Pou5f3, SoxB1 and Nanog remodel chromatin on High Nucleosome Affinity Regions at Zygotic Genome Activation.

Purpose: Find out the consequences of Pou5f3/Oct4 and Nanog binding to the nucleosome distribution before and after Zygotic Genome Activation Methods: MNase-seq of the wild-type(WT), Pou5f3 (MZspg) and Nanog (MZnanog) mutant zebrafish embryos at 512-cell stage and dome stage. Results: Nanog, Sox19b and Pou5f3 bind to the High Nucleosome Affinity Regions (HNARs). HNARs are spanning over 600 bp, featuring high in vivo and predicted in vitro nucleosome occupancy and high predicted propeller twist DNA shape value. We suggest a two-step model, where the same intrinsic DNA properties of HNAR promote both high nucleosome occupancy and differential binding of TFs. In the first step, already prior to ZGA, Pou5f3 and Nanog reduce nucleosome occupancy on HNARs genome-wide. In the second step, Nanog and Pou5f3/SoxB1 complex maintain open chromatin state on the subset of HNARs, acting synergistically. Nanog binds to the HNAR center, while the Pou5f3/SoxB1 complex stabilizes the flanks. Overall design: Genome-wide nucleosome occupancy in 512-cell and dome staged wild-type (WT), MZspg (Pou5f3) and MZnanog (Nanog) zebrafish mutant embryos was assayed by MNase treatment followed by deep sequencing (MNase-seq), using Illumina HiSeq 2500.

研究目的：明确Pou5f3/Oct4与Nanog结合对合子基因组激活（Zygotic Genome Activation, ZGA）前后核小体分布的影响。
实验方法：对512细胞期与圆顶期的野生型（wild-type, WT）、Pou5f3突变型（MZspg）及Nanog突变型（MZnanog）斑马鱼胚胎开展微球菌核酸酶测序（MNase-seq）实验。
研究结果：Nanog、Sox19b与Pou5f3可结合至高核小体亲和区域（High Nucleosome Affinity Regions, HNARs）。HNARs的跨度可达600 bp，兼具较高的体内核小体占据率、体外预测核小体占据率，以及较高的预测DNA螺旋扭转形状值。本研究提出双阶段模型：HNARs所具备的固有DNA特性，可同时促进高核小体占据率与转录因子（Transcription Factor, TF）的差异化结合。第一阶段，在合子基因组激活之前，Pou5f3与Nanog即可在全基因组范围内降低HNARs上的核小体占据率；第二阶段，Nanog与Pou5f3/SoxB1复合物可发挥协同作用，维持部分HNARs的开放染色质状态：Nanog结合于HNAR中心区域，而Pou5f3/SoxB1复合物则稳定其侧翼区域。
实验整体设计：采用Illumina HiSeq 2500测序平台，通过微球菌核酸酶处理结合深度测序（MNase-seq），对512细胞期与圆顶期的野生型（WT）、MZspg（Pou5f3）及MZnanog（Nanog）斑马鱼胚胎的全基因组核小体占据情况进行检测。