Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.

BAllC and BAllCools: Efficient Formatting and Operating for Single-Cell DNA Methylation Data.

Motivation: With single-cell DNA methylation studies yielding vast datasets, existing data formats struggle with the unique challenges of storage and efficient operations, highlighting a need for improved solutions. Results: BAllC (Binary All Cytosines) emerges as a tailored binary format for methylation data, addressing these challenges. BAllCools, its complementary software toolkit, enhances parsing, indexing, and querying capabilities, promising superior operational speeds and reduced storage needs. Availability: https://github.com/jksr/ballcools.

Human Immune Cell Epigenomic Signatures in Response to Infectious Diseases and Chemical Exposures.

Variations in DNA methylation patterns in human tissues have been linked to various environmental exposures and infections. Here, we identified the DNA methylation signatures associated with multiple exposures in nine major immune cell types derived from peripheral blood mononuclear cells (PBMCs) at single-cell resolution. We performed methylome sequencing on 111,180 immune cells obtained from 112 individuals who were exposed to different viruses, bacteria, or chemicals. Our analysis revealed 790,662 differentially methylated regions (DMRs) associated with these exposures, which are mostly individual CpG sites. Additionally, we integrated methylation and ATAC-seq data from same samples and found strong correlations between the two modalities. However, the epigenomic remodeling in these two modalities are complementary. Finally, we identified the minimum set of DMRs that can predict exposures. Overall, our study provides the first comprehensive dataset of single immune cell methylation profiles, along with unique methylation biomarkers for various biological and chemical exposures.

Comparative epigenome analysis using Infinium DNA methylation BeadChips.

The arrival of the Infinium DNA methylation BeadChips for mice and other nonhuman mammalian species has outpaced the development of the informatics that supports their use for epigenetics study in model organisms. Here, we present informatics infrastructure and methods to allow easy DNA methylation analysis on multiple species, including domesticated animals and inbred laboratory mice (in SeSAMe version 1.16.0+). First, we developed a data-driven analysis pipeline covering species inference, genome-specific data preprocessing and regression modeling. We targeted genomes of 310 species and 37 inbred mouse strains and showed that genome-specific preprocessing prevents artifacts and yields more accurate measurements than generic pipelines. Second, we uncovered the dynamics of the epigenome evolution in different genomic territories and tissue types through comparative analysis. We identified a catalog of inbred mouse strain-specific methylation differences, some of which are linked to the strains' immune, metabolic and neurological phenotypes. By streamlining DNA methylation array analysis for undesigned genomes, our methods extend epigenome research to broad species contexts.

PyComplexHeatmap: a Python package to visualize multimodal genomics data.

Python has emerged as a robust programming language increasingly employed in genomics data analysis, largely due to its comprehensive deep learning libraries and proficiency in handling large-scale data, such as single-cell multi-omics datasets. Although Python has become a prominent data science ecosystem for bioinformatics, there remains a growing demand for advanced heatmap visualization and assembly tools, which are not sufficiently addressed by existing Python-based data visualization libraries. We present PyComplexHeatmap, an all-inclusive Python library for heatmap visualization, inspired by the ComplexHeatmap package currently available in R. PyComplexHeatmap is built upon the matplotlib library and features a versatile, modular interface that seamlessly integrates with other Python-based data science tools, such as Pandas, NumPy, and genomics tools, such as Scanpy, in a standard-compliant manner. This library caters to the requirements of exquisite rendering of multimodal matrix data, incorporating both textual and graphical annotations, thereby enabling efficient integrative analysis of multimodal data and associated metadata.

DNA methylation dynamics and dysregulation delineated by high-throughput profiling in the mouse.

We have developed a mouse DNA methylation array that contains 296,070 probes representing the diversity of mouse DNA methylation biology. We present a mouse methylation atlas as a rich reference resource of 1,239 DNA samples encompassing distinct tissues, strains, ages, sexes, and pathologies. We describe applications for comparative epigenomics, genomic imprinting, epigenetic inhibitors, patient-derived xenograft assessment, backcross tracing, and epigenetic clocks. We dissect DNA methylation processes associated with differentiation, aging, and tumorigenesis. Notably, we find that tissue-specific methylation signatures localize to binding sites for transcription factors controlling the corresponding tissue development. Age-associated hypermethylation is enriched at regions of Polycomb repression, while hypomethylation is enhanced at regions bound by cohesin complex members. Apc Min/+ polyp-associated hypermethylation affects enhancers regulating intestinal differentiation, while hypomethylation targets AP-1 binding sites. This Infinium Mouse Methylation BeadChip (version MM285) is widely accessible to the research community and will accelerate high-sample-throughput studies in this important model organism.

Autonomous climbing: An effective exercise mode with beneficial outcomes of aerobic exercise and resistance training.

AIMS: To assess the effects of three specific exercise training modes, aerobic exercise (A), resistance training (R) and autonomous climbing (AC), aimed at proposing a cross-training method, on improving the physical, molecular and metabolic characteristics of mice without many side effects. MATERIALS AND METHODS: Seven-week-old male mice were randomly divided into four groups: control (C), aerobic exercise (A), resistance training (R), and autonomous climbing (AC) groups. Physical changes in mice were tracked and analysed to explore the similarities and differences of these three exercise modes. Histochemistry, quantitative real-time PCR (RT-PCR), western blot (WB) and metabolomics analysis were performed to identify the underlying relationships among the three training modes. KEY FINDINGS: Mice in the AC group showed better body weight control, glucose and energy homeostasis. Molecular markers of myogenesis, hypertrophy, antidegradation and mitochondrial function were highly expressed in the muscle of mice after autonomous climbing. The serum metabolomics landscape and enriched pathway comparison indicated that the aerobic oxidation pathway (pentose phosphate pathway, galactose metabolism and fatty acid degradation) and amino acid metabolism pathway (tyrosine, arginine and proline metabolism) were significantly enriched in group AC, suggesting an increased muscle mitochondrial function and protein balance ability of mice after autonomous climbing. SIGNIFICANCE: We propose a new exercise mode, autonomous climbing, as a convenient but effective training method that combines the beneficial effects of aerobic exercise and resistance training.

