PML modulates H3.3 targeting to telomeric and centromeric repeats in mouse fibroblasts.

Targeted deposition of histone variant H3.3 into chromatin is paramount for proper regulation of chromatin integrity, particularly in heterochromatic regions including repeats. We have recently shown that the promyelocytic leukemia (PML) protein prevents H3.3 from being deposited in large heterochromatic PML-associated domains (PADs). However, to what extent PML modulates H3.3 loading on chromatin in other areas of the genome remains unexplored. Here, we examined the impact of PML on targeting of H3.3 to genes and repeat regions that reside outside PADs. We show that loss of PML increases H3.3 deposition in subtelomeric, telomeric, pericentric and centromeric repeats in mouse embryonic fibroblasts, while other repeat classes are not affected. Expression of major satellite, minor satellite and telomeric non-coding transcripts is altered in Pml-null cells. In particular, telomeric Terra transcripts are strongly upregulated, in concordance with a marked reduction in H4K20me3 at these sites. Lastly, for most genes H3.3 enrichment or gene expression outcomes are independent of PML. Our data argue towards the importance of a PML-H3.3 axis in preserving a heterochromatin state at centromeres and telomeres.

The specific alteration of histone methylation profiles by DZNep during early zebrafish development.

Early embryo development constitutes a unique opportunity to study acquisition of epigenetic marks, including histone methylation. This study investigates the in vivo function and specificity of 3-deazaneplanocin A (DZNep), a promising anti-cancer drug that targets polycomb complex genes. One- to two-cell stage embryos were cultured with DZNep, and subsequently evaluated at the post-mid blastula transition stage for H3K27me3, H3K4me3 and H3K9me3 occupancy and enrichment at promoters using ChIP-chip microarrays. DZNep affected promoter enrichment of H3K27me3 and H3K9me3, whereas H3K4me3 remained stable. Interestingly, DZNep induced a loss of H3K27me3 and H3K9me3 from a substantial number of promoters but did not prevent de novo acquisition of these marks on others, indicating gene-specific targeting of its action. Loss/gain of H3K27me3 on promoters did not result in changes in gene expression levels until 24h post-fertilization. In contrast, genes gaining H3K9me3 displayed strong and constant down-regulation upon DZNep treatment. H3K9me3 enrichment on these gene promoters was observed not only in the proximal area as expected, but also over the transcription start site. Altered H3K9me3 profiles were associated with severe neuronal and cranial phenotypes at day 4-5 post-fertilization. Thus, DZNep was shown to affect enrichment patterns of H3K27me3 and H3K9me3 at promoters in a gene-specific manner.

Epigenetic priming of inflammatory response genes by high glucose in adipose progenitor cells.

Cellular metabolism confers wide-spread epigenetic modifications required for regulation of transcriptional networks that determine cellular states. Mesenchymal stromal cells are responsive to metabolic cues including circulating glucose levels and modulate inflammatory responses. We show here that long term exposure of undifferentiated human adipose tissue stromal cells (ASCs) to high glucose upregulates a subset of inflammation response (IR) genes and alters their promoter histone methylation patterns in a manner consistent with transcriptional de-repression. Modeling of chromatin states from combinations of histone modifications in nearly 500 IR genes unveil three overarching chromatin configurations reflecting repressive, active, and potentially active states in promoter and enhancer elements. Accordingly, we show that adipogenic differentiation in high glucose predominantly upregulates IR genes. Our results indicate that elevated extracellular glucose levels sensitize in ASCs an IR gene expression program which is exacerbated during adipocyte differentiation. We propose that high glucose exposure conveys an epigenetic 'priming' of IR genes, favoring a transcriptional inflammatory response upon adipogenic stimulation. Chromatin alterations at IR genes by high glucose exposure may play a role in the etiology of metabolic diseases.

Epigenetic complexity during the zebrafish mid-blastula transition.

