H3.3K122A results in a neomorphic phenotype in mouse embryonic stem cells (CUT&RUN)

The histone variant H3.3 facilitates mRNA transcription activation and suppression at cis-regulatory elements such as promoters and enhancers, with histone epigenetic modifications including acetylation or methylation as context-defining features. Canonical histone H3 and histone variant H3.3 are post-translationally modified with the genomic distribution of these marks denoting transcription features and with more recent evidence suggesting that these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications with the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the residue H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mES cells, but were unsuccessful. Through multi-omic profiling of mutant cell lines harbor two of four or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality while H3.3-null mES cells are viable and pluripotent, albeit with reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue of H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells.