SRSF2 plays an unexpected role as reader of m5C on mRNA, linking epitranscriptomics to cancer.

A common mRNA modification is 5-methylcytosine (m5C), whose role in gene-transcript processing and cancer remains unclear. Here, we identify serine/arginine-rich splicing factor 2 (SRSF2) as a reader of m5C and impaired SRSF2 m5C binding as a potential contributor to leukemogenesis. Structurally, we identify residues involved in m5C recognition and the impact of the prevalent leukemia-associated mutation SRSF2P95H. We show that SRSF2 binding and m5C colocalize within transcripts. Furthermore, knocking down the m5C writer NSUN2 decreases mRNA m5C, reduces SRSF2 binding, and alters RNA splicing. We also show that the SRSF2P95H mutation impairs the ability of the protein to read m5C-marked mRNA, notably reducing its binding to key leukemia-related transcripts in leukemic cells. In leukemia patients, low NSUN2 expression leads to mRNA m5C hypomethylation and, combined with SRSF2P95H, predicts poor outcomes. Altogether, we highlight an unrecognized mechanistic link between epitranscriptomics and a key oncogenesis driver.

Dynamic changes in RNA m6A and 5 hmC influence gene expression programs during macrophage differentiation and polarisation.

RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m6A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m6A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity.

SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma.

Cancer stem cells (CSCs) have been reported in various cancers, including in skin squamous-cell carcinoma (SCC). The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here we find that Sox2, a transcription factor expressed in various types of embryonic and adult stem cells, was the most upregulated transcription factor in the CSCs of squamous skin tumours in mice. SOX2 is absent in normal epidermis but begins to be expressed in the vast majority of mouse and human pre-neoplastic skin tumours, and continues to be expressed in a heterogeneous manner in invasive mouse and human SCCs. In contrast to other SCCs, in which SOX2 is frequently genetically amplified, the expression of SOX2 in mouse and human skin SCCs is transcriptionally regulated. Conditional deletion of Sox2 in the mouse epidermis markedly decreases skin tumour formation after chemical-induced carcinogenesis. Using green fluorescent protein (GFP) as a reporter of Sox2 transcriptional expression (SOX2-GFP knock-in mice), we showed that SOX2-expressing cells in invasive SCC are greatly enriched in tumour-propagating cells, which further increase upon serial transplantations. Lineage ablation of SOX2-expressing cells within primary benign and malignant SCCs leads to tumour regression, consistent with the critical role of SOX2-expressing cells in tumour maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to tumour regression and decreases the ability of cancer cells to be propagated upon transplantation into immunodeficient mice, supporting the essential role of SOX2 in regulating CSC functions. Transcriptional profiling of SOX2-GFP-expressing CSCs and of tumour epithelial cells upon Sox2 deletion uncovered a gene network regulated by SOX2 in primary tumour cells in vivo. Chromatin immunoprecipitation identified several direct SOX2 target genes controlling tumour stemness, survival, proliferation, adhesion, invasion and paraneoplastic syndrome. We demonstrate that SOX2, by marking and regulating the functions of skin tumour-initiating cells and CSCs, establishes a continuum between tumour initiation and progression in primary skin tumours.

Playing TETris with DNA modifications.

Methylation of the fifth carbon of cytosine was the first epigenetic modification to be discovered in DNA. Recently, three new DNA modifications have come to light: hydroxymethylcytosine, formylcytosine, and carboxylcytosine, all generated by oxidation of methylcytosine by Ten Eleven Translocation (TET) enzymes. These modifications can initiate full DNA demethylation, but they are also likely to participate, like methylcytosine, in epigenetic signalling per se. A scenario is emerging in which coordinated regulation at multiple levels governs the participation of TETs in a wide range of physiological functions, sometimes via a mechanism unrelated to their enzymatic activity. Although still under construction, a sophisticated picture is rapidly forming where, according to the function to be performed, TETs ensure epigenetic marking to create specific landscapes, and whose improper build-up can lead to diseases such as cancer and neurodegenerative disorders.

FOXP1 is a regulator of quiescence in healthy human CD4+ T cells and is constitutively repressed in T cells from patients with lymphoproliferative disorders.