The zebrafish developmental transcription program is determined by temporal post-translational histone modifications established in a step-wise and combinatorial manner on specific promoters around the time of zygotic genome activation (ZGA). Here, we characterize this increasing epigenetic complexity before, during and after ZGA. H3K4me3/H3K27me3 co-enrichment prevails over H3K4me3/H3K9me3 at the time of ZGA. Whereas most H3K4me3-marked promoters are devoid of transcriptionally repressive H3K9me3 or H3K27me3, the latter marks rarely occur in absence of H3K4me3. On co-enriched genomic regions, H3K4me3 and H3K27me3 can overlap regardless of H3K9me3 enrichment, but H3K4me3 and H3K9me3 are mutually exclusive. H3K4me3 and H3K9me3 may however overlap only when H3K27me3 also marks the overlapping domain, suggesting that H3K27me3 may modulate chromatin states. On metagenes, H3K27me3 enrichment correlates with local alteration in H3K4me3 density, and co-enrichment in H3K9me3 is linked to alterations in both H3K27me3 and H3K4me3 profiles. This suggests physical proximity of these marks and supports a view of existence of bi- or tri-valent chromatin domains. Thus enrichment in trimethylated H3K9 or H3K27 is associated with local remodeling of chromatin manifested by changes in H3K4me3 density. We propose that metagenes can provide information on the multivalency of chromatin sates.

Regional performance variation in external validation of four prediction models for severity of COVID-19 at hospital admission: An observational multi-centre cohort study.

BACKGROUND: Prediction models should be externally validated to assess their performance before implementation. Several prediction models for coronavirus disease-19 (COVID-19) have been published. This observational cohort study aimed to validate published models of severity for hospitalized patients with COVID-19 using clinical and laboratory predictors. METHODS: Prediction models fitting relevant inclusion criteria were chosen for validation. The outcome was either mortality or a composite outcome of mortality and ICU admission (severe disease). 1295 patients admitted with symptoms of COVID-19 at Kings Cross Hospital (KCH) in London, United Kingdom, and 307 patients at Oslo University Hospital (OUH) in Oslo, Norway were included. The performance of the models was assessed in terms of discrimination and calibration. RESULTS: We identified two models for prediction of mortality (referred to as Xie and Zhang1) and two models for prediction of severe disease (Allenbach and Zhang2). The performance of the models was variable. For prediction of mortality Xie had good discrimination at OUH with an area under the receiver-operating characteristic (AUROC) 0.87 [95% confidence interval (CI) 0.79-0.95] and acceptable discrimination at KCH, AUROC 0.79 [0.76-0.82]. In prediction of severe disease, Allenbach had acceptable discrimination (OUH AUROC 0.81 [0.74-0.88] and KCH AUROC 0.72 [0.68-0.75]). The Zhang models had moderate to poor discrimination. Initial calibration was poor for all models but improved with recalibration. CONCLUSIONS: The performance of the four prediction models was variable. The Xie model had the best discrimination for mortality, while the Allenbach model had acceptable results for prediction of severe disease.

A Bayesian mixed modeling approach for estimating heritability.

BACKGROUND: A Bayesian mixed model approach using integrated nested Laplace approximations (INLA) allows us to construct flexible models that can account for pedigree structure. Using these models, we estimate genome-wide patterns of DNA methylation heritability (h 2 ), which are currently not well understood, as well as h 2 of blood lipid measurements. METHODS: We included individuals from the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study with Infinium 450 K cytosine-phosphate-guanine (CpG) methylation and blood lipid data pre- and posttreatment with fenofibrate in families with up to three-generation pedigrees. For genome-wide patterns, we constructed 1 model per CpG with methylation as the response variable, with a random effect to model kinship, and age and gender as fixed effects. RESULTS: In total, 425,791 CpG sites pre-, but only 199,027 CpG sites posttreatment were found to have nonzero heritability. Across these CpG sites, the distributions of h 2 estimates are similar in pre- and posttreatment (pre: median = 0.31, interquartile range [IQR] = 0.16; post: median = 0.34, IQR = 0.20). Blood lipid h 2 estimates were similar pre- and posttreatment with overlapping 95% credibility intervals. Heritability was nonzero for treatment effect, that is, the difference between pre- and posttreatment blood lipids. Estimates for triglycerides h 2 are 0.48 (pre), 0.42 (post), and 0.21 (difference); likewise for high-density lipoprotein cholesterol h 2 the estimates are 0.61, 0.68, and 0.10. CONCLUSIONS: We show that with INLA, a fully Bayesian approach to estimate DNA methylation h 2 is possible on a genome-wide scale. This provides uncertainty assessment of the estimates, and allows us to perform model selection via deviance information criterion (DIC) to identify CpGs with strong evidence for nonzero heritability.

Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells.

BACKGROUND & AIMS: Liver fibrogenesis - scarring of the liver that can lead to cirrhosis and liver cancer - is characterized by hepatocyte impairment, capillarization of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cell (HSC) activation. To date, the molecular determinants of a healthy human liver cell phenotype remain largely uncharacterized. Here, we assess the transcriptome and the genome-wide promoter methylome specific for purified, non-cultured human hepatocytes, LSECs and HSCs, and investigate the nature of epigenetic changes accompanying transcriptional changes associated with activation of HSCs. MATERIAL AND METHODS: Gene expression profile and promoter methylome of purified, uncultured human liver cells and culture-activated HSCs were respectively determined using Affymetrix HG-U219 genechips and by methylated DNA immunoprecipitation coupled to promoter array hybridization. Histone modification patterns were assessed at the single-gene level by chromatin immunoprecipitation and quantitative PCR. RESULTS: We unveil a DNA-methylation-based epigenetic relationship between hepatocytes, LSECs and HSCs despite their distinct ontogeny. We show that liver cell type-specific DNA methylation targets early developmental and differentiation-associated functions. Integrative analysis of promoter methylome and transcriptome reveals partial concordance between DNA methylation and transcriptional changes associated with human HSC activation. Further, we identify concordant histone methylation and acetylation changes in the promoter and putative novel enhancer elements of genes involved in liver fibrosis. CONCLUSIONS: Our study provides the first epigenetic blueprint of three distinct freshly isolated, human hepatic cell types and of epigenetic changes elicited upon HSC activation.

Epigenetic marking of the zebrafish developmental program.

A characteristic of anamniote development is a relatively long period of embryonic cell divisions in the absence of on-going transcription. In zebrafish, this period lasts for 10 cell cycles, or ∼3-h postfertilization, after which zygotic genome activation (ZGA) takes place during the midblastula transition. How the embryo establishes transcriptional competence and how ZGA is spatially and temporally regulated have not been examined until recently. We review here recent data on the transitions in DNA methylation and posttranslational histone modifications occurring during early zebrafish development, as the embryo acquires transcriptional competence and initiates its own gene expression program. We also address models accounting for the origin of epigenetic states detected in early embryos. From these observations, a concept of epigenetic prepatterning of the embryonic gene expression program prior to the onset of ZGA is emerging. The recent data collectively start shedding light on how ZGA may be programmed and regulated.

Developmental features of DNA methylation during activation of the embryonic zebrafish genome.

