Cell type- and chromosome-specific chromatin landscapes and DNA replication programs of Drosophila testis stem cells [scRNA-seq]
Stem cells have the unique ability to self-renew and differentiate into specialized cell types. Among the many factors influencing cell fate, epigenetic mechanisms, like histones, play a crucial role in regulating genomic programs like gene transcription and DNA replication, which are integral to a cell's identity. However, the transcriptional, chromatin, and DNA replication timing profiles of stem cells within in vivo tissues remain poorly understood. The Drosophila testis serves as an excellent in vivo model for studying stem cells, as it contains two stem cell populations: the germline stem cells (GSC) and somatic cyst stem cells (CySC). Despite this, a comprehensive understanding of genome regulation in these cell types has been limited by the small number of stem cells and heterogeneity of the testis. In this study, we developed cell-specific genomic techniques to analyze the transcriptome, histone modification patterns, and replication timing profiles of GSCs and CySCs. Our single cell RNA-sequencing validated previous cell-biological and genetic studies on the role of intercellular communication between GSCs and CySCs while revealing high expression of chromatin regulators in GSCs. To characterize chromatin landscapes, we developed a cell-specific chromatin profiling assay to map H3K4me3, H3K27me3, and H3K9me3. These posttranslational modifications define euchromatic, facultative heterochromatic, and constitutive heterochromatic domains, respectively. Finally, we determined the cell-specific replication timing profiles of GSCs and CySCs, integrating our in vivo datasets with published in vitro data. Our results reveal that the replication program of GSCs is distinct from somatic lineages and that these differences align with variations in chromatin patterns. Collectively, our integrated genomic datasets build a framework for understanding genome-wide regulatory differences in two in vivo stem cell populations, demonstrating the power of combining transcriptomic, chromatin, and replication datasets to uncover cell-specific features of genome regulation. Overall design: Drosophila melanogaster testis tumors were produced by overexpressing the Unpaired (Upd) ligand via two genetic strategies. Upd was either overexpressed using the germline-specific nanos promoter or cyst cell-specific traffic jam promoter. From each genetic strategy, approximately 18-20 testis pairs were dissected and dissociated to create a single cell suspension. Two replicates of each genetic type (four samples total) were proccessed for single cell RNA-sequencing using the 10X Genomics platform.