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Molecular characterization of Mybbp1a as a co-repressor on the Period2 promoter.

Hara Y, Onishi Y, Oishi K, Miyazaki K, Fukamizu A, Ishida N - Nucleic Acids Res. (2009)

Bottom Line: We found that Mybbp1a functions as a co-repressor of Per2 expression and repressed Per2 promoter activity in reporter assays.Furthermore, Mybbp1a binding to the Per2 promoter correlated with the start of the down-regulation of Per2 expression and with the dimethylation of histone H3 Lys9, to which it could also bind.These findings suggest that Mybbp1a and mCRY1 can form complexes on the Per2 promoter that function as negative regulators of Per2 expression.

View Article: PubMed Central - PubMed

Affiliation: Clock Cell Biology, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.

ABSTRACT
The circadian clock comprises transcriptional feedback loops of clock genes. Cryptochromes are essential components of the negative feedback loop in mammals as they inhibit CLOCK-BMAL1-mediated transcription. We purified mouse CRY1 (mCRY1) protein complexes from Sarcoma 180 cells to determine their roles in circadian gene expression and discovered that Myb-binding protein 1a (Mybbp1a) interacts with mCRY1. Mybbp1a regulates various transcription factors, but its role in circadian gene expression is unknown. We found that Mybbp1a functions as a co-repressor of Per2 expression and repressed Per2 promoter activity in reporter assays. Chromatin immunoprecipitation (ChIP) assays revealed endogenous Mybbp1a binding to the Per2 promoter that temporally matched that of mCRY1. Furthermore, Mybbp1a binding to the Per2 promoter correlated with the start of the down-regulation of Per2 expression and with the dimethylation of histone H3 Lys9, to which it could also bind. These findings suggest that Mybbp1a and mCRY1 can form complexes on the Per2 promoter that function as negative regulators of Per2 expression.

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Mybbp1a represses transcriptional activity of Per2 promoter. (A) Schematic representation of reporter gene containing mouse Per2 promoter (Per2 promoter-Luc). Arrow and open box indicate transcription start site (TSS) and E2-box, respectively. (B) Repression of Per2 promoter activity by Mybbp1a in NIH3T3 cells. Co-transfection with increasing amounts of Mybbp1a expression plasmid (0.15, 0.30, 0.60 and 1.2 μg) and Per2 promoter-Luc without CLOCK and BMAL1 (left panel). Co-transfection with increasing amounts of Mybbp1a expression plasmid (0.5, 1.0 and 1.5 μg) and Per2 promoter-Luc with CLOCK and BMAL1 (right panel). Symbols (+) or (–), presence or absence of expression plasmids, respectively. Expression levels were calculated relative to luciferase activities without Mybbp1a and CLOCK-BMAL1. Note difference in y-axis scale between left and right panels. (C) Binding of Mybbp1a to Per2 promoter. NIH3T3 cells were transfected with Flag-tagged Mybbp1a and then analyzed by ChIP assays using indicated antibodies, followed by PCR amplification with primers for Per2 promoter. Symbols (+) or (–), presence or absence of Flag-Mybbp1a-expression plasmids, respectively. (D) Identification of endogenous proteins on Per2 promoter region. NIH3T3 cells were analyzed by ChIP assays using either anti-Mybbp1a or anti-mCRY1 antibodies. Preimmune: preimmune serum of same animals in which antisera were raised. No Ab: without antibody. G3PDH primers served as negative control.
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Figure 3: Mybbp1a represses transcriptional activity of Per2 promoter. (A) Schematic representation of reporter gene containing mouse Per2 promoter (Per2 promoter-Luc). Arrow and open box indicate transcription start site (TSS) and E2-box, respectively. (B) Repression of Per2 promoter activity by Mybbp1a in NIH3T3 cells. Co-transfection with increasing amounts of Mybbp1a expression plasmid (0.15, 0.30, 0.60 and 1.2 μg) and Per2 promoter-Luc without CLOCK and BMAL1 (left panel). Co-transfection with increasing amounts of Mybbp1a expression plasmid (0.5, 1.0 and 1.5 μg) and Per2 promoter-Luc with CLOCK and BMAL1 (right panel). Symbols (+) or (–), presence or absence of expression plasmids, respectively. Expression levels were calculated relative to luciferase activities without Mybbp1a and CLOCK-BMAL1. Note difference in y-axis scale between left and right panels. (C) Binding of Mybbp1a to Per2 promoter. NIH3T3 cells were transfected with Flag-tagged Mybbp1a and then analyzed by ChIP assays using indicated antibodies, followed by PCR amplification with primers for Per2 promoter. Symbols (+) or (–), presence or absence of Flag-Mybbp1a-expression plasmids, respectively. (D) Identification of endogenous proteins on Per2 promoter region. NIH3T3 cells were analyzed by ChIP assays using either anti-Mybbp1a or anti-mCRY1 antibodies. Preimmune: preimmune serum of same animals in which antisera were raised. No Ab: without antibody. G3PDH primers served as negative control.

