Genome-wide comparison of DNA hydroxymethylation in mouse embryonic stem cells and neural progenitor cells by a new comparative hMeDIP-seq method

The genome-wide distribution patterns of the ‘6th base’ 5-hydroxymethylcytosine (5hmC) in many tissues and cells have recently been revealed by hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by high throughput sequencing or tiling arrays. However, it has been challenging to directly compare different data sets and samples using data generated by this method. Here, we report a new comparative hMeDIP-seq method, which involves barcoding different input DNA samples at the start and then performing hMeDIP-seq for multiple samples in one hMeDIP reaction. This approach extends the barcode technology from simply multiplexing the DNA deep sequencing outcome and provides significant advantages for quantitative control of all experimental steps, from unbiased hMeDIP to deep sequencing data analysis. Using this improved method, we profiled and compared the DNA hydroxymethylomes of mouse ES cells (ESCs) and mouse ESC-derived neural progenitor cells (NPCs). We identified differentially hydroxymethylated regions (DHMRs) between ESCs and NPCs and uncovered an intricate relationship between the alteration of DNA hydroxymethylation and changes in gene expression during neural lineage commitment of ESCs. Presumably, the DHMRs between ESCs and NPCs uncovered by this approach may provide new insight into the function of 5hmC in gene regulation and neural differentiation. Thus, this newly developed comparative hMeDIP-seq method provides a cost-effective and user-friendly strategy for direct genome-wide comparison of DNA hydroxymethylation across multiple samples, lending significant biological, physiological and clinical implications

METTL14 regulates chromatin bivalent domains in mouse embryonic stem cells

METTL14 (methyltransferase-like 14) is an RNA-binding protein that partners with METTL3 to mediate N6-methyladenosine (m6A) methylation. Recent studies identified a function for METTL3 in heterochromatin in mouse embryonic stem cells (mESCs), but the molecular function of METTL14 on chromatin in mESCs remains unclear. Here, we show that METTL14 specifically binds and regulates bivalent domains, which are marked by trimethylation of histone H3 lysine 27 (H3K27me3) and lysine 4 (H3K4me3). Knockout of Mettl14 results in decreased H3K27me3 but increased H3K4me3 levels, leading to increased transcription. We find that bivalent domain regulation by METTL14 is independent of METTL3 or m6A modification. METTL14 enhances H3K27me3 and reduces H3K4me3 by interacting with and probably recruiting the H3K27 methyltransferase polycomb repressive complex 2 (PRC2) and H3K4 demethylase KDM5B to chromatin. Our findings identify an METTL3-independent role of METTL14 in maintaining the integrity of bivalent domains in mESCs, thus indicating a mechanism of bivalent domain regulation in mammals

Circadian Regulator-Mediated Molecular Subtypes Depict the Features of Tumor Microenvironment and Indicate Prognosis in Head and Neck Squamous Cell Carcinoma

Introduction. Circadian rhythm is involved in multiple biological activities and implicated in cancer development. However, the role of circadian rhythm in head and neck squamous cell carcinoma (HNSCC) has not been fully interpreted yet. Herein, the present study set out to explore the significance of circadian regulator genes (CRGs) in HNSCC. Materials and Methods. The molecular landscape and clinical significance of 13 CRGs in HNSCC were explored based on The Cancer Genome Atlas (TCGA). The biological functions of PER3, a key CRG, were validated by cellular experiments. The correlation of CRGs with microenvironment, pathway activities, and prognosis was determined by bioinformatic algorithms. A novel circadian score was introduced to evaluate the circadian modification pattern of each patient and further validated in an independent cohort from the Gene Expression Omnibus (GEO) dataset. Results. CRGs presented high heterogeneity in HNSCC at both genomic and transcriptomic levels. Specifically, PER3 indicated a better prognosis and inhibited HNSCC cell proliferation. Moreover, HNSCC tissues displayed three circadian regulator patterns with distinct clinical outcomes, transcriptomic characteristics, and microenvironment features. Circadian score was an independent risk factor and exhibited excellent predictive efficiency in both the training cohort from the TCGA database and the validation cohort from the GEO database. Conclusions. CRGs played an indispensable role in HNSCC development. An in-depth exploration of circadian rhythm would improve the understanding of HNSCC carcinogenesis and confer novel insights for future clinical practices

Tumour suppressor TET2 safeguards enhancers from aberrant DNA methylation and epigenetic reprogramming in ERα-positive breast cancer cells

Aberrant DNA methylation is an epigenetic hallmark of malignant tumours. The DNA methylation level is regulated by not only DNA methyltransferases (DNMTs) but also Ten-Eleven Translocation (TET) family proteins. However, the exact role of TET genes in breast cancer remains controversial. Here, we uncover that the ERα-positive breast cancer patients with high TET2 mRNA expression had better overall survival rates. Consistently, knockout of TET2 promotes the tumorigenesis of ERα-positive MCF7 breast cancer cells. Mechanistically, TET2 loss leads to aberrant DNA methylation (gain of 5mC) at a large proportion of enhancers, accompanied by significant reduction in H3K4me1 and H3K27ac enrichment. By analysing the epigenetically reprogrammed enhancers, we identify oestrogen responsive element (ERE) as one of the enriched motifs of transcriptional factors. Importantly, TET2 loss impairs 17beta-oestradiol (E2)-induced transcription of the epigenetically reprogrammed EREs-associated genes through attenuating the binding of ERα. Taken together, these findings shed light on our understanding of the epigenetic mechanisms underlying the enhancer reprogramming during breast cancer pathogenesis

Cytokine status and significant increase of IL-6 and sIL-6R in the aqueous humor of diabetic cataract patients revealed by quantitative multiplexed assays

