Proteomic insights into circadian transcription regulation: novel E-box interactors revealed by proximity labeling

Abstract

Circadian clocks (∼24 h) are responsible for daily physiological, metabolic, and behavioral changes. Central to these oscillations is the regulation of gene transcription. Previous research has identified clock protein complexes that interact with the transcriptional machinery to orchestrate circadian transcription, but technological constraints have limited the identification of de novo proteins. Here we use a novel genomic locus-specific quantitative proteomics approach to provide a new perspective on time of day-dependent protein binding at a critical chromatin locus involved in circadian transcription: the E-box. Using proximity labeling proteomics at the E-box of the clock-controlled Dbp gene in mouse fibroblasts, we identified 69 proteins at this locus at the time of BMAL1 binding. This method successfully enriched BMAL1 as well as HDAC3 and HISTONE H2A.V/Z, known circadian regulators. New E-box proteins include the MINK1 kinase and the transporters XPO7 and APPL1, whose depletion in human U-2 OS cells results in disrupted circadian rhythms, suggesting a role in the circadian transcriptional machinery. Overall, our approach uncovers novel circadian modulators and provides a new strategy to obtain a complete temporal picture of circadian transcriptional regulation.

Keywords: DNA–protein proximity labeling; circadian clock; circadian transcription; enhancer–enhancer interactions; protein–protein interactions; proteomics.

Figure 1.

Proximity labeling of E-box proteins using the CASPEX method. (A) Schematic description of the method. The sgRNAs target the dCAS9–APEX2 fusion protein downstream from the E-box of the first intron of the Dbp gene. After induction, dCAS9–APEX2 binds the Dbp gene, and upon addition of biotin-phenol and H₂O₂, the APEX2 peroxidase induces biotinylation of proteins in close proximity. This reaction occurs simultaneously at the 200 repeats of the Dbp gene expressed in this Dbp(luc) array NIH3T3 mouse cell line. The biotinylated proteins are then purified using their affinity for streptavidin and subsequently identified by LC-MS/MS. (B) Immunodetection of dCAS9–APEX2 and validation of its enzymatic activity. dCAS9–APEX2 was immunodetected in nuclear lysates of Dbp(luc) array NIH3T3 mouse cells using an anti-CAS9 antibody. In the same samples, biotinylated proteins were detected using horseradish peroxidase (HRP)-conjugated streptavidin followed by chemiluminescence imaging. All cells were treated with biotin-phenol, and H₂O₂ was added where indicated. (C) Circadian period of different cell lines. Period values were extracted using Chronostar software from 3 day bioluminescence time series recorded 24 h after synchronization and dCAS9–APEX2 induction. No significant differences were detected. One-way ANOVA, n = 5, P > 0.05; i.e., not significant (NS). (D) Example of confocal microscopy fluorescence images of dCAS9–APEX2 (anti-CAS9 antibody) and biotinylated proteins (streptavidin, Alexa fluor 514 conjugate) in Dbp(luc) array NIH3T3 mouse cells expressing both dCAS9–APEX2 and guide sgRNAs 24 h after dCAS9–APEX2 induction and fixed after addition of APEX2 substrates (biotin-phenol and H₂O₂). High-intensity aggregates were observed in the nucleus, similar to the BMAL1-YFP aggregates on the Dbp(luc) array in the NIH3T3 cell model (Stratmann et al. 2012). (E,F) Quantification of aggregates of dCAS9–APEX2 (E) and biotinylated proteins (F) from confocal microscopy images (as shown in D). For each field of view, the percentage of nuclei containing an aggregate of dCAS9–APEX2 or biotinylated proteins was quantified and is expressed as log₂ fold change compared with the control cell type (dCAS9–APEX2 noGuide). n = 3–4 biological replicates, each with five to 12 fields of view per cell line. (G) Chromatin immunoprecipitation experiment on NIH3T3 Dbp(luc) array cells 24 h after treatment with doxycycline (purple) or solvent (gray). Enrichment of Dbp's first intron E-box was analyzed by qPCR (expressed as fold enrichment compared with the respective solvent-treated sample) for immunoprecipitation with CAS9 antibody. n = 3 biological replicates.

Figure 2.

Identification of proteins located at the E-box of the Dbp gene using mass spectrometry. (A) Experimental pipeline. (B) Number of identified proteins in total, per cell type, and overlapping between cell types. Proteins were considered reliably identified if they were detected in three or more replicates of a cell type and the standard deviation between replicates of the same cell type was <1. (C) Fold change of the detection intensity of all samples compared with the mean intensity of noGuide for the 1685 proteins that were detected in all three cell types. Warm colors indicate a positive fold change compared with noGuide cells, and cold colors indicate a negative fold change (three or more replicates per cell type). (D) Volcano plot of the log₂ fold change of proteins versus the −log₁₀ Q-value in guide1 and guide2 compared with noGuide cells. The proteins with significant enrichment (i.e., fold change of >2 and Q-value < 0.1) are highlighted (three or more replicates per cell type). (E) Abundance (in log₂ LFQ intensity) of the 153 proteins that were detected in both guide1 and guide2 in three or more replicates but had detection in less than three replicates of noGuide cells (three or more replicates per cell type).

Figure 3.

(A–D) Identified proteins and their functional roles. Log₂ LFQ intensity values of the proteins identified in guide1 and guide2 compared with noGuide control cells. (ND) Not detected; statistical significance is defined as fold change of ≥2 and a Q-value < 0.1.

Figure 4.

Protein location on the E-box of the Dbp gene. Chromatin immunoprecipitation experiment on NIH3T3 Dbp(luc) array cells 24 h after synchronization. Enrichment of Dbp’s first intron E-box was analyzed by qPCR, expressed as fold enrichment compared with the corresponding IgG, for immunoprecipitation with BMAL1 antibody (A), RUNX2 antibody (B), and SMAD1 antibody (C). n = 3 biological replicates; t-test, P < 0.05.

Figure 5.

Depleting identified E-box-enriched proteins affects circadian dynamics. Real-time bioluminescence monitoring of U-2 OS Bmal1 luciferase cells after synchronization with dexamethasone. (A) Knockdown of Runx2. (Left) Representative time series. (Middle) Change in Bmal1 luciferase signal intensity compared with nonsilencing control as a function of the percentage of RUNX2 transcript. (Right) Error of fit to a sinosoidal curve as a function of the percentage of RUNX2 transcript. The red line represents a fit of the data to a monophase decay curve. (B–D) Knockdown of APPL1 (B), XPO7 (C), and MINK1 (D). (Left) Representative time series. (Right) Change in period length compared with the nonsilencing controls as a function of the shRNA construct. The dotted red line represents the mean change in period length for all shRNA combined. Dots represent the results of independent experiments.