Rbpj expression in regulatory T cells is critical for restraining T2 responses.

The transcriptional regulator Rbpj is involved in T-helper (T) subset polarization, but its function in T cells remains unclear. Here we show that T-specific Rbpj deletion leads to splenomegaly and lymphadenopathy despite increased numbers of T cells with a polyclonal TCR repertoire. A specific defect of Rbpj-deficient T cells in controlling T2 polarization and B cell responses is observed, leading to the spontaneous formation of germinal centers and a T2-associated immunoglobulin class switch.

DNA (de)methylation in embryonic stem cells controls CTCF-dependent chromatin boundaries.

Coordinated changes of DNA (de)methylation, nucleosome positioning, and chromatin binding of the architectural protein CTCF play an important role for establishing cell-type-specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes, we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the and dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC), or carboxylated (5caC) forms.

Corrigendum: Genome-wide DNA-methylation landscape defines specialization of regulatory T cells in tissues.

This corrects the article DOI: 10.1038/ni.3799.

Genome-wide DNA-methylation landscape defines specialization of regulatory T cells in tissues.

Regulatory T cells (T cells) perform two distinct functions: they maintain self-tolerance, and they support organ homeostasis by differentiating into specialized tissue T cells. We found that epigenetic modifications defined the molecular characteristics of tissue T cells. Tagmentation-based whole-genome bisulfite sequencing revealed more than 11,000 regions that were methylated differentially in pairwise comparisons of tissue T cell populations and lymphoid T cells.

Measurement of Cellular Behavior by Electrochemical Impedance Sensing.

There is a great demand for label-free in vitro assays in a high-throughput context, in order to measure cell viability and analyze cellular functions like cell migration or cell differentiation under noninvasive conditions. Here, we describe impedance measurement to quantify dynamic changes on cell morphology in real time. In order to monitor physiological changes, cells are grown in tissue culture vessels where gold electrodes are incorporated at the bottom.

Transcriptional programs that control expression of the autoimmune regulator gene Aire.

Aire is a transcriptional regulator that induces promiscuous expression of thousands of genes encoding tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs that regulate its own expression have remained elusive. Here we comprehensively analyzed both cis-acting and trans-acting regulatory mechanisms and found that the Aire locus was insulated by the global chromatin organizer CTCF and was hypermethylated in cells and tissues that did not express Aire.

Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is a valuable method to investigate protein-DNA interactions in vivo. Since its discovery it has been indispensable to identify binding sites and patterns of a variety of DNA-interacting proteins, such as transcription factors and regulators, modified histones, and epigenetic modifiers. The Polycomb repressors were the first proteins that have been mapped using this technique, which provided the mechanistic basis for the understanding of their biological function.

Tet1 and Tet2 Protect DNA Methylation Canyons against Hypermethylation.

DNA methylation is a dynamic epigenetic modification with an important role in cell fate specification and reprogramming. The Ten eleven translocation (Tet) family of enzymes converts 5-methylcytosine to 5-hydroxymethylcytosine, which promotes passive DNA demethylation and functions as an intermediate in an active DNA demethylation process. Tet1/Tet2 double-knockout mice are characterized by developmental defects and epigenetic instability, suggesting a requirement for Tet-mediated DNA demethylation for the proper regulation of gene expression during differentiation.

Epigenetic regulatory functions of DNA modifications: 5-methylcytosine and beyond.

The chemical modification of DNA bases plays a key role in epigenetic gene regulation. While much attention has been focused on the classical epigenetic mark, 5-methylcytosine, the field garnered increased interest through the recent discovery of additional modifications. In this review, we focus on the epigenetic regulatory roles of DNA modifications in animals.

Chronic inflammation induces a novel epigenetic program that is conserved in intestinal adenomas and in colorectal cancer.

Chronic inflammation represents a major risk factor for tumor formation, but the underlying mechanisms have remained largely unknown. Epigenetic mechanisms can record the effects of environmental challenges on the genome level and could therefore play an important role in the pathogenesis of inflammation-associated tumors. Using single-base methylation maps and transcriptome analyses of a colitis-induced mouse colon cancer model, we identified a novel epigenetic program that is characterized by hypermethylation of DNA methylation valleys that are characterized by low CpG density and active chromatin marks.

Loss of Tet enzymes compromises proper differentiation of embryonic stem cells.

Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and are dynamically expressed during development. Whereas loss of individual Tet enzymes or combined deficiency of Tet1/2 allows for embryogenesis, the effect of complete loss of Tet activity and 5hmC marks in development is not established. We have generated Tet1/2/3 triple-knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential.

RNA-interference components are dispensable for transcriptional silencing of the drosophila bithorax-complex.

Background: Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.

Embryonic carcinoma cells show specific dielectric resistance profiles during induced differentiation.

