The single-cell chromatin accessibility landscape in mouse perinatal testis development.

Spermatogenesis depends on an orchestrated series of developing events in germ cells and full maturation of the somatic microenvironment. To date, the majority of efforts to study cellular heterogeneity in testis has been focused on single-cell gene expression rather than the chromatin landscape shaping gene expression. To advance our understanding of the regulatory programs underlying testicular cell types, we analyzed single-cell chromatin accessibility profiles in more than 25,000 cells from mouse developing testis. We showed that single-cell sequencing assay for transposase-accessible chromatin (scATAC-Seq) allowed us to deconvolve distinct cell populations and identify cis-regulatory elements (CREs) underlying cell-type specification. We identified sets of transcription factors associated with cell type-specific accessibility, revealing novel regulators of cell fate specification and maintenance. Pseudotime reconstruction revealed detailed regulatory dynamics coordinating the sequential developmental progressions of germ cells and somatic cells. This high-resolution dataset also unveiled previously unreported subpopulations within both the Sertoli and Leydig cell groups. Further, we defined candidate target cell types and genes of several genome-wide association study (GWAS) signals, including those associated with testosterone levels and coronary artery disease. Collectively, our data provide a blueprint of the 'regulon' of the mouse male germline and supporting somatic cells.

scATAC-Seq reveals heterogeneity associated with spermatogonial differentiation in cultured male germline stem cells.

Spermatogonial stem cells are the most primitive spermatogonia in testis, which can self-renew to maintain the stem cell pool or differentiate to give rise to germ cells including haploid spermatids. All-trans-retinoic acid (RA), a bioactive metabolite of vitamin A, plays a fundamental role in initiating spermatogonial differentiation. In this study, single-cell ATAC-seq (scATAC-seq) was used to obtain genome-wide chromatin maps of cultured germline stem cells (GSCs) that were in control and RA-induced differentiation states. We showed that different subsets of GSCs can be distinguished based on chromatin accessibility of self-renewal and differentiation signature genes. Importantly, both progenitors and a subset of stem cells are able to respond to RA and give rise to differentiating cell subsets with distinct chromatin accessibility profiles. In this study, we identified regulatory regions that undergo chromatin remodeling and are associated with the retinoic signaling pathway. Moreover, we reconstructed the differentiation trajectory and identified novel transcription factor candidates enriched in different spermatogonia subsets. Collectively, our work provides a valuable resource for understanding the heterogeneity associated with differentiation and RA response in GSCs.

Transcriptomic and epigenomic profiling of young and aged spermatogonial stem cells reveals molecular targets regulating differentiation.

Spermatogonial stem cells (SSC), the foundation of spermatogenesis and male fertility, possess lifelong self-renewal activity. Aging leads to the decline in stem cell function and increased risk of paternal age-related genetic diseases. In the present study, we performed a comparative genomic analysis of mouse SSC-enriched undifferentiated spermatogonia (Oct4-GFP+/KIT-) and differentiating progenitors (Oct4-GFP+/KIT+) isolated from young and aged testes. Our transcriptome data revealed enormous complexity of expressed coding and non-coding RNAs and alternative splicing regulation during SSC differentiation. Further comparison between young and aged undifferentiated spermatogonia suggested these differentiation programs were affected by aging. We identified aberrant expression of genes associated with meiosis and TGF-ß signaling, alteration in alternative splicing regulation and differential expression of specific lncRNAs such as Fendrr. Epigenetic profiling revealed reduced H3K27me3 deposition at numerous pro-differentiation genes during SSC differentiation as well as aberrant H3K27me3 distribution at genes in Wnt and TGF-ß signaling upon aging. Finally, aged undifferentiated spermatogonia exhibited gene body hypomethylation, which is accompanied by an elevated 5hmC level. We believe this in-depth molecular analysis will serve as a reference for future analysis of SSC aging.

MicroRNA-29b/Tet1 regulatory axis epigenetically modulates mesendoderm differentiation in mouse embryonic stem cells.

Ten eleven translocation (Tet) family-mediated DNA oxidation on 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) represents a novel epigenetic modification that regulates dynamic gene expression during embryonic stem cells (ESCs) differentiation. Through the role of Tet on 5hmC regulation in stem cell development is relatively defined, how the Tet family is regulated and impacts on ESCs lineage development remains elusive. In this study, we show non-coding RNA regulation on Tet family may contribute to epigenetic regulation during ESCs differentiation, which is suggested by microRNA-29b (miR-29b) binding sites on the Tet1 3' untranslated region (3' UTR). We demonstrate miR-29b increases sharply after embyoid body (EB) formation, which causes Tet1 repression and reduction of cellular 5hmC level during ESCs differentiation. Importantly, we show this miR-29b/Tet1 regulatory axis promotes the mesendoderm lineage formation both in vitro and in vivo by inducing the Nodal signaling pathway and repressing the key target of the active demethylation pathway, Tdg. Taken together, our findings underscore the contribution of small non-coding RNA mediated regulation on DNA demethylation dynamics and the differential expressions of key mesendoderm regulators during ESCs lineage specification. MiR-29b could potentially be applied to enrich production of mesoderm and endoderm derivatives and be further differentiated into desired organ-specific cells.

