Nucleosome organization dynamics during fertilization reveals regulation factors of zygotic gene activation in mouse (MNase-seq)

Chromatin remodeling is essential for epigenome reprogramming after fertilization. However, the underlying mechanisms of chromatin remodeling remain to be explored. Here, we investigated the dynamic changes in nucleosome occupancy and positioning in pronucleus-stage zygotes using low-input MNase-seq. We observed distinct features of inheritance and reconstruction of nucleosome position in both male and female genomes. Genome-wide de novo nucleosome occupancy in the paternal genome was observed as early as 1 hour after the injection of sperm. The nucleosome positioning pattern was continually rebuilt to form nucleosome depletion regions (NDRs) at promoters and transcription factor (TF) binding sites. NDRs formed more quickly on the promoters of genes involved in zygotic genome activation (ZGA), and their formation is closely connected with histone acetylation, but not transcription elongation or DNA replication. Importantly, we found that NDR establishment on the binding motifs of specific TFs might be associated with their potential pioneer functions in ZGA. Further investigations suggested that the predicted factors MLX and RFX1 played important roles in regulating minor and major ZGA, respectively. Our data not only elucidate the nucleosome positioning dynamics in both male and female pronuclei following fertilization, but also provide an efficient method for identifying key transcription regulators during development. Overall design: We generated the nucleosome profiles using ultra-low input MNase-seq in mouse gametes and pronucleus. For mouse gametes, we generated sperm, oocyte and round spermatid (RS) MNase-seq profiles. For sperm fertilized pronucleus, we separated them into male pronucleus and female pronucleus, collected the samples at 0.5h, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 12h after fertilization. For RS fertilized pronucleus, we generated the samples at 0.5h, 1h, 2h and 3h after fertilization. To investigate whether block transcription, DNA replication or histone acetylation affect the nucleosome positioning, we generated MNase-seq profiles for pronucleus samples at 6h treated with a-amantin, aphicolin, Hdac1/2 and JQ1. To explore the function of potential ZGA factors on nucleosome positioning, we generated MNase-seq profiles for pronucleus samples at 8h treated with siMlx, siRfx1 treatment.