1. Volume 48, Issue 2, Pages 277-287 (October 2012)
Circadian Dbp Transcription Relies on Highly Dynamic BMAL1-CLOCK Interaction with E Boxes and Requires the Proteasome 
Markus Stratmann, David Michael Suter, Nacho Molina, Felix Naef, Ueli Schibler 
Molecular Cell 
Volume 48, Issue 2, Pages (October 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1 Generation and Analysis of a Dbp-Luciferase Array Cell Line
(A) Schematic representation of the Dbp-luc construct used for array generation.
(B) Copy number determination by quantitative real-time PCR, using a genomic Dbp probe (mean ± standard deviation [SD] of three independent experiments). The value obtained from NIH 3T3 cells was set to two copies.
(C) Luciferase expression of array cells and cells expressing Bmal1-luc. Values represent counts per minute for Bmal1-luc cells and counts per second for array cells.
(D) Quantification of Dbp pre-mRNA levels by real-time RT-PCR (mean ± SD of three independent experiments). The value obtained for NIH 3T3 cells was set to 1.
(E and F) DNA-FISH with a Dbp-specific probe. Scale bar, 5 μm.
(G) Quantification of the volume of FISH structures at 12 and 24 hr after Dex shock (mean ± standard error of the mean [SEM]; ten cells were analyzed per time point). Asterisks indicate p <
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
BMAL1-CLOCK Binding to Regulatory Sites in Dbp Is Circadian In Vivo
(A) Time-lapse series of an array cell stably expressing YFP-BMAL1. Arrows indicate arrays bound by YFP-BMAL1.
(B) Temporal binding profile of YFP-BMAL1 to the Dbp array (mean of 12 cells ± SEM). Dbp pre-mRNA levels were measured by real-time RT-PCR (mean ± SD of three independent experiments; dashed line, double-plotted values form first day).
(C) Colocalization of YFP-BMAL1 and mKate-CLOCK to the array 24 hr after Dex shock.
(D) VENUS-CLOCK spot-positive cells 24 and 36 hr after Dex shock. Eighty cells were analyzed per time point.
(E) FLIP profiles of bleached and nonbleached YFP-BMAL1-expressing cells 20, 28, and 36 hr after Dex shock. The prebleach fluorescence levels in individual cells were set to 1.
(F) Quantification of bleaching kinetics around the clock. Plotted values represent time to reach half of the initial fluorescence (mean ± SD; ten cells were measured per time point). Asterisks indicate p <
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
BMAL1 and CLOCK Are Interdependent Activators of Dbp Transcription
(A) Luciferase recordings of NIH 3T3 cells cotransfected with a Dbp-luc reporter plasmid and a control siRNA or Clock-specific siRNA.
(B) Percentage of YFP-BMAL1 spot-positive array cells after transfection with either a control siRNA or a Clock-specific siRNA, together with a tomato red fluorescent protein expression plasmid. Cells were synchronized 48 hr after transfection, and spot-positive cells were counted 24 hr later. Thirty tomato-positive and -negative cells were analyzed per condition.
(C) Immunostaining of Bmal1+/+ and Bmal1−/− primary fibroblasts with a CLOCK-specific antibody (24 hr after Dex shock). Scale bar, 5 μm.
(D) Quantification of CLOCK nuclear fluorescence levels in Bmal1+/+ and Bmal1−/− primary fibroblasts.
(E) ChIP on chromatin isolated from Bmal1+/+ and Bmal1−/− primary fibroblasts with a CLOCK-specific antibody. Quantification of ChIP was performed by real-time PCR using a Dbp promoter probe (DbpUP) and a GAPDH probe as a control. The value obtained for Bmal1+/+ fibroblasts 24 hr after Dex shock was set to 1.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Highly Dynamic Recruitment of BMAL1 to Dbp
(A) High temporal resolution imaging of YFP-BMAL1 binding to the array between 24 and 25.5 hr after Dex shock.
(B–D) (B) Quantification of binding profiles of YFP-BMAL1 to the array in untreated (C) TSA-treated (100 nM) and (D) MG132-treated (10 μM) array cells. The array intensity was plotted over the intensity of a random region in the nucleoplasm. As a control, the intensity of a random region in the nucleoplasm was plotted over a second random region in the nucleoplasm.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
The Residence Time of BMAL1 on E Boxes in Dbp Is Determined by the Proteasome
(A) Photobleaching (FLIP) of cells in the absence (upper panel) or presence (lower panel) of MG132.
(B) Quantification of spot bleaching kinetics. Plotted values represent the mean ± SD of at least ten cells. Numbers indicate the time (seconds) to reach half of the initial fluorescence.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
The Transcription of Early BMAL1-CLOCK Target Genes Requires a Functional Proteasome
(A and B) (A) Luciferase recordings of array cells and (B) of Bmal1-luc cells with and without proteasome inhibitor. DMSO or MG132 was added 24 hr after Dex shock (arrow).
(C–E) Real-time RT-PCR quantification of mRNA levels of control and MG132-treated cells over the course of 8 hr (mean ± SD of three independent experiments). (C) Early-peaking BMAL1-CLOCK target genes, (D) late-peaking BMAL1-CLOCK target genes, (E) genes whose expression is independent of BMAL1-CLOCK. The highest value obtained for control cells was set to 1.
(F) Protein accumulation of circadian repressors in DMSO- and MG132-treated cells.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Proteasome Inhibition Reduces Burst Frequency and Burst Size of Dbp Transcription
(A) Schematic representation of the transgenes present as a single copy in the cell lines used to monitor Dbp- and Bmal1-luc expression in individual cells: bsd, blasticidine resistance; F2A, 2A peptide from foot and mouth disease virus; ARE, AU-rich element; polyA, polyadenylation signal.
(B) Examples of single cell bioluminescence traces of Dbp-luc (upper panel) and Bmal1-luc cells (lower panel) with and without MG132. The arrow indicates time of MG132 addition.
(C and D) (C) Mean burst frequency and (D) mean burst size of Dbp-luc and Bmal1-luc cells with and without MG132 (mean of 34 cells for each condition ± SD).
(E) Model of transcriptional activation of Dbp. BMAL1-CLOCK heterodimers associate with E boxes in Dbp. At this time, BMAL1 is phosphorylated (P), monoubiquitylated (U), and sumoylated, and the heterodimer is capable of recruiting the transcription machinery, resulting in a burst of Dbp transcription. BMAL1 is subsequently polyubiquitylated, which targets the activator complex for proteasomal degradation. After the clearance of BMAL1-CLOCK from E boxes, a new cycle of BMAL1-CLOCK association and transcriptional initiation can ensue.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions