Transcriptome analysis in mouse ES cells deficient for Zfp352 or both Zfp352/Zfp353

Mouse ES cell population contains a minor sub-population expressing the genes that are specifically expressed in 2-cell stage embryos. This sub-population, consists of the 2-cell-gene labelled cells (2CLCs), is generated by the transient activation of the 2-cell specific genes initiated by the master regulator Dux. However, the mechanism regulating the transient expression remains largely unclear. Here we reported a novel function of Zfp352, one of the 2-cell specific genes, in the regulation of the 2CLC sub-population. Zfp352 encodes zinc-finger transcription factor belonging to the Klf family. Zfp352 is transiently activated by Dux after the activation of Zscan4c and expresses in a subset of 2CLC subpopulation. Interestingly, in the reporter assay, the transcriptional activation of Zscan4c by Dux is strongly repressed by the co-expression of Zfp352. However, knockout of Zfp352 resulted in the repression of a subset of the 2-cell specific genes. These data suggest the dual roles of Zfp352 in the regulation of the transient activation of the 2-cell specific genes. Overall design: In the EB5 mouse ES cell line (derived from male E14tg2a mESCs, 129Ola, Oct3/4IRES-BSD-pA/+, deposited to RIKEN Cell Bank) , only the Zfp352 gene or both Zfp352/Zfp353 genes were deleted using the CRISPR-Cas9 method. Total RNA was extracted using the QuickGene RNA cultured cell kit S (Kurabo). After checking the quality of the extracted RNA using the BioAnalyzer 2100 (RIN > 8), the library DNA was prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina) and sequenced with Illumina NextSeq 500 (Illumina) using Nextseq 500/550 High Output v2.5 Kit (Illumina) to obtain single end 75 nt reads.