Differentiation-coupled formation of core clock protein interactions in mouse embryonic stem cells

In mammals, circadian rhythms are absent from embryonic stem (ES) cells and emerge during cellular differentiation, yet the profile of endogenous clock protein complexes in undifferentiated ES cells and differentiating cells remains unclear. Here we introduced 3×FLAG tags into endogenous Per1, Cry1, or Bmal1 locus in mouse ES cells to investigate core clock protein interactions before and during differentiation. The established 3×FLAG knock-in ES cells expressed the expected 3×FLAG-fusion proteins and displayed normal differentiation-coupled development of circadian gene expression rhythms. mRNA expression levels of core clock genes (Per1, Per2, Cry1, Cry2, Clock and Bmal1) were comparable between knock-in and parental cells across differentiation. The expression profiles of core clock proteins showed that PER1, CRY1/2, BMAL1, CKIδ but not CLOCK were expressed even in undifferentiated ES cells. Anti-FLAG co-immunoprecipitation revealed a progressive change in complex assembly: BMAL1 begins to interact with CLOCK, PER1, CRY1/2 and CKIδ from differentiation day 14 onward, coinciding with the emergence of circadian gene expression rhythms. In contrast, PER1 binds to CRY1/2 and CKIδ throughout the differentiation time course, including undifferentiated ES cells. These findings reveal the differentiation-coupled formation of the core clock protein complex, particularly at the interface between transcriptional activators and repressors, providing a mechanism for ensuring the emergence of robust circadian rhythms during ES cell differentiation.