Co-occurrence and Mutual Exclusivity Analysis of DNA Methylation Reveals Distinct Subtypes in Multiple Cancers.

Co-occurrence and mutual exclusivity (COME) of DNA methylation refer to two or more genes that tend to be positively or negatively correlated in DNA methylation among different samples. Although COME of gene mutations in pan-cancer have been well explored, little is known about the COME of DNA methylation in pan-cancer. Here, we systematically explored the COME of DNA methylation profile in diverse human cancer. A total of 5,128,332 COME events were identified in 14 main cancers types in The Cancer Genome Atlas (TCGA). We also identified functional epigenetic modules of the zinc finger gene family in six cancer types by integrating the gene expression and DNA methylation data and the frequently occurred COME network. Interestingly, most of the genes in those functional epigenetic modules are epigenetically repressed. Strikingly, those frequently occurred COME events could be used to classify the patients into several subtypes with significant different clinical outcomes in six cancers as well as pan-cancer (p-value ≤ = 0.05). Moreover, we observed significant associations between different COME subtypes and clinical features (e.g., age, gender, histological type, neoplasm histologic grade, and pathologic stage) in distinct cancers. Taken together, we identified millions of COME events of DNA methylation in pan-cancer and detected functional epigenetic COME events that could separate tumor patients into different subtypes, which may benefit the diagnosis and prognosis of pan-cancer.

DNMIVD: DNA methylation interactive visualization database.

Aberrant DNA methylation plays an important role in cancer progression. However, no resource has been available that comprehensively provides DNA methylation-based diagnostic and prognostic models, expression-methylation quantitative trait loci (emQTL), pathway activity-methylation quantitative trait loci (pathway-meQTL), differentially variable and differentially methylated CpGs, and survival analysis, as well as functional epigenetic modules for different cancers. These provide valuable information for researchers to explore DNA methylation profiles from different aspects in cancer. To this end, we constructed a user-friendly database named DNA Methylation Interactive Visualization Database (DNMIVD), which comprehensively provides the following important resources: (i) diagnostic and prognostic models based on DNA methylation for multiple cancer types of The Cancer Genome Atlas (TCGA); (ii) meQTL, emQTL and pathway-meQTL for diverse cancers; (iii) Functional Epigenetic Modules (FEM) constructed from Protein-Protein Interactions (PPI) and Co-Occurrence and Mutual Exclusive (COME) network by integrating DNA methylation and gene expression data of TCGA cancers; (iv) differentially variable and differentially methylated CpGs and differentially methylated genes as well as related enhancer information; (v) correlations between methylation of gene promoter and corresponding gene expression and (vi) patient survival-associated CpGs and genes with different endpoints. DNMIVD is freely available at http://www.unimd.org/dnmivd/. We believe that DNMIVD can facilitate research of diverse cancers.

Integrative analysis identifies potential DNA methylation biomarkers for pan-cancer diagnosis and prognosis.

DNA methylation status is closely associated with diverse diseases, and is generally more stable than gene expression, thus abnormal DNA methylation could be important biomarkers for tumor diagnosis, treatment and prognosis. However, the signatures regarding DNA methylation changes for pan-cancer diagnosis and prognosis are less explored. Here we systematically analyzed the genome-wide DNA methylation patterns in diverse TCGA cancers with machine learning. We identified seven CpG sites that could effectively discriminate tumor samples from adjacent normal tissue samples for 12 main cancers of TCGA (1216 samples, AUC > 0.99). Those seven potential diagnostic biomarkers were further validated in the other 9 different TCGA cancers and 4 independent datasets (AUC > 0.92). Three out of the seven CpG sites were correlated with cell division, DNA replication and cell cycle. We also identified 12 CpG sites that can effectively distinguish 26 different cancers (7605 samples), and the result was repeatable in independent datasets as well as two disparate tumors with metastases (micro-average AUC > 0.89). Furthermore, a series of potential signatures that could significantly predict the prognosis of tumor patients for 7 different cancer were identified via survival analysis (p-value < 1e-4). Collectively, DNA methylation patterns vary greatly between tumor and adjacent normal tissues, as well as among different types of cancers. Our identified signatures may aid the decision of clinical diagnosis and prognosis for pan-cancer and the potential cancer-specific biomarkers could be used to predict the primary site of metastatic breast and prostate cancers.

In Vitro Modeling of Human Germ Cell Development Using Pluripotent Stem Cells.

Due to differences across species, the mechanisms of cell fate decisions determined in mice cannot be readily extrapolated to humans. In this study, we developed a feeder- and xeno-free culture protocol that efficiently induced human pluripotent stem cells (iPSCs) into PLZF+/GPR125+/CD90+ spermatogonium-like cells (SLCs). These SLCs were enriched with key genes in germ cell development such as MVH, DAZL, GFRα1, NANOS3, and DMRT1. In addition, a small fraction of SLCs went through meiosis in vitro to develop into haploid cells. We further demonstrated that this chemically defined induction protocol faithfully recapitulated the features of compromised germ cell development of PSCs with NANOS3 deficiency or iPSC lines established from patients with non-obstructive azoospermia. Taken together, we established a powerful experimental platform to investigate human germ cell development and pathology related to male infertility.