Histone Chaperones Coupled to DNA Replication and Transcription Control Divergent Chromatin Elements to Safeguard Cell Identity [CUT&Run]

The manipulation of DNA replication and transcription can be harnessed to control cell fate. Central to the regulation of these DNA templated processes are histone chaperones, which in turn are emerging as cell fate regulators. Histone chaperones are a group of proteins with diverse functions that are primarily involved in escorting histones to assemble nucleosomes and maintain the chromatin landscape. Whether distinct histone chaperone pathways control cell fate, and whether they function using related mechanisms, remains unclear. To address this, we performed a screen to assess the requirement of diverse histone chaperones in the self-renewal of hematopoietic stem and progenitor cells. Remarkably, all candidates were required to maintain cell fate to differing extents, with no clear correlation with their specific histone partners or DNA templated process. Among all the histone chaperones, the loss of the transcription-coupled histone chaperone SPT6 most strongly promoted differentiation, even more so than the major replication-coupled chromatin assembly factor complex CAF-1. To directly compare how DNA replication- and transcription-coupled histone chaperones maintain stem cell self-renewal, we generated an isogenic dual inducible system to perturb each pathway individually. We found that both SPT6 and CAF-1 perturbations required cell cycle progression to induce differentiation, with distinct effects on chromatin accessibility, transcription factor activity, and gene expression. Specifically, CAF-1 depletion increased accessibility at heterochromatic loci with a pronounced effect on H3K27me3 elements leading to aberrant multilineage gene expression. In contrast, SPT6 loss influenced promoter elements and triggered a more canonical differentiation state, that is in part dependent on the activity of AP-1 transcription factors. Thus, CAF-1 and SPT6 histone chaperones maintain cell fate through the control of distinct chromatin elements, providing a paradigm for how different histone chaperone pathways can be manipulated to alter cell fate.