Dnmt3L antagonizes DNA methylation at bivalent promoters and favors DNA methylation at gene bodies in ESCs.

The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methyltransferase that cooperates with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESCs), but its function in these cells is unknown. Through genome-wide analysis of Dnmt3L knockdown in ESCs, we found that Dnmt3L is a positive regulator of methylation at the gene bodies of housekeeping genes and, more surprisingly, is also a negative regulator of methylation at promoters of bivalent genes. Dnmt3L is required for the differentiation of ESCs into primordial germ cells (PGCs) through the activation of the homeotic gene Rhox5. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyltransferases Dnmt3a and Dnmt3b to maintain low methylation levels at the H3K27me3 regions. Thus, in ESCs, Dnmt3L counteracts the activity of de novo DNA methylases to maintain hypomethylation at promoters of bivalent developmental genes.

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. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cancer.

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. We compare our method to other state-of-art approaches, and show that it achieves higher sensitivity and specificity. By applying 2OMe-seq to HeLa cells, we show that it is able to recover the majority of the annotated 2΄-OMe sites on ribosomal RNA. By performing knockdown of the Fibrillarin methyltransferase in mouse embryonic stem cells (ESCs) we show the ability of 2OMe-seq to capture 2΄-O-Methylation level variations. Moreover, using 2OMe-seq data we here report the discovery of 12 previously unannotated 2΄-OMe sites across 18S and 28S rRNAs, 11 of which are conserved in both human and mouse cells, and assigned the respective snoRNAs for all sites. Our approach expands the repertoire of methods for transcriptome-wide mapping of RNA post-transcriptional modifications, and promises to provide novel insights into the role of this modification.

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. By loss-of-function experiments we found that lincRNA CCR492 is required for G1/S progression, localizes in the cell cytoplasm and contains 4 let-7 microRNA recognition elements (MREs). Mechanistically, CCR492 functions as a competing endogenous RNA (ceRNA) to antagonize the function of let-7 microRNAs, leading to the de-repression of c-Myc. Moreover, we show that ectopic expression of CCR492 along with a constitutively active H-Ras promotes cell transformation. Thus, we identified a new lincRNA expressed as an early gene in mammalian cells to regulate the cell-cycle progression by upregulating c-Myc expression.

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. Although the functional role of Tet1 has been largely studied, its regulation is poorly understood. Here we show that Tet1 gene is regulated, both in mouse and human ESCs, by the stemness specific factors Oct3/4, Nanog and by Myc. Thus Tet1 is integrated in the pluripotency transcriptional network of ESCs. We found that Tet1 is switched off by cell proliferation in adult cells and tissues with a consequent genome-wide reduction of 5hmC, which is more evident in hypermethylated regions and promoters. Tet1 downmodulation is mediated by the Polycomb repressive complex 2 (PRC2) through H3K27me3 histone mark deposition. This study expands the knowledge about Tet1 involvement in stemness circuits in ESCs and provides evidence for a transcriptional relationship between Tet1 and PRC2 in adult proliferating cells improving our understanding of the crosstalk between the epigenetic events mediated by these factors.

Molecular characterization of toxigenic Clostridium difficile in a northern Italian hospital.

Clostridium difficile is responsible for more than 90 % of cases of antibiotic-associated diarrhea and pseudomembranous colitis. The most important virulence factors are two toxins called enterotoxin A and cytotoxin B; some C. difficile strains contain the C. difficile binary toxin (CDT). The aim of our study was to prospectively analyze C. difficile clinical isolates in a single center to determine the molecular features of collected strains. Among the 252 isolates, 217 were A + B + (86.1 %), 33 were A + B + cdt + (13.1 %) and 2 were A - B + (0.8 %). There were 15 different ribotypes with a predominance of 018.

FOXA1 regulates alternative splicing in prostate cancer.

Dysregulation of alternative splicing in prostate cancer is linked to transcriptional programs activated by AR, ERG, FOXA1, and MYC. Here, we show that FOXA1 functions as the primary orchestrator of alternative splicing dysregulation across 500 primary and metastatic prostate cancer transcriptomes. We demonstrate that FOXA1 binds to the regulatory regions of splicing-related genes, including HNRNPK and SRSF1. By controlling trans-acting factor expression, FOXA1 exploits an "exon definition" mechanism calibrating alternative splicing toward dominant isoform production. This regulation especially impacts splicing factors themselves and leads to a reduction of nonsense-mediated decay (NMD)-targeted isoforms. Inclusion of the NMD-determinant FLNA exon 30 by FOXA1-controlled oncogene SRSF1 promotes cell growth in vitro and predicts disease recurrence. Overall, we report a role for FOXA1 in rewiring the alternative splicing landscape in prostate cancer through a cascade of events from chromatin access, to splicing factor regulation, and, finally, to alternative splicing of exons influencing patient survival.

Integrative genomic and transcriptomic analyses illuminate the ontology of HER2-low breast carcinomas.

