Systematic discovery of potential regulators of transcriptional variability reveals NAP1L1 as a transcriptional stabilizer in mouse ESCs

Embryonic stem cells (ESCs) maintain their capacity to generate all different cell types of the developing embryo. When grown in serum, ESCs display transcriptional heterogeneity of several key pluripotency-related genes (e.g. Nanog). This transcriptional variability was suggested to support ESC pluripotency by enabling them to differentiate along different lineages simultaneously. We hypothesized that expression heterogeneity is a regulated process, mediated by transcriptional (de)stabilizers. To test this systematically, and to identify potential ESC regulators of transcriptional variability, we compared single-cell RNA-seq (scRNA-seq) with ChIP-seq datasets to identify variable and stable genes in ESCs. One of the potential ‘stabilizer’ proteins we identified is Nucleosome Assembly Protein 1-Like 1 (NAP1L1), a member of the NAP1 family. NAP1L1 participates in many cellular functions, including nucleosome maintenance, chromatin remodeling, cell division, and transcription. To test its potential ‘stabilizer’ function, we recruited NAP1L1 to the Nanog promoter via fusion with dCAS9. Immunofluorescence (IF) and single-molecule fluorescence in situ hybridization (smFISH) analyses of Nanog expression revealed that NAP1L1 directly stabilizes both RNA and protein expression levels. To further explore NAP1L1’s function genome-wide, we generated Nap1l1-knock-out (KO) ESCs. scRNA-seq experiments comparing Nap1l1-KO and WT ESCs, revealed increased gene expression variability in Nap1l1-KO cells. To reveal the potential mechanism by which NAP1L1 exerts its stabilizing activity we identified NAP1L1-interacting partners by co-IP followed by Mass-Spectrometry experiments, revealing DPPA3 as a key interacting partner. Taken together, we provide a systematic discovery platform for regulators of transcriptional variability and identify NAP1L1 as a transcriptional stabilizer in ESCs.