The forkhead box P1 (FOXP1) transcription factor has been shown to regulate the generation and maintenance of quiescent naïve murine T cells. In humans, FOXP1 expression has been correlated with overall survival in patients with peripheral T-cell lymphoma (PTCL), although its regulatory role in T-cell function is currently unknown. We found that FOXP1 is normally expressed in all human leukocyte subpopulations. Focusing on primary human CD4+ T cells, we show that nuclear expression of FOXP1 predominates in naïve cells with significant downregulation detected in memory cells from blood and tonsils. FOXP1 is repressed following in vitro T-cell activation of naïve T cells, and later re-established in memory CD4+ T cells, albeit at lower levels. DNA methylation analysis revealed that epigenetic mechanisms participate in regulating the human FOXP1 gene. ShRNA-mediated FOXP1 repression induces CD4+ T cells to enter the cell cycle, acquire memory-like markers and upregulate helper T-cell differentiation genes. In patients with lymphoproliferative disorders, FOXP1 expression is constitutionally repressed in the clonal T cells in parallel with overexpression of helper T-cell differentiation genes. Collectively, these data identify FOXP1 as an essential transcriptional regulator for primary human CD4+ T cells and suggest its potential important role in the development of PTCL.

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.

TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS.

TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with O-GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3-OGT co-localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3-OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step-wise model, involving TET-OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation.

DNA methylation profiling identifies epigenetic dysregulation in pancreatic islets from type 2 diabetic patients.

In addition to genetic predisposition, environmental and lifestyle factors contribute to the pathogenesis of type 2 diabetes (T2D). Epigenetic changes may provide the link for translating environmental exposures into pathological mechanisms. In this study, we performed the first comprehensive DNA methylation profiling in pancreatic islets from T2D and non-diabetic donors. We uncovered 276 CpG loci affiliated to promoters of 254 genes displaying significant differential DNA methylation in diabetic islets. These methylation changes were not present in blood cells from T2D individuals nor were they experimentally induced in non-diabetic islets by exposure to high glucose. For a subgroup of the differentially methylated genes, concordant transcriptional changes were present. Functional annotation of the aberrantly methylated genes and RNAi experiments highlighted pathways implicated in ß-cell survival and function; some are implicated in cellular dysfunction while others facilitate adaptation to stressors. Together, our findings offer new insights into the intricate mechanisms of T2D pathogenesis, underscore the important involvement of epigenetic dysregulation in diabetic islets and may advance our understanding of T2D aetiology.

A comprehensive overview of Infinium HumanMethylation450 data processing.

Infinium HumanMethylation450 beadarray is a popular technology to explore DNA methylomes in health and disease, and there is a current explosion in the use of this technique. Despite experience acquired from gene expression microarrays, analyzing Infinium Methylation arrays appeared more complex than initially thought and several difficulties have been encountered, as those arrays display specific features that need to be taken into consideration during data processing. Here, we review several issues that have been highlighted by the scientific community, and we present an overview of the general data processing scheme and an evaluation of the different normalization methods available to date to guide the 450K users in their analysis and data interpretation.

Citrullination of DNMT3A by PADI4 regulates its stability and controls DNA methylation.

DNA methylation is a central epigenetic modification in mammals, with essential roles in development and disease. De novo DNA methyltransferases establish DNA methylation patterns in specific regions within the genome by mechanisms that remain poorly understood. Here we show that protein citrullination by peptidylarginine deiminase 4 (PADI4) affects the function of the DNA methyltransferase DNMT3A. We found that DNMT3A and PADI4 interact, from overexpressed as well as untransfected cells, and associate with each other's enzymatic activity. Both in vitro and in vivo, PADI4 was shown to citrullinate DNMT3A. We identified a sequence upstream of the PWWP domain of DNMT3A as its primary region citrullinated by PADI4. Increasing the PADI4 level caused the DNMT3A protein level to increase as well, provided that the PADI4 was catalytically active, and RNAi targeting PADI4 caused reduced DNMT3A levels. Accordingly, pulse-chase experiments revealed stabilization of the DNMT3A protein by catalytically active PADI4. Citrullination and increased expression of native DNMT3A by PADI4 were confirmed in PADI4-knockout MEFs. Finally, we showed that PADI4 overexpression increases DNA methyltransferase activity in a catalytic-dependent manner and use bisulfite pyrosequencing to demonstrate that PADI4 knockdown causes significant reduction of CpG methylation at the p21 promoter, a known target of DNMT3A and PADI4. Protein citrullination by PADI4 thus emerges as a novel mechanism for controlling a de novo DNA methyltransferase. Our results shed new light on how post-translational modifications might contribute to shaping the genomic CpG methylation landscape.