Mentions: Although Mybbp1a binds to several transcription factors, which indicates a role in transcriptional regulation, the function of Mybbp1a in circadian gene expression remains unknown. To determine whether Mybbp1a regulates Per2 gene expression, we performed reporter assays in NIH3T3 cells in which Per2 is a core oscillator for maintenance of the circadian clock. We cotransfected the Per2-luciferase reporter (Figure 3A) with a Mybbp1a expression plasmid. Figure 3B shows that Mybbp1a dose-dependently repressed Per2 promoter activity (left panel). We then tested the effect of Mybbp1a on CLOCK-BMAL1-dependent transactivation (Figure 3B, right panel). Increasing amounts of Mybbp1a dose-dependently repressed Per2 promoter activity regardless of the presence of CLOCK-BMAL1. We also examined whether or not Mybbp1a represses Per2 promoter activity via an E2-box. We used a reporter containing a mutant E2-box that lacks transcriptional activation by CLOCK-BMAL1 (31) (Supplementary Figure S5A). A mutation of the E2-box (mut E2-box reporter) resulted in the same transcriptional repression (up to 44%) as the wild type (wt E2-box reporter) (Supplementary Figure S5B, left panel).Figure 3.


Molecular characterization of Mybbp1a as a co-repressor on the Period2 promoter.

Hara Y, Onishi Y, Oishi K, Miyazaki K, Fukamizu A, Ishida N - Nucleic Acids Res. (2009)

