Fig 2: Binding of CREB and PER2 to the CRE-element of the Per1 promoter and interaction between CREB and PER2. (A) Top panel: Chromatin immunoprecipitation (ChIP) of CREB on the CRE-element of the Per1 promoter in wt (black bars) and Per2 KO (grey bars) fibroblast cell lines after forskolin treatment. Bottom panel: Control ChIP on an unrelated promoter region of Per1. Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, *p < 0.05, **p < 0.01. Two-way ANOVA indicates a significantly different time profile between the two genotypes, **p < 0.01. (B) Top panel: ChIP of pSer-133 CREB on the CRE-element of the Per1 promoter in wt (black bars) and Per2 KO (grey bars) fibroblast cell lines after forskolin treatment. Bottom panel: Control ChIP on an unrelated promoter region of Per1. Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, *p < 0.05. Two-way ANOVA indicates a significantly different time profile between the two genotypes, **p < 0.01. (C) Top panel: ChIP of PER2 on the CRE-element of the Per1 promoter in wt (black bars) and Per2 KO (grey bars) fibroblast cell lines after forskolin treatment. Bottom panel: Control ChIP on an unrelated promoter region of Per1. Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, *p < 0.05. Two-way ANOVA indicates a significantly different time profile between the two genotypes, **p < 0.01. (D) Western blot (left panel) was performed on 10% of the total input used for the immunoprecipitation using a CREB antibody as bait (right panel). PER2 co-precipitates with CREB in NIH 3T3 cell extracts, the pSer-133 antibody was used to confirm the forskolin induction. kD = kilo Dalton. (E) IP with an antibody recognizing CREB in SCN extracts, which co-precipitated with PER2 before (D) and after a 15 min light pulse (LP) at ZT14. The pSer-133 antibody was used to confirm light induction. Input control with lamin B (bottom panel), kD = kilo Dalton. *Unspecific band recognized by the αPER2 antibody in tissue extracts.

Fig 3: Binding of CRTC1 to the Per1 promoter and interaction with PER2 and CREB. (A) Left panel: Chromatin immunoprecipitation (ChIP) of CRTC1 on the CRE-element of the Per1 promoter in wt (black bars) and Per2 KO (grey bars) fibroblast cell lines after forskolin treatment. Right panel: Control ChIP on an unrelated promoter region of Per1. Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, *p < 0.05, **p < 0.01. Two-way ANOVA indicates a significantly different time profile between the two genotypes, **p < 0.01. (B) Western blot of wt and Per2-/- (Per2 KO) cells before and 25 min after forskolin treatment. CRTC1 is induced by forskolin independently of Per2. CREB is phosphorylated (pSer133) in both genotypes after the stimulus. (C) Immunoprecipitation (IP) of CRTC1 pulls down PER2 independently of forskolin treatment. (D) IP of CREB pulls down CRTC1 after forskolin treatment in wt cells, but in Per2-/- cells, the interaction between CREB and CRTC1 is forskolin independent. (E) IP of CRTC1 in SCN extracts before and after a 15 min light pulse (LP) at ZT14. Co-IP of PER2 is independent of LP. (F) Western blot of wt and Per2-/- SCN tissue performed as in B on samples collected before and after LP, * unspecific band recognized by the αPER2 antibody in tissue extracts. CREB is phosphorylated in both genotypes after the light pulse. (G) IP of CREB pulls down CRTC1 in wt SCN extracts after LP, but in Per2-/- SCN the CREB: CRTC1 interaction is light-independent. kD = kilo Dalton.

Fig 4: Modulation of CBP-mediated chromatin acetylation of histone H3K27 by PER2. (A) Chromatin immunoprecipitation (ChIP) of acetylated histone H3 (AcH3K27) on the CRE-element of the Per1 promoter in wt (black bars) and Per2 KO (grey bars) fibroblast cell lines after forskolin treatment. Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, *p < 0.05, **p < 0.01. Two-way ANOVA indicates a significantly different time profile between the two genotypes, **p < 0.01. (B) The ChIP of RNA polymerase II (RNA Pol II) on the CRE-element of the Per1 promoter in wt (black bars) and Per2 KO (grey bars) fibroblast cell lines after forskolin treatment. Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, **p < 0.01. Two-way ANOVA indicates a significantly different time profile between the two genotypes, ***p < 0.001. (C) ChIP with an antibody against AcH3K27 on the Cre-element of the cFos promoter in wt (black bars) and Per2 KO (grey bars) fibroblast cell lines after forskolin treament. Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, ***p < 0.001. (D) ChIP of RNA Pol II on the CRE-element of the cFos promoter in wt (black bars) and Per2 KO (grey bars) fibroblast cell lines after forskolin treatment. Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, *p < 0.05, ***p < 0.001. (E) Immunohistochemistry (IHC) on wt and Per2 KO SCN before and after a light pulse (LP) at ZT14. AcH3K27 (red signal) is stronger after LP in wt compared to Per2 KO SCN. Scale bar: 121 µm. (F) Quantification of 3 independent Western blot experiments of wt and Per2 KO SCN before and after an LP at ZT14. Acetylation of H3K27 is increased after an LP with a tendentially lower increase in Per2 KO. Two-way ANOVA, p = 0.059.

Fig 5: Model of PER2 modulating the assembly of the CREB/CRTC1/CBP transcriptional complex. Left: In the early dark phase at ZT14, PER2 interacts with CREB and CRTC1. On the other hand, CREB and CRTC1 do not dimerize at this stage, and all the elements do not bind to the CRE element in the Per1 promoter. CBP is not recruited to the regulatory region of Per1 and the histone H3K27 is not acetylated. Per1 is not transcribed through this pathway. Right: In the SCN or in cells a light pulse or forskolin treatment initiates a kinase cascade that leads to CREB phosphorylation and assembly of the CREB: CRTC1: CBP complex which is facilitated by PER2. As a consequence of this assembly, histone H3K27 is acetylated (grey star) and the Per1 gene is transcribed. The hatched oval with question mark indicates that most likely additional factors contribute to the initiation of transcription by the RNA polymerase II (POLII). Figure was generated using Adobe Illustrator version 24.3.1.