Non-canonical miRNA-RNA base-pairing impedes tumor suppressor activity of miR-16.

Uveal melanoma (UM), the most common primary intraocular tumor in adults, has been extensively characterized by omics technologies during the last 5 yr. Despite the discovery of gene signatures, the molecular actors driving cancer aggressiveness are not fully understood, and UM is still associated with very poor overall survival (OS) at the metastatic stage. By defining the miR-16 interactome, we revealed that miR-16 mainly interacts via non-canonical base-pairing to a subset of RNAs, promoting their expression levels. Consequently, the canonical miR-16 activity, involved in the RNA decay of oncogenes, such as <i>cyclin D3</i>, is impaired. This non-canonical base-pairing can explain both the derepression of miR-16 targets and the promotion of oncogene expression observed in patients with poor OS in two cohorts. miR-16 activity, assessment using our RNA signature, discriminates the patient's OS as effectively as current methods. To the best of our knowledge, this is the first time that a predictive signature has been composed of genes belonging to the same mechanism (miR-16) in UM. Altogether, our results strongly suggest that UM is a miR-16 disease.

5-methylcytosine turnover: Mechanisms and therapeutic implications in cancer.

DNA methylation at the fifth position of cytosine (5mC) is one of the most studied epigenetic mechanisms essential for the control of gene expression and for many other biological processes including genomic imprinting, X chromosome inactivation and genome stability. Over the last years, accumulating evidence suggest that DNA methylation is a highly dynamic mechanism driven by a balance between methylation by DNMTs and TET-mediated demethylation processes. However, one of the main challenges is to understand the dynamics underlying steady state DNA methylation levels. In this review article, we give an overview of the latest advances highlighting DNA methylation as a dynamic cycling process with a continuous turnover of cytosine modifications. We describe the cooperative actions of DNMT and TET enzymes which combine with many additional parameters including chromatin environment and protein partners to govern 5mC turnover. We also discuss how mathematical models can be used to address variable methylation levels during development and explain cell-type epigenetic heterogeneity locally but also at the genome scale. Finally, we review the therapeutic implications of these discoveries with the use of both epigenetic clocks as predictors and the development of epidrugs that target the DNA methylation/demethylation machinery. Together, these discoveries unveil with unprecedented detail how dynamic is DNA methylation during development, underlying the establishment of heterogeneous DNA methylation landscapes which could be altered in aging, diseases and cancer.

The N-terminal domain of TET1 promotes the formation of dense chromatin regions refractory to transcription.

TET (ten-eleven translocation) enzymes initiate active cytosine demethylation via the oxidation of 5-methylcytosine. TET1 is composed of a C-terminal domain, which bears the catalytic activity of the enzyme, and a N-terminal region that is less well characterized except for the CXXC domain responsible for the targeting to CpG islands. While cytosine demethylation induced by TET1 promotes transcription, this protein also interacts with chromatin-regulating factors that rather silence this process, the coordination between these two opposite functions of TET1 being unclear. In the present work, we uncover a new function of the N-terminal part of the TET1 protein in the regulation of the chromatin architecture. This domain of the protein promotes the establishment of a compact chromatin architecture displaying reduced exchange rate of core histones and partial dissociation of the histone linker. This chromatin reorganization process, which does not rely on the CXXC domain, is associated with a global shutdown of transcription and an increase in heterochromatin-associated histone epigenetic marks. Based on these findings, we propose that the dense chromatin organization generated by the N-terminal domain of TET1 could contribute to restraining the transcription enhancement induced by the DNA demethylation activity of this enzyme.

TET2-mediated epigenetic reprogramming of breast cancer cells impairs lysosome biogenesis.

Methylation and demethylation of cytosines in DNA are believed to act as keystones of cell-specific gene expression by controlling the chromatin structure and accessibility to transcription factors. Cancer cells have their own transcriptional programs, and we sought to alter such a cancer-specific program by enforcing expression of the catalytic domain (CD) of the methylcytosine dioxygenase TET2 in breast cancer cells. The TET2 CD decreased the tumorigenic potential of cancer cells through both activation and repression of a repertoire of genes that, interestingly, differed in part from the one observed upon treatment with the hypomethylating agent decitabine. In addition to promoting the establishment of an antiviral state, TET2 activated 5mC turnover at thousands of MYC-binding motifs and down-regulated a panel of known MYC-repressed genes involved in lysosome biogenesis and function. Thus, an extensive cross-talk between TET2 and the oncogenic transcription factor MYC establishes a lysosomal storage disease-like state that contributes to an exacerbated sensitivity to autophagy inducers.

