAFLD) and alters the fetal metabolome. These changes are accompanied by altered acetylation of histone H3 (H3K14ac). However, the mechanism behind this alteration in acetylation remains unknown. As SIRT1 is both a lysine deacetylase and a crucial sensor of cellular metabolism, we hypothesized that SIRT1 may be involved in fetal epigenomic alterations. Here we show that in utero exposure to a MHFD, but not maternal obesity per se, increases fetal H3K14ac with concomitant decreased SIRT1 expression and diminished in vitro protein and histone deacetylase activity. MHFD increased H3K14ac and DBC1‐SIRT1 complex formation in fetal livers, both of which were abrogated with diet reversal despite persistent maternal obesity. Moreover, MHFD was associated with altered expression of known downstream effectors deregulated in NAFLD and modulated by SIRT1 (e.g., PPARA, PPARG, SREBF1, CYP7A1, FASN, and SCD). Finally, ex vivo purified SIRT1 retains deacetylase activity on an H3K14ac peptide substrate with preferential activity toward acetylated histone H3; mutagenesis of the catalytic domain of SIRT1 (H363Y) abrogates H3K14ac deacetylation. Our data implicate SIRT1 as a likely molecular mediator of the fetal epigenome and metabolome under MHFD conditions.—Suter, M. A., Chen, A., Burdine, M. S., Choudhury, M., Harris, R. A., Lane, R. H., Friedman, J. E., Grove, K. L., Tackett, A. J., Aagaard, K. M. A maternal high‐fat diet modulates fetal SIRT1 histone and protein deacetylase activity in nonhuman primates. FASEB J. 26, 5106–5114 (2012). www.fasebj.org

Transcriptome profiling of microRNA by Next-Gen deep sequencing reveals known and novel miRNA species in the lipid fraction of human breast milk.

While breast milk has unique health advantages for infants, the mechanisms by which it regulates the physiology of newborns are incompletely understood. miRNAs have been described as functioning transcellularly, and have been previously isolated in cell-free and exosomal form from bodily liquids (serum, saliva, urine) and tissues, including mammary tissue. We hypothesized that breast milk in general, and milk fat globules in particular, contain significant numbers of known and limited novel miRNA species detectable with massively parallel sequencing. Extracted RNA from lactating mothers before and following short-term treatment with recombinant human growth hormone (rhGH) was smRNA-enriched. smRNA-Seq was performed to generate 124,110,646 36-nt reads. Of these, 31,102,927 (25%) exactly matched known human miRNAs; with relaxing of stringency, 74,716,151 (60%) matched known miRNAs including 308 of the 1018 (29%) mature miRNAs (miRBase 16.0). These miRNAs are predicted to target 9074 genes; the 10 most abundant of these predicted to target 2691 genes with enrichment for transcriptional regulation of metabolic and immune responses. We identified 21 putative novel miRNAs, of which 12 were confirmed in a large validation set that included cohorts of lactating women consuming enriched diets. Of particular interest, we observed that expression of several novel miRNAs were altered by the perturbed maternal diet, notably following a high-fat intake (p<0.05). Our findings suggest that known and novel miRNAs are enriched in breast milk fat globules, and expression of several novel miRNA species is regulated by maternal diet. Based on robust pathway mapping, our data supports the notion that these maternally secreted miRNAs (stable in the milk fat globules) play a regulatory role in the infant and account in part for the health benefits of breast milk. We further speculate that regulation of these miRNA by a high fat maternal diet enables modulation of fetal metabolism to accommodate significant dietary challenges.

Colonic mucosal DNA methylation, immune response, and microbiome patterns in Toll-like receptor 2-knockout mice

The connection between intestinal micro‐biota and host physiology is increasingly becoming recognized. The details of this dynamic interaction, however, remain to be explored. Toll‐like receptor 2 (Tlr2) is important for its role in bacterial recognition, intestinal inflammation, and obesity‐related metabolic changes. Therefore, we sought to determine the epigenomic and metagenomic consequences of Tlr2 deficiency in the colonic mucosa of mice to gain insights into biological pathways that shape the interface between the gut micro‐biota and the mammalian host. Colonic mucosa from wild type (WT) and Tlr2−/− C57BL/6 mice was interrogated by microarrays specific for DNA methylation and gene expression. The mucosal microbiome was studied by next‐generation pyrosequencing of bacterial 16S rRNA The expression of genes involved in immune processes was significantly modified by the absence of Tlr2, a number of which correlated with DNA methylation changes. The epigenomic and transcriptomic modifications associated with alteration in mucosal microbial composition. Several bacterial species, including members of the Firmicutes were significantly different in abundance between WT and Tlr2−/− animals. This manuscript highlights the intimate interrelationships between expression of immune‐related genes and immunity pathways in the host with compositional and functional differences of the mammalian microbiome.—Kellermayer, R., Dowd, S. E., Harris, R. A., Balasa, A., Schaible, T. D., Wolcott, R. D., Tatevian, N., Szigeti, R., Li, Z., Versalovic, J., Smith, C. W. Colonic mucosal DNA methylation, immune response, and microbiome patterns in Toll‐like receptor 2‐knockout mice. FASEB J. 25, 1449–1460 (2011). www.fasebj.org