Spatial organization of different epigenomic marks was used to infer functions of the epigenome. It remains unclear what can be learned from the temporal changes of the epigenome. Here, we developed a probabilistic model to cluster genomic sequences based on the similarity of temporal changes of multiple epigenomic marks during a cellular differentiation process. We differentiated mouse embryonic stem (ES) cells into mesendoderm cells. At three time points during this differentiation process, we used high-throughput sequencing to measure seven histone modifications and variants--H3K4me1/2/3, H3K27ac, H3K27me3, H3K36me3, and H2A.Z; two DNA modifications--5-mC and 5-hmC; and transcribed mRNAs and noncoding RNAs (ncRNAs). Genomic sequences were clustered based on the spatiotemporal epigenomic information. These clusters not only clearly distinguished gene bodies, promoters, and enhancers, but also were predictive of bidirectional promoters, miRNA promoters, and piRNAs. This suggests specific epigenomic patterns exist on piRNA genes much earlier than germ cell development. Temporal changes of H3K4me2, unmethylated CpG, and H2A.Z were predictive of 5-hmC changes, suggesting unmethylated CpG and H3K4me2 as potential upstream signals guiding TETs to specific sequences. Several rules on combinatorial epigenomic changes and their effects on mRNA expression and ncRNA expression were derived, including a simple rule governing the relationship between 5-hmC and gene expression levels. A Sox17 enhancer containing a FOXA2 binding site and a Foxa2 enhancer containing a SOX17 binding site were identified, suggesting a positive feedback loop between the two mesendoderm transcription factors. These data illustrate the power of using epigenome dynamics to investigate regulatory functions.

N6-methyladenosine (m6A), the most prevalent internal RNA modification on mammalian messenger RNAs, regulates the fates and functions of modified transcripts through m6A-specific binding proteins1-5. In the nervous system, m6A is abundant and modulates various neural functions6-11. Whereas m6A marks groups of mRNAs for coordinated degradation in various physiological processes12-15, the relevance of m6A for mRNA translation in vivo remains largely unknown. Here we show that, through its binding protein YTHDF1, m6A promotes protein translation of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory. Mice with genetic deletion of Ythdf1 show learning and memory defects as well as impaired hippocampal synaptic transmission and long-term potentiation. Re-expression of YTHDF1 in the hippocampus of adult Ythdf1-knockout mice rescues the behavioural and synaptic defects, whereas hippocampus-specific acute knockdown of Ythdf1 or Mettl3, which encodes the catalytic component of the m6A methyltransferase complex, recapitulates the hippocampal deficiency. Transcriptome-wide mapping of YTHDF1-binding sites and m6A sites on hippocampal mRNAs identified key neuronal genes. Nascent protein labelling and tether reporter assays in hippocampal neurons showed that YTHDF1 enhances protein synthesis in a neuronal-stimulus-dependent manner. In summary, YTHDF1 facilitates translation of m6A-methylated neuronal mRNAs in response to neuronal stimulation, and this process contributes to learning and memory.

N7-methylguanosine (m7G) is a positively charged, essential modification at the 5' cap of eukaryotic mRNA, regulating mRNA export, translation, and splicing. m7G also occurs internally within tRNA and rRNA, but its existence and distribution within eukaryotic mRNA remain to be investigated. Here, we show the presence of internal m7G sites within mammalian mRNA. We then performed transcriptome-wide profiling of internal m7G methylome using m7G-MeRIP sequencing (MeRIP-seq). To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively converts internal m7G sites into abasic sites, inducing misincorporation at these sites during reverse transcription. This base-resolution m7G-seq enabled transcriptome-wide mapping of m7G in human tRNA and mRNA, revealing distribution features of the internal m7G methylome in human cells. We also identified METTL1 as a methyltransferase that installs a subset of m7G within mRNA and showed that internal m7G methylation could affect mRNA translation.

