A molecular pathway for cancer cachexia-induced muscle atrophy revealed at single-nucleus resolution.

Cancer cachexia is a prevalent and often fatal wasting condition that cannot be fully reversed with nutritional interventions. Muscle atrophy is a central component of the syndrome, but the mechanisms whereby cancer leads to skeletal muscle atrophy are not well understood. We performed single-nucleus multi-omics on skeletal muscles from a mouse model of cancer cachexia and profiled the molecular changes in cachexic muscle. Our results revealed the activation of a denervation-dependent gene program that upregulates the transcription factor myogenin. Further studies showed that a myogenin-myostatin pathway promotes muscle atrophy in response to cancer cachexia. Short hairpin RNA inhibition of myogenin or inhibition of myostatin through overexpression of its endogenous inhibitor follistatin prevented cancer cachexia-induced muscle atrophy in mice. Our findings uncover a molecular basis of muscle atrophy associated with cancer cachexia and highlight potential therapeutic targets for this disorder.

Single-nucleus transcriptional and chromatin accessible profiles reveal critical cell types and molecular architecture underlying chicken sex determination.

INTRODUCTION: Understanding the sex determination mechanisms in birds has great significance for the biological sciences and production in the poultry industry. Sex determination in chickens is a complex process that involves fate decisions of supporting cells such as granulosa or Sertoli cells. However, a systematic understanding of the genetic regulation and cell commitment process underlying sex determination in chickens is still lacking. OBJECTIVES: We aimed to dissect the molecular characteristics associated with sex determination in the gonads of chicken embryos. METHODS: Single-nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) analysis were conducted on the gonads of female and male chickens at embryonic day 3.5 (E3.5), E4.5, and E5.5. RESULTS: Here, we provided a time-course transcriptional and chromatin accessible profiling of gonads during chicken sex determination at single-cell resolution. We uncovered differences in cell composition and developmental trajectories between female and male gonads and found that the divergence of transcription and accessibility in gonadal cells first emerged at E5.5. Furthermore, we revealed key cell-type-specific transcription factors (TFs) and regulatory networks that drive lineage commitment. Sex determination signaling pathways, dominated by BMP signaling, are preferentially activated in males during gonadal development. Further pseudotime analysis of the supporting cells indicated that granulosa cells were regulated mainly by the TEAD gene family and that Sertoli cells were driven by the DMRT1 regulons. Cross-species analysis suggested high conservation of both cell types and cell-lineage-specific TFs across the six vertebrates. CONCLUSIONS: Overall, our study will contribute to accelerating the development of sex manipulation technology in the poultry industry and the application of chickens as a unique model for studying cell fate decisions.

Complementation testing identifies genes mediating effects at quantitative trait loci underlying fear-related behavior.

Knowing the genes involved in quantitative traits provides an entry point to understanding the biological bases of behavior, but there are very few examples where the pathway from genetic locus to behavioral change is known. To explore the role of specific genes in fear behavior, we mapped three fear-related traits, tested fourteen genes at six quantitative trait loci (QTLs) by quantitative complementation, and identified six genes. Four genes, Lamp, Ptprd, Nptx2, and Sh3gl, have known roles in synapse function; the fifth, Psip1, was not previously implicated in behavior; and the sixth is a long non-coding RNA, 4933413L06Rik, of unknown function. Variation in transcriptome and epigenetic modalities occurred preferentially in excitatory neurons, suggesting that genetic variation is more permissible in excitatory than inhibitory neuronal circuits. Our results relieve a bottleneck in using genetic mapping of QTLs to uncover biology underlying behavior and prompt a reconsideration of expected relationships between genetic and functional variation.

Single-nucleus RNA and ATAC sequencing analyses provide molecular insights into early pod development of peanut fruit.

