Modeling methyl-sensitive transcription factor motifs with an expanded epigenetic alphabet.

BACKGROUND: Transcription factors bind DNA in specific sequence contexts. In addition to distinguishing one nucleobase from another, some transcription factors can distinguish between unmodified and modified bases. Current models of transcription factor binding tend not to take DNA modifications into account, while the recent few that do often have limitations. This makes a comprehensive and accurate profiling of transcription factor affinities difficult. RESULTS: Here, we develop methods to identify transcription factor binding sites in modified DNA. Our models expand the standard A/C/G/T DNA alphabet to include cytosine modifications. We develop Cytomod to create modified genomic sequences and we also enhance the MEME Suite, adding the capacity to handle custom alphabets. We adapt the well-established position weight matrix (PWM) model of transcription factor binding affinity to this expanded DNA alphabet. Using these methods, we identify modification-sensitive transcription factor binding motifs. We confirm established binding preferences, such as the preference of ZFP57 and C/EBPß for methylated motifs and the preference of c-Myc for unmethylated E-box motifs. CONCLUSIONS: Using known binding preferences to tune model parameters, we discover novel modified motifs for a wide array of transcription factors. Finally, we validate our binding preference predictions for OCT4 using cleavage under targets and release using nuclease (CUT&RUN) experiments across conventional, methylation-, and hydroxymethylation-enriched sequences. Our approach readily extends to other DNA modifications. As more genome-wide single-base resolution modification data becomes available, we expect that our method will yield insights into altered transcription factor binding affinities across many different modifications.

Biological roles of RNA m7G modification and its implications in cancer.

M7G modification, known as one of the common post-transcriptional modifications of RNA, is present in many different types of RNAs. With the accurate identification of m7G modifications within RNAs, their functional roles in the regulation of gene expression and different physiological functions have been revealed. In addition, there is growing evidence that m7G modifications are crucial in the emergence of cancer. Here, we review the most recent findings regarding the detection techniques, distribution, biological functions and Regulators of m7G. We also summarize the connections between m7G modifications and cancer development, drug resistance, and tumor microenvironment as well as we discuss the research's future directions and trends.

The chromatin and single-cell transcriptional landscapes of CD4 T cells in inflammatory bowel disease link risk loci with a proinflammatory Th17 cell population.

Introduction: The imbalance between Th17 and regulatory T cells in inflammatory bowel diseases (IBD) promotes intestinal epithelial cell damage. In this scenario, T helper cell lineage commitment is accompanied by dynamic changes to the chromatin that facilitate or repress gene expression. Methods: Here, we characterized the chromatin landscape and heterogeneity of intestinal and peripheral CD4 T cellsfrom IBD patients using in house ATAC-Seq and single cell RNA-Seq libraries. Results: We show that chromatin accessibility profiles of CD4 T cells from inflamed intestinal biopsies relate to genes associated with a network of inflammatory processes. After integrating the chromatin profiles of tissue-derived CD4 T cells and in-vitro polarized CD4 T cell subpopulations, we found that the chromatin accessibility changes of CD4 T cells were associated with a higher predominance of pathogenic Th17 cells (pTh17 cells) in inflamed biopsies. In addition, IBD risk loci in CD4 T cells were colocalized with accessible chromatin changes near pTh17-related genes, as shown in intronic STAT3 and IL23R regions enriched in areas of active intestinal inflammation. Moreover, single cell RNA-Seq analysis revealed a population of pTh17 cells that co-expresses Th1 and cytotoxic transcriptional programs associated with IBD severity. Discussion: Altogether, we show that cytotoxic pTh17 cells were specifically associated with IBD genetic variants and linked to intestinal inflammation of IBD patients.

Cerebrospinal fluid methylome-based liquid biopsies for accurate malignant brain neoplasm classification.

