Andrew H. Reiner

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.

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

BackgroundZygotic 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.ResultsWe 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.ConclusionsOur 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.

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

The molecular background for the similarity of mesenchymal progenitor cells from various tissues is unknown. We characterize DNA methylation profiles of RefSeq promoters in relation to gene expression and differentiation in progenitors from adipose tissue, bone marrow, and muscle. Our data support the view of a common origin of mesenchymal progenitors.

Fast genomic μChIP-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 (μChIP) assay for 1,000 cells in combination with microarrays to produce genome-scale surveys of histone modifications. μChIP-chip reliably reproduces data obtained by large-scale assays: H3K9ac and H3K9m3 enrichment profiles are conserved and nucleosome-free regions are revealed.

Chromatin Environment of Histone Variant H3.3 Revealed by Quantitative Imaging and Genome-scale Chromatin and DNA Immunoprecipitation

Histone variant H3.3 is loaded onto chromatin in a replication-independent manner, but the epigenetic environment of H3.3 is unclear. Quantitative imaging and chromatin immunoprecipitation show that in mesenchymal stem cells H3.3 targets lineage-priming genes with a potential for activation facilitated by a permissive chromatin environment.

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.

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.

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.