Journal: Scientific Reports

Article Title: PER2 mediates CREB-dependent light induction of the clock gene Per1

doi: 10.1038/s41598-021-01178-6

Figure Lengend Snippet: Induction of Per1 , Per2 , and cFos gene expression after light or forskolin stimulation. The diagram top left indicates the light treatment protocol of mice ( A , C , E ) and the top right diagram shows the forskolin treatment of cells ( B , D , F ). ( A ) Fold change of Per1 (black bars) and Per2 (grey bars) pre-mRNA expression in the SCN after a 15 min light pulse (LP) applied at ZT14. Values are the mean ± SEM. Student's t-test with Welch's correction, n = 3, * p < 0.05, ** p < 0.01. ( B ) Fold change of Per1 (black bars) and Per2 (grey bars) pre-mRNA expression in NIH 3T3 cells after forskolin treatment. Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, * p < 0.05, ** p < 0.01. ( C ) Fold change of Per1 (top panel) and Per2 (bottom panel) pre-mRNA expression in the SCN of wild type (wt, black bars) and Per2 knock-out (KO, grey bars) mice after a 15 min light pulse (LP) applied at ZT14. Values are the mean ± SEM. Student's t-test with Welch's correction, n = 3, * p < 0.05, ** p < 0.01. ( D ) Fold change of Per1 (top panel) and Per2 (bottom panel) pre-mRNA expression in immortalized wt or Per2 KO fibroblast cells after forskolin treatment. Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, * p < 0.05, ** p < 0.01. ( E ) Fold change of cFos expression in the SCN of wt (black bars) and Per2 KO (grey bars) mice after a 15 min light pulse (LP) applied at ZT14. Values are the mean ± SEM. Student's t-test with Welch's correction, n = 3, * p < 0.05, ** p < 0.01. ( F ) Fold change of cFos expression in immortalized wt or Per2 KO fibroblast cells after forskolin treatment. Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, * p < 0.05.

Article Snippet: Sh RNA Per2 Vector used for silencing Per2 in cell lines. (Origene cat #TL501620).

Techniques: Gene Expression, Expressing, Knock-Out

Journal: Scientific Reports

Article Title: PER2 mediates CREB-dependent light induction of the clock gene Per1

doi: 10.1038/s41598-021-01178-6

Figure Lengend Snippet: 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.

Article Snippet: Sh RNA Per2 Vector used for silencing Per2 in cell lines. (Origene cat #TL501620).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Control, Western Blot, Immunoprecipitation

Journal: Scientific Reports

Article Title: PER2 mediates CREB-dependent light induction of the clock gene Per1

doi: 10.1038/s41598-021-01178-6

Figure Lengend Snippet: 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.

Article Snippet: Sh RNA Per2 Vector used for silencing Per2 in cell lines. (Origene cat #TL501620).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Control, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Scientific Reports

Article Title: PER2 mediates CREB-dependent light induction of the clock gene Per1

doi: 10.1038/s41598-021-01178-6

Figure Lengend Snippet: Modulation of the interaction between CREB and CBP by PER2. ( A ) Left panel: Chromatin immunoprecipitation (ChIP) of CBP 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 a promoter region of Per1 without a CRE-element . 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, left panel: **** p < 0.0001, right panel: ** p < 0.01. ( B ) Immunohistochemistry (IHC) of wt and Per2 -/- cells before and after forskolin treatment. CBP (red signal) is induced after forskolin treatment in both genotypes. Scale bar: 17 µm. ( C ) IP of CREB pulls down CBP in wt but not Per2 -/- cells after forskolin treatment, kD = kilo Dalton. ( D ) IHC of wt and Per2 -/- SCN before and after a light pulse (LP) at ZT14. CBP (red signal) is induced after LP in both genotypes. Scale bar: 7 µm. ( E ) IP of CREB pulls down CBP in wt SCN extracts after LP, but not in Per2 -/- SCN extracts. kD = kilo Dalton.

Article Snippet: Sh RNA Per2 Vector used for silencing Per2 in cell lines. (Origene cat #TL501620).

Techniques: Chromatin Immunoprecipitation, Control, Immunohistochemistry

Journal: Scientific Reports

Article Title: PER2 mediates CREB-dependent light induction of the clock gene Per1

doi: 10.1038/s41598-021-01178-6

Figure Lengend Snippet: FRET analysis of the interaction between PER2 and the KID/KIX domains of CREB/CBP. ( A ) Top panel: Schematic diagram of the ICAP construct that is an indicator of CREB: CBP dimerization due to phosphorylation on the KID (CREB) domain at serine 133. Bottom panel: Proposed model of PER2 mediated interaction between the KID and KIX domains. Modified from using PowerPoint software for Mac version 16.16.27 ( B ) % of cellular response to forskolin after 3 min in NIH 3T3 cells transfected either with scrambled (scr) shRNA or an shRNA directed against Per2 (shPer2). Values are the mean ± SD. Student's t-test with Welch's correction, n = 3, *** p < 0.001. ( C ) The FRET/CFP signal ratio changes in response to forskolin treatment in cells transfected with either a scr control (red) or shPer2 (blue) expressing construct. Values are the mean ± SD. Two-way ANOVA revealed a significant difference between the curves, n = 50, **** p < 0.0001. ( D ) Time elapsed to reach half of the total FRET/CFP signal is significantly shorter in scr control transfected cells than shPer2 transfected cells. Values are the mean ± SD. Student's t-test with Welch's correction, n = 50, * p < 0.05. ( E ) KID/KIX dimerization is significantly lower in shPer2 cells than scr control cells. Values are the mean ± SD. Two-way ANOVA, n = 50, * p < 0.05.

Article Snippet: Sh RNA Per2 Vector used for silencing Per2 in cell lines. (Origene cat #TL501620).

Techniques: Construct, Phospho-proteomics, Modification, Software, Transfection, shRNA, Control, Expressing

Journal: Scientific Reports

Article Title: PER2 mediates CREB-dependent light induction of the clock gene Per1

doi: 10.1038/s41598-021-01178-6

Figure Lengend Snippet: 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.

Article Snippet: Sh RNA Per2 Vector used for silencing Per2 in cell lines. (Origene cat #TL501620).

Techniques: Chromatin Immunoprecipitation, Immunohistochemistry, Western Blot

Journal: Scientific Reports

Article Title: PER2 mediates CREB-dependent light induction of the clock gene Per1

doi: 10.1038/s41598-021-01178-6

Figure Lengend Snippet: 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.

Article Snippet: Sh RNA Per2 Vector used for silencing Per2 in cell lines. (Origene cat #TL501620).

Techniques: Phospho-proteomics, Generated

Journal: Scientific Reports

Article Title: PER2 mediates CREB-dependent light induction of the clock gene Per1

doi: 10.1038/s41598-021-01178-6

Figure Lengend Snippet: List of primers used in the paper (5′–3′; FAM: 6-fluorescin, BHQ1: black hole quencher 1).

Article Snippet: Sh RNA Per2 Vector used for silencing Per2 in cell lines. (Origene cat #TL501620).

Techniques: Sequencing, Amplification

Journal: Scientific Reports

Article Title: PER2 mediates CREB-dependent light induction of the clock gene Per1

doi: 10.1038/s41598-021-01178-6

Figure Lengend Snippet: List of antibodies used in the paper and relative applications.

Article Snippet: Sh RNA Per2 Vector used for silencing Per2 in cell lines. (Origene cat #TL501620).

Techniques: Western Blot, Immunofluorescence