Exposing the DNA methylome iceberg.

DNA methylation was the first epigenetic modification discovered. Until recently, comprehensive coverage of the composition and distribution of methylated cytosines across the genome was lacking. Technological advances, however, are providing methylation maps that can reveal the genomic distribution of DNA methylation in different cell states or phenotypes. The emerging picture includes extensive gene body methylation that is highly conserved in eukaryotes, the presence of DNA methylation in previously unappreciated sequence contexts, and the discovery of another modified DNA base, 5-hydroxymethylcytosine. These new data point to the role of DNA methylation both in gene silencing and gene activation; reconciliation of these seemingly contradictory roles will be essential to fully unravel the biological function of DNA methylation in eukaryotes. Here we review how these recently exposed features of the DNA methylome are challenging previously held dogmas in the field.

TET2 deficiency inhibits mesoderm and hematopoietic differentiation in human embryonic stem cells.

Ten-eleven-translocation 2 (TET2) belongs to the TET protein family that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and plays a central role in normal and malignant adult hematopoiesis. Yet the role of TET2 in human hematopoietic development remains largely unknown. Here, we show that TET2 expression is low in human embryonic stem cell (ESC) lines and increases during hematopoietic differentiation. shRNA-mediated TET2 knockdown had no effect on the pluripotency of various ESCs. However, it skewed their differentiation into neuroectoderm at the expense of endoderm and mesoderm both in vitro and in vivo. These effects were rescued by reintroducing the targeted TET2 protein. Moreover, TET2-driven differentiation was dependent on NANOG transcriptional factor. Indeed, TET2 bound to NANOG promoter and in TET2-deficient cells the methylation of the NANOG promoter correlated with a decreased in NANOG expression. The altered differentiation resulting from TET2 knockdown in ESCs led to a decrease in both the number and the cloning capacities of hematopoietic progenitors. These defects were due to an increased apoptosis and an altered gene expression profile, including abnormal expression of neuronal genes. Intriguingly, when TET2 was knockdown in hematopoietic cells, it increased hematopoietic development. In conclusion, our work suggests that TET2 is involved in different stages of human embryonic development, including induction of the mesoderm and hematopoietic differentiation.

Role of the polycomb repressive complex 2 in acute promyelocytic leukemia.

Epigenetic changes are common alterations in cancer cells. Here, we have investigated the role of Polycomb group proteins in the establishment and maintenance of the aberrant silencing of tumor suppressor genes during transformation induced by the leukemia-associated PML-RARalpha fusion protein. We show that in leukemic cells knockdown of SUZ12, a key component of Polycomb repressive complex 2 (PRC2), reverts not only histone modification but also induces DNA demethylation of PML-RARalpha target genes. This results in promoter reactivation and granulocytic differentiation. Importantly, the epigenetic alterations caused by PML-RARalpha can be reverted by retinoic acid treatment of primary blasts from leukemic patients. Our results demonstrate that the direct targeting of Polycomb group proteins by an oncogene plays a key role during carcinogenesis.

The histone demethylase Kdm3a is essential to progression through differentiation.

Histone demethylation has important roles in regulating gene expression and forms part of the epigenetic memory system that regulates cell fate and identity by still poorly understood mechanisms. Here, we examined the role of histone demethylase Kdm3a during cell differentiation, showing that Kdm3a is essential for differentiation into parietal endoderm-like (PE) cells in the F9 mouse embryonal carcinoma model. We identified a number of target genes regulated by Kdm3a during endoderm differentiation; among the most dysregulated were the three developmental master regulators Dab2, Pdlim4 and FoxQ1. We show that dysregulation of the expression of these genes correlates with Kdm3a H3K9me2 demethylase activity. We further demonstrate that either Dab2 depletion or Kdm3a depletion prevents F9 cells from fully differentiating into PE cells, but that ectopic expression of Dab2 cannot compensate for Kdm3a knockdown; Dab2 is thus necessary, but insufficient on its own, to promote complete terminal differentiation. We conclude that Kdm3a plays a crucial role in progression through PE differentiation by regulating expression of a set of endoderm differentiation master genes. The emergence of Kdm3a as a key modulator of cell fate decision strengthens the view that histone demethylases are essential to cell differentiation.

