DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.

The correct establishment of DNA methylation patterns during mouse early development is essential for cell fate specification. However, the molecular targets as well as the mechanisms that determine the specificity of the de novo methylation machinery during differentiation are not completely elucidated. Here we show that the DNMT3B-dependent DNA methylation of key developmental regulatory regions at epiblast-like cells (EpiLCs) provides an epigenetic priming that ensures flawless commitment at later stages.

The acetyltransferase p300 is recruited in trans to multiple enhancer sites by lncSmad7.

The histone acetyltransferase p300 (also known as KAT3B) is a general transcriptional coactivator that introduces the H3K27ac mark on enhancers triggering their activation and gene transcription. Genome-wide screenings demonstrated that a large fraction of long non-coding RNAs (lncRNAs) plays a role in cellular processes and organ development although the underlying molecular mechanisms remain largely unclear (1,2). We found 122 lncRNAs that interacts directly with p300.

Genome-Wide Analysis of Smad7-Mediated Transcription in Mouse Embryonic Stem Cells.

Smad7 has been identified as a negative regulator of the transforming growth factor TGF-β pathway by direct interaction with the TGF-β type I receptor (TβR-I). Although Smad7 has also been shown to play TGF-β unrelated functions in the cytoplasm and in the nucleus, a comprehensive analysis of its nuclear function has not yet been performed. Here, we show that in ESCs Smad7 is mainly nuclear and acts as a general transcription factor regulating several genes unrelated to the TGF-β pathway.

Cannabinoid Receptor Modulation of Neurogenesis: ST14A Striatal Neural Progenitor Cells as a Simplified In Vitro Model.

The endocannabinoid system (ECS) is involved in the modulation of several basic biological processes, having widespread roles in neurodevelopment, neuromodulation, immune response, energy homeostasis and reproduction. In the adult central nervous system (CNS) the ECS mainly modulates neurotransmitter release, however, a substantial body of evidence has revealed a central role in regulating neurogenesis in developing and adult CNS, also under pathological conditions. Due to the complexity of investigating ECS functions in neural progenitors in vivo, we tested the suitability of the ST14A striatal neural progenitor cell line as a simplified in vitro model to dissect the role and the mechanisms of ECS-regulated neurogenesis, as well as to perform ECS-targeted pharmacological approaches.

The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia.

Dihydroorotate Dehydrogenase (DHODH) is a key enzyme of the pyrimidine biosynthesis, whose inhibition can induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors had shown promising in vitro and in vivo activity on solid tumors, but their effectiveness was not confirmed in clinical trials, probably because cancer cells exploited the pyrimidine salvage pathway to survive. Here, we investigated the antileukemic activity of MEDS433, the DHODH inhibitor developed by our group, against AML.

Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration.

Intragenic DNA methylation prevents spurious transcription initiation.

In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation.

High-throughput single-base resolution mapping of RNA 2΄-O-methylated residues.

Functional characterization of the transcriptome requires tools for the systematic investigation of RNA post-transcriptional modifications. 2΄-O-methylation (2΄-OMe) of the ribose moiety is one of the most abundant post-transcriptional modifications of RNA, although its systematic analysis is difficult due to the lack of reliable high-throughput mapping methods. We describe here a novel high-throughput approach, named 2OMe-seq, that enables fast and accurate mapping at single-base resolution, and relative quantitation, of 2΄-OMe modified residues.

The long intergenic non-coding RNA CCR492 functions as a let-7 competitive endogenous RNA to regulate c-Myc expression.

In mammals the cell-cycle progression through the G1 phase is a tightly regulated process mediated by the transcriptional activation of early genes in response to mitogenic stimuli, whose dysregulation often leads to tumorigenesis. We here report the discovery by RNA-seq of cell-cycle regulated (CCR) long intergenic non-coding RNAs (lincRNAs), potentially involved in the control of the cell-cycle progression. We identified 10 novel lincRNAs expressed in response to serum treatment in mouse embryonic fibroblasts (MEFs) and in BALB/c fibroblasts, comparably to early genes.

TET1 is controlled by pluripotency-associated factors in ESCs and downmodulated by PRC2 in differentiated cells and tissues.

Ten-eleven translocation (Tet) genes encode for a family of hydroxymethylase enzymes involved in regulating DNA methylation dynamics. Tet1 is highly expressed in mouse embryonic stem cells (ESCs) where it plays a critical role the pluripotency maintenance. Tet1 is also involved in cell reprogramming events and in cancer progression.

Single-Base Resolution Analysis of 5-Formyl and 5-Carboxyl Cytosine Reveals Promoter DNA Methylation Dynamics.

Ten eleven translocation (Tet) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be further excised by thymine-DNA glycosylase (Tdg). Here, we present a genome-wide approach, named methylation-assisted bisulfite sequencing (MAB-seq), that enables single-base resolution mapping of 5fC and 5caC and measures their abundance.

Snai1 promotes ESC exit from the pluripotency by direct repression of self-renewal genes.

Although much is known about the pluripotency self-renewal circuitry, the molecular events that lead embryonic stem cells (ESCs) exit from pluripotency and begin differentiation are largely unknown. We found that the zinc finger transcription factor Snai1, involved in gastrulation and epithelial-mesenchymal transition, is already expressed in the inner cell mass of the preimplantation blastocysts. In ESCs, Snai1 does not respond to TGFβ or BMP4 signaling but it is induced by retinoic acid treatment, which induces the binding, on the Snai1 promoter, of the retinoid receptors RARγ and RXRα, the dissociation of the Polycomb repressor complex 2 which results in the decrease of H3K27me3, and the increase of histone H3K4me3.

Myc and max genome-wide binding sites analysis links the Myc regulatory network with the polycomb and the core pluripotency networks in mouse embryonic stem cells.

Myc is a master transcription factor that has been demonstrated to be required for embryonic stem cell (ESC) pluripotency, self-renewal, and inhibition of differentiation. Although recent works have identified several Myc-targets in ESCs, the list of Myc binding sites is largely incomplete due to the low sensitivity and specificity of the antibodies available. To systematically identify Myc binding sites in mouse ESCs, we used a stringent streptavidin-based genome-wide chromatin immunoprecipitation (ChIP-Seq) approach with biotin-tagged Myc (Bio-Myc) as well as a ChIP-Seq of the Myc binding partner Max.

Genome-wide analysis identifies a functional association of Tet1 and Polycomb repressive complex 2 in mouse embryonic stem cells.

Background: Ten-Eleven Translocation (TETs)proteins mediate the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet1 is expressed at high levels in mouse embryonic stem cells (ESCs), where it mediates the induction of 5hmC decoration on gene-regulatory elements. While the function of Tet1 is known, the mechanisms of its specificity remain unclear.