Creb5 controls its own expression and directly induces the joint-interzone regulatory program [ATACseq_Sox9_GFP]

Our prior work indicated that the transcription factor Creb5 plays a crucial role in the genesis of synovial joints. Here, we perform an integrative analysis of the transcriptome, chromatin accessibility, and Creb5-chromatin occupancy in Creb5-expressing synovial joint progenitor cells. This analysis revealed that Creb5 directly binds to both its own two promoters and to the regulatory regions of the early joint markers, Gdf5 and Sfrp2; each of whose expression in the joint-interzone is Creb5-dependent. Functional enhancer analysis indicated that Creb5 binding sites in its own two promoters are necessary for these sequences to drive transgene expression in a pattern that mimics endogenous Creb5 expression. While Creb5 directly regulates both Gdf5 and Sfrp2 expression in the inner joint-interzone by binding to either CREs or TREs in their regulatory elements, Creb5 activates Barx1 expression specifically in the outer joint-interzone; and thus initiates both the formation of the joint-interzone and regionalization of cell fates within this tissue. Guided by fluorescence of both tdTomato and EGFP, we dissected the developing knee joints of E14.5 Creb53XHA-P2A-tdTomato/+; Sox9iresEGFP/+ embryos. Following collagenase treatment and filtration to isolate single cells, we sorted the cells from the developing knees into three populations: those that express either Creb5HA-tdTomato, Sox9-ires-EGFP, or both Creb5HA-tdTomato and Sox9-ires-EGFP. To identify Creb5-dependent regulatory regions in knee joint progenitor cells we compared chromatin accessibility in Fluorescence-Activated Cell (FAC) sorted Creb5+Sox9+, Creb5+Sox9-, and Creb5-Sox9+ cells, isolated from the developing knees of E14.5 Creb53XHA-P2A-tdTomato/+; Sox9 iresEGFP/+ embryos from two independent litters by ATAC-Seq.