A multiomics dataset for the study of RNA modifications in human macrophage differentiation and polarisation.

RNA modifications have emerged as central regulators of gene expression programs. Amongst RNA modifications are N6-methyladenosine (m6A) and RNA 5-hydroxymethylcytosine (5hmC). While m6A is established as a versatile regulator of RNA metabolism, the functions of RNA 5hmC are unclear. Despite some evidence linking RNA modifications to immunity, their implications in gene expression control in macrophage development and functions remain unclear. Here we present a multi-omics dataset capturing different layers of the gene expression programs driving macrophage differentiation and polarisation. We obtained mRNA-Seq, m6A-IP-Seq, 5hmC-IP-Seq, Polyribo-Seq and LC-MS/MS data from monocytes and resting-, pro- and anti-inflammatory-like macrophages. We present technical validation showing high quality and correlation between samples for all datasets, and evidence of biological consistency of modelled macrophages at the transcriptomic, epitranscriptomic, translational and proteomic levels. This multi-omics dataset provides a resource for the study of RNA m6A and 5hmC in the context of macrophage biology and spans the gene expression process from transcripts to proteins.

Epigenetic silencing of BEND4, a novel DNA damage repair gene, is a synthetic lethal marker for ATM inhibitor in pancreatic cancer.

Synthetic lethality is a novel model for cancer therapy. To understand the function and mechanism of BEN domain-containing protein 4 (BEND4) in pancreatic cancer, eight cell lines and a total of 492 cases of pancreatic neoplasia samples were included in this study. Methylation-specific polymerase chain reaction, CRISPR/Cas9, immunoprecipitation assay, comet assay, and xenograft mouse model were used. BEND4 is a new member of the BEN domain family. The expression of BEND4 is regulated by promoter region methylation. It is methylated in 58.1% (176/303) of pancreatic ductal adenocarcinoma (PDAC), 33.3% (14/42) of intraductal papillary mucinous neoplasm, 31.0% (13/42) of pancreatic neuroendocrine tumor, 14.3% (3/21) of mucinous cystic neoplasm, 4.3% (2/47) of solid pseudopapillary neoplasm, and 2.7% (1/37) of serous cystic neoplasm. BEND4 methylation is significantly associated with late-onset PDAC (> 50 years, P < 0.01) and tumor differentiation (P < 0.0001), and methylation of BEND4 is an independent poor prognostic marker (P < 0.01) in PDAC. Furthermore, BEND4 plays tumor-suppressive roles in vitro and in vivo. Mechanistically, BEND4 involves non-homologous end joining signaling by interacting with Ku80 and promotes DNA damage repair. Loss of BEND4 increased the sensitivity of PDAC cells to ATM inhibitor. Collectively, the present study revealed an uncharacterized tumor suppressor BEND4 and indicated that methylation of BEND4 may serve as a potential synthetic lethal marker for ATM inhibitor in PDAC treatment.

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.

Regulation of mammalian DNA methyltransferases: a route to new mechanisms.

DNA methyltransferases (DNMTs) establish and maintain DNA methylation patterns at specific regions of the genome, thereby contributing to gene regulation. It is becoming evident that an intricate web of pathways target DNMTs to these genomic regions. Here, we review the understanding of these regulatory mechanisms and provide an overview of the new findings, emphasizing the emerging scenario in which several levels of regulation are coordinated to control DNMTs. The mechanisms involved include the dynamic interplay between interdependent post-translational modifications that regulate DNMTs, post-transcriptional regulation by miRNAs and the emerging role of non-coding RNA in targeting mammalian DNMTs. The analysis of these mechanisms is imperative to the understanding of the role of DNA methylation in regulating gene expression during development and in disease.

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.