Project description:A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur prior to any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Illumina Expresson BeadChip arrays (MouseRef-8v2.0) were used to assess gene expression changes in neural stem cells (n=5; derived from mouse embryonic stem cells) and their differentiated neuronal progeny (n=3; FACS-purified based on Tau-RedStar expression).

Project description:Epigenetic priming factors establish a permissive epigenetic landscape which is not required until a later developmental or physiological time point, temporally uncoupling the presence of these factors with their phenotypic effects. One classic example of epigenetic priming is in the context of bivalent chromatin, found in pluripotent stem cells and early embryos at key developmental gene promoters marked by both activating-associated H3K4me3 and repressive-associated H3K27me3 histone modifications. It is currently unknown how these bivalent domains are targeted, or precisely how they impact on lineage commitment. Here we show that the small heterodimerising non-enzymatic DNA binding proteins Developmental Pluripotency Associated 2 (Dppa2) and 4 (Dppa4) act as epigenetic priming factors to establish bivalency at a subset of developmental genes. Dppa2/4 localise to the +1 nucleosome position of bivalent genes and while they are not required for pluripotency in embryonic stem cells (ESCs), double knockout cells fail to exit pluripotency and to differentiate efficiently, with delays in upregulating bivalently marked lineage genes. Proteomics reveal that Dppa2/4 interact on chromatin with members of the COMPASS and Polycomb complexes important for H3K4me3 and H3K27me3 deposition, respectively. Epigenetic profiling reveals a striking loss of H3K4me3, H3K27me3, and their associated enzymatic machinery at a significant subset of bivalent promoters in Dppa2/4 mutants, in addition to loss of H2A.Z and chromatin accessibility. In wild-type ESCs, these “Dppa2/4-dependent” bivalent promoters are characterised by low H3K4me3 enrichment and breadth, near-absent expression levels and initiating but not elongating RNA polymerase. Notably, Dppa2/4-dependent promoters are less evolutionarily conserved suggesting that they lack additional safeguard measures to maintain bivalency at these genes in the absence of Dppa2/4. Concomitantly with the loss of bivalency, Dppa2/4-dependent bivalent promoters gain DNA methylation and consequently are no longer able to be effectively activated upon ESC differentiation, leading to defects in cell fate acquisition. Our findings reveal a targeting principle for bivalency to developmental gene promoters poising them for future lineage specific gene activation.

Project description:BACKGROUND: The polycomb group protein Ezh2 is an epigenetic repressor of transcription originally found to prevent untimely differentiation of pluripotent embryonic stem cells. We previously demonstrated that Ezh2 is also expressed in multipotent neural stem cells (NSCs). We showed that Ezh2 expression is downregulated during NSC differentiation into astrocytes or neurons. However, high levels of Ezh2 remained present in differentiating oligodendrocytes until myelinating. This study aimed to elucidate the target genes of Ezh2 in NSCs and in premyelinating oligodendrocytes (pOLs). METHODOLOGY/PRINCIPAL FINDINGS: We performed chromatin immunoprecipitation followed by high-throughput sequencing to detect the target genes of Ezh2 in NSCs and pOLs. We found 1532 target genes of Ezh2 in NSCs. During NSC differentiation, the occupancy of these genes by Ezh2 was alleviated. However, when the NSCs differentiated into oligodendrocytes, 393 of these genes remained targets of Ezh2. Analysis of the target genes indicated that the repressive activity of Ezh2 in NSCs concerns genes involved in stem cell maintenance, in cell cycle control and in preventing neural differentiation. Among the genes in pOLs that were still repressed by Ezh2 were most prominently those associated with neuronal and astrocytic committed cell lineages. Suppression of Ezh2 activity in NSCs caused loss of stem cell characteristics, blocked their proliferation and ultimately induced apoptosis. Suppression of Ezh2 activity in pOLs resulted in derangement of the oligodendrocytic phenotype, due to re-expression of neuronal and astrocytic genes, and ultimately in apoptosis. CONCLUSIONS/SIGNIFICANCE: Our data indicate that the epigenetic repressor Ezh2 in NSCs is crucial for proliferative activity and maintenance of neural stemness. During differentiation towards oligodendrocytes, Ezh2 repression continues particularly to suppress other neural fate choices. Ezh2 is completely downregulated during differentiation towards neurons and astrocytes allowing transcription of these differentiation programs. The specific fate choice towards astrocytes or neurons is apparently controlled by epigenetic regulators other than Ezh2. Examination of Ezh2 target sites in 2 different primary cells types

Project description:Purpose: The aim of this study is (1) to identify the chromatin occupancy of the epigenetic regulator Smchd1 in neural stem cells (NSCs) derived from E14.5 mouse brain; (2) to profile key epigenetic marks H3K4me3, H3K27me3 and DNA methylation in wild type and Smchd1 null NSCs; (3) to identify the chromatin occupancy of Ctcf in wild type and Smchd1 null NSCs. Methods: Chromatin immunoprecipitation for Smchd1, H3K4me3, H3K27me3 and Ctcf was performed essentially as in (Nelson et al. 2006). Briefly, nuclei were isolated from formaldehyde crosslinked NSCs and chromatin was fragmented by sonication. Chromatin immunoprecipitation was performed with corresponding antibodies for Smchd1, H3K4me3 and H3K27me3. DNA was extracted from the immunoprecipitated fraction following reverse-crosslinking. Isolated DNA was used to generate sequencing libraries with Illumina's TruSeq DNA Sample Preparation Kit or Ovation Ultralow system (NuGen) according to manufacturer's instruction. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. To examine the level of DNA methylation, genomic DNA was extracted using an AllPrep DNA/RNA Mini Kit (Qiagen) and methylated DNA was isolated via binding to the methyl-CpG binding domain of human MBD2 protein coupled beads using the MethylMiner methylated DNA enrichment kit (Life Technologies) according to the manufacturer’s instructions. Isolated DNA was used to generate sequencing libraries as for the ChIP-seq experiment with Illumina’s TruSeq DNA Sample Preparation Kit according to manufacturer's instruction and sequenced on the Illumina HiSeq 2000 platform for 49 bp single-end reads. Sequencing analysis was performed as described for the ChIP-seq experiments. Chromatin occupancy of the epigenetic regulator Smchd1 in neural stem cells (NSCs) derived from E14.5 mouse brain was determined by Smchd1 ChIP-seq. Enrichment of H3K4me3 and H3K27me3 in wild type and Smchd1 null NSCs were assessed by H3K4me3 and H3K27me3 ChIP-seq, respectively. DNA methylation in wild type and Smchd1 null NSCs was assessed by MBD-seq. Chromatin occupancy of Ctcf in wild type and Smchd1 null NSCs was determined by by Ctcf ChIP-seq.

Dataset Information

An epigenetic signature of developmental potential in neural stem cells and early neurons [ChIP-seq]

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