Whole-genome profiles of the epigenetic modification 5-hydroxymethylcytosine (5-hmC) are robust diagnostic biomarkers in adult patients with cancer. We investigated if 5-hmC profiles would serve as novel prognostic markers in neuroblastoma, a clinically heterogeneous pediatric cancer. Because this DNA modification facilitates active gene expression, we hypothesized that 5-hmC profiles would identify transcriptomic networks driving the clinical behavior of neuroblastoma.

TET2 is a dioxygenase that catalyses multiple steps of 5-methylcytosine oxidation. Although TET2 mutations frequently occur in various types of haematological malignancies, the mechanism by which they increase risk for these cancers remains poorly understood. Here we show that Tet2-/- mice develop spontaneous myeloid, T- and B-cell malignancies after long latencies. Exome sequencing of Tet2-/- tumours reveals accumulation of numerous mutations, including Apc, Nf1, Flt3, Cbl, Notch1 and Mll2, which are recurrently deleted/mutated in human haematological malignancies. Single-cell-targeted sequencing of wild-type and premalignant Tet2-/- Lin-c-Kit+ cells shows higher mutation frequencies in Tet2-/- cells. We further show that the increased mutational burden is particularly high at genomic sites that gained 5-hydroxymethylcytosine, where TET2 normally binds. Furthermore, TET2-mutated myeloid malignancy patients have significantly more mutational events than patients with wild-type TET2. Thus, Tet2 loss leads to hypermutagenicity in haematopoietic stem/progenitor cells, suggesting a novel TET2 loss-mediated mechanism of haematological malignancy pathogenesis.

DNA N6-methyldeoxyadenosine (6mA) is a well-known prokaryotic DNA modification that has been shown to exist and play epigenetic roles in eukaryotic DNA. Here we report that 6mA accumulates up to ∼0.1-0.2% of total deoxyadenosine during early embryogenesis of vertebrates, but diminishes to the background level with the progression of the embryo development. During this process a large fraction of 6mAs locate in repetitive regions of the genome.

N6-methyladenosine (m6A) on mRNAs is critical for various biological processes, yet whether m6A regulates drug resistance remains unknown. Here we show that developing resistant phenotypes during tyrosine kinase inhibitor (TKI) therapy depends on m6A reduction resulting from FTO overexpression in leukemia cells. This deregulated FTO-m6A axis pre-exists in naïve cell populations that are genetically homogeneous and is inducible/reversible in response to TKI treatment. Cells with mRNA m6A hypomethylation and FTO upregulation demonstrate more TKI tolerance and higher growth rates in mice. Either genetic or pharmacological restoration of m6A methylation through FTO deactivation renders resistant cells sensitive to TKIs. Mechanistically, the FTO-dependent m6A demethylation enhances mRNA stability of proliferation/survival transcripts bearing m6A and subsequently leads to increased protein synthesis. Our findings identify a novel function for the m6A methylation in regulating cell fate decision and demonstrate that dynamic m6A methylome is an additional epigenetic driver of reversible TKI-tolerance state, providing a mechanistic paradigm for drug resistance in cancer.

Traditional epitranscriptomics relies on capturing a single RNA modification by antibody or chemical treatment, combined with short-read sequencing to identify its transcriptomic location. This approach is labor-intensive and may introduce experimental artifacts. Direct sequencing of native RNA using Oxford Nanopore Technologies (ONT) can allow for directly detecting the RNA base modifications, although these modifications might appear as sequencing errors. The percent Error of Specific Bases (%ESB) was higher for native RNA than unmodified RNA, which enabled the detection of ribonucleotide modification sites. Based on the %ESB differences, we developed a bioinformatic tool, epitranscriptional landscape inferring from glitches of ONT signals (ELIGOS), that is based on various types of synthetic modified RNA and applied to rRNA and mRNA. ELIGOS is able to accurately predict known classes of RNA methylation sites (AUC > 0.93) in rRNAs from Escherichiacoli, yeast, and human cells, using either unmodified in vitro transcription RNA or a background error model, which mimics the systematic error of direct RNA sequencing as the reference. The well-known DRACH/RRACH motif was localized and identified, consistent with previous studies, using differential analysis of ELIGOS to study the impact of RNA m6A methyltransferase by comparing wild type and knockouts in yeast and mouse cells. Lastly, the DRACH motif could also be identified in the mRNA of three human cell lines. The mRNA modification identified by ELIGOS is at the level of individual base resolution. In summary, we have developed a bioinformatic software package to uncover native RNA modifications.

