Craigen, Evelyn1; Clark, Brian1
1Washington University in St. Louis
Purpose:The seven cell types of the mature vertebrate retina derive from a single pool of retinal progenitor cells (RPCs) and are born in a highly stereotyped, evolutionarily conserved and partially overlapping temporal birth order. The temporally-restricted specification of retinal cell fates is closely related to gene expression changes in RPCs as they shift their developmental competence. The transcription factors (TFs) associated with different retinal cell fates are well-characterized, but the noncoding DNA sequences where these TFs bind, act, and are regulated—cis-regulatory elements (CREs)—are only beginning to be uncovered.
Methods: To overcome limitations with current methods for studying CREs, we have combined the assay for transposase-accessible chromatin (ATAC) with self-transcribing active regulatory region sequencing (STARR-seq) to directly and quantitatively measure genome-wide CRE activity in RPCs over the course of mouse retinal development. We generated a library of accessible DNA sequences in sorted RPCs from embryonic day 18 (E18) and cloned them into a self-transcribing enhancer reporter to read out CRE activity in the form of RNA transcripts. We then electroporated our library of accessible sequences from E18 RPCs into mouse retinal explants from either early (E14) or late (post-natal day 0, or P0) developmental ages.
Results: We have identified almost 33,000 unique DNA sequences that are differentially active late in development—a period when late-born cell types such as rod photoreceptors, bipolar cells, and Müller glia are specified. By correlating the activity of these CREs with gene expression changes in RPCs, as well as cross-referencing our sequences with existing evolutionary conservation, chromatin conformation, and TF binding data sets, we have prioritized putative regulators of retinal cell fate specification for functional study. To biologically link our CRE activity to cell fate, we perform in vivo lineage tracing in RPCs to visualize whether a given active sequence confers cell type bias upon specification. We also perturb CRE activity in vivo via CRISPR interference (CRISPRi) to assess resulting changes in cell type proportions and retinal organization.
Conclusions: ATAC-STARR-seq, also referred to as high-resolution dissection of regulatory activity (HiDRA), is an unbiased method for measuring genome-wide CRE activity that provides a wealth of information regarding the complex regulatory network governing temporally-restricted cell fate in the vertebrate retina.
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