Reduction of RUNX1 transcription factor activity by a CBFA2T3-mimicking peptide: application to B cell precursor acute lymphoblastic leukemia.

BACKGROUND: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia. METHODS: We worked with BCP-ALL mononuclear bone marrow patients' cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models. RESULTS: We demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients' cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located - 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation. CONCLUSIONS: Altogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein.

The hydroxymethylome of multiple myeloma identifies FAM72D as a 1q21 marker linked to proliferation.

Cell identity relies on the cross-talk between genetics and epigenetics and their impact on gene expression. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) is the first step of an active DNA demethylation process occurring mainly at enhancers and gene bodies and, as such, participates in processes governing cell identity in normal and pathological conditions. Although genetic alterations are well documented in multiple myeloma (MM), epigenetic alterations associated with this disease have not yet been thoroughly analyzed. To gain insight into the biology of MM, genome-wide 5hmC profiles were obtained and showed that regions enriched in this modified base overlap with MM enhancers and super enhancers and are close to highly expressed genes. Through the definition of a MM-specific 5hmC signature, we identified FAM72D as a poor prognostic gene located on 1q21, a region amplified in high risk myeloma. We further uncovered that FAM72D functions as part of the FOXM1 transcription factor network controlling cell proliferation and survival and we evidenced an increased sensitivity of cells expressing high levels of FOXM1 and FAM72 to epigenetic drugs targeting histone deacetylases and DNA methyltransferases.

Interplay between transcription regulators RUNX1 and FUBP1 activates an enhancer of the oncogene c-KIT and amplifies cell proliferation.

Runt-related transcription factor 1 (RUNX1) is a well-known master regulator of hematopoietic lineages but its mechanisms of action are still not fully understood. Here, we found that RUNX1 localizes on active chromatin together with Far Upstream Binding Protein 1 (FUBP1) in human B-cell precursor lymphoblasts, and that both factors interact in the same transcriptional regulatory complex. RUNX1 and FUBP1 chromatin localization identified c-KIT as a common target gene. We characterized two regulatory regions, at +700 bp and +30 kb within the first intron of c-KIT, bound by both RUNX1 and FUBP1, and that present active histone marks. Based on these regions, we proposed a novel FUBP1 FUSE-like DNA-binding sequence on the +30 kb enhancer. We demonstrated that FUBP1 and RUNX1 cooperate for the regulation of the expression of the oncogene c-KIT. Notably, upregulation of c-KIT expression by FUBP1 and RUNX1 promotes cell proliferation and renders cells more resistant to the c-KIT inhibitor imatinib mesylate, a common therapeutic drug. These results reveal a new mechanism of action of RUNX1 that implicates FUBP1, as a facilitator, to trigger transcriptional regulation of c-KIT and to regulate cell proliferation. Deregulation of this regulatory mechanism may explain some oncogenic function of RUNX1 and FUBP1.

Reading cytosine modifications within chromatin.

Zinc-finger and homeodomain transcription factors have been shown in vitro to bind to recognition motifs containing a methylated CpG. However, accessing these motifs in vivo might be seriously impeded by the inclusion of DNA in nucleosomes and by the condensed structure adopted by chromatin formed on methylated DNA. Here, we discuss how oxidation of 5-methylcytosine into 5-hydroxymethylcytosine could provide the initial destabilizing clue for such transcription factors to get access to nucleosomal DNA and read epigenetic information.

Coupling Exonuclease Digestion with Selective Chemical Labeling forBase-resolution Mapping of 5-Hydroxymethylcytosine in Genomic DNA.

This protocol is designed to obtain base-resolution information on the level of 5-hydroxymethylcytosine (5hmC) in CpGs without the need for bisulfite modification. It relies on (i) the capture of hydroxymethylated sequences by a procedure known as 'selective chemical labeling' (see Szulwach et al., 2012 ) and (ii) the digestion of the captured DNA by exonucleases. After Illumina sequencing of the digested DNA fragments, an ad hoc bioinformatic pipeline extracts the information for further downstream analysis.

