Abstract
SummaryMammalian gametogenesis involves dramatic and tightly regulated chromatin remodeling, whose regulatory pathways remain largely unexplored. Here, we generate a comprehensive high-resolution structural and functional atlas of mouse spermatogenesis by combining in situ chromosome conformation capture sequencing (Hi-C), RNA sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) of CCCTC-binding factor (CTCF) and meiotic cohesins, coupled with confocal and super-resolution microscopy. Spermatogonia presents well-defined compartment patterns and topological domains. However, chromosome occupancy and compartmentalization are highly re-arranged during prophase I, with cohesins bound to active promoters in DNA loops out of the chromosomal axes. Compartment patterns re-emerge in round spermatids, where cohesin occupancy correlates with transcriptional activity of key developmental genes. The compact sperm genome contains compartments with actively transcribed genes but no fine-scale topological domains, concomitant with the presence of protamines. Overall, we demonstrate how genome-wide cohesin occupancy and transcriptional activity is associated with three-dimensional (3D) remodeling during spermatogenesis, ultimately reprogramming the genome for the next generation.
Highlights
Mammalian genomes are packaged into a tailored chromatin structure, the regulation of which depends on several superimposed layers of organization, including epigenetic modifications and the higher-order organization of chromatin compartments inside the nucleus
Dynamic Overall Chromatin Structure Reorganization during Spermatogenesis To unveil changes in chromosome conformation during spermatogenesis, we developed a reproducible fluorescence-activated cell sorting (FACS) protocol to obtain, based on DNA content and chromatin complexity, highly enriched (90.4% average enrichment) cell fractions for Spg, primary spermatocytes at the leptonema-zygonema (L/Z) and pachynema-diplonema (P/D) stages, round spermatids (RSs), and sperm (Figures 1B, 1C, and S1) (STAR Methods)
Genome organization changed during spermatogenesis (Figures 1F and 2AÀ2F), as reflected by the analysis of distancedependent interaction frequencies (Figure 2C) and inter- and intra-chromosomal interaction ratios (Figure 2D)
Summary
Mammalian genomes are packaged into a tailored chromatin structure, the regulation of which depends on several superimposed layers of organization, including epigenetic modifications (of both the DNA and nucleosomes) and the higher-order organization of chromatin compartments inside the nucleus This organization is achieved by chromatins folding into loops, topologically associating domains (TADs), and compartments (A and B), which can influence transcriptional activity (Dixon et al, 2012; Lieberman-Aiden et al, 2009; Rao et al, 2014). The highly compartmentalized folding of the genome in interphase is lost during mitosis, when chromosomes are linearly organized in consecutive chromatin loops (Gibcus et al, 2018; Naumova et al, 2013). How the higher-order chromatin organization is configured during all stages of spermatogenesis, and how insulator proteins and
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