Mouse models of colitis have been used to study the pathogenesis of inflammatory bowel disease (IBD) and for pre-clinical development of therapeutic agents. Various epigenetic pathways have been shown to play important regulatory roles in IBD. Reversible N6-methyladenosine (m6A) methylation represents a new layer of post-transcriptional gene regulation that affects a variety of biological processes. We aim to study how deletion of a critical component of m6A writer complex, METTL14, in T cells affects the development of colitis.

The regulation of islet cell biology is critical for glucose homeostasis1.N6 -methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification in mammals2. Here we report that the m6A landscape segregates human type 2 diabetes (T2D) islets from controls significantly better than the transcriptome and that m6A is vital for β-cell biology. m6A-sequencing in human T2D islets reveals several hypomethylated transcripts involved in cell-cycle progression, insulin secretion, and the Insulin/IGF1-AKT-PDX1 pathway. Depletion of m6A levels in EndoC-βH1 induces cell-cycle arrest and impairs insulin secretion by decreasing AKT phosphorylation and PDX1 protein levels. β-cell specific Mettl14 knock-out mice, which display reduced m6A levels, mimic the islet phenotype in human T2D with early diabetes onset and mortality due to decreased β-cell proliferation and insulin degranulation. Our data underscore the significance of RNA methylation in regulating human β-cell biology, and provide a rationale for potential therapeutic targeting of m6A modulators to preserve β-cell survival and function in diabetes.

Internal N6-methyladenosine (m6A) modification is one of the most common and abundant modifications of RNA. However, the biological roles of viral RNA m6A remain elusive. Here, using human metapneumovirus (HMPV) as a model, we demonstrate that m6A serves as a molecular marker for innate immune discrimination of self from non-self RNAs. We show that HMPV RNAs are m6A methylated and that viral m6A methylation promotes HMPV replication and gene expression. Inactivating m6A addition sites with synonymous mutations or demethylase resulted in m6A-deficient recombinant HMPVs and virion RNAs that induced increased expression of type I interferon, which was dependent on the cytoplasmic RNA sensor RIG-I, and not on melanoma differentiation-associated protein 5 (MDA5). Mechanistically, m6A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I and facilitates the conformational change of RIG-I, leading to enhanced interferon expression. Furthermore, m6A-deficient recombinant HMPVs triggered increased interferon in vivo and were attenuated in cotton rats but retained high immunogenicity. Collectively, our results highlight that (1) viruses acquire m6A in their RNA as a means of mimicking cellular RNA to avoid detection by innate immunity and (2) viral RNA m6A can serve as a target to attenuate HMPV for vaccine purposes.

Transcription is a highly dynamic process that generates single-stranded DNA (ssDNA) in the genome as 'transcription bubbles'. Here we describe a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, based on the fast and specific reaction between N3-kethoxal and guanines in ssDNA. KAS-seq allows rapid (within 5 min), sensitive and genome-wide capture and mapping of ssDNA produced by transcriptionally active RNA polymerases or other processes in situ using as few as 1,000 cells. KAS-seq enables definition of a group of enhancers that are single-stranded and enrich unique sequence motifs. These enhancers are associated with specific transcription-factor binding and exhibit more enhancer-promoter interactions than typical enhancers do. Under conditions that inhibit protein condensation, KAS-seq uncovers a rapid release of RNA polymerase II (Pol II) from a group of promoters. KAS-seq thus facilitates fast and accurate analysis of transcription dynamics and enhancer activities simultaneously in both low-input and high-throughput manner.

