Creb5 controls its own expression and directly induces the joint-interzone regulatory program [CutandRun_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. To specifically identify Creb5 binding sites in the articular perichondrium of the zeugopods, we crossed Creb53XHA-P2A-tdTomato/+ mice with Hoxa11EGFP/+ mice to obtain E14.5 Creb53XHA-P2A-tdTomato/+; Hoxa11EGFP/+ embryos; employed FAC sorting to isolate cells that expressed both Creb5HA-tdTomato & Hoxa11EGFP from either forelimbs or hindlimbs; and performed either Creb5- or HA- or IgG-(control) Cut&Run-Seq. Creb5-Cut&Run-Seq was also performed with either Creb5+Sox9- cells or Creb5+Sox9+ cells (isolated from E14.5 Creb53XHA-P2A-tdTomato/+; Sox9iresEGFP/+ embryos).