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: 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, 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: 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

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

Journal: Molecular Neurodegeneration

Article Title: Aβ-induced degradation of BMAL1 and CBP leads to circadian rhythm disruption in Alzheimer’s disease

doi: 10.1186/s13024-015-0007-x

Figure Lengend Snippet: Regulation of BMAL1 protein stability by Aβ-mediated sumoylation. (A & B) Aβ affected the stability of BMAL1 protein. HT22 cells were incubated with CHX (100 μg/ml) with vehicle, or CHX (100 μg/ml) with Aβ (2 μM) for the indicated times. The protein level of BMAL1 was analyzed by immunoblotting with anti-BMAL1 antibody and then quantified by densitometry. Data are represented as mean ± SEM. ** P < 0.01, *** P < 0.001. (C & D) Effects of Aβ on the interaction between BMAL1 and SUMO1. (C) HT22 cells were incubated with vehicle or Aβ (2 μM) for 24 h after cell synchronization. (D) Both littermate and 5XFAD mice brains were prepared for immunoprecipitation. Each sample was immunoprecipitated using the anti-BMAL1 antibody. Confirmation by western blot analysis with anti-SUMO-1 antibody showed that the immunoprecipitated BMAL1 was physically interacted with SUMO1. This interaction was greater with Aβ treatment. (E) The transfection of Sumo1 siRNA into HT22 cells rescued Aβ-induced BMAL1 degradation compared to control siRNA. (F) Densitometric analysis of BMAL1 protein from three independent experiments. BMAL1 was normalized to α-Tubulin. ** P < 0.01, # P < 0.05, $ P < 0.05. (G) The transfection of BMAL1 K259R into HT22 cells decreased Aβ-induced BMAL1 degradation compared to BMAL1 WT. (H) Densitometric analysis of BMAL1 protein from three independent experiments. BMAL1 was normalized to GAPDH. *** P < 0.001, # P < 0.05.

Article Snippet: The control siRNA (SC-37007) and siRNA against SUMO1 (SC-36574) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). cDNA expression vectors for pcDNATM 3.1-mouse CBP, Per2-Luc, Myc-BMAL1, VC-BMAL1 and VN-CBP constructs were generously gifted from Dr. Kyungjin Kim (Seoul National University, Korea).

Techniques: Incubation, Western Blot, Immunoprecipitation, Transfection, Control

Journal: Cell reports

Article Title: Cell-Autonomous Regulation of Astrocyte Activation by the Circadian Clock Protein BMAL1

doi: 10.1016/j.celrep.2018.09.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Gfap – Rabbit polyclonal, 1:5000 Dako/Agilent Cat# Z0334; RRID:AB_10013482 Gfap- Mouse monoclonal, 1:1000 (Co-stain only) Novus Cat# NBP1-05197; RRID:AB_1555288 Bmal1- Rabbit Monoclonal, 1:2000 (IF/IHC only) Novus Cat# NB100-2288; RRID:AB_10000794 Bmal1- Rabbit polyclonal, 1:1000 (WB only) Bethyl Cat# A302-616A; RRID:AB_10555918 MAP2- Mouse monoclonal, 1:1000 EMD/Milipore Cat# AB5622; RRID:AB_91939 Ki67- Rat monoclonal, SolA15, 1:500 ThermoFisher Cat# 14-5698-82; RRID:AB_2016711 NeuN- Mouse monoclonal, 1:1000 EMD/Millipore Cat# MAB377; RRID:AB_2298772 P42/44 MAPK ERK 1/2- Rabbit monoclonal, 1:1000 (WB) Cell Signaling Technology Cat# 9102; RRID:AB_330744 Bacterial and Virus Strains AAV8-GFAP-eGFP UNC Viral Vector Core N/A AAV8-GFAP-GFP-Cre UNC Viral Vector Core N/A Chemicals, Peptides, and Recombinant Proteins Lipofectamine RNAiMAX transfection reagent ThermoFisher/Life Cat# 13778075 N-acetyl-L-cysteine Sigma Cat# A7250 Tamoxifen Sigma Cat# T5648 L-Buthionine sulfoxime Sigma Cat# 5082280001 Poly-D-lysine hydrobromide Sigma Cat# P6407 Critical Commercial Assays BioGEE Glutathionylation assay kit ThermoFisher Cat# G36000 High Capacity RNA-to-cDNA Kit Applied Biosystems Cat# 4387406 GSH/GSSG Ratio Detection Assay Kit Abcam Cat# ab138881 Taqman Universal PCR Master Mix Applied Biosystems Cat# 4304437 PureLink RNA Mini Kit Ambion/Life Cat# 12183025 Deposited Data Agilent 4x44k mouse microarray v2 data from Nestin-Bmal1, Per1;Per2 double mutant, and control cortex at CT 6 and 18.

Techniques: Plasmid Preparation, Recombinant, Transfection, Detection Assay, Microarray, Mutagenesis, Expressing, Software