Limits...
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.

Show MeSH

Related in: MedlinePlus

Specific binding of Mybbp1a to histone H3 dimethylated Lys9. Pull-down assays of lysates from COS-1 cells transfected with Flag-Mybbp1a using histone H3 N-terminal peptides that were modified or not at Lys9. Bound proteins eluted from beads previously bound to peptides were analyzed by Western blotting against anti-Flag antibody. Control experiments were performed with lysates from COS-1 cells transfected with empty vector (pFlag-vector). K9(Me)2, dimethylated Lys9; Unmodified, unmodified Lys9; K9(Ac), acetylated Lys9 (indicated on top).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651808&req=5

Figure 6: Specific binding of Mybbp1a to histone H3 dimethylated Lys9. Pull-down assays of lysates from COS-1 cells transfected with Flag-Mybbp1a using histone H3 N-terminal peptides that were modified or not at Lys9. Bound proteins eluted from beads previously bound to peptides were analyzed by Western blotting against anti-Flag antibody. Control experiments were performed with lysates from COS-1 cells transfected with empty vector (pFlag-vector). K9(Me)2, dimethylated Lys9; Unmodified, unmodified Lys9; K9(Ac), acetylated Lys9 (indicated on top).

Mentions: We postulated that Mybbp1a binds to the histone H3 N-terminal tail, because Mybbp1a binding to the Per2 promoter correlated with histone H3 dimethylated Lys9 (Figure 4B). We performed pull-down assays using histone H3 N-terminal peptide-immobilized beads to confirm this notion. Figure 6 shows that Mybbp1a preferentially bound to the dimethylated peptide at Lys9. On the other hand, Mybbp1a also relatively weakly bound to the acetylated and unmodified peptide at Lys9. To further confirm these results, we quantified the band intensity of bound Mybbp1a to each peptide. We found that the intensity of bands of Mybbp1a bound to the peptide dimethylated at Lys9 was 2-fold higher than that of Mybbp1a bound to the acetylated and unmodified peptide at Lys9 (Supplementary Figure S6), confirming that Mybbp1a preferentially binds to histone H3 dimethylated Lys9. This result was consistent with the finding that the temporal binding of Mybbp1a to the Per2 promoter correlated with the dimethylation of histone H3 Lys9.Figure 6.


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)

Specific binding of Mybbp1a to histone H3 dimethylated Lys9. Pull-down assays of lysates from COS-1 cells transfected with Flag-Mybbp1a using histone H3 N-terminal peptides that were modified or not at Lys9. Bound proteins eluted from beads previously bound to peptides were analyzed by Western blotting against anti-Flag antibody. Control experiments were performed with lysates from COS-1 cells transfected with empty vector (pFlag-vector). K9(Me)2, dimethylated Lys9; Unmodified, unmodified Lys9; K9(Ac), acetylated Lys9 (indicated on top).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Specific binding of Mybbp1a to histone H3 dimethylated Lys9. Pull-down assays of lysates from COS-1 cells transfected with Flag-Mybbp1a using histone H3 N-terminal peptides that were modified or not at Lys9. Bound proteins eluted from beads previously bound to peptides were analyzed by Western blotting against anti-Flag antibody. Control experiments were performed with lysates from COS-1 cells transfected with empty vector (pFlag-vector). K9(Me)2, dimethylated Lys9; Unmodified, unmodified Lys9; K9(Ac), acetylated Lys9 (indicated on top).
Mentions: We postulated that Mybbp1a binds to the histone H3 N-terminal tail, because Mybbp1a binding to the Per2 promoter correlated with histone H3 dimethylated Lys9 (Figure 4B). We performed pull-down assays using histone H3 N-terminal peptide-immobilized beads to confirm this notion. Figure 6 shows that Mybbp1a preferentially bound to the dimethylated peptide at Lys9. On the other hand, Mybbp1a also relatively weakly bound to the acetylated and unmodified peptide at Lys9. To further confirm these results, we quantified the band intensity of bound Mybbp1a to each peptide. We found that the intensity of bands of Mybbp1a bound to the peptide dimethylated at Lys9 was 2-fold higher than that of Mybbp1a bound to the acetylated and unmodified peptide at Lys9 (Supplementary Figure S6), confirming that Mybbp1a preferentially binds to histone H3 dimethylated Lys9. This result was consistent with the finding that the temporal binding of Mybbp1a to the Per2 promoter correlated with the dimethylation of histone H3 Lys9.Figure 6.

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