Objective This study aimed to investigate inflammatory cytokine expression profiles in the aqueous humor (AH) of diabetic cataract (DC) patients. Methods A quantitative multiplexed antibody assay was performed to measure the expression levels of 40 inflammatory cytokines in AH samples from DC and age-related cataract (ARC) patients. Bioinformatics analysis was used to examine the functions of the cytokines. Enzyme-linked immunosorbent assays (ELISAs) and western blots were performed to verify the data. Results The multiplexed antibody assay revealed that the expression levels of IL-6, sIL-6R, IL-17A, IL-8, MCP-1, TNF-β, RANTES, TIMP-1, and TIMP-2 were higher in the AH of DC patients compared with ARC patients. However, IL-1ra and IL-1a expression levels were lower in the DC patient AH samples. Pathway analysis indicated that IL-6 and sIL-6R belong to the class I helical cytokine family, which is associated with many biological functions. ELISA and western blot results confirmed that IL-6R and IL-6 expression levels were significantly higher in DC patients compared with ARC patients. Conclusions Our results revealed the status of 40 inflammatory cytokines in the AH by quantitative multiplexed assays. Additionally, IL-6 and sIL-6R were expressed markedly higher in DC compared with ARC, which may play critical roles in DC pathophysiology

The oxidative demethylase ALKBH3 marks hyperactive gene promoters in human cancer cells

Background: The oxidative DNA demethylase ALKBH3 targets single-stranded DNA (ssDNA) in order to perform DNA alkylation damage repair. ALKBH3 becomes upregulated during tumorigenesis and is necessary for proliferation. However, the underlying molecular mechanism remains to be understood. Methods: To further elucidate the function of ALKBH3 in cancer, we performed ChIP-seq to investigate the genomic binding pattern of endogenous ALKBH3 in PC3 prostate cancer cells coupled with microarray experiments to examine the expression effects of ALKBH3 depletion. Results: We demonstrate that ALKBH3 binds to transcription associated locations, such as places of promoter-proximal paused RNA polymerase II and enhancers. Strikingly, ALKBH3 strongly binds to the transcription initiation sites of a small number of highly active gene promoters. These promoters are characterized by high levels of transcriptional regulators, including transcription factors, the Mediator complex, cohesin, histone modifiers, and active histone marks. Gene expression analysis showed that ALKBH3 does not directly influence the transcription of its target genes, but its depletion induces an upregulation of ALKBH3 non-bound inflammatory genes. Conclusions: The genomic binding pattern of ALKBH3 revealed a putative novel hyperactive promoter type. Further, we propose that ALKBH3 is an intrinsic DNA repair protein that suppresses transcription associated DNA damage at highly expressed genes and thereby plays a role to maintain genomic integrity in ALKBH3-overexpressing cancer cells. These results raise the possibility that ALKBH3 may be a potential target for inhibiting cancer progression. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0180-0) contains supplementary material, which is available to authorized users

A Specific LSD1/KDM1A Isoform Regulates Neuronal Differentiation through H3K9 Demethylation

Lysine-specific demethylase 1 (LSD1) has been reported to repress and activate transcription by mediating histone H3K4me1/2 and H3K9me1/2 demethylation, respectively. The molecular mechanism that underlies this dual substrate specificity has remained unknown. Here we report that an isoform of LSD1, LSD1+8a, does not have the intrinsic capability to demethylate H3K4me2. Instead, LSD1+8a mediates H3K9me2 demethylation in collaboration with supervillin (SVIL), a new LSD1+8a interacting protein. LSD1+8a knockdown increases H3K9me2, but not H3K4me2, levels at its target promoters and compromises neuronal differentiation. Importantly, SVIL co-localizes to LSD1+8a-bound promoters, and its knockdown mimics the impact of LSD1+8a loss, supporting SVIL as a cofactor for LSD1+8a in neuronal cells. These findings provide insight into mechanisms by which LSD1 mediates H3K9me demethylation and highlight alternative splicing as a means by which LSD1 acquires selective substrate specificities (H3K9 versus H3K4) to differentially control specific gene expression programs in neurons

Transcriptional Profiling and miRNA-Target Network Analysis Identify Potential Biomarkers for Efficacy Evaluation of Fuzheng-Huayu Formula-Treated Hepatitis B Caused Liver Cirrhosis

Fuzheng-Huayu (FZHY) formula has been found to have a satisfactory effect on hepatitis B-caused cirrhosis (HBC) treatment. However, the efficacy evaluation of FZHY is often challenging. In this study, a randomized, double-blind and placebo-controlled trial was used to evaluate the therapeutic efficacy of FZHY in HBC treatment. In the trial, 35 medical indexes were detected, and 14 indexes had a statistically-significant difference before compared to after the trial. Importantly, the Child-Pugh score also demonstrated FZHY having therapeutic efficacy. Furthermore, the microRNA (miRNA) profiles of 12 serum samples were detected in FZHY groups, and 112 differential-expressed (DE) miRNAs were determined. Using predicted miRNA targets, 13 kernel miRNAs were identified from the established miRNA-target network. Subsequently, quantitative Real-time Polymerase Chain Reaction (qRT-PCR) was used to validate the expression level of 13 identified miRNAs in the trials. The results showed that nine miRNAs have a statistically-significant difference before compared to after FZHY treatment. By means of a logistic regression model, a miRNA panel with hsa-miR-18a-5p, -326, -1182 and -193b-5p was established, and it can clearly improve the accuracy of the efficacy evaluation of FZHY. This study suggested that the particular miRNAs can act as potential biomarkers and obviously increase the diagnostic accuracy for drug evaluation in HBC treatment progression