BACKGROUND: Resolving the differential diagnosis between brain metastases (BM), glioblastomas (GBM), and central nervous system lymphomas (CNSL) is an important dilemma for the clinical management of the main three intra-axial brain tumor types. Currently, treatment decisions require invasive diagnostic surgical biopsies that carry risks and morbidity. This study aimed to utilize methylomes from cerebrospinal fluid (CSF), a biofluid proximal to brain tumors, for reliable non-invasive classification that addresses limitations associated with low target abundance in existing approaches. METHODS: Binomial GLMnet classifiers of tumor type were built, in fifty iterations of 80% discovery sets, using CSF methylomes obtained from 57 BM, GBM, CNSL, and non-neoplastic control patients. Publicly-available tissue methylation profiles (N = 197) on these entities and normal brain parenchyma were used for validation and model optimization. RESULTS: Models reliably distinguished between BM (area under receiver operating characteristic curve [AUROC] = 0.93, 95% confidence interval [CI]: 0.71-1.0), GBM (AUROC = 0.83, 95% CI: 0.63-1.0), and CNSL (AUROC = 0.91, 95% CI: 0.66-1.0) in independent 20% validation sets. For validation, CSF-based methylome signatures reliably distinguished between tumor types within external tissue samples and tumors from non-neoplastic controls in CSF and tissue. CSF methylome signals were observed to align closely with tissue signatures for each entity. An additional set of optimized CSF-based models, built using tumor-specific features present in tissue data, showed enhanced classification accuracy. CONCLUSIONS: CSF methylomes are reliable for liquid biopsy-based classification of the major three malignant brain tumor types. We discuss how liquid biopsies may impact brain cancer management in the future by avoiding surgical risks, classifying unbiopsiable tumors, and guiding surgical planning when resection is indicated.

Single-nucleus multiomic atlas of frontal cortex in amyotrophic lateral sclerosis with a deep learning-based decoding of alternative polyadenylation mechanisms.

The understanding of how different cell types contribute to amyotrophic lateral sclerosis (ALS) pathogenesis is limited. Here we generated a single-nucleus transcriptomic and epigenomic atlas of the frontal cortex of ALS cases with C9orf72 (C9) hexanucleotide repeat expansions and sporadic ALS (sALS). Our findings reveal shared pathways in C9-ALS and sALS, characterized by synaptic dysfunction in excitatory neurons and a disease-associated state in microglia. The disease subtypes diverge with loss of astrocyte homeostasis in C9-ALS, and a more substantial disturbance of inhibitory neurons in sALS. Leveraging high depth 3'-end sequencing, we found a widespread switch towards distal polyadenylation (PA) site usage across ALS subtypes relative to controls. To explore this differential alternative PA (APA), we developed APA-Net, a deep neural network model that uses transcript sequence and expression levels of RNA-binding proteins (RBPs) to predict cell-type specific APA usage and RBP interactions likely to regulate APA across disease subtypes.

The cell-free DNA methylome captures distinctions between localized and metastatic prostate tumors.

Metastatic prostate cancer remains a major clinical challenge and metastatic lesions are highly heterogeneous and difficult to biopsy. Liquid biopsy provides opportunities to gain insights into the underlying biology. Here, using the highly sensitive enrichment-based sequencing technology, we provide analysis of 60 and 175 plasma DNA methylomes from patients with localized and metastatic prostate cancer, respectively. We show that the cell-free DNA methylome can capture variations beyond the tumor. A global hypermethylation in metastatic samples is observed, coupled with hypomethylation in the pericentromeric regions. Hypermethylation at the promoter of a glucocorticoid receptor gene NR3C1 is associated with a decreased immune signature. The cell-free DNA methylome is reflective of clinical outcomes and can distinguish different disease types with 0.989 prediction accuracy. Finally, we show the ability of predicting copy number alterations from the data, providing opportunities for joint genetic and epigenetic analysis on limited biological samples.

Sensitive and reproducible cell-free methylome quantification with synthetic spike-in controls.

Cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) identifies genomic regions with DNA methylation, using a protocol adapted to work with low-input DNA samples and with cell-free DNA (cfDNA). We developed a set of synthetic spike-in DNA controls for cfMeDIP-seq to provide a simple and inexpensive reference for quantitative normalization. We designed 54 DNA fragments with combinations of methylation status (methylated and unmethylated), fragment length (80 bp, 160 bp, 320 bp), G + C content (35%, 50%, 65%), and fraction of CpG dinucleotides within the fragment (1/80 bp, 1/40 bp, 1/20 bp). Using 0.01 ng of spike-in controls enables training a generalized linear model that absolutely quantifies methylated cfDNA in MeDIP-seq experiments. It mitigates batch effects and corrects for biases in enrichment due to known biophysical properties of DNA fragments and other technical biases.

