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T(H)1-mediated diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA) improve during pregnancy, coinciding with increasing levels of the pregnancy hormone progesterone (P4), highlighting P4 as a potential mediator of this immunomodulation. Here, we performed detailed characterization of how P4 affects the chromatin and transcriptomic landscape during early human T(H)1 differentiation, utilizing both ATAC-seq and RNA-seq. Time series analysis of the earlier events (0.5-24 hrs) during T(H)1 differentiation revealed that P4 counteracted many of the changes induced during normal differentiation, mainly by downregulating key regulatory genes and their upstream transcription factors (TFs) involved in the initial T-cell activation. Members of the AP-1 complex such as FOSL1, FOSL2, JUN and JUNB were particularly affected, in both in promoters and in distal regulatory elements. Moreover, the changes induced by P4 were significantly enriched for disease-associated changes related to both MS and RA, revealing several shared upstream TFs, where again JUN was highlighted to be of central importance. Our findings support an immune regulatory role for P4 during pregnancy by impeding T-cell activation, a crucial checkpoint during pregnancy and in T-cell mediated diseases, and a central event prior to T-cell lineage commitment. Indeed, P4 is emerging as a likely candidate involved in disease modulation during pregnancy and further studies evaluating P4 as a potential treatment option are needed. Funding Agencies|Swedish Foundation for Strategic ResearchSwedish Foundation for Strategic Research [SB16-0011]

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is an aggres-sive malignancy resulting from overproduction of immature T-cells in the thymus and is typified by widespread alterations in DNA methyl-ation. As survival rates for relapsed T-ALL remain dismal (10 to 25%), development of targeted therapies to prevent relapse is key to improv-ing prognosis. Whereas mutations in the DNA demethylating enzyme TET2 are frequent in adult T-cell malignancies, TET2 mutations in T-ALL are rare. Here, we analyzed RNA-sequencing data of 321 primary T-ALLs, 20 T-ALL cell lines, and 25 normal human tissues, revealing that TET2 is transcriptionally repressed or silenced in 71% and 17% of T-ALL, respec-tively. Furthermore, we show that TET2 silencing is often associated with hypermethylation of the TET2 promoter in primary T-ALL. Impor-tantly, treatment with the DNA demethylating agent, 5-azacytidine (5-aza), was significantly more toxic to TET2-silenced T-ALL cells and resulted in stable re-expression of the TET2 gene. Additionally, 5-aza led to up-regulation of methylated genes and human endogenous ret-roviruses (HERVs), which was further enhanced by the addition of phys-iological levels of vitamin C, a potent enhancer of TET activity. Together, our results clearly identify 5-aza as a potential targeted therapy for TET2-silenced T-ALL.

An amendment to this paper has been published and can be accessed via the original article. Funding Agencies|Nordforsk; Swedish Cancer Foundation [17 0542, 15 0532]; European CommissionEuropean Commission Joint Research Centre [305033]; Swedish Research CouncilSwedish Research Council [2015-02575, 2015-03495, 2015-03807]; Clinical Cancer Research, Jonkoping, Sweden; Torsten Soderberg Foundation; East Gothia Regional Fund

N-6-methyladenine (6mdA) is a widespread DNA modification in bacteria. More recently, 6mdA has also been characterized in mammalian DNA. However, measurements of 6mdA abundance and profiles are often very dissimilar between studies, even when performed on DNA from identical mammalian cell types. Using comprehensive bioinformatics analyses of published data and novel experimental approaches, we reveal that efforts to assay 6mdA in mammals have been severely compromised by bacterial contamination, RNA contamination, technological limitations, and antibody nonspecificity. These complications render 6mdA an exceptionally problematic DNA modification to study and have resulted in erroneous detection of 6mdA in several mammalian systems. Together, our results strongly imply that the evidence published to date is not sufficient to support the presence of 6mdA in mammals. Funding Agencies|Swedish Research CouncilSwedish Research Council [2015-03495]; LiU-Cancer Network [2016-007]; Swedish Cancer SocietySwedish Cancer Society [CAN 2017/625]

