Cellular differentiation involves profound remodeling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNAi screens targeting chromatin factors during transcription factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Remarkably, subunits of the chromatin assembly factor-1 (CAF-1) complex emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPSC formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 as a novel regulator of somatic cell identity during transcription factor-induced cell fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

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Image-based phenotypic screening relies on the extraction of multivariate information from cells cultured under a large variety of conditions. Technical advances in high-throughput microscopy enable screening in increasingly complex and biologically relevant model systems. To this end, organoids hold great potential for high-content screening because they recapitulate many aspects of parent tissues and can be derived from patient material. However, screening is substantially more difficult in organoids than in classical cell lines from both technical and analytical standpoints. In this review, we present an overview of studies employing organoids for screening applications. We discuss the promises and challenges of small-molecule treatments in organoids and give practical advice on designing, running, and analyzing high-content organoid-based phenotypic screens.