BACKGROUND: The "HER2-low" nomenclature identifies breast carcinomas (BCs) displaying a HER2 score of 1+/2+ in immunohistochemistry and lacking ERBB2 amplification. Whether HER2-low BCs (HLBCs) constitute a distinct entity is debated. METHODS: We performed DNA and RNA high-throughput analysis on 99 HLBC samples (n = 34 cases with HER2 score 1+/HLBC-1, n = 15 cases with HER2 score 2+ and ERBB2 not amplified/HLBC-2N, and n = 50 cases with score 2+ and ERBB2 copy number in the equivocal range/HLBC-2E). We compared the mutation rates with data from 1317 samples in the Memorial Sloan-Kettering Cancer Center (MSKCC) BC cohort and gene expression data with those from an internal cohort of HER2-negative and HER2-positive BCs. RESULTS: The most represented mutations affected PIK3CA (31/99, 31%), GATA3 (18/99, 18%), TP53 (17/99, 17%), and ERBB2 (8/99, 8%, private to HLBC-2E). Tumor mutational burden was significantly higher in HLBC-1 compared to HLBC-2E/N (P = 0.04). Comparison of mutation spectra revealed that HLBCs were different from both HER2-negative and HER2-positive BCs, with HLBC-1 resembling more HER2-negative tumors and HLBC-2 mutationally related to HER2-addicted tumors. Potentially actionable alterations (annotated by using OncoKB/ESCAT classes) affected 52 patients. Intra-group gene expression revealed overlapping features between HLBC-1 and control HER2-negative BCs, whereas the HLBC-2E tumors showed the highest diversity overall. The RNA-based class discovery analysis unveiled four subsets of tumors with (i) lymphocyte activation, (ii) unique enrichment in HER2-related features, (iii) stromal remodeling alterations, and (iv) actionability of PIK3CA mutations (LAURA classification). CONCLUSIONS: HLBCs harbor distinct genomic features when compared with HER2-positive and HER2-negative BCs; however, differences across IHC classes were also unveiled thus dissecting the full picture of heterogeneity across HER2-low disease. The HLBC-2E category harbors most distinctive features, whereas HLBC-1 seems superimposable to HER2-negative disease. Further studies are needed to ascertain whether the four genomic-driver classes of the LAURA classification hold prognostic and/or predictive implications.

HaTSPiL: A modular pipeline for high-throughput sequencing data analysis.

BACKGROUND: Next generation sequencing methods are widely adopted for a large amount of scientific purposes, from pure research to health-related studies. The decreasing costs per analysis led to big amounts of generated data and to the subsequent improvement of software for the respective analyses. As a consequence, many approaches have been developed to chain different software in order to obtain reliable and reproducible workflows. However, the large range of applications for NGS approaches entails the challenge to manage many different workflows without losing reliability. METHODS: We here present a high-throughput sequencing pipeline (HaTSPiL), a Python-powered CLI tool designed to handle different approaches for data analysis with a high level of reliability. The software relies on the barcoding of filenames using a human readable naming convention that contains any information regarding the sample needed by the software to automatically choose different workflows and parameters. HaTSPiL is highly modular and customisable, allowing the users to extend its features for any specific need. CONCLUSIONS: HaTSPiL is licensed as Free Software under the MIT license and it is available at https://github.com/dodomorandi/hatspil.

Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale for DNA demethylation analysis.

Active DNA demethylation is mediated by ten-eleven translocation (TET) proteins that progressively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). We have developed a methylation-assisted bisulfite sequencing (MAB-seq) method that enables direct genome-scale mapping and quantification of 5fC and 5caC marks together at single-base resolution. In bisulfite sequencing (BS), unmethylated cytosine residues (Cs), 5fCs and 5caCs, are converted to uracil and cannot be discriminated from each other. The pretreatment of the DNA with the CpG methylation enzyme M.SssI, which converts only the Cs to 5mCs, protects Cs but not 5fCs and 5caCs, which enables direct detection of 5fCs and 5caCs as uracils. Here we also describe an adapted version of the protocol to perform reduced-representation MAB-seq (RRMAB-seq) that provides increased coverage on CpG-rich regions, thus reducing the execution costs and increasing the feasibility of the technique. The main advantage of MAB-seq is to reduce the number of chemical/enzymatic DNA treatments required before bisulfite treatment and to avoid the need for prohibitive sequencing coverage, thus making it more reliable and affordable than subtractive approaches. The method presented here is the ideal tool for studying DNA demethylation dynamics in any biological system. Overall timing is ∼3 d for library preparation.

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. Application of this method to mouse embryonic stem cells (ESCs) shows the occurrence of 5fC and 5caC residues on the hypomethylated promoters of highly expressed genes, which is increased upon Tdg silencing, revealing active DNA demethylation on these promoters. Genome-wide mapping of Tdg reveals extensive colocalization with Tet1 on active promoters. These regions were found to be methylated by Dnmt1 and Dnmt3a and demethylated by a Tet-dependent mechanism. Our work demonstrates the DNA methylation dynamics that occurs on the promoters of the expressed genes and provides a genomic reference map of 5fC and 5caC in ESCs.

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. RESULTS: We perform a genome-wide comparative analysis of 5hmC in pluripotent ESCs, as well as in differentiated embryonic and adult cells. We find that 5hmC co-localization with Polycomb repressive complex 2 (PRC2) is specific to ESCs and is absent in differentiated cells. Tet1 in ESCs is distributed on bivalent genes in two independent pools: one with Sin3a centered at non-hydroxymethylated transcription start sites and another centered downstream from these sites. This latter pool of Tet1 co-localizes with 5hmC and PRC2. Through co-immunoprecipitation experiments, we show that Tet1 forms a complex with PRC2 specifically in ESCs. Genome-wide analysis of 5hmC profiles in ESCs following knockdown of the PRC2 subunit Suz12 shows a reduction of 5hmC within promoter sequences, specifically at H3K27me3-positive regions of bivalent promoters. CONCLUSIONS: In ESCs, PRC2 recruits Tet1 to chromatin at H3K27me3 positive regions of the genome, with 5hmC enriched in a broad peak centered 455 bp after the transcription start site and dependent on the PRC2 component Suz12. These results suggest that PRC2-dependent recruitment of Tet1 contributes to epigenetic plasticity throughout cell differentiation.