Cytosine modifications modulate the chromatin architecture of transcriptional enhancers.

Epigenetic mechanisms are believed to play key roles in the establishment of cell-specific transcription programs. Accordingly, the modified bases 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been observed in DNA of genomic regulatory regions such as enhancers, and oxidation of 5mC into 5hmC by Ten-eleven translocation (TET) proteins correlates with enhancer activation. However, the functional relationship between cytosine modifications and the chromatin architecture of enhancers remains elusive. To gain insights into their function, 5mC and 5hmC levels were perturbed by inhibiting DNA methyltransferases and TETs during differentiation of mouse embryonal carcinoma cells into neural progenitors, and chromatin characteristics of enhancers bound by the pioneer transcription factors FOXA1, MEIS1, and PBX1 were interrogated. In a large fraction of the tested enhancers, inhibition of DNA methylation was associated with a significant increase in monomethylation of H3K4, a characteristic mark of enhancer priming. In addition, at some specific enhancers, 5mC oxidation by TETs facilitated chromatin opening, a process that may stabilize MEIS1 binding to these genomic regions.

Inactivation of the Nuclear Orphan Receptor COUP-TFII by Small Chemicals.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII/NR2F2) is an orphan member of the nuclear receptor family of transcription factors whose activities are modulated upon binding of small molecules into an hydrophobic ligand-binding pocket (LBP). Although the LBP of COUP-TFII is filled with aromatic amino-acid side chains, alternative modes of ligand binding could potentially lead to regulation of the orphan receptor. Here, we screened a synthetic and natural compound library in a yeast one-hybrid assay and identified 4-methoxynaphthol as an inhibitor of COUP-TFII. This synthetic inhibitor was able to counteract processes either positively or negatively regulated by COUP-TFII in different mammalian cell systems. Hence, we demonstrate that the true orphan receptor COUP-TFII can be targeted by small chemicals which could be used to study the physiological functions of COUP-TFII or to counteract detrimental COUP-TFII activities in various pathological conditions.

5-hydroxymethylcytosine marks postmitotic neural cells in the adult and developing vertebrate central nervous system.

The epigenetic mark 5-hydroxymethylcytosine (5hmC) is a cytosine modification that is abundant in the central nervous system of mammals and which results from 5-methylcytosine oxidation by TET enzymes. Such a mark is suggested to play key roles in the regulation of chromatin structure and gene expression. However, its precise functions still remain poorly understood and information about its distribution in non-mammalian species is still lacking. Here, the distribution of 5hmC was investigated in the brain of adult zebrafish, African claw frog, and mouse in a comparative manner. We show that zebrafish neurons are endowed with high levels of 5hmC, whereas quiescent or proliferative neural progenitors show low to undetectable levels of the modified cytosine. In the brain of larval and juvenile Xenopus, 5hmC is also detected in neurons, while ventricular proliferative cells do not display this epigenetic mark. Similarly, 5hmC is enriched in neurons compared to neural progenitors of the ventricular zone in the mouse developing cortex. Interestingly, 5hmC colocalized with the methylated DNA binding protein MeCP2 and with the active chromatin histone modification H3K4me2 in mouse neurons. Taken together, our results show an evolutionarily conserved cerebral distribution of 5hmC between fish and tetrapods and reinforce the idea that 5hmC fulfills major functions in the control of chromatin activity in vertebrate neurons. J. Comp. Neurol. 525:478-497, 2017. © 2016 Wiley Periodicals, Inc.

A Point Mutation in a lincRNA Upstream of GDNF Is Associated to a Canine Insensitivity to Pain: A Spontaneous Model for Human Sensory Neuropathies.

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.

Changes in Gene Expression and Estrogen Receptor Cistrome in Mouse Liver Upon Acute E2 Treatment.

