1. Specificity in Circadian Clock Feedback from Targeted Reconstitution of the NuRD Corepressor 
Jin Young Kim, Pieter Bas Kwak, Charles J. Weitz 
Molecular Cell 
Volume 56, Issue 6, Pages (December 2014)
DOI: /j.molcel
Copyright © 2014 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2014 56, 738-748DOI: (10.1016/j.molcel.2014.10.017)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 Mouse PER Complexes Include the NuRD Corepressor
(A) Native PER complexes are >1.2 mDa in mass. BN-PAGE profile of nuclear PER complexes from wild-type and PER2-FH mouse livers obtained early in circadian negative feedback (CT 14). Image shows immunoblots of BN-PAGE gel probed with antibodies to PER1, PER2, PER3, or CRY2 to monitor native PER complexes or antibody to HA to monitor PER complexes from PER2-FH mice. Positions of size markers are shown at left; 1.2 mDa, IgM hexamer; 1.0 mDa, IgM pentamer; 0.72 mDa, Apoferritin band 1. Top of image corresponds to top of running gel. Longer exposure reveals a minor band at ∼800 kDa.
(B) Silver-stained SDS-PAGE of immunoaffinity-purified PER complexes from PER2-FH mouse liver (CT14) and parallel wild-type (Wt) control purification. Arrow marks diffuse band of PER proteins (from variable phosphorylation). Approximately 30 specific bands were detected (see Table S1 for mass spectrometry results).
(C) Immunoblots of nuclear extracts from wild-type mouse livers (CT14) probed with antibodies against the proteins indicated on the right after immunoprecipitation (IP) with control IgG or PER2 IgG. Components of the core PER complex (including CLOCK-BMAL1 target) served as positive controls. CoREST, a component of different corepressor complex that includes HDAC1, served as negative control, and IgG heavy chain (IgG-HC) served as positive control for immunoprecipitation.
(D) NuRD subunits are present in large PER complexes. Left panel, silver-stained BN-PAGE gel showing immunoaffinity-purified PER complexes (PER2-FH) and parallel control purification from wild-type mice (Wt). Arrow marks the expected size of the PER complex. Right panels, immunoblots of the BN-PAGE gel probed with the indicated antibodies against core clock proteins or NuRD subunbits. Protein size markers as in (A).
See also Figures S1, S2, and S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

4. Figure 2
Opposite Circadian Clock Phenotypes after Depletion of Different PER-Associated NuRD Subunits
(A–D) MTA2, representative of three independent experiments.
(E–H) CHD4, representative of four independent experiments. Analysis after depletion of CHD4, as in (A)–(D) above.
(A) Immunoblot showing partial depletion of MTA2 by siRNA in circadian bioluminescent reporter cells. U2AF65 (U2 small nuclear ribonucleoprotein particle auxiliary factor), loading control.
(B) Real-time circadian oscillations of bioluminescence in synchronized U2OS reporter cells after delivery of MTA2 siRNA (red) or control siRNA (black). Traces were aligned at day 1.
(C) Circadian periods after MTA2 depletion (mean ± SEM; n = 3 for each condition; two-tailed t test).
(D) Effect of MTA2 depletion on transcription of CLOCK-BMAL1 target genes (left) and control genes (right) by quantitative RT-PCR of the respective pre-mRNAs (mean ± SEM, triplicate; normalized to control; representative of three independent experiments).
See also Figures S3 and S7.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

5. Figure 3 A Split NuRD Complex in the Circadian Clock
(A) Immunoblots showing coimmunoprecipitation of the indicated proteins with control IgG or CLOCK antibody at CT6 (activation phase) or CT14 (repression phase). U2AF65, loading control for input; IgG-HC, positive control for immunoprecipitation. Representative of four independent experiments.
(B–D) Genetic evidence for a split NuRD complex in the circadian clock feedback loop. Representative of three independent experiments.
(B) Immunoblot showing liver nuclear extracts (CT14) from wild-type and Bmal1−/− mice probed for the proteins indicated at right. Levels of PER and CLOCK are reduced in Bmal1−/− mice, whereas levels of NuRD subunits MBD2 and CHD4 are unaffected.
(C) Coimmunoprecipitation from the extracts in (B) of proteins indicated at right with CHD4 antibody or IgG control. The association of CHD4 with PER2 observed in wild-type mice was undetectable in Bmal1−/− mice.
(D) Coimmunoprecipitation from the extracts in (B) of proteins indicated at right with MBD2 antibody or IgG control. The association of MBD2 with PER2 observed in wild-type mice was intact in Bmal1−/− mice. The reduced signal for MBD2 is expected because of the reduction in PER2 levels in Bmal1−/− mice.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