Additional file 1: Supplementary Figure 1. DNA methylation quality control and pre-processing. (A) Density plot showing expected bi-modal distribution of DNA methylation values in all samples except one (dashed line) (top) and fraction of failed probe positions per sample based on detection P-values (P > 0.01) (bottom). (B) Plot showing the variability cutoff below which probes were excluded from linear regression studies of association (see methods). Supplementary Figure 2. No association between parental phenotype and infant DNA methylation levels. (A) Manhattan plot showing lack of association (FDRADJUSTED = 0.05) between infant DNA methylation levels and maternal homeostatic model assessment insulin resistance (HOMA-IR) score. (B) Manhattan plots showing lack of association (FDRADJUSTED = 0.05) between DNA methylation levels and infant body mass index, infant fat mass index and infant fat-free mass index. (C) Manhattan plots showing lack of association (FDRADJUSTED = 0.05) between DNA methylation levels and paternal body mass index, paternal fat mass index and paternal fat-free mass index.

Additional file 3: Figure S1. Promoter characteristics of aberrantly expressed genes in Trim28MommeD9/+ mice. Genome-wide expression analysis (Illumina MouseRef-8 v2.0 Expression BeadChip) was performed using RNA from the livers of 4-week-old male Trim28MommeD9/+ individuals (n = 4) and their wild-type littermates (n = 4). Promoter classification analysis was performed on genes classed as upregulated (n = 59) and downregulated (n = 225) by the GenomeStudio Gene Expression Module (Illumina). (a) Promoters were classified as low (LCP), intermediate (ICP) or high (HCP) CpG density. (b) Promoters were classified as having histone 3 lysine 4 trimethylation (K4), histone 3 lysine 27 trimethylation (K27), both marks (K4 + K27) or neither mark. (PDF 47 KB)

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Contains Tables S1–S4. (PDF 79 kb)

Models of 174 diseases based on epigenetic marker enrichments in GWAS genes (excluding rheumatoid arthritis presented in the main text). Compressed folder (ZIP), which can be found as DataS2.zip at: https://figshare.com/articles/DataS2_zip/7976552. (DOCX 14 kb)

Additional file 4: Figure S2. No correlation between body weight and open field activity. The body weights of 10 Trim28+/+ mice and 14 Trim28MommeD9/+ mice were plotted against their activity in an open field test (Squares). (PDF 5 KB)

of Reduced levels of two modifiers of epigenetic gene silencing, Dnmt3a and Trim28, cause increased phenotypic noise

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Additional file 1: Table S1. List of genes in the MommeD9 linked interval. (DOCX 16 KB)

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Random walk betweenness centrality of cell types for validation of MCDM in joint tissue. Contains supplementary table describing ligand-receptor based centrality analyses. (PDF 115 kb)

Contains Figures S1–S14. (PDF 2063 kb)

Additional file 2: Table S2. Gene expression analysis of Trim28MommeD9/+ mice. (DOCX 25 KB)

Recent progress in interpreting comprehensive genetic and epigenetic profiles for human cellular states has contributed new insights into the developmental origins of disease, elucidated novel signalling pathways and enhanced drug discovery programs. A similar comprehensive approach to decoding the epigenetic readouts from chemical challenges in vivo would yield new paradigms for monitoring and assessing environmental exposure in model systems and humans.