Peanut (Arachis hypogaea L.) is an important leguminous oil and economic crop that produces flowers aboveground and fruits belowground. Subterranean fruit-pod development, which significantly affects peanut production, involves complex molecular mechanisms that likely require the coordinated regulation of multiple genes in different tissues. To investigate the molecular mechanisms that underlie peanut fruit-pod development, we characterized the anatomical features of early fruit-pod development and integrated single-nucleus RNA-sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) data at the single-cell level. We identified distinct cell types, such as meristem, embryo, vascular tissue, cuticular layer, and stele cells within the shell wall. These specific cell types were used to examine potential molecular changes unique to each cell type during pivotal stages of fruit-pod development. snRNA-seq analyses of differentially expressed genes revealed cell-type-specific insights that were not previously obtainable from transcriptome analyses of bulk RNA. For instance, we identified MADS-box genes that contributes to the formation of parenchyma cells and gravity-related genes that are present in the vascular cells, indicating an essential role for the vascular cells in peg gravitropism. Overall, our single-nucleus analysis provides comprehensive and novel information on specific cell types, gene expression, and chromatin accessibility during the early stages of fruit-pod development. This information will enhance our understanding of the mechanisms that underlie fruit-pod development in peanut and contribute to efforts aimed at improving peanut production.

Single-Nucleus ATAC-seq for Mapping Chromatin Accessibility in Individual Cells of Murine Hearts.

During the last decade a wide range of single-cell and single-nucleus next-generation sequencing techniques have been developed, which revolutionized detection of rare cell populations, enabling creation of comprehensive cell atlases of complex organs and tissues. State-of-the-art methods do not only allow classical transcriptomics of individual cells but also comprise a number of epigenetic approaches, including assessment of chromatin accessibility by single-nucleus Assay for Transposase Accessible Chromatin ATAC-seq (snATAC-seq). The snATAC-seq assay detects "open chromatin," a term for low nucleosome occupancy of genomic regions, which is a prerequisite for effective transcription factor binding. Information about open chromatin at the single-nucleus level helps to recognize epigenetic changes, sometimes before transcription of respective genes occurs. snATAC-seq detects cellular heterogeneity in otherwise still transcriptionally and/or morphologically homogeneous cell populations. Chromatin accessibility assays may be used to detect epigenetic changes in cardiac lineages during heart development, chromatin landscape changes during aging, and epigenetic alterations in heart diseases. Here, we provide an optimized protocol for snATAC-seq of murine hearts. We describe isolation of single nuclei from snap-frozen hearts, provide hints for preparation of libraries suitable for snATAC-seq next-generation sequencing (NGS) using the Chromium 10× platform, and give general recommendations for downstream analysis using conventional bioinformatic pipelines and packages. The protocol should serve as a beginner's guide to generate high-quality snATAC-seq datasets and to perform chromatin accessibility analysis of individual heart-derived cell nuclei.

Chromatin accessibility analysis and architectural profiling of human kidneys reveal key cell types and a regulator of diabetic kidney disease.

Diabetes is the leading cause of kidney disease that progresses to kidney failure. However, the key molecular and cellular pathways involved in diabetic kidney disease (DKD) pathogenesis are largely unknown. Here, we performed a comparative analysis of adult human kidneys by examining cell type-specific chromatin accessibility by single-nucleus ATAC-seq (snATAC-seq) and analyzing three-dimensional chromatin architecture via high-throughput chromosome conformation capture (Hi-C method) of paired samples. We mapped the cell type-specific and DKD-specific open chromatin landscape and found that genetic variants associated with kidney diseases were significantly enriched in the proximal tubule- (PT) and injured PT-specific open chromatin regions in samples from patients with DKD. BACH1 was identified as a core transcription factor of injured PT cells; its binding target genes were highly associated with fibrosis and inflammation, which were also key features of injured PT cells. Additionally, Hi-C analysis revealed global chromatin architectural changes in DKD, accompanied by changes in local open chromatin patterns. Combining the snATAC-seq and Hi-C data identified direct target genes of BACH1, and indicated that BACH1 binding regions showed increased chromatin contact frequency with promoters of their target genes in DKD. Thus, our multi-omics analysis revealed BACH1 target genes in injured PTs and highlighted the role of BACH1 as a novel regulator of tubular inflammation and fibrosis.