RURAL LEFT-BEHIND CHILDREN ARE SERIOUSLY AFFLICTED WITH INTESTINAL HELMINTH INFECTIONS IN SOUTHERN SICHUAH, CHINA.

Rural children are seriously afflicted with intestinal helminth infections in China. Of note, the term rural children includes rural left-behind children (LBC) and rural non-left-behind children (NLBC); the difference in the prevalence of intestinal helminths between the 2 groups remains unclear. In this study, Gulin and Xuyong counties in southern Sichuan were chosen for investigation in 2019. The Kato Katz thick smear method was used to detect the presence of intestinal helminth eggs in rural children. For children aged 3-6 yr, the adhesive tape perianal swab method was used to detect Enterobius vermicularis and tapeworm eggs. Statistical differences in infection rates between the 2 groups were determined by the chi-square test. In total, 1,608 rural children, 911 LBC and 697 NLBC, participated in the investigation. Six species of intestinal helminths were detected. A total of 358 (39.3%) and 130 (18.7%) intestinal helminth positives were found among LBC and NLBC, respectively; the former had a higher (P < 0.05) infection level. Moreover, an analysis of double worm infection rates among intestinal helminth positive LBC and NLBC showed a difference between the 2 groups that was also statistically significant. These surveys indicated that the risk of intestinal helminth infection was substantially higher and the severity of infection much worse in rural LBC in southern Sichuan. More attention should be paid to the parasitic infection of LBC.

Tumor-Naïve Multimodal Profiling of Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma.

PURPOSE: Circulating tumor DNA (ctDNA) enables personalized treatment strategies in oncology by providing a noninvasive source of clinical biomarkers. In patients with low ctDNA abundance, tumor-naïve methods are needed to facilitate clinical implementation. Here, using locoregionally confined head and neck squamous cell carcinoma (HNSCC) as an example, we demonstrate tumor-naïve detection of ctDNA by simultaneous profiling of mutations and methylation. EXPERIMENTAL DESIGN: We conducted CAncer Personalized Profiling by deep Sequencing (CAPP-seq) and cell-free Methylated DNA ImmunoPrecipitation and high-throughput sequencing (cfMeDIP-seq) for detection of ctDNA-derived somatic mutations and aberrant methylation, respectively. We analyzed 77 plasma samples from 30 patients with stage I-IVA human papillomavirus-negative HNSCC as well as plasma samples from 20 risk-matched healthy controls. In addition, we analyzed leukocytes from patients and controls. RESULTS: CAPP-seq identified mutations in 20 of 30 patients at frequencies similar to that of The Tumor Genome Atlas (TCGA). Differential methylation analysis of cfMeDIP-seq profiles identified 941 ctDNA-derived hypermethylated regions enriched for CpG islands and HNSCC-specific methylation patterns. Both methods demonstrated an association between ctDNA abundance and shorter fragment lengths. In addition, mutation- and methylation-based ctDNA abundance was highly correlated (r > 0.85). Patients with detectable pretreatment ctDNA by both methods demonstrated significantly worse overall survival (HR = 7.5; P = 0.025) independent of clinical stage, with lack of ctDNA clearance post-treatment strongly correlating with recurrence. We further leveraged cfMeDIP-seq profiles to validate a prognostic signature identified from TCGA samples. CONCLUSIONS: Tumor-naïve detection of ctDNA by multimodal profiling may facilitate biomarker discovery and clinical use in low ctDNA abundance applications.

DNA hypomethylating agents increase activation and cytolytic activity of CD8+ T cells.

We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8+ T cells. In vivo HMA treatment promotes CD8+ T cell tumor infiltration and suppresses tumor growth via CD8+ T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8+ T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8+ T cell subpopulations, increasing both the number and abundance of a granzyme Bhigh, perforinhigh effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.