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

Embryonic stem cells (ESCs) exhibit transcriptional heterogeneity in key pluripotency regulators, such as Nanog , suggesting functional roles for variability in cell state plasticity and regulation. While the importance of this variability is well-recognized, the specific molecular regulators that stabilize or destabilize these fluctuations remain poorly understood. We developed an integrative pipeline combining single-cell RNA sequencing (scRNA-seq) with ChIP-seq datasets to identify candidate regulators of gene expression variability in ESCs. This approach identified Nucleosome Assembly Protein 1-Like 1 (NAP1L1), a member of the NAP1 histone chaperone family, as a potential transcriptional stabilizer. Targeted recruitment of NAP1L1 to the Nanog promoter using a dCas9 fusion protein stabilized NANOG expression in ESCs. Genome-wide single-cell transcriptomic analysis of wild-type and Nap1l1 -knockout ESCs revealed increased transcriptional variability upon NAP1L1 loss, supporting a global role in transcriptional stabilization. Co-immunoprecipitation followed by mass spectrometry identified Developmental Pluripotency Associated 3 (DPPA3) as a key interacting partner of NAP1L1, suggesting a mechanistic basis for its function. Our study establishes a systematic framework for discovering regulators of transcriptional variability and identifies NAP1L1 as a transcriptional stabilizer that modulates gene expression heterogeneity in ESCs.