Epigenetic modifications, including DNA methylation and histone modifications, are reprogrammed considerably following fertilization during mammalian early embryonic development.
More...Epigenetic modifications, including DNA methylation and histone modifications, are reprogrammed considerably following fertilization during mammalian early embryonic development. Incomplete epigenetic reprogramming is a major factor leading to poor developmental outcome in embryos generated by assisted reproductive technologies, such as somatic cell nuclear transfer. However, the role of histone modifications in preimplantation development is poorly understood. Here, we show that co-knockdown (cKD) of Hdac1 and 2 (but not individually) resulted in developmental failure during the morula to blastocyst transition. This outcome was also confirmed with the use of small-molecule Hdac1/2-specific inhibitor FK228. We observed reduced cell proliferation and increased incidence of apoptosis in cKD embryos, which were likely caused by increased acetylation of Trp53. Importantly, both RNA-seq and immunostaining analysis revealed a failure of lineage specification to generate trophectoderm and pluripotent cells. Among many gene expression changes, a substantial decrease of Cdx2 may be partly accounted for by the aberrant Hippo pathway occurring in cKD embryos. In addition, we observed an increase in global DNA methylation, consistent with increased DNA methyltransferases and Uhrf1. Interestingly, deficiency of Rbbp4 and 7 (both are core components of several Hdac1/2-containing epigenetic complexes) results in similar phenotypes as those of cKD embryos. Overall, Hdac1 and 2 play redundant functions required for lineage specification, cell viability and accurate global DNA methylation, each contributing to critical developmental programs safeguarding a successful preimplantation development.
Overall design: Mouse zygotes were injected with nonspecific control siRNA (NC), Sin3a siRNA (Sin3a KD) or Hdac1/2 siRNA (Hdac1/2 cKD). At E2.75, embryos were collected (60 embryos per sample, n=3). Total RNA was isolated from embryos using Picopure RNA isolation kit (Life Technologies, Grand Island, NY, USA) according to the manufacturer's instruction. Before RNA extraction, 2 × 106 copies of RFP and GFP mRNA was added. mRNAs were separated with oligo(dT)25 beads, and was used to prepare sequencing libraries with NEB Next Ultra RNA Library Prep Kit for Illumina (New England Biolabs). Briefly, mRNA was fragmented and reverse transcribed. The cDNA library was subject to end repair, poly(A)-tailing, adaptor ligation, and PCR amplification of 12–15 cycles for sequencing library construction. The library was sequenced by Illumina Hiseq X Ten and RNA-seq reads were assigned directly to transcripts and counted with Salmon (https://combine-lab.github.io/salmon/).
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