6. Figure 4 A Split NuRD Complex at E-Box Sites of Per Genes
Circadian cycles of PER2, BMAL1, and NuRD subunits at (A) Per2 E-box and (B) Per1 E-box. Shown are ChIP assays from mouse livers obtained across a circadian cycle and performed with the antibodies indicated in each panel (mean ± SEM of triplicate assay; some error bars are too small to be seen; normalized to parallel control IgG ChIP; each representative of three independent experiments).
See also Figures S5, S6, and S7.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

7. Figure 5
CHD4 Promotes RNA Pol II Binding to Promoters of CLOCK-BMAL1 Target Genes
(A) Immunoblot showing steady-state levels of the indicated proteins after depletion of CHD4 by siRNA in unsynchronized circadian reporter fibroblasts. Decreased level of CHD4 had no detectable effect on expression levels of CLOCK, BMAL1, or PER2. U2AF65, loading control.
(B) Binding of CLOCK, BMAL1, and RNA Polymerase II (total RNA Pol II) to promoter regions of Per2 (top) and Per1 (bottom) genes. Shown are ChIP assays from unsynchronized fibroblasts after introduction of control siRNA (black) or CHD4 siRNA (gray; mean ± SEM of triplicate assay; normalized to parallel control IgG ChIP; representative of three independent experiments). Reduction in RNA Pol II at Per2 and Per1 promoters was highly significant (p < and p < 0.001, respectively, by one-tailed t test; n = 3 independent experiments for each).
(C) No detectable effect of CHD4 at control gene promoters. Binding of CLOCK, BMAL1, and RNA Pol II to promoter regions of noncircadian control genes. Shown are ChIP assays from unsynchronized fibroblasts after introduction of control siRNA (black) or CHD4 siRNA (gray), performed with the antibodies indicated at bottom of each panel (mean ± SEM of triplicate assay; normalized to parallel control IgG ChIP; representative of three independent experiments).
See also Figures S6 and S7.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

8. Figure 6
PER Complexes without CLOCK-BMAL1 Lack CHD4 and MTA2 but Include Other NuRD Subunits
(A) Enrichment of PER complexes not bound to CLOCK-BMAL1. Mouse liver (CT14) nuclear extracts (Input) were adsorbed to control IgG (mock depletion) or CLOCK antibody (to deplete PER complexes containing CLOCK-BMAL1), and the unbound fraction (“depleted with”) was probed by immunoblotting with the indicated antibodies.
(B) Coimmunoprecipitation of indicated proteins with IgG control antibody or PER2 antibody from mock-depleted (left lanes) or CLOCK-depleted extracts (right lanes).
(A and B) Representative of four independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

9. Figure 7
A Mechanism for Specificity of Circadian Clock Feedback: Targeted Reconstitution of the NuRD Corepressor by the PER Complex
(A) Circadian transcriptional activation phase: CLOCK-BMAL1 with NuRD components CHD4 and MTA2 at E-box site of circadian target gene. CHD4 in this configuration functions to promote CLOCK-BMAL1 transcriptional activity, while MTA2 is important for subsequent assembly or function of the NuRD corepressor.
(B) Circadian negative feedback phase: newly formed PER complex brings the remaining NuRD components to CLOCK-BMAL1 at the E-box, thereby reconstituting a functional NuRD transcriptional corepressor. Full repressor activity of the PER complex is thus generated only upon correct targeting of CLOCK-BMAL1.
(C) Circadian negative feedback phase: PER complex incorporates multiple repressive effector complexes. In addition to NuRD, the PER complex includes SIN3-HDAC (Duong et al., 2011), helicases DDX5, DHX9, and SETX, factors that are important for transcriptional termination (Padmanabhan et al., 2012), and the HP1γ-SUV39h histone methyltransferase, which is recruited several hours later than the others (Duong & Weitz, 2014). How the activities of the multiple repressors in the PER complex are integrated during negative feedback is not yet clear.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions