Unraveling the cohesin-chromatin interface: identifying protein interactions that modulate chromosome structure and function

Abstract

Background: The evolutionarily conserved cohesin complex is a pleiotropic regulator of chromosome structure and function, participating in sister chromatid cohesion, transcriptional regulation of genes, DNA replication, and DNA repair. Cohesin uses ATP hydrolysis to dynamically extrude DNA loops that bring together cis-regulatory elements and thus regulate gene expression. Some DNA loops are anchored by the binding of CTCF insulator proteins which can stall extruding cohesin complexes, however many DNA loops that connect enhancers and promoters lack CTCF and it is unclear how cohesin is stabilized at these cis-regulatory sites. While cohesin has been found to co-purify with a number of proteins, some of which regulate cohesin function, our current knowledge of cohesin activity is incomplete. Identification of transient or less stable interactions between cohesin and chromatin-associated proteins is crucial for understanding regulation of gene expression and chromosome structure.

Results: Here we utilize a TurboID proximity labeling and mass spectrometry approach for identifying cohesin-interacting proteins. We identify > 400 cohesin-interacting proteins in NIH-3T3 cells, including previously known and potentially novel cohesin interactors. Among the cohesin interactors were chromatin remodeling complexes and histone-modifying complexes. Interactions between seven of these chromatin regulating complexes and cohesin were confirmed with co-immunoprecipitations performed in multiple cell lines. The SWI/SNF complex was found to co-purify with cohesin and SWI/SNF co-occupied enhancers and promoters with cohesin. To investigate the functional relevance of the cohesin-SWI/SNF interaction, we assessed whether the binding of cohesin to the genome is regulated by SWI/SNF or vice versa. Acute small molecule perturbations of SWI/SNF altered the amount of both SWI/SNF and cohesin on chromatin, particularly affecting cohesin binding to CTCF sites.

Conclusions: This work represents the most comprehensive investigation of cohesin-interacting proteins to date. These results identify a physical link between cohesin and a vast number of chromatin-associated proteins inside of cells, including chromatin remodeling complexes and histone-modifying complexes. Furthermore, these results indicate SWI/SNF activity stabilizes cohesin on chromatin particularly at insulator sites. These cohesin interactome data are a resource for future studies aimed at characterizing the functional interactions between cohesin and numerous chromatin-associated proteins in regulating chromosome structure and gene control.

Keywords: Chromatin; Cohesin; Remodeling; Structure.

Fig. 1

Identification of the cohesin interactome with proximity ligation mass spectrometry. A Schematic of the experimental setup for cohesin proximity ligation followed by mass spectrometry. Transgenic NIH-3T3 mouse fibroblast cell lines were generated containing a 3xHA-TurboID or RAD21-3xHA-TurboID transgene with a tetracycline responsive element (TRE). Cell lines were treated with doxycycline for 36 h to promote expression of the transgenes. Additionally, RAD21-3xHA-TurboID cells that were not treated with doxycycline serve as a No TurboID control. Following doxycycline treatment, cells were treated ± biotin for 2 h. Cells were collected, lysed, and nuclear proteins were extracted. A streptavidin enrichment was performed, and a quality control check was done on 10% of the protein sample by western blotting for biotinylated proteins, while the remaining 90% of the streptavidin enriched sample was used for LC–MS/MS. Proteins identified by mass spectrometry were analyzed by two methods to identify proteins enriched relative to controls. Proteins enriched in the RAD21-3xHA-TurboID biotin treated sample relative to the: (1) 3xHA-TurboID biotin treated sample (Tier 1; p < 0.05, FC > 2); or (2) No TurboID biotin treated sample (Tier 2; p < 0.05, FC > 2). Each condition was performed in triplicate. B Co-purification of RAD21-3xHA-TurboID protein with endogenous cohesin subunits. IgG (negative control), anti-SMC3, and anti-HA antibodies were used to purify cohesin complexes in nuclear extracts from RAD21-3xHA-TurboID expressing cells under high stringency conditions. Cohesin subunits were detected by western blotting (SMC3, SMC1, RAD21, and HA). Arrows indicate endogenous RAD21 (black) and transgenic RAD21-3xHA-TurboID proteins (green). Box contains a depiction of core cohesin complex with RAD21-3xHA-TurboID. C Venn diagram showing the overlap of proteins detected in the Tier 1 cohesin interactome, Tier 2 cohesin interactome, and cohesin IP-MS experiments [–17]. D, E Volcano plots showing proteins detected in the Tier 1 (D) and Tier 2 (E) cohesin interactomes. Proteins significantly enriched in Tier 1 and Tier 2 shown in red and blue, respectively. Known cohesin subunits and interactors are indicated with green dots and labels