BackgroundThe DNA methylation profile of mammalian cell lines differs from the primary tissue from which they were derived, exhibiting increasing divergence from the in vivo methylation profile with extended time in culture. Few studies have directly examined the initial epigenetic and transcriptional consequences of adaptation of primary mammalian cells to culture, and the potential mechanisms through which this epigenetic dysregulation occurs is unknown.ResultsWe demonstrate that adaptation of mouse embryonic fibroblast, MEFS, to cell culture results in a rapid reprogramming of epigenetic and transcriptional states. We observed global 5-hydroxymethylcytosine (5hmC) erasure within three days of culture initiation. Loss of genic 5hmC was independent of global 5-methylcytosine (5mC) levels and could be partially rescued by addition of Vitamin C. Significantly, 5hmC loss was not linked to concomitant changes in transcription. Discrete promoter-specific gains of 5mC were also observed within seven days of culture initiation. Against this background of global 5hmC loss we identified a handful of developmentally important genes that maintained their 5hmC profile in culture, including the imprinted loci Gnas and H19. Similar outcomes were identified in the adaption of CD4+ T-cells to culture.ConclusionsWe report a dramatic and novel consequence of adaptation of mammalian cells to culture in which global loss of 5hmC occurs; suggesting rapid concomitant loss of methylcytosine dioxygenase activity. The observed epigenetic and transcriptional re-programming occurs much earlier than previously assumed, and has significant implications for the use of cell lines as faithful mimics of in vivo epigenetic and physiological processes. We thank Professors Adrian Bird and Nicholas Hastie for their comments on our manuscript. JT and RO are funded by IMI-MARCAR (under grant agreement number (115001) (MARCAR project)). Work in RM's lab is supported by the MRC, IMI-MARCAR and the BBSRC. This work in RM's lab was also initially funded by the Breakthrough Breast Cancer charity. Work in MB's lab was supported by Linkoping University strategic research funding and the Ake Wibergs fund (3772738). Work in SP's lab is supported by the BBSRC.

The dynamic expansion of CAG.CTG repeats in otherwise unrelated genes is responsible for a growing number of late-onset progressive disorders, including Huntington disease, myotonic dystrophy type 1 (DM1) and the spinocerebellar ataxias. As toxicity increases with repeat length, the intergenerational expansion of unstable CAG.CTG repeats leads to anticipation, an earlier age-at-onset in successive generations in these disorders. Crucially, disease associated alleles are also somatically unstable and continue to expand throughout the lifetime of the individual. In addition, evidence suggests that c/s-acting elements may be major modifiers of instability. Here it was found that the toxicity of expanded polyQ-encoding CAG.CTG tracts correlates with both the expandability of the underlying CAG.CTG repeat and the GC content of the genomic DNA flanking sequences. PolyQ toxicity does not correlate with properties of mRNA or protein sequences, or with polyQ location within the gene or protein. These data thus strongly suggest that the observed inter-locus differences in polyQ. toxicity are not mediated by protein context effects, but that the rate at which somatic expansion of the DNA delivers proteins to their cytotoxic state is a critical factor in expanded polyQ-disease age-at-onset. Using human and mouse cell lines transgenic for an expanded human DM1 locus, it was found that an expanded CTGhz repeat alone is not sufficient for instability. Moreover, by generating mouse cell lines stably transfected with both a stable and unstable expanded CTG[142] repeat, it was possible to assay the effect of cis-elements on these two loci in the same cell line over time. The sequences flanking the unstable repeat were hypermethylated, whereas the sequences flanking the stable transgenic repeat were unmethylated, suggesting an association between CpG methylation and repeat instability. However, methylation of the stable transgenic repeat failed to induce instability. In addition, it was revealed that transcription of an expanded repeat was not sufficient to induce instability. Analysis of genome-wide CAG.CTG microsatellite instability revealed a significant correlation between flanking sequence GC content and microsatellite mutability. This association was most significant for short (< 7 repeats) microsatellites and for those microsatellites located within exons. However, comparison of microsatellite lengths in the human and chimpanzee genomes revealed a complex association between flanking GC content and misalignment mutations at microsatellite loci, suggesting that the modifying effect of flanking GC content on expanded repeat instability may be specific to the expanded repeat disease loci In conclusion, this work suggests that the rate of somatic repeat expansion is a major modifier of disease progression, and that cis-acting elements in turn, modify repeat instability.