BACKGROUND: Zygotic genome activation (ZGA) occurs at the mid-blastula transition (MBT) in zebrafish and is a period of extensive chromatin remodeling. Genome-scale gametic demethylation and remethylation occurs after fertilization, during blastula stages, but how ZGA relates to promoter DNA methylation states is unknown. Using methylated DNA immunoprecipitation coupled to high-density microarray hybridization, we characterize genome-wide promoter DNA methylation dynamics before, during and after ZGA onset, in relation to changes in post-translational histone modifications and gene expression. RESULTS: We show methylation of thousands of promoters before ZGA and additional methylation after ZGA, finding more dynamic methylation -1 to 0 kb upstream of the transcription start site than downstream. The MBT is marked by differential methylation of high and low CpG promoters, and we identify hypomethylated promoters that are mostly CG-rich and remain hypomethylated through the MBT. Hypomethylated regions constitute a platform for H3K4me3, whereas H3K9me3 preferentially associates with methylated regions. H3K27me3 associates with either methylation state depending on its coincidence with H3K4me3 or H3K9me3. Cohorts of genes differentially expressed through the MBT period display distinct promoter methylation patterns related to CG content rather than transcriptional fate. Lastly, although a significant proportion of genes methylated in sperm are unmethylated in embryos, over 90% of genes methylated in embryos are also methylated in sperm. CONCLUSIONS: Our results suggest a pre-patterning of developmental gene expression potential by a combination of DNA hypomethylation and H3K4 trimethylation on CG-rich promoters, and are consistent with a transmission of DNA methylation states from gametes to early embryos.

Prepatterning of developmental gene expression by modified histones before zygotic genome activation.

A hallmark of anamniote vertebrate development is a window of embryonic transcription-independent cell divisions before onset of zygotic genome activation (ZGA). Chromatin determinants of ZGA are unexplored; however, marking of developmental genes by modified histones in sperm suggests a predictive role of histone marks for ZGA. In zebrafish, pre-ZGA development for ten cell cycles provides an opportunity to examine whether genomic enrichment in modified histones is present before initiation of transcription. By profiling histone H3 trimethylation on all zebrafish promoters before and after ZGA, we demonstrate here an epigenetic prepatterning of developmental gene expression. This involves pre-ZGA marking of transcriptionally inactive genes involved in homeostatic and developmental regulation by permissive H3K4me3 with or without repressive H3K9me3 or H3K27me3. Our data suggest that histone modifications are instructive for the developmental gene expression program.

Tiling histone H3 lysine 4 and 27 methylation in zebrafish using high-density microarrays.

BACKGROUND: Uncovering epigenetic states by chromatin immunoprecipitation and microarray hybridization (ChIP-chip) has significantly contributed to the understanding of gene regulation at the genome-scale level. Many studies have been carried out in mice and humans; however limited high-resolution information exists to date for non-mammalian vertebrate species. PRINCIPAL FINDINGS: We report a 2.1-million feature high-resolution Nimblegen tiling microarray for ChIP-chip interrogations of epigenetic states in zebrafish (Danio rerio). The array covers 251 megabases of the genome at 92 base-pair resolution. It includes ∼15 kb of upstream regulatory sequences encompassing all RefSeq promoters, and over 5 kb in the 5' end of coding regions. We identify with high reproducibility, in a fibroblast cell line, promoters enriched in H3K4me3, H3K27me3 or co-enriched in both modifications. ChIP-qPCR and sequential ChIP experiments validate the ChIP-chip data and support the co-enrichment of trimethylated H3K4 and H3K27 on a subset of genes. H3K4me3- and/or H3K27me3-enriched genes are associated with distinct transcriptional status and are linked to distinct functional categories. CONCLUSIONS: We have designed and validated for the scientific community a comprehensive high-resolution tiling microarray for investigations of epigenetic states in zebrafish, a widely used developmental and disease model organism.

Histone H3 lysine 27 methylation asymmetry on developmentally-regulated promoters distinguish the first two lineages in mouse preimplantation embryos.

First lineage specification in the mammalian embryo leads to formation of the inner cell mass (ICM) and trophectoderm (TE), which respectively give rise to embryonic and extraembryonic tissues. We show here that this first differentiation event is accompanied by asymmetric distribution of trimethylated histone H3 lysine 27 (H3K27me3) on promoters of signaling and developmentally-regulated genes in the mouse ICM and TE. A genome-wide survey of promoter occupancy by H3K4me3 and H3K27me3 indicates that both compartments harbor promoters enriched in either modification, and promoters co-enriched in trimethylated H3K4 and H3K27 linked to developmental and signaling functions. The majority of H3K4/K27me3 co-enriched promoters are distinct between the two lineages, primarily due to differences in the distribution of H3K27me3. Derivation of embryonic stem cells leads to significant losses and gains of H3K4/K27me3 co-enriched promoters relative to the ICM, with distinct contributions of (de)methylation events on K4 and K27. Our results show histone trimethylation asymmetry on promoters in the first two developmental lineages, and highlight an epigenetic skewing associated with embryonic stem cell derivation.

