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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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Identification of protein components of TAP-purified mCRY1 complexes. (A) Protein complexes eluted after final TAP-purification. Numbers on left side indicate bands excised from gels. Blue characters indicate bands analyzed by MS. Identified bands are marked with asterisks. Numbers on right side represent molecular weight markers. Bands corresponding to purified mCRY1 are indicated by arrowhead. Gel concentrations are indicated on top. (B) Characterization of proteins that interacted with mCRY1 identified by MS analysis.
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Figure 1: Identification of protein components of TAP-purified mCRY1 complexes. (A) Protein complexes eluted after final TAP-purification. Numbers on left side indicate bands excised from gels. Blue characters indicate bands analyzed by MS. Identified bands are marked with asterisks. Numbers on right side represent molecular weight markers. Bands corresponding to purified mCRY1 are indicated by arrowhead. Gel concentrations are indicated on top. (B) Characterization of proteins that interacted with mCRY1 identified by MS analysis.

Mentions: Purified mCRY1 protein complexes were concentrated by trichloroacetic acid (TCA) precipitation, resolved by SDS–PAGE and visualized by silver staining (Silver Stain MS Kit, Wako Pure Chemical). Excised bands on gels (Figure 1A) were digested in situ with trypsin. The tryptic digest was analyzed by either MALDI-TOF-MS (Voyager-DE STR; Applied Biosystems) or nano LC-MS/MS (MAGIC 2002 nano LC; Michrom Bioresources Inc.) and Q-Tof 2 (Waters Micromass). The MALDI-TOF-MS and nano LC-MS/MS data were searched against the public NCBI databases using MS-Fit (http://prospector.ucsf.edu/) and Mascot software (MatrixScience), respectively. Any matches found were manually evaluated and confirmed.Figure 1.


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)

Identification of protein components of TAP-purified mCRY1 complexes. (A) Protein complexes eluted after final TAP-purification. Numbers on left side indicate bands excised from gels. Blue characters indicate bands analyzed by MS. Identified bands are marked with asterisks. Numbers on right side represent molecular weight markers. Bands corresponding to purified mCRY1 are indicated by arrowhead. Gel concentrations are indicated on top. (B) Characterization of proteins that interacted with mCRY1 identified by MS analysis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651808&req=5

Figure 1: Identification of protein components of TAP-purified mCRY1 complexes. (A) Protein complexes eluted after final TAP-purification. Numbers on left side indicate bands excised from gels. Blue characters indicate bands analyzed by MS. Identified bands are marked with asterisks. Numbers on right side represent molecular weight markers. Bands corresponding to purified mCRY1 are indicated by arrowhead. Gel concentrations are indicated on top. (B) Characterization of proteins that interacted with mCRY1 identified by MS analysis.
Mentions: Purified mCRY1 protein complexes were concentrated by trichloroacetic acid (TCA) precipitation, resolved by SDS–PAGE and visualized by silver staining (Silver Stain MS Kit, Wako Pure Chemical). Excised bands on gels (Figure 1A) were digested in situ with trypsin. The tryptic digest was analyzed by either MALDI-TOF-MS (Voyager-DE STR; Applied Biosystems) or nano LC-MS/MS (MAGIC 2002 nano LC; Michrom Bioresources Inc.) and Q-Tof 2 (Waters Micromass). The MALDI-TOF-MS and nano LC-MS/MS data were searched against the public NCBI databases using MS-Fit (http://prospector.ucsf.edu/) and Mascot software (MatrixScience), respectively. Any matches found were manually evaluated and confirmed.Figure 1.

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