Single-Cell Transcriptomic and Epigenetic Analyses of Mouse Mammary Development Starting with the Embryo.

Cancers are caricatures of normal development. Yet, for most organs we are only beginning to learn about the molecular events underlying the embryonic antecedents of organogenesis and when differentiation into the cell types found in the adult actually begins. Here, we will focus on the powerful single-cell RNA sequencing and Assay for Transposase Accessible DNA by DNA sequencing (ATAC-seq) that we and others have been using to decipher the key regulators and signal transduction pathways involved in normal mammary development. We will first describe the techniques we use to identify, dissect, and isolate embryonic mammary rudiments and their constituent cells. We then describe the methods we have employed to perform single-cell RNA-seq and single-nucleus ATAC-seq using the small number of cells obtainable from mouse embryos. Finally, we will discuss the bioinformatic techniques we have used to interpret the vast amount of data obtained with these methods.

AMULET: a novel read count-based method for effective multiplet detection from single nucleus ATAC-seq data.

Detecting multiplets in single nucleus (sn)ATAC-seq data is challenging due to data sparsity and limited dynamic range. AMULET (ATAC-seq MULtiplet Estimation Tool) enumerates regions with greater than two uniquely aligned reads across the genome to effectively detect multiplets. We evaluate the method by generating snATAC-seq data in the human blood and pancreatic islet samples. AMULET has high precision, estimated via donor-based multiplexing, and high recall, estimated via simulated multiplets, compared to alternatives and identifies multiplets most effectively when a certain read depth of 25K median valid reads per nucleus is achieved.

Isolation of Nuclei from Mouse Dorsal Root Ganglia for Single-nucleus Genomics.

Primary somatosensory neurons, whose cell bodies reside in the dorsal root ganglion (DRG) and trigeminal ganglion, are specialized to transmit sensory information from the periphery to the central nervous system. Our molecular understanding of peripheral sensory neurons has been limited by both their heterogeneity and low abundance compared with non-neuronal cell types in sensory ganglia. We describe a protocol to isolate nuclei from mouse DRGs using iodixanol density gradient centrifugation, which enriches for neuronal nuclei while still sampling non-neuronal cells such as satellite glia and Schwann cells. This protocol is compatible with a range of downstream applications such as single-nucleus transcriptional and epigenomic assays.

Single-nucleus RNA and ATAC sequencing reveals the impact of chromatin accessibility on gene expression in Arabidopsis roots at the single-cell level.

Similar to other complex organisms, plants consist of diverse and specialized cell types. The gain of unique biological functions of these different cell types is the consequence of the establishment of cell-type-specific transcriptional programs. As a necessary step in gaining a deeper understanding of the regulatory mechanisms controlling plant gene expression, we report the use of single-nucleus RNA sequencing (sNucRNA-seq) and single-nucleus assay for transposase accessible chromatin sequencing (sNucATAC-seq) technologies on Arabidopsis roots. The comparison of our single-nucleus transcriptomes to the published protoplast transcriptomes validated the use of nuclei as biological entities to establish plant cell-type-specific transcriptomes. Furthermore, our sNucRNA-seq results uncovered the transcriptomes of additional cell subtypes not identified by single-cell RNA-seq. Similar to our transcriptomic approach, the sNucATAC-seq approach led to the distribution of the Arabidopsis nuclei into distinct clusters, suggesting the differential accessibility of chromatin between groups of cells according to their identity. To reveal the impact of chromatin accessibility on gene expression, we integrated sNucRNA-seq and sNucATAC-seq data and demonstrated that cell-type-specific marker genes display cell-type-specific patterns of chromatin accessibility. Our data suggest that the differential chromatin accessibility is a critical mechanism to regulate gene activity at the cell-type level.