Fig. 2

The cohesin interactome includes chromatin regulating complexes. A Gene Ontology (GO) analysis of biological processes enriched in the Tier 1 and Tier 2 cohesin interactomes. The size of dots represents the ratio of genes detected within the GO term and the color intensity of dots represents -log10(FDR). All significant terms can be found in Supplemental Table 2. B Genes detected within the Tier 1 (red) or Tier 2 (blue) cohesin interactome are indicated for the “Chromatin remodeling” GO term. C The percentage of proteins detected in the top seven chromatin regulating complexes are shown for the Tier 1 (red) and Tier 2 (blue) cohesin interactomes. The Mediator complex is known to interact with cohesin and is shown as an example. D Functions of the top seven chromatin regulating complexes and their variant forms. E Validation of cohesin-chromatin regulating complex interactions by co-IP and western blotting. NIH-3T3 nuclear lysates were prepared and SMC3 and IgG immunoprecipitations were performed under low stringency conditions. Colored bars to right of blot indicate whether the chromatin regulating complex proteins were detected in the Tier 1 (red) or Tier 2 (blue) cohesin interactomes, or not detected (gray). F Validation of cohesin-chromatin regulating complex interactions in a second cell type, mESCs

Fig. 3

Cohesin co-localizes with SWI/SNF at enhancers, promoters, and CTCF sites. A Genome browser view of the Sox2 gene and super enhancer. ChIP-seq signal for cohesin (RAD21), SWI/SNF (BRG1), H3K4me3 (promoters), H3K27ac (enhancers), and CTCF (insulators) is shown. DNA interactions were detected by Micro-C and are shown at the top with a contact frequency heatmap and arcs for DNA loops (5 kb resolution). RAD21 peaks overlapping BRG1 peaks highlighted in purple, RAD21 peaks not overlapping BRG1 highlighted in green, and BRG1 peaks not overlapping RAD21 highlighted in orange. B Heatmaps showing ChIP-seq signal for cohesin (RAD21), SWI/SNF (BRG1), H3K4me3 (promoters), H3K27ac (enhancers), and CTCF (insulators) at sites where cohesin peaks do not overlap SWI/SNF peaks (top), sites where cohesin peaks overlap SWI/SNF peaks (middle), and sites where SWI/SNF peaks do not overlap cohesin peaks (bottom). Signal is z-score normalized. C UpSet plot of cohesin peaks overlapping CTCF, H3K27ac, H3K4me3, and/or BRG1

Fig. 4

Altered cohesin occupancy at CTCF sites and enhancers upon SWI/SNF perturbation. A The SWI/SNF cBAF complex and three small molecules that inhibit or lead to degradation of the BRG1 subunit (dark orange). The ACBI1 PROTAC (red) targets the bromodomain of BRG1, recruiting an E3 ubiquitin ligase for poly-ubiquitination and proteasomal degradation. PFI-3 (purple) targets the bromodomain of the BRG1 subunit. BRM014 (blue) targets the ATPase active site of BRG1. The levels of SWI/SNF subunits shown in light orange were assessed following ACBI1 PROTAC treatment. B Genome browser views of RAD21 peaks that increase (up) or decrease (down) following ACBI1 PROTAC treatment. ChIP-seq signal for RAD21 with DMSO or ACBI1 treatment, BRG1, H3K4me3, H3K27ac, and CTCF is shown. Differential RAD21 peaks are indicated with a gray box. C Violin plots showing differential RAD21 signal in ACBI1 PROTAC treated mESCs relative to DMSO treated mESCs. The RAD21 peaks with significantly increased signal following ACBI1 treatment are shown in red, while peaks with significantly decreased signal are shown in gray. D Violin plots showing BRG1 (orange), H3K27ac (pink), and CTCF (dark blue) ChIP-seq signal at all RAD21 peaks, peaks with increased RAD21 signal (up) following ACBI1 treatment, and peaks with decreased RAD21 signal (down) following ACBI1 treatment. Significance determined by non-parametric Mann–Whitney test (****p-value < 0.0001). E Percentage of differential RAD21 peaks (up on left, down on right) upon ACBI1 treatment that overlap with the anchors of DNA loops detected by Micro-C, and percentage of non-differential RAD21 peaks with similar RAD21 signal intensity shown as a control. F Same as (C) for PFI-3 treatment. The RAD21 peaks with significantly increased signal following PFI-3 treatment are shown in purple, while peaks with significantly decreased signal are shown in gray. G Same as (D) for all RAD21 peaks and peaks with significantly different RAD21 signal following PFI-3 treatment (^n.s.not significant, *p-value < 0.01). H Same as (E) for PFI-3 treatment. I Same as (B) for BRM014 treatment. DNA loop anchor and long-range DNA interaction is depicted on top. J Same as (C) for BRM014 treatment. The RAD21 peaks with significantly increased signal following BRM014 treatment are shown in blue. K Same as (D) for all RAD21 peaks and peaks with increased RAD21 signal (up) following BRM014 treatment (****p-value < 0.0001). L Same as (E) for BRM014 treatment

Fig. 5

SWI/SNF perturbation alters cohesin binding to chromatin. A Chromatin bound (C) and nucleoplasmic (N) fractions were obtained following DMSO, ACBI1, PFI-3 and BRM014 treatment and levels of SWI/SNF and cohesin subunit proteins were measured by western blot. B Box plot depicting the percentage of chromatin bound protein when extracted with 300 mM NaCl for ACBI1 (n = 5), PFI-3 (n = 4), and BRM014 (n = 5) with DMSO controls. For each box plot, the bottom of the box represents the first quartile, the top represents the third quartile, and the line in the middle is the median. Bars extend to the minimum and maximum values