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

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

Yasuhiro Hara et al. Nucleic Acids Res. 2009 Mar.

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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Figures

Figure 1.
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.

Figure 2.
Figure 2.

Interaction between Mybbp1a and mCRY1 confirmed by reciprocal co-precipitation. (A) TAP-tagged mCRY1 co-transfected with Flag-Mybbp1a into COS-1 cells. Pull-down assays were performed using IgG beads with high-affinity to TAP-tagged protein. (B) Flag-Mybbp1a co-transfected with V5-His-tagged mCRY1 into COS-1 cells. Cell lysates were immunoprecipitated (IP) with anti-Flag antibody. Symbols (+) or (–), presence or absence of Flag-Mybbp1a-expression plasmids, respectively.

Figure 3.
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.

Figure 4.
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).

Figure 5.
Figure 5.

Temporal expression profile of Mybbp1a mRNA in mouse liver. Mice were maintained under 12 h light: 12 h dark cycles (light on at ZT 0) and liver samples were obtained at each time point. Levels of Mybbp1a mRNA (top panel) were determined using real-time RT-PCR. Expression levels were normalized to β-actin mRNA. Values are means ± SEM of three mice per time point. Expression profiles of Per2 and Cry1 mRNA are also shown in middle and bottom panels, respectively.

Figure 6.
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).

Figure 7.
Figure 7.

Regulation of circadian transcription through histone tail modification. Mybbp1a and mCRY1 (and CLOCK) form complexes on Per2 promoter and repress Per2 transcription although Mybbp1a did not directly bind to mCRY1. After Mybbp1a-independent histone H3 Lys9 dimethylation, Mybbp1a binds to histone H3 dimethylated Lys9. Mybbp1a–mCRY1 complexes then recruit unknown chromatin remodeling factor(s) (indicated as X, e.g. histone deacetylase) to Per2 promoter and support establishment of transcription-permissive chromatin independently of E2-box. Consequently, Per2 gene expression is repressed.

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