ZFP982 confers mouse embryonic stem cell characteristics by regulating expression of Nanog, Zfp42, and Dppa3

Abstract Background: Understanding the genetic underpinnings of protein networks conferring stemness is of broad interest for basic and translational research. Methods: We used multi-omics analyses to identify and characterize stemness genes, and focused on the zinc finger protein 982 (Zfp982) that regulates stemness through the expression of Nanog, Zfp42, and Dppa3 in mouse embryonic stem cells (mESC). Results: Zfp982 was expressed in stem cells, and bound to chromatin through a GCAGAGKC motif, for example near the stemness genes Nanog, Zfp42, and Dppa3. Nanog and Zfp42 were direct targets of ZFP982 that decreased in expression upon knockdown and increased upon overexpression of Zfp982. We show that ZFP982 expression strongly correlated with stem cell characteristics, both on the transcriptional and morphological levels. Zfp982 expression decreased with progressive differentiation into ecto-, endo- and mesodermal cell lineages, and knockdown of Zfp982 correlated with morphological and transcriptional features of differentiated cells. Zfp982 showed transcriptional overlap with members of the Hippo signaling pathway, one of which was Yap1, the major co-activator of Hippo signaling. Despite the observation that ZFP982 and YAP1 interacted and localized predominantly to the cytoplasm upon differentiation, the localization of YAP1 was not influenced by ZFP982 localization. Conclusions: Together, our study identified ZFP982 as a transcriptional regulator of early stemness genes, and since ZFP982 is under the control of the Hippo pathway, underscored the importance of the context-dependent Hippo signals for stem cell characteristics. Keywords: Hippo pathway; Nanog; Pluripotency; Stemness; Yap1; ZFP982. Overall design: Assesment of ZFP982 direct targets via ChIP of mESCs which transfected with ZFP982-MYC tagged expression vector. 2. Materials and methods 2.3. ChIP and ChIP-seq Chromatin-immunoprecipitation (ZFP982-ChIP-seq) was performed from chromatin of mESC transfected with pEXP-Empty and pEXP-ZFP982, using the MYC-Tag according to NEXSON protocol [26]. For MYC-Tag ChIP, cells were fixed for 5 min at room temperature with 1 % methanol-free formaldehyde (Thermo Scientific, #28906). Cells were lysed using ice-cold Farnham lab buffer supplemented with a complete protease inhibitor cocktail (#4693159001, Roche, Switzerland). Chromatin was sheared with Bioruptor (Diagenode, Belgium) at low power for three cycles 15 s on, 30 s off. ChIP-sequencing was performed on an Illumina HiSeq 2500 (single-end, read length of 50 bp, 50 Mio/reads per sample). Quality control of sequencing reads was performed using FastQC [27]. CG bias and ChIP to input efficiency were controlled by computeCGBias and bamFingerprint [28]. Reads were mapped to the UCSC-main RefSeq GRCm38/mm10 reference genome using Bowtie2 [29,30]. Samples were deduplicated using MarkDuplicates (http://broadinstitute.github.io/picard/). Signal extraction scaling (SES) was used for data normalization [31] and log2 (ratio of the number of reads) of ChIP to input was calculated by bamCompare using a pseudocount of 1 and a bin size of 25 bp. ComputeMatrix, plotHeatmap, and deepTools2 were used for plotting. Motif analysis was performed using MEME-ChIP version 4.11.2 [32]. The Galaxy Platform (https://usegalaxy.eu) was used for ChIP-seq analyses [33]. For the published ChIP-seq datasets of histone marks (Fig. 4E, S5) in mESC (GSE135318 [34]) fastq files were downloaded and reanalyzed as described. Individual replicates were averaged after normalization. Further information about the workflow and Bioinformatic analysis can be found at https://github.com/Vogel-lab/ZFP982-confers-mESC-characteristics.