DNA replication timing of the human β-globin domain is controlled by histone modification at the origin

The human β-globin genes constitute a large chromosomal domain that is developmentally regulated. In nonerythroid cells, these genes replicate late in S phase, while in erythroid cells, replication is early. The replication origin is packaged with acetylated histones in erythroid cells, yet is associated with deacetylated histones in nonerythroid cells. Recruitment of histone acetylases to this origin brings about a transcription-independent shift to early replication in lymphocytes. In contrast, tethering of a histone deacetylase in erythroblasts causes a shift to late replication. These results suggest that histone modification at the origin serves as a binary switch for controlling replication timing

Islet cells share promoter hypomethylation independently of expression, but exhibit cell-type-specific methylation in enhancers

DNA methylation at promoters is an important determinant of gene expression. Earlier studies suggested that the insulin gene promoter is uniquely unmethylated in insulin-expressing pancreatic β-cells, providing a classic example of this paradigm. Here we show that islet cells expressing insulin, glucagon, or somatostatin share a lack of methylation at the promoters of the insulin and glucagon genes. This is achieved by rapid demethylation of the insulin and glucagon gene promoters during differentiation of Neurogenin3+ embryonic endocrine progenitors, regardless of the specific endocrine cell-type chosen. Similar methylation dynamics were observed in transgenic mice containing a human insulin promoter fragment, pointing to the responsible cis element. Whole-methylome comparison of human α- and β-cells revealed generality of the findings: genes active in one cell type and silent in the other tend to share demethylated promoters, while methylation differences between α- and β-cells are concentrated in enhancers. These findings suggest an epigenetic basis for the observed plastic identity of islet cell types, and have implications for β-cell reprogramming in diabetes and diagnosis of β-cell death using methylation patterns of circulating DNA

IFNG-AS1 Enhances Interferon Gamma Production in Human Natural Killer Cells

Summary: Long, non-coding RNAs (lncRNAs) are involved in the regulation of many cellular processes. The lncRNA IFNG-AS1 was found to strongly influence the responses to several pathogens in mice by increasing interferon gamma (IFNγ) secretion. Studies have looked at IFNG-AS1 in T cells, yet IFNG-AS1 function in natural killer cells (NKs), an important source of IFNγ, remains unknown. Here, we show a previously undescribed sequence of IFNG-AS1 and report that it may be more abundant in cells than previously thought. Using primary human NKs and an NK line with IFNG-AS1 overexpression, we show that IFNG-AS1 is quickly induced upon NK cell activation, and that IFNG-AS1 overexpression leads to increased IFNγ secretion. Taken together, our work expands IFNG-AS1's activity to the innate arm of the type I immune response, helping to explain its notable effect in animal models of disease. : Molecular Biology; Molecular Mechanism of Gene Regulation; Immunology; Immune Response Subject Areas: Molecular Biology, Molecular Mechanism of Gene Regulation, Immunology, Immune Respons

Aberrant DNA Methylation in ES Cells

<div><p>Both mouse and human embryonic stem cells can be differentiated in vitro to produce a variety of somatic cell types. Using a new developmental tracing approach, we show that these cells are subject to massive aberrant CpG island de novo methylation that is exacerbated by differentiation in vitro. Bioinformatics analysis indicates that there are two distinct forms of abnormal de novo methylation, global as opposed to targeted, and in each case the resulting pattern is determined by molecular rules correlated with local pre-existing histone modification profiles. Since much of the abnormal methylation generated in vitro appears to be stably maintained, this modification may inhibit normal differentiation and could predispose to cancer if cells are used for replacement therapy. Excess CpG island methylation is also observed in normal placenta, suggesting that this process may be governed by an inherent program.</p></div

Resetting de novo methylation in vivo.

<p>Blastocysts injected with ES cells carrying a GFP expression vector were transplanted into pseudo-pregnant mice. Whole embryos were isolated at 16 dpc and sorted for GFP⁺ and GFP⁻ cells. DNA from these cells was then treated with bisulfite and deep-sequenced (Ion Torrent) at four different specific CpG island sequences. <b>a</b>. 7,000 individual molecules of island A containing nine individual CpGs with yellow indicating methylation. <b>b</b>. Graph showing percent methylation for islands A, B, C and D.</p

De novo methylation is proportional to H3K27me3 concentration.

<p><b>a</b>. IMS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096090#pone-0096090-g001" target="_blank">Fig. 1</a>) of all 9,500 constitutively unmethylated CpG islands in undifferentiated, endoderm-differentiated ES cells and adult tissue DNA graphed as a function of H3K27me3 density <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096090#pone.0096090-Mikkelsen1" target="_blank">[22]</a>. Each point represents the average IMS within a 5 unit span. <b>b</b>. Average methylation levels of all constitutively unmethylated CpG islands in endoderm (IMS), NPCs (%) and adult tissue (%) were graphed against H3K27me3 density partitioned into ten bins. The X-axis also shows average H3K27me3 levels in each percentile. <b>c</b>. Methylation levels in adult tissue, ES differentiated into endoderm and NPCs of all constitutively unmethylated CpG islands with above background concentrations (>2) of H3K27me3 graphed against their H3K4me3 density in ES.</p

Excess methylation in human ES cells.

<p>DNA from normal human fetal tissues, undifferentiated and in vitro differentiated ES cells were subject to mDIP microarray analysis. Heat map shows 950(IMS>0.75) in at least one of the differentiated ES cell types out of 13,000 CpG islands constitutively unmethylated (IMS<0) in fetal tissue samples. Retinoic acid treated cells and embryoid bodies were derived from CSES2. An estimate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096090#pone.0096090-Straussman1" target="_blank">[10]</a> for the average percent methylation in fetal tissues as compared to in vitro differentiated cells is also shown.</p