Transcriptional regulation by the estrogen receptor-α (ER) has been investigated mainly in breast cancer cell lines, but estrogens such as 17ß-estradiol (E2) exert numerous extrareproductive effects, particularly in the liver, where E2 exhibits both protective metabolic and deleterious thrombotic actions. To analyze the direct and early transcriptional effects of estrogens in the liver, we determined the E2-sensitive transcriptome and ER cistrome in mice after acute administration of E2 or placebo. These analyses revealed the early induction of genes involved in lipid metabolism, which fits with the crucial role of ER in the prevention of liver steatosis. Characterization of the chromatin state of ER binding sites (BSs) in mice expressing or not ER demonstrated that ER is not required per se for the establishment and/or maintenance of chromatin modifications at the majority of its BSs. This is presumably a consequence of a strong overlap between ER and hepatocyte nuclear factor 4α BSs. In contrast, 40% of the BSs of the pioneer factor forkhead box protein a (Foxa2) were dependent upon ER expression, and ER expression also affected the distribution of nucleosomes harboring dimethylated lysine 4 of Histone H3 around Foxa2 BSs. We finally show that, in addition to a network of liver-specific transcription factors including CCAAT/enhancer-binding protein and hepatocyte nuclear factor 4α, ER might be required for proper Foxa2 function in this tissue.

Single-CpG resolution mapping of 5-hydroxymethylcytosine by chemical labeling and exonuclease digestion identifies evolutionarily unconserved CpGs as TET targets.

Conventional techniques for single-base resolution mapping of epigenetic modifications of DNA such as 5-hydroxymethylcytosine (5hmC) rely on the sequencing of bisulfite-modified DNA. Here we present an alternative approach called SCL-exo which combines selective chemical labeling (SCL) of 5hmC in genomic DNA with exonuclease (exo) digestion of the bead-trapped modified DNA molecules. Associated with a straightforward bioinformatic analysis, this new procedure provides an unbiased and fast method for mapping this epigenetic mark at high resolution. Implemented on mouse genomic DNA from in vitro-differentiated neural precursor cells, SCL-exo sheds light on an intrinsic lack of conservation of hydroxymethylated CpGs across vertebrates.

Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells.

Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-?1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxy)methylation, and cell fate determination.

Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities.

Estradiol signaling is ideally suited for analyzing the molecular and functional linkages between the different layers of information directing transcriptional regulations: the DNA sequence, chromatin modifications, and the spatial organization of the genome. Hence, the estrogen receptor (ER) can bind at a distance from its target genes and engages timely and spatially coordinated processes to regulate their expression. In the context of the coordinated regulation of colinear genes, identifying which ER binding sites (ERBSs) regulate a given gene still remains a challenge. Here, we investigated the coordination of such regulatory events at a 2-Mb genomic locus containing the estrogen-sensitive trefoil factor (TFF) cluster of genes in breast cancer cells. We demonstrate that this locus exhibits a hormone- and cohesin-dependent reduction in the plasticity of its three-dimensional organization that allows multiple ERBSs to be dynamically brought to the vicinity of estrogen-sensitive genes. Additionally, by using triplex-forming oligonucleotides, we could precisely document the functional links between ER engagement at given ERBSs and the regulation of particular genes. Hence, our data provide evidence of a formerly suggested cooperation of enhancers toward gene regulation and also show that redundancy between ERBSs can occur.

Peroxisome proliferator-activated receptor γ regulates genes involved in insulin/insulin-like growth factor signaling and lipid metabolism during adipogenesis through functionally distinct enhancer classes.

The nuclear receptor peroxisome proliferator-activated receptor (PPAR) is a transcription factor whose expression is induced during adipogenesis and that is required for the acquisition and control of mature adipocyte functions. Indeed, PPAR induces the expression of genes involved in lipid synthesis and storage through enhancers activated during adipocyte differentiation. Here, we show that PPAR also binds to enhancers already active in preadipocytes as evidenced by an active chromatin state including lower DNA methylation levels despite higher CpG content. These constitutive enhancers are linked to genes involved in the insulin/insulin-like growth factor signaling pathway that are transcriptionally induced during adipogenesis but to a lower extent than lipid metabolism genes, because of stronger basal expression levels in preadipocytes. This is consistent with the sequential involvement of hormonal sensitivity and lipid handling during adipocyte maturation and correlates with the chromatin structure dynamics at constitutive and activated enhancers. Interestingly, constitutive enhancers are evolutionary conserved and can be activated in other tissues, in contrast to enhancers controlling lipid handling genes whose activation is more restricted to adipocytes. Thus, PPAR utilizes both broadly active and cell type-specific enhancers to modulate the dynamic range of activation of genes involved in the adipogenic process.