Dynamic Reorganization of Nucleosome Positioning in Somatic Cells after Transfer into Porcine Enucleated Oocytes

Abstract

SummaryThe nucleosome, the fundamental structural unit of chromatin, is a critical regulator of gene expression. The mechanisms governing changes to nucleosome occupancy and positioning during somatic cell reprogramming remain poorly understood. We established a method for generating genome-wide nucleosome maps of porcine embryonic fibroblasts (PEF), reconstructed 1-cell embryos generated by somatic cell nuclear transfer (SCNT), and fertilized zygotes (FZ) using MNase sequencing with only 1,000 cells. We found that donor PEF chromatin, especially X chromosome, became more open after transfer into porcine oocytes and nucleosome occupancy decreased in promoters but increased in the genic regions. Nucleosome arrangements around transcriptional start sites of genes with different expression levels in somatic cells tended to become transcriptionally silent in SCNT; however, some pluripotency genes adopted transcriptionally active nucleosome arrangements. FZ and SCNT had similar characteristics, unlike PEF. This study reveals the dynamics and importance of nucleosome positioning and chromatin organization early after reprogramming.

Identification of Chromatin Accessibility During The Early Stage of Pig SCNT Embryo Reprogramming by ATAC-Seq

“…Despite this low level of accessibility, some accessible chromatin sites could still be detected. However, our results suggest that these accessibility sites were not highly expressed genes in somatic cells; furthermore, the X chromosome was in an inactive state, which is consistent with previous studies [17,30], indicating that large-scale chromatin remodeling had occurred at an early stage of embryo reprogramming. Given that inhibiting the small-scale transcription of the zygotic genome before activation can lead to the arrest of embryonic development [31], we speculate that accessible chromatin in the embryo 10 h after activation may be related to subsequent embryonic developmental events.…”

“…Radical changes in gene expression patterns in cells are critically important for SCNT [15,16]. Chromatin nucleosome positioning has been reported to undergo large-scale dynamic reorganization in the rst few hours after the activation of the SCNT embryo [17], suggesting that chromatin has already been reprogrammed at the early stage of embryo activation.…”

Transcriptome Analysis of In Vitro Fertilization and Parthenogenesis Activation during Early Embryonic Development in Pigs

“…Cumulus oocyte complexes (COCs) with uniform cytoplasm and several layer of cumulus cells were selected, and 3-5 layers of these cumulus cells were selected and then washed twice in wash buffer. In vitro maturation, conducted in 4-cell dishes in TCM-199 culture medium, included incubation at 38.5 • C with 5% CO 2 in air, at maximum humidity, for 42-44 h. Mature oocytes were chosen with first polar body and uniform cytoplasm for the next step [34].…”

“…Eighteen hours after fertilization, fluorescence stains (5 µg/mL H33342, 10-20 min) were used to observe prokaryotic formation. One female pronucleus, one male pronucleus, two polar bodies, and non-agglutinated sperm heads were used as the standard for normal fertilization, and then according to the type of pronucleus formation recorded the number of embryos of various types [34,35].…”

Aberrant nucleosome organization in mouse SCNT embryos revealed by ULI-MNase-seq

“…Nucleosome remodeling occurs quickly after fertilization, which is necessary for embryonic development ( Oliva, 2006 ; Wang et al., 2022 ). This process also occurs during SCNT embryonic development ( Tao et al., 2017 ; Wen et al., 2014 ), but how the nucleosome position changes after the donor cell nucleus is transferred into enucleated oocytes and the underlying mechanism is largely unexplored. Here, we performed genome-wide profiling of nucleosome occupancy and positioning in early mouse SCNT embryos using ultra-low-input micrococcal nuclease digestion-based high-throughput sequencing (ULI-MNase-seq) method.…”