MicroRNA (miR)-106b-5p has been reported to act as both an oncogene and tumor suppressor in several tumors. The aim of the present study was to investigate the biological function of miR-106b-5p in osteosarcoma (OS). miR-106b-5p expression was observed to be significantly increased in OS tissues and cell lines. MTT assay and flow cytometry analysis determined that miR-106b-5p inhibitor transfection suppressed OS cell proliferation and induced cell cycle G0/G1 phase arrest. Furthermore, bioinformatics analysis and a luciferase reporter assay demonstrated that cyclin-dependent kinase inhibitor 1A (CDKN1A) was a potential target of miR-106b-5p. p21 protein expression was found to be significantly increased by miR-106b-5p downregulation in OS cells. Further analysis demonstrated that CDKN1A was downregulated in OS tissues and was negatively correlated with miR-106b-5p expression. Furthermore, upregulation of CDKN1A expression mimicked, whilst CDKN1A knockdown reversed the suppressive effects of miR-106b-5p inhibitor on OS cell proliferation and cell cycle progression. In summary, the present data suggested that miR-106b-5p promotes cell proliferation and cell cycle progression by directly targeting CDKN1A in OS.

N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m6A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m6A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC) in an m6A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m6A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m6A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m6A readers in post-transcriptional gene regulation and cancer biology.

DNA methylation in the promoters of plant genes sometimes leads to transcriptional repression, and the loss of DNA methylation in methyltransferase mutants results in altered gene expression and severe developmental defects. However, many cases of naturally occurring DNA methylation variations have been reported, whereby altered expression of differentially methylated genes is responsible for agronomically important traits. The ability to manipulate plant methylomes to generate epigenetically distinct individuals could be invaluable for breeding and research purposes. Here, we describe "epimutagenesis," a method to rapidly generate DNA methylation variation through random demethylation of the Arabidopsis thaliana genome. This method involves the expression of a human ten-eleven translocation (TET) enzyme, and results in widespread hypomethylation that can be inherited to subsequent generations, mimicking mutants in the maintenance of DNA methyltransferase met1. Application of epimutagenesis to agriculturally significant plants may result in differential expression of alleles normally silenced by DNA methylation, uncovering previously hidden phenotypic variations.

Protein aggregation is the hallmark of neurodegeneration, but the molecular mechanisms underlying late-onset Alzheimer's disease (AD) are unclear. Here we integrated transcriptomic, proteomic and epigenomic analyses of postmortem human brains to identify molecular pathways involved in AD. RNA sequencing analysis revealed upregulation of transcription- and chromatin-related genes, including the histone acetyltransferases for H3K27ac and H3K9ac. An unbiased proteomic screening singled out H3K27ac and H3K9ac as the main enrichments specific to AD. In turn, epigenomic profiling revealed gains in the histone H3 modifications H3K27ac and H3K9ac linked to transcription, chromatin and disease pathways in AD. Increasing genome-wide H3K27ac and H3K9ac in a fly model of AD exacerbated amyloid-β42-driven neurodegeneration. Together, these findings suggest that AD involves a reconfiguration of the epigenome, wherein H3K27ac and H3K9ac affect disease pathways by dysregulating transcription- and chromatin-gene feedback loops. The identification of this process highlights potential epigenetic strategies for early-stage disease treatment.

The dynamic and reversible N6-methyladenosine (m6A) RNA modification installed and erased by N6-methyltransferases and demethylases regulates gene expression and cell fate. We show that the m6A demethylase ALKBH5 is highly expressed in glioblastoma stem-like cells (GSCs). Silencing ALKBH5 suppresses the proliferation of patient-derived GSCs. Integrated transcriptome and m6A-seq analyses revealed altered expression of certain ALKBH5 target genes, including the transcription factor FOXM1. ALKBH5 demethylates FOXM1 nascent transcripts, leading to enhanced FOXM1 expression. Furthermore, a long non-coding RNA antisense to FOXM1 (FOXM1-AS) promotes the interaction of ALKBH5 with FOXM1 nascent transcripts. Depleting ALKBH5 and FOXM1-AS disrupted GSC tumorigenesis through the FOXM1 axis. Our work uncovers a critical function for ALKBH5 and provides insight into critical roles of m6A methylation in glioblastoma.

