Interactome mapping defines BRG1, a component of the SWI/SNF chromatin remodeling complex, as a new partner of the transcriptional regulator CTCF

Abstract

The highly conserved zinc finger CCCTC-binding factor (CTCF) regulates genomic imprinting and gene expression by acting as a transcriptional activator or repressor of promoters and insulator of enhancers. The multiple functions of CTCF are accomplished by co-association with other protein partners and are dependent on genomic context and tissue specificity. Despite the critical role of CTCF in the organization of genome structure, to date, only a subset of CTCF interaction partners have been identified. Here we present a large-scale identification of CTCF-binding partners using affinity purification and high-resolution LC-MS/MS analysis. In addition to functional enrichment of specific protein families such as the ribosomal proteins and the DEAD box helicases, we identified novel high-confidence CTCF interactors that provide a still unexplored biochemical context for CTCF's multiple functions. One of the newly validated CTCF interactors is BRG1, the major ATPase subunit of the chromatin remodeling complex SWI/SNF, establishing a relationship between two master regulators of genome organization. This work significantly expands the current knowledge of the human CTCF interactome and represents an important resource to direct future studies aimed at uncovering molecular mechanisms modulating CTCF pleiotropic functions throughout the genome.

Keywords: BRG1; CTCF; Interactomics; SWI/SNF; chromatin; mass spectrometry (MS); protein–protein interaction; proteomics; transcription factor; transcriptional regulation.

Figure 1.

Schematic workflow of the immunoprecipitation-MS approach to identify the CTCF-interacting proteins. Protein complexes were purified by WiT49 whole cell extract by a two-step affinity purification with an anti-CTCF followed by protein A/G pulldown. Following elution and tryptic digestion, the resulting peptides were subjected to nano-LC–MS/MS in technical replicates for protein identification. Selected preys were then validated by co-immunoprecipitation and Western blotting (WB).

Figure 2.

High-confidence interaction partners of CTCF. 76 of the 91 candidate CTCF-interacting proteins were mapped on a single interconnected network constructed by NetworkAnalyst using the literature-curated IMEx Interactome database and visualized as clusters identified by using the ClusterMaker2 plug-in of Cytoscape 3.6.0. The Markov clustering algorithm was used for network clustering.

Figure 3.

Functionally grouped network of enriched molecular function categories for the identified proteins generated by using the ClueGO cytoscape plug-in. The proportion of shared proteins between terms was evaluated using kappa statistics. GO terms are represented as nodes whose size represents the term enrichment significance. Partially overlapping functionally related groups are represented as squares, whereas nonoverlapping terms are represented as circles. Clusters including more than two terms are numbered as clusters 1 (green), 2 (blue), and 3 (yellow). The group number resulting from ClueGO associations of GO terms is indicated for each cluster (Table S2). Nonclustered terms including groups 0–7 are colored pink.

Figure 4.

Co-immunoprecipitations between CTCF and endogenous DDX5 and BRG1. A, anti-CTCF-immunoprecipitated samples were blotted with anti-BRG1 and anti-DDX5 antibodies. Anti-CTCF was used as positive control. Input, 2% of the cell lysate used for immunoprecipitation. B, anti-BRG1-immunoprecipitated samples were blotted with anti-CTCF and anti-DDX5 antibodies. Anti-BRG1 was used as positive control. Input, 2% of the cell lysate used for immunoprecipitation. C, anti-DDX5-immunoprecipitated samples were blotted with anti-CTCF and anti-BRG1 antibodies. Anti-DDX5 was used as positive control. Input, 4% of the cell lysate used for immunoprecipitation.

Figure 5.

ChIP-Seq co-localizations between CTCF, BRG1, and DDX5. The Venn diagrams show the overlap of CTCF and BRG1 (A), CTCF and DDX5 (B), BRG1 and DDX5 (C), and CTCF, BRG1, and DDX5 (D) ChIP-seq peaks.

Figure 6.

Associations between co-localized regions: genes as a function of the distance to TSS (±2 kb). Co-occupied sites are reported as blue for CTCF–BRG1 (A), CTCF–DDX5 (B), and CTCF–BRG1–DDX5 (C). In all graphs, sites occupied by CTCF alone are reported as green bars.