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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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Temporal binding of Mybbp1a to Per2 promoter correlates with mCRY1 binding. (A) Temporal expression profile of Mybbp1a mRNA in NIH3T3 cells. Cells were stimulated with dexamethasone and then total RNA isolated at various time points was analyzed by RT-PCR. Products of PCR were resolved by electrophoresis in 2% agarose gels and stained with ethidium bromide (top panels). Levels of mRNA were normalized to G3PDH expression and peak values of individual curves were set to 1 (bottom panel). (B) Oscillatory binding of Mybbp1a to the Per2 promoter. NIH3T3 cells were stimulated with dexamethasone, and then analyzed at each time point by ChIP assays using indicated antibodies and primers for Per2 promoter. Products of PCR were resolved by electrophoresis in 2% agarose gels and stained with ethidium bromide (top panels). Relative band intensities were normalized to input intensities. Peak values of individual curves were set to 1 (bottom panel).
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Figure 4: Temporal binding of Mybbp1a to Per2 promoter correlates with mCRY1 binding. (A) Temporal expression profile of Mybbp1a mRNA in NIH3T3 cells. Cells were stimulated with dexamethasone and then total RNA isolated at various time points was analyzed by RT-PCR. Products of PCR were resolved by electrophoresis in 2% agarose gels and stained with ethidium bromide (top panels). Levels of mRNA were normalized to G3PDH expression and peak values of individual curves were set to 1 (bottom panel). (B) Oscillatory binding of Mybbp1a to the Per2 promoter. NIH3T3 cells were stimulated with dexamethasone, and then analyzed at each time point by ChIP assays using indicated antibodies and primers for Per2 promoter. Products of PCR were resolved by electrophoresis in 2% agarose gels and stained with ethidium bromide (top panels). Relative band intensities were normalized to input intensities. Peak values of individual curves were set to 1 (bottom panel).

Mentions: Circadian oscillators are operative even in cell lines cultured in vitro (4,37,38). We examined the role of Mybbp1a on the expression of Per2 in the cell-autonomous clock after inducing circadian gene expression in NIH3T3 cells with dexamethasone (13). Reverse transcription (RT)-PCR showed that Mybbp1a mRNA expression temporally fluctuated (Figure 4A), but without a circadian rhythm. In contrast, the transcription of Per2 oscillated with a robust circadian rhythm and rhythmic Per2 expression peaked at 30 and 54 h. The overall fluctuation in the mRNA level of Mybbp1a seemed to be generated from a primary response to dexamethasone stimulation. Thus, we examined the temporal expression profile of Mybbp1a mRNA in livers of mice maintained under light/dark cycles (Figure 5). Circadian fluctuation was absent in the Mybbp1a mRNA (Figure 5, top panel). Conversely, the rhythms of Per2 and Cry1 expression were robustly circadian and rhythmic Per2 and Cry1 mRNA expression peaked at Zeitgeber times (ZT) 14 and ZT 17, respectively (Figure 5, middle and bottom panels). Therefore, we concluded that Mybbp1a mRNA did not oscillate in a circadian manner.Figure 4.


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)

Temporal binding of Mybbp1a to Per2 promoter correlates with mCRY1 binding. (A) Temporal expression profile of Mybbp1a mRNA in NIH3T3 cells. Cells were stimulated with dexamethasone and then total RNA isolated at various time points was analyzed by RT-PCR. Products of PCR were resolved by electrophoresis in 2% agarose gels and stained with ethidium bromide (top panels). Levels of mRNA were normalized to G3PDH expression and peak values of individual curves were set to 1 (bottom panel). (B) Oscillatory binding of Mybbp1a to the Per2 promoter. NIH3T3 cells were stimulated with dexamethasone, and then analyzed at each time point by ChIP assays using indicated antibodies and primers for Per2 promoter. Products of PCR were resolved by electrophoresis in 2% agarose gels and stained with ethidium bromide (top panels). Relative band intensities were normalized to input intensities. Peak values of individual curves were set to 1 (bottom panel).
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Figure 4: Temporal binding of Mybbp1a to Per2 promoter correlates with mCRY1 binding. (A) Temporal expression profile of Mybbp1a mRNA in NIH3T3 cells. Cells were stimulated with dexamethasone and then total RNA isolated at various time points was analyzed by RT-PCR. Products of PCR were resolved by electrophoresis in 2% agarose gels and stained with ethidium bromide (top panels). Levels of mRNA were normalized to G3PDH expression and peak values of individual curves were set to 1 (bottom panel). (B) Oscillatory binding of Mybbp1a to the Per2 promoter. NIH3T3 cells were stimulated with dexamethasone, and then analyzed at each time point by ChIP assays using indicated antibodies and primers for Per2 promoter. Products of PCR were resolved by electrophoresis in 2% agarose gels and stained with ethidium bromide (top panels). Relative band intensities were normalized to input intensities. Peak values of individual curves were set to 1 (bottom panel).
Mentions: Circadian oscillators are operative even in cell lines cultured in vitro (4,37,38). We examined the role of Mybbp1a on the expression of Per2 in the cell-autonomous clock after inducing circadian gene expression in NIH3T3 cells with dexamethasone (13). Reverse transcription (RT)-PCR showed that Mybbp1a mRNA expression temporally fluctuated (Figure 4A), but without a circadian rhythm. In contrast, the transcription of Per2 oscillated with a robust circadian rhythm and rhythmic Per2 expression peaked at 30 and 54 h. The overall fluctuation in the mRNA level of Mybbp1a seemed to be generated from a primary response to dexamethasone stimulation. Thus, we examined the temporal expression profile of Mybbp1a mRNA in livers of mice maintained under light/dark cycles (Figure 5). Circadian fluctuation was absent in the Mybbp1a mRNA (Figure 5, top panel). Conversely, the rhythms of Per2 and Cry1 expression were robustly circadian and rhythmic Per2 and Cry1 mRNA expression peaked at Zeitgeber times (ZT) 14 and ZT 17, respectively (Figure 5, middle and bottom panels). Therefore, we concluded that Mybbp1a mRNA did not oscillate in a circadian manner.Figure 4.

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