A single-nucleus RNA-sequencing pipeline to decipher the molecular anatomy and pathophysiology of human kidneys

Defining cellular and molecular identities within the kidney is necessary to understand its organization and function in health and disease. Here we demonstrate a reproducible method with minimal artifacts for single-nucleus Droplet-based RNA sequencing (snDrop-Seq) that we use to resolve thirty distinct cell populations in human adult kidney. We define molecular transition states along more than ten nephron segments spanning two major kidney regions. We further delineate cell type-specific expression of genes associated with chronic kidney disease, diabetes and hypertension, providing insight into possible targeted therapies. This includes expression of a hypertension-associated mechano-sensory ion channel in mesangial cells, and identification of proximal tubule cell populations defined by pathogenic expression signatures. Our fully optimized, quality-controlled transcriptomic profiling pipeline constitutes a tool for the generation of healthy and diseased molecular atlases applicable to clinical samples

Global DNA methylation and transcriptional analyses of human ESC-derived cardiomyocytes.

With defined culture protocol, human embryonic stem cells (hESCs) are able to generate cardiomyocytes in vitro, therefore providing a great model for human heart development, and holding great potential for cardiac disease therapies. In this study, we successfully generated a highly pure population of human cardiomyocytes (hCMs) (>95% cTnT(+)) from hESC line, which enabled us to identify and characterize an hCM-specific signature, at both the gene expression and DNA methylation levels. Gene functional association network and gene-disease network analyses of these hCM-enriched genes provide new insights into the mechanisms of hCM transcriptional regulation, and stand as an informative and rich resource for investigating cardiac gene functions and disease mechanisms. Moreover, we show that cardiac-structural genes and cardiac-transcription factors have distinct epigenetic mechanisms to regulate their gene expression, providing a better understanding of how the epigenetic machinery coordinates to regulate gene expression in different cell types

5-Formylcytosine could be a semi-permanent base in specific genome sites

5-Formyl-2’-deoxycytosine (fdC) is a recently discovered epigenetic base in the genome of stem cells, with yet unknown functions. Sequencing data show that the base is enriched in CpG islands of promoters and hence likely involved in the regulation of transcription during cellular differentiation. fdC is known to be recognized and excised by the enzyme thymine-DNA-glycosylase (Tdg). As such, fdC is believed to function as an intermediate during active demethylation. In order to understand the function of the new epigenetic base fdC, it is important to analyze its formation and removal at defined genomic sites. Here, we report a new method that combines sequence-specific chemical derivatization of fdC with droplet digital PCR that enables such analysis. We show initial data, indicating that the repair protein Tdg removes only 50% of the fdCs at a given genomic site, arguing that fdC is a semi-permanent base

Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells

Generation of human induced pluripotent stem cells (hiPSCs) by the expression of specific transcription factors depends on successful epigenetic reprogramming to a pluripotent state. Although hiPSCs and human embryonic stem cells (hESCs) display a similar epigenome, recent reports demonstrated the persistence of specific epigenetic marks from the somatic cell type of origin and aberrant methylation patterns in hiPSCs. However, it remains unknown whether the use of different somatic cell sources, encompassing variable levels of se- lection pressure during reprogramming, influences the level of epigenetic aberrations in hiPSCs. In this work, we characterized the epigenomic integrity of 17 hiPSC lines derived from six different cell types with varied reprogramming efficiencies. We demonstrate that epigenetic aberrations are a general feature of the hiPSC state and are independent of the somatic cell source. Interestingly, we observe that the reprogramming efficiency of somatic cell lines inversely correlates with the amount of methylation change needed to acquire pluripotency. Additionally, we determine that both shared and line- specific epigenetic aberrations in hiPSCs can directly translate into changes in gene expression in both the pluripotent and differenti- ated states. Significantly, our analysis of different hiPSC lines from multiple cell types of origin allow us to identify a reprogramming- specific epigenetic signature comprised of nine aberrantly methyl- ated genes that is able to segregate hESC and hiPSC lines regardless of the somatic cell source or differentiation state

5-Formylcytosin ist vermutlich eine semi-permanente Base an definierten Genompositionen

Die epigenetische Base 5-Formyl-2’-desoxycytosin (fdC) wurde kürzlich im Genom von Stammzellen entdeckt. Ihre Funktion ist bisher jedoch unbekannt. Daten aus Genomsequenzierungen zeigen eine Anreicherung in CpG Inseln von Promotoren, weshalb eine Beteiligung an der Regulation der Transkription während der zellulären Differenzierung angenommen wird. Auch ist fdC dafür bekannt von dem Enzym Thymin-DNA-Glykosylase (Tdg) erkannt und ausgeschnitten zu werden. Folglich wird angenommen, dass fdC als Intermediat während der aktiven Demethylierung auftritt. Um die Funktion der neuen epigenetischen Base fdC zu verstehen, ist es von großer Bedeutung dessen Bildung und Entfernung an definierten genomischen Positionen analysieren zu können. Im Folgenden beschreiben wir eine neue derartige Methode, die sequenzspezifische, chemische Derivatisierung von fdC mit Droplet Digital PCR kombiniert. Erste Ergebnisse zeigen, dass das Reparaturprotein Tdg nur 50% der fdCs an einer bestimmten Position im Genom entfernt, was auf semi-permanente Eigenschaften dieser Base hinweist

Epi-fingerprinting and epi-interventions for improved crop production and food quality

Increasing crop production at a time of rapid climate change represents the greatest challenge facing contemporary agricultural research. Our understanding of the genetic control of yield derives from controlled field experiments designed to minimise environmental variance. In spite of these efforts there is substantial residual variability among plants attributable to Genotype x Environment (GxE) interactions. Recent advances in the field of epigenetics have revealed a plethora of gene control mechanisms that could account for much of this unassigned variation. These systems act as a regulatory interface between the perception of the environment and associated alterations in gene expression. Direct intervention of epigenetic control systems hold the enticing promise of creating new sources of variability that could enhance crop performance. Equally, understanding the relationship between various epigenetic states and responses of the crop to specific aspects of the growing environment (epigenetic fingerprinting) could allow for a more tailored approach to plant agronomy. In this review, we explore the many ways in which epigenetic interventions and epigenetic fingerprinting can be deployed for the improvement of crop production and quality

A lexicon of DNA modifications: their roles in embryo development and the germline

5-methylcytosine (5mC) on CpG dinucleotides has been viewed as the major epigenetic modification in eukaryotes for a long time. Apart from 5mC, additional DNA modifications have been discovered in eukaryotic genomes. Many of these modifications are thought to be solely associated with DNA damage. However, growing evidence indicates that some base modifications, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), and N6-methadenine (6mA), may be of biological relevance, particularly during early stages of embryo development. Although abundance of these DNA modifications in eukaryotic genomes can be low, there are suggestions that they cooperate with other epigenetic markers to affect DNA-protein interactions, gene expression, defense of genome stability and epigenetic inheritance. Little is still known about their distribution in different tissues and their functions during key stages of the animal lifecycle. This review discusses current knowledge and future perspectives of these novel DNA modifications in the mammalian genome with a focus on their dynamic distribution during early embryonic development and their potential function in epigenetic inheritance through the germ line

Comparative cellular analysis of motor cortex in human, marmoset and mouse

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals(1). Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.Cardiovascular Aspects of Radiolog