GSE157650
SIN3A regulates porcine early embryonic development by modulating CCNB1 expression
SIN3A is the central scaffold protein of the SIN3/HDAC transcriptional repressor complex. We previously found that SIN3A participates in the mouse preimplantation development by finetuning HDAC1 expression. However, it remains unresolved if this functional significance of SIN3A was conserved in other mammals. The objective of this work was thus to characterize the expression profiles and the functional role of SIN3A in preimplantation development using non-rodent animal models. RNA-seq results show a large amount of SIN3A mRNA is present in oocytes and early embryos prior to embryonic genome activation and a low amount thereafter, suggesting a maternal origin of SIN3A in all species examined. Interestingly, immunofluorescence data show that SIN3A protein level peaks at 4-cell stage in pigs compared with morula stage in cattle, suggesting a differential role of SIN3A among species. Indeed, SIN3A depletion in early embryos causes a developmental arrest at 2-cell stage in pigs but does not affect bovine early embryonic development. In contrast with mouse data, SIN3A depletion results in only a slight decrease and even no difference in HDAC1 expression in porcine and bovine early embryos, respectively. In addition, HDAC1 knockdown does not cause 2-cell block, however, leads to a reduced blastocyst rate, suggesting the effect of SIN3A depletion on porcine early embryos is independent of HDAC1. By using un-biased RNA-seq approach, we found that CCNB1 transcript level is dramatically reduced. Moreover, CCNB1 knockdown results in a similar phenotype as SIN3A depletion. Injection of exogenous CCNB1 into SIN3A-depleted embryos could partly rescue embryonic development beyond 2-cell stage. In conclusion, our results indicate SIN3A plays an essential role in porcine early embryonic development, which probably involving the regulation of CCNB1 expression. Overall design: 2-cell porcine embryos (32 h after parthenogenetic activation) were harvested from NC and KD groups (n=3; 20 embryos/group/replicate). Total RNA extraction was performed using Picopure RNA isolation kit (Life Technologies, Grand Island, NY, USA) based on the manufacturer's manual. Before RNA isolation, equal amount of GFP and RFP mRNA  were added to each group as a spike-in control. mRNAs separation was achieved using oligo(dT)25 beads. Sequencing libraries were constructed with NEB Next Ultra RNA Library Prep Kit for Illumina (New England Biolabs) according to the instruction. In brief, mRNAs were fragmented followed by reverse transcription. The cDNA library underwent end repair, poly(A)-tailing, adaptor ligation, and PCR amplification for 12–15 cycles to prepare sequencing libraries. Libraries were sequenced using Illumina Hiseq X Ten at the Novogene Co. Ltd.