DNA modifications such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are epigenetic marks known to affect global gene expression in mammals. Given their prevalence in the human genome, close correlation with gene expression and high chemical stability, these DNA epigenetic marks could serve as ideal biomarkers for cancer diagnosis. Taking advantage of a highly sensitive and selective chemical labeling technology, we report here the genome-wide profiling of 5hmC in circulating cell-free DNA (cfDNA) and in genomic DNA (gDNA) of paired tumor and adjacent tissues collected from a cohort of 260 patients recently diagnosed with colorectal, gastric, pancreatic, liver or thyroid cancer and normal tissues from 90 healthy individuals. 5hmC was mainly distributed in transcriptionally active regions coincident with open chromatin and permissive histone modifications. Robust cancer-associated 5hmC signatures were identified in cfDNA that were characteristic for specific cancer types. 5hmC-based biomarkers of circulating cfDNA were highly predictive of colorectal and gastric cancers and were superior to conventional biomarkers and comparable to 5hmC biomarkers from tissue biopsies. Thus, this new strategy could lead to the development of effective, minimally invasive methods for diagnosis and prognosis of cancer from the analyses of blood samples.

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen triggers an unfolded protein response (UPR) for stress adaptation, the failure of which induces cell apoptosis and tissue/organ damage. The molecular switches underlying how the UPR selects for stress adaptation over apoptosis remain unknown. Here, we discovered that accumulation of unfolded/misfolded proteins selectively induces N6-adenosine-methyltransferase-14 (METTL14) expression. METTL14 promotes C/EBP-homologous protein (CHOP) mRNA decay through its 3' UTR N6-methyladenosine (m6A) to inhibit its downstream pro-apoptotic target gene expression. UPR induces METTL14 expression by competing against the HRD1-ER-associated degradation (ERAD) machinery to block METTL14 ubiquitination and degradation. Therefore, mice with liver-specific METTL14 deletion are highly susceptible to both acute pharmacological and alpha-1 antitrypsin (AAT) deficiency-induced ER proteotoxic stress and liver injury. Further hepatic CHOP deletion protects METTL14 knockout mice from ER-stress-induced liver damage. Our study reveals a crosstalk between ER stress and mRNA m6A modification pathways, termed the ERm6A pathway, for ER stress adaptation to proteotoxicity.

Anti-PD-1/PD-L1 immunotherapy has achieved impressive therapeutic outcomes in patients with multiple cancer types. However, the underlined molecular mechanism(s) for moderate response rate (15-25%) or resistance to PD-1/PD-L1 blockade remains not completely understood. Here, we report that inhibiting the deubiquitinase, USP8, significantly enhances the efficacy of anti-PD-1/PD-L1 immunotherapy through reshaping an inflamed tumor microenvironment (TME). Mechanistically, USP8 inhibition increases PD-L1 protein abundance through elevating the TRAF6-mediated K63-linked ubiquitination of PD-L1 to antagonize K48-linked ubiquitination and degradation of PD-L1. In addition, USP8 inhibition also triggers innate immune response and MHC-I expression largely through activating the NF-κB signaling. Based on these mechanisms, USP8 inhibitor combination with PD-1/PD-L1 blockade significantly activates the infiltrated CD8+ T cells to suppress tumor growth and improves the survival benefit in several murine tumor models. Thus, our study reveals a potential combined therapeutic strategy to utilize a USP8 inhibitor and PD-1/PD-L1 blockade for enhancing anti-tumor efficacy.