Repression of chondrogenesis through binding of notch signaling proteins HES-1 and HEY-1 to N-box domains in the COL2A1 enhancer site

Objective—Notch signaling is implicated in repressing mesenchymal stem cell (MSC) chondrogenic differentiation. The mechanism of this repression and how this is modulated to permit chondrogenesis has not been elucidated. Methods—Notch intracellular domain (NICD) protein levels were monitored via western blot throughout chondrogenic differentiation of human in MSC pellet cultures. Overexpression of Notch signaling components and their effect on chondrogenesis was achieved by transfecting plasmids coding for NICD, hairy and enhancer of split 1 (Hes-1) and hairy and enhancer of split related-2 (HERP-2/Hey-1). Col2a1 and aggrecan expression was monitored via quantitative PCR. Chromatin immunoprecipitation (chIP) was utilized to test whether Hes-1 and Hey-1 bind putative N-box domains in intron 1 of Col2a1. Results—NICD protein levels were reduced during chondrogenesis of hMSC, which was mediated by TGFβ3. Col2a1 gene expression was repressed following overexpression of NICD (2-fold), Hes-1 (3-fold) and markedly by Hey-1 (80-fold). Hey-1 repressed aggrecan expression 10-fold, while NICD and Hes-1 had no effect. chIP studies show that endogenous Hes-1 and Hey-1 bind to two putative N-box domains adjacent to and as part of the Sox9 enhancer binding site located in intron 1 of Col2a1. The Hes-1 co-repressor protein transducin like enhancer (TLE) was displaced during chondrogenic differentiation and following TGFβ3 treatment. Conclusion—These results reveal novel mechanisms by which Notch signaling represses gene expression and further define the role of TGFβ to promote chondrogenic differentiation.