Journal: Molecular and Cellular Biology

Article Title: BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

doi: 10.1128/mcb.00337-06

Figure Lengend Snippet: FIG. 1. Subcellular distribution of truncated BMAL1 mutants and their effects on nuclear translocation of CLOCK. (A) Myc-tagged BMAL1 and GPF-CLOCK were introduced alone (upper panels) or together (lower panel) into HeLa cells. Arrowhead indicates the nucleus of a cell expressing both proteins. (B) Schematic diagram of the GFP-or Myc-tagged BMAL1 constructs. The putative NLS, bHLH, PAS, and TADs are indicated as purple, blue, yellow, and red boxes, respectively. (C) Putative molecular sizes of truncated BMAL1 mutants confirmed by immunoblot analysis with anti-Myc antibody (9E10). (D) Representative fluorescence images demonstrating the subcellular localization of each construct and coexpressed wild-type CLOCK. The GFP-fused BMAL1 constructs and Myc-tagged wild-type CLOCK were coexpressed in NIH 3T3 cells, which were stained with 9E10 to visualize the distribution of wild-type CLOCK. (E) Quantitation of the subcellular distribution of BMAL1 (solid bars) and CLOCK (checked bars). The subcellular localization was categorized as nuclear (blue), cytoplasmic and nuclear (gray), and cytoplasmic (red). The data were obtained from two independent experiments, and in each experiment more than 100 cells were counted.

Article Snippet: Antibodies specific for CLOCK and CRY1 were purchased from Santa Cruz.

Techniques: Translocation Assay, Expressing, Construct, Western Blot, Staining, Quantitation Assay

Journal: Molecular and Cellular Biology

Article Title: BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

doi: 10.1128/mcb.00337-06

Figure Lengend Snippet: FIG. 5. CLOCK and BMAL1 reduce each other’s stability. (A) Subcellular fractionation of exogenous CLOCK and BMAL1 in NIH 3T3 cells transfected with HA-tagged Bmal1 and/or Myc-tagged Clock. (B) Effect of the proteasome inhibitor MG132 on the stability of CLOCK and BMAL1. Cells were transfected with a fixed amount of Clock DNA (600 ng) and the indicated amounts of Bmal1 DNA. At 36 h after transfection, they were incubated for 5 h with or without 50 M MG132. (C) Ubiquitination of CLOCK and BMAL1. Cells were cotransfected with fixed amounts of Myc-Clock (600 ng) and increasing amounts of Bmal1 and vice versa in the presence of HA-ubiquitin. Cells were incubated with 50 M MG132 for 5 h, and corresponding cell extracts were subjected to immunoprecipitation with anti-Myc or anti-BMAL1 antibodies. Immuno- precipitated proteins were then analyzed by immunoblotting with anti-HA antibodies. (D) Role of BMAL1 shuttling in the decreased CLOCK stability. Wild-type and BMAL1-deficient MEFs transfected with Myc-Clock (600 ng) were treated with CHX (30 g/ml) alone or with LMB (10 ng/ml) for 5 h and then subjected to immunoblotting with anti-Myc antibodies. (E) Stability of endogenous CLOCK in wild-type and BMAL1- deficient MEFs. BMAL1-deficient cells transfected with wild-type Bmal1 or NESmut Bmal1 were exposed to CHX (30 g/ml) for the indicated times, and CLOCK levels were assessed by immunoblotting at the indicated times after drug treatment. (F) Stability of endogenous BMAL1 in wild-type and Clock/Clock mutant MEFs. BMAL1 levels were assessed by the same experimental procedure as the CLOCK analysis, except that one set of Clock mutant cells were incubated with MG132 (50 M) together with CHX (30 g/ml) until cells were harvested at the indicated time points.

Article Snippet: Antibodies specific for CLOCK and CRY1 were purchased from Santa Cruz.