GermlncRNA: a unique catalogue of long non-coding RNAs and associated regulations in male germ cell development.

Spermatogenic failure is a major cause of male infertility, which affects millions of couples worldwide. Recent discovery of long non-coding RNAs (lncRNAs) as critical regulators in normal and disease development provides new clues for delineating the molecular regulation in male germ cell development. However, few functional lncRNAs have been characterized to date. A major limitation in studying lncRNA in male germ cell development is the absence of germ cell-specific lncRNA annotation. Current lncRNA annotations are assembled by transcriptome data from heterogeneous tissue sources; specific germ cell transcript information of various developmental stages is therefore under-represented, which may lead to biased prediction or fail to identity important germ cell-specific lncRNAs. GermlncRNA provides the first comprehensive web-based and open-access lncRNA catalogue for three key male germ cell stages, including type A spermatogonia, pachytene spermatocytes and round spermatids. This information has been developed by integrating male germ transcriptome resources derived from RNA-Seq, tiling microarray and GermSAGE. Characterizations on lncRNA-associated regulatory features, potential coding gene and microRNA targets are also provided. Search results from GermlncRNA can be exported to Galaxy for downstream analysis or downloaded locally. Taken together, GermlncRNA offers a new avenue to better understand the role of lncRNAs and associated targets during spermatogenesis. Database URL: http://germlncrna.cbiit.cuhk.edu.hk/

MicroRNAs mediated targeting on the Yin-yang dynamics of DNA methylation in disease and development.

For decades, DNA methylation at the 5 position of cytosine (5mC) catalyzed by DNA methyltransferases (DNMTs) is a well-known epigenetic modification in mammalian genome, where it modulates chromatin remodeling and transcriptional silencing. The discovery of Ten-eleven translocation (TET) enzymes that oxidize 5mC to 5-hydroxymethylcytosine (5hmC) prompts a new era of DNA demethylation research. It is now established that in DNA demethylation pathway 5mC is first converted to 5-hydroxymethylcytosine (5hmC), then 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) through TETs. Conversion to unmethylated cytosine (5C) is further facilitated by excision mechanism through thymine-DNA glycosylase (TDG) or base excision repair (BER) pathway. Our understanding of DNMTs and TETs on epigenetic dynamics of cytosine methylation has led to a completion of the methylation (Yin) - demethylation (Yang) cycle on epigenetic modifications on cytosine. However, the regulations on DNA demethylation pathway remain largely unknown. In this review, we provide the recent advances on epigenetic dynamics of DNA demethylation and its potential control from the prespective of small non-coding RNA-mediated regulation. Specifically, we will illustrate how microRNAs contribute to active DNA demethylation control in normal and disease development based on recent findings in stem cells and cancer. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

Long noncoding RNAs in spermatogenesis: insights from recent high-throughput transcriptome studies.

Spermatogenesis is a complex developmental process in which undifferentiated spermatogonia are differentiated into spermatocytes and spermatids through two rounds of meiotic division and finally giving rise to mature spermatozoa (sperm). These processes involve many testis- or male germ cell-specific gene products that undergo strict developmental regulations. As a result, identifying critical, regulatory genes controlling spermatogenesis provide the clues not only to the regulatory mechanism of spermatogenesis at the molecular level, but also to the identification of candidate genes for infertility or contraceptives development. Despite the biological importance in male germ cell development, the underlying mechanisms of stage-specific gene regulation and cellular transition during spermatogenesis remain largely elusive. Previous genomic studies on transcriptome profiling were largely limited to protein-coding genes. Importantly, protein-coding genes only account for a small percentage of transcriptome; the majority are noncoding transcripts that do not translate into proteins. Although small noncoding RNAs (ncRNAs) such as microRNAs, siRNAs, and Piwi-interacting RNAs are extensively investigated in male germ cell development, the role of long ncRNAs (lncRNAs), commonly defined as ncRNAs longer than 200 bp, is relatively unexplored. Herein, we summarize recent transcriptome studies on spermatogenesis and show examples that a subset of noncoding transcript population, known as lncRNAs, constitutes a novel regulatory target in spermatogenesis.