Mybbp1a represses transcriptional activity of Per2 promoter. (A) Schematic representation of reporter gene containing mouse Per2 promoter (Per2 promoter-Luc). Arrow and open box indicate transcription start site (TSS) and E2-box, respectively. (B) Repression of Per2 promoter activity by Mybbp1a in NIH3T3 cells. Co-transfection with increasing amounts of Mybbp1a expression plasmid (0.15, 0.30, 0.60 and 1.2 μg) and Per2 promoter-Luc without CLOCK and BMAL1 (left panel). Co-transfection with increasing amounts of Mybbp1a expression plasmid (0.5, 1.0 and 1.5 μg) and Per2 promoter-Luc with CLOCK and BMAL1 (right panel). Symbols (+) or (–), presence or absence of expression plasmids, respectively. Expression levels were calculated relative to luciferase activities without Mybbp1a and CLOCK-BMAL1. Note difference in y-axis scale between left and right panels. (C) Binding of Mybbp1a to Per2 promoter. NIH3T3 cells were transfected with Flag-tagged Mybbp1a and then analyzed by ChIP assays using indicated antibodies, followed by PCR amplification with primers for Per2 promoter. Symbols (+) or (–), presence or absence of Flag-Mybbp1a-expression plasmids, respectively. (D) Identification of endogenous proteins on Per2 promoter region. NIH3T3 cells were analyzed by ChIP assays using either anti-Mybbp1a or anti-mCRY1 antibodies. Preimmune: preimmune serum of same animals in which antisera were raised. No Ab: without antibody. G3PDH primers served as negative control.
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Figure 3: Mybbp1a represses transcriptional activity of Per2 promoter. (A) Schematic representation of reporter gene containing mouse Per2 promoter (Per2 promoter-Luc). Arrow and open box indicate transcription start site (TSS) and E2-box, respectively. (B) Repression of Per2 promoter activity by Mybbp1a in NIH3T3 cells. Co-transfection with increasing amounts of Mybbp1a expression plasmid (0.15, 0.30, 0.60 and 1.2 μg) and Per2 promoter-Luc without CLOCK and BMAL1 (left panel). Co-transfection with increasing amounts of Mybbp1a expression plasmid (0.5, 1.0 and 1.5 μg) and Per2 promoter-Luc with CLOCK and BMAL1 (right panel). Symbols (+) or (–), presence or absence of expression plasmids, respectively. Expression levels were calculated relative to luciferase activities without Mybbp1a and CLOCK-BMAL1. Note difference in y-axis scale between left and right panels. (C) Binding of Mybbp1a to Per2 promoter. NIH3T3 cells were transfected with Flag-tagged Mybbp1a and then analyzed by ChIP assays using indicated antibodies, followed by PCR amplification with primers for Per2 promoter. Symbols (+) or (–), presence or absence of Flag-Mybbp1a-expression plasmids, respectively. (D) Identification of endogenous proteins on Per2 promoter region. NIH3T3 cells were analyzed by ChIP assays using either anti-Mybbp1a or anti-mCRY1 antibodies. Preimmune: preimmune serum of same animals in which antisera were raised. No Ab: without antibody. G3PDH primers served as negative control.
Mentions: Although Mybbp1a binds to several transcription factors, which indicates a role in transcriptional regulation, the function of Mybbp1a in circadian gene expression remains unknown. To determine whether Mybbp1a regulates Per2 gene expression, we performed reporter assays in NIH3T3 cells in which Per2 is a core oscillator for maintenance of the circadian clock. We cotransfected the Per2-luciferase reporter (Figure 3A) with a Mybbp1a expression plasmid. Figure 3B shows that Mybbp1a dose-dependently repressed Per2 promoter activity (left panel). We then tested the effect of Mybbp1a on CLOCK-BMAL1-dependent transactivation (Figure 3B, right panel). Increasing amounts of Mybbp1a dose-dependently repressed Per2 promoter activity regardless of the presence of CLOCK-BMAL1. We also examined whether or not Mybbp1a represses Per2 promoter activity via an E2-box. We used a reporter containing a mutant E2-box that lacks transcriptional activation by CLOCK-BMAL1 (31) (Supplementary Figure S5A). A mutation of the E2-box (mut E2-box reporter) resulted in the same transcriptional repression (up to 44%) as the wild type (wt E2-box reporter) (Supplementary Figure S5B, left panel).Figure 3.

Bottom Line: We found that Mybbp1a functions as a co-repressor of Per2 expression and repressed Per2 promoter activity in reporter assays.Furthermore, Mybbp1a binding to the Per2 promoter correlated with the start of the down-regulation of Per2 expression and with the dimethylation of histone H3 Lys9, to which it could also bind.These findings suggest that Mybbp1a and mCRY1 can form complexes on the Per2 promoter that function as negative regulators of Per2 expression.

View Article: PubMed Central - PubMed

Affiliation: Clock Cell Biology, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.

ABSTRACT
The circadian clock comprises transcriptional feedback loops of clock genes. Cryptochromes are essential components of the negative feedback loop in mammals as they inhibit CLOCK-BMAL1-mediated transcription. We purified mouse CRY1 (mCRY1) protein complexes from Sarcoma 180 cells to determine their roles in circadian gene expression and discovered that Myb-binding protein 1a (Mybbp1a) interacts with mCRY1. Mybbp1a regulates various transcription factors, but its role in circadian gene expression is unknown. We found that Mybbp1a functions as a co-repressor of Per2 expression and repressed Per2 promoter activity in reporter assays. Chromatin immunoprecipitation (ChIP) assays revealed endogenous Mybbp1a binding to the Per2 promoter that temporally matched that of mCRY1. Furthermore, Mybbp1a binding to the Per2 promoter correlated with the start of the down-regulation of Per2 expression and with the dimethylation of histone H3 Lys9, to which it could also bind. These findings suggest that Mybbp1a and mCRY1 can form complexes on the Per2 promoter that function as negative regulators of Per2 expression.

Show MeSH
Related in: MedlinePlus