Induction of differentiation in cancer stem cells by drug treatment represents an important approach for cancer therapy. The understanding of the mechanisms that regulate such a forced exit from malignant pluripotency is fundamental to enhance our knowledge of tumour stability. Certain nucleoside analogues, such as 2'-deoxy-5-azacytidine and 1β-arabinofuranosylcytosine, can induce the differentiation of the embryonic cancer stem cell line NTERA 2 D1 (NT2).

Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development.

Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in various embryonic and adult tissues. Mice mutant for either Tet1 or Tet2 are viable, raising the question of whether these enzymes have overlapping roles in development. Here we have generated Tet1 and Tet2 double-knockout (DKO) embryonic stem cells (ESCs) and mice.

MYC-induced epigenetic activation of GATA4 in lung adenocarcinoma.

Human lung cancer is a disease with high incidence and accounts for most cancer-related deaths in both men and women. Metastasis is a common event in non-small cell lung carcinoma (NSCLC), diminishing the survival chance of the patients with this type of tumor. It has been shown that MYC is involved in the development of metastasis from NSCLC, but the mechanism underlying this switch remained to be identified.

Hydroxylation of 5-methylcytosine by TET2 maintains the active state of the mammalian HOXA cluster.

Differentiation is accompanied by extensive epigenomic reprogramming, leading to the repression of stemness factors and the transcriptional maintenance of activated lineage-specific genes. Here we use the mammalian Hoxa cluster of developmental genes as a model system to follow changes in DNA modification patterns during retinoic acid-induced differentiation. We find the inactive cluster to be marked by defined patterns of 5-methylcytosine (5mC).

Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila.

RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21-30 nucleotide) RNAs. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive.

Genome-wide promoter DNA methylation dynamics of human hematopoietic progenitor cells during differentiation and aging.

DNA methylation plays an important role in the self-renewal of hematopoietic stem cells and in the commitment to the lymphoid or myeloid lineages. Using purified CD34⁺ hematopoietic progenitor cells and differentiated myeloid cell populations from the same human samples, we obtained detailed methylation profiles at distinct stages of hematopoiesis. We identified a defined set of differentiation-related genes that are methylated in CD34⁺ hematopoietic progenitor cells but show pronounced DNA hypomethylation in monocytes and in granulocytes.

Epigenetically deregulated microRNA-375 is involved in a positive feedback loop with estrogen receptor alpha in breast cancer cells.

Estrogen receptor α (ERα) upregulation causes abnormal cell proliferation in about two thirds of breast cancers, yet understanding of the underlying mechanisms remains incomplete. Here, we show that high expression of the microRNA miR-375 in ERα-positive breast cell lines is a key driver of their proliferation. miR-375 overexpression was caused by loss of epigenetic marks including H3K9me2 and local DNA hypomethylation, dissociation of the transcriptional repressor CTCF from the miR-375 promoter, and interactions of ERα with regulatory regions of miR-375.

Biology of polycomb and trithorax group proteins.

Cellular phenotypes can be ascribed to different patterns of gene expression. Epigenetic mechanisms control the generation of different phenotypes from the same genotype. Thus differentiation is basically a process driven by changes in gene activity during development, often in response to transient factors or environmental stimuli.

Noncoding RNA synthesis and loss of Polycomb group repression accompanies the colinear activation of the human HOXA cluster.

The ratio of noncoding to protein coding DNA rises with the complexity of the organism, culminating in nearly 99% of nonprotein coding DNA in humans. Nevertheless, a large portion of these regions is transcribed, creating the alleged paradox that noncoding RNA (ncRNA) represents the largest output of the human genome. Such a complex scenario may include epigenetic mechanisms where ncRNAs would be involved in chromatin regulation.

Chromatin Immunoprecipitation (ChIP) of Protein Complexes: Mapping of Genomic Targets of Nuclear Proteins in Cultured Cells.

INTRODUCTIONThis protocol describes the use of chromatin immunoprecipitation technology (ChIP) to analyze interactions of proteins or protein complexes with DNA in vivo. In this approach, the material is fixed with formaldehyde to preserve DNA-protein and protein-protein associations, the cells are lysed, and the chromatin is cut and solubilized. The chromatin suspension is immunoprecipitated with an antibody against the protein(s) of interest, and the coimmunoprecipitated DNA fragments are analyzed.

Epigenome changes in active and inactive polycomb-group-controlled regions.

The Polycomb group (PcG) of proteins conveys epigenetic inheritance of repressed transcriptional states. In Drosophila, the Polycomb repressive complex 1 (PRC1) maintains the silent state by inhibiting the transcription machinery and chromatin remodelling at core promoters. Using immunoprecipitation of in vivo formaldehyde-fixed chromatin in phenotypically diverse cultured cell lines, we have mapped PRC1 components, the histone methyl transferase (HMT) Enhancer of zeste (E(z)) and histone H3 modifications in active and inactive PcG-controlled regions.