Promoter DNA methylation patterns of differentiated cells are largely programmed at the progenitor stage.

Mesenchymal stem cells (MSCs) isolated from various tissues share common phenotypic and functional properties. However, intrinsic molecular evidence supporting these observations has been lacking. Here, we unravel overlapping genome-wide promoter DNA methylation patterns between MSCs from adipose tissue, bone marrow, and skeletal muscle, whereas hematopoietic progenitors are more epigenetically distant from MSCs as a whole. Commonly hypermethylated genes are enriched in signaling, metabolic, and developmental functions, whereas genes hypermethylated only in MSCs are associated with early development functions. We find that most lineage-specification promoters are DNA hypomethylated and harbor a combination of trimethylated H3K4 and H3K27, whereas early developmental genes are DNA hypermethylated with or without H3K27 methylation. Promoter DNA methylation patterns of differentiated cells are largely established at the progenitor stage; yet, differentiation segregates a minor fraction of the commonly hypermethylated promoters, generating greater epigenetic divergence between differentiated cell types than between their undifferentiated counterparts. We also show an effect of promoter CpG content on methylation dynamics upon differentiation and distinct methylation profiles on transcriptionally active and inactive promoters. We infer that methylation state of lineage-specific promoters in MSCs is not a primary determinant of differentiation capacity. Our results support the view of a common origin of mesenchymal progenitors.

Chromatin environment of histone variant H3.3 revealed by quantitative imaging and genome-scale chromatin and DNA immunoprecipitation.

In contrast to canonical histones, histone variant H3.3 is incorporated into chromatin in a replication-independent manner. Posttranslational modifications of H3.3 have been identified; however, the epigenetic environment of incorporated H3.3 is unclear. We have investigated the genomic distribution of epitope-tagged H3.3 in relation to histone modifications, DNA methylation, and transcription in mesenchymal stem cells. Quantitative imaging at the nucleus level shows that H3.3, relative to replicative H3.2 or canonical H2B, is enriched in chromatin domains marked by histone modifications of active or potentially active genes. Chromatin immunoprecipitation of epitope-tagged H3.3 and array hybridization identified 1649 H3.3-enriched promoters, a fraction of which is coenriched in H3K4me3 alone or together with H3K27me3, whereas H3K9me3 is excluded, corroborating nucleus-level imaging data. H3.3-enriched promoters are predominantly CpG-rich and preferentially DNA methylated, relative to the proportion of methylated RefSeq promoters in the genome. Most but not all H3.3-enriched promoters are transcriptionally active, and coenrichment of H3.3 with repressive H3K27me3 correlates with an enhanced proportion of expressed genes carrying this mark. H3.3-target genes are enriched in mesodermal differentiation and signaling functions. Our data suggest that in mesenchymal stem cells, H3.3 targets lineage-priming genes with a potential for activation facilitated by H3K4me3 in facultative association with H3K27me3.

Fast genomic muChIP-chip from 1,000 cells.

Genome-wide location analysis of histone modifications and transcription factor binding relies on chromatin immunoprecipitation (ChIP) assays. These assays are, however, time-consuming and require large numbers of cells, hindering their application to the analysis of many interesting cell types. We report here a fast microChIP (muChIP) assay for 1,000 cells in combination with microarrays to produce genome-scale surveys of histone modifications. muChIP-chip reliably reproduces data obtained by large-scale assays: H3K9ac and H3K9m3 enrichment profiles are conserved and nucleosome-free regions are revealed.