Techniques: Fractionation, Transfection, Incubation, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

doi: 10.1128/mcb.00337-06

Figure Lengend Snippet: FIG. 6. BMAL1-dependent proteolysis of CLOCK is closely related to its transcriptional activity. (A) Intact BMAL1 but not functionally defective BMAL1 induces drastic decreases in CLOCK levels. NIH 3T3 cells were transfected with Myc-Clock alone or together with wild-type Bmal1 or Bmal1 mutants and then analyzed by immunoblotting with the indicated antibodies. (B) Confirmation of protein-protein interactions between CLOCK and BMAL1 mutants. Cells were cotransfected with wild-type CLOCK and Myc-tagged Bmal1 constructs with various defects and then immunoprecipitated with anti-Myc antibodies. The immunoprecipitates were analyzed by immunoblotting with anti-CLOCK or anti-Myc. (C) BMAL1-dependent CLOCK degradation is prevented when CLOCK has functional defects or is coexpressed with CRY1. Cells were transfected with Myc-tagged Clock constructs encoding wild-type or transcriptionally inactive mutants [N (52-855) lacking the DNA-binding domain and C (1-480) lacking TAD] alone or together with Bmal1 and/or Cry1 and then analyzed by immunoblotting with the indicated antibodies. (D) The CLOCK mutants lacking DNA-binding domain or TAD colocalize with BMAL1 in the nucleus. Localization of the Myc-tagged CLOCK mutants and BMAL1 was detected with anti-Myc and anti-BMAL1 antibodies and visualized with TRITC and FITC, respectively. (E) Relative Per1 promoter-dependent luciferase reporter activity induced by combined transfection with the various forms of Clock and Bmal1. The rightmost bar is the result from cells transfected with wild-type Clock and Bmal1 and exposed to 50 M MG132 for 5 h. Relative luciferase reporter activity is expressed as fold of the control. Each value is the mean the SEM of three replicates from a single assay. Similar results were obtained from replicated experiments.

Article Snippet: Antibodies specific for CLOCK and CRY1 were purchased from Santa Cruz.

Techniques: Activity Assay, Transfection, Western Blot, Protein-Protein interactions, Construct, Immunoprecipitation, Functional Assay, Binding Assay, Luciferase, Control

Journal: Molecular and Cellular Biology

Article Title: BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

doi: 10.1128/mcb.00337-06

Figure Lengend Snippet: FIG. 7. Circadian regulation of the abundance and the stability of CLOCK and BMAL1 in vivo. (A) Real-time measurement of Per2 promoter-controlled destabilized luciferase (Per2-Pro-dsLuc) activity in live cells. Wild-type MEFs (WT) and Cry-deficient MEFs (Crys/) carrying Per2-Pro-dsLuc with the Per2 promoter were synchronized by treatment with 1 M Dex, and their luciferase activities were monitored in real time (see Materials and Methods). (B) Changes in the levels of BMAL1, CLOCK, and CRY1 during the circadian cycle. Each protein was analyzed at the times corresponding to the maximal and minimal transcription of Per2-Pro-dsLuc, respectively (32 and 44 h). (C) Relative mRNA levels of Bmal1 and Clock at the same circadian time points (32 h and 44 h) in wild-type and Cry-deficient cells. mRNA levels in the wild-type (■) and Cry-deficient cells () were quantified by real-time PCR (see Materials and Methods). The data are presented as means the SEM (n 3). (D) Transient expression of CRY1 and CRY2 in Cry-deficient cells increases endogenous CLOCK and BMAL1 levels. The cellular levels of the indicated proteins were analyzed by immunoblotting at 24 h after transfection with Cry-expressing plasmids. (E) Nuclear accumulation of CRY1 is correlated with accumulation of BMAL1 and CLOCK during the circadian cycle. The levels of each protein in nuclear and cytoplasmic fractions of wild-type MEFs were assessed by immunoblotting at 32 and 44 h after Dex treatment. (F) Massive degradation of BMAL1 via the ubiquitin-proteasome pathway during the transcriptionally active phase. Wild-type cells were treated with CHX (30 g/ml), MG132 (50 M), and LMB (10 ng/ml) 4 h before the indicated times and were subjected to immunoblotting with the indicated antibodies.

Article Snippet: Antibodies specific for CLOCK and CRY1 were purchased from Santa Cruz.

Techniques: In Vivo, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Ubiquitin Proteomics