27Conventional methods for profiling the molecular content of biological samples fail to resolve 28 heterogeneity that is present at the level of single cells. these data represent a rich resource for future methods aimed at defining cell types and states. 47They will advance our understanding of developmental biology, and constitute a major step 48 towards a comprehensive, single-cell molecular atlas of a whole animal.

Over a million DNA regulatory elements have been cataloged in the human genome, but linking these elements to the genes that they regulate remains challenging. We introduce Cicero, a statistical method that connects regulatory elements to target genes using single cell chromatin accessibility data. We apply Cicero to investigate how thousands of dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of co-accessible regulatory elements linked by Cicero meet criteria of "chromatin hubs", in that they are physically proximal, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of gene expression. Pseudotemporal analysis revealed a subset of elements bound by MYOD in myoblasts that exhibit early opening, potentially serving as the initial sites of recruitment of chromatin remodeling and histonemodifying enzymes. The methodological framework described here constitutes a powerful new approach for elucidating the architecture, grammar and mechanisms of cis-regulation on a genome-wide basis.

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