Transcription factors form a ternary complex with NIPBL/MAU2 to localize cohesin at enhancers

Abstract

While the cohesin complex is a key player in genome architecture, how it localizes to specific chromatin sites is not understood. Recently, we and others have proposed that direct interactions with transcription factors lead to the localization of the cohesin-loader complex (NIPBL/MAU2) within enhancers. Here, we identify two clusters of LxxLL motifs within the NIPBL sequence that regulate NIPBL dynamics, interactome, and NIPBL-dependent transcriptional programs. One of these clusters interacts with MAU2 and is necessary for the maintenance of the NIPBL-MAU2 heterodimer. The second cluster binds specifically to the ligand-binding domains of steroid receptors. For the glucocorticoid receptor (GR), we examine in detail its interaction surfaces with NIPBL and MAU2. Using AlphaFold2 and molecular docking algorithms, we uncover a GR-NIPBL-MAU2 ternary complex and describe its importance in GR-dependent gene regulation. Finally, we show that multiple transcription factors interact with NIPBL-MAU2, likely using interfaces other than those characterized for GR.

Figure 1:. LxxLL mutations alter NIPBL dynamics and basal transcriptome.

(A) Domain organization of Mus musculus NIPBL (mNIPBL) including the ectopic FLAG-Halo protein tags at the N-terminus of the protein. The three annotated LxxLL motif clusters are indicated as C1, C2, and C3. (B) AIUPred disorder score for mNIPBL. (C) Conservation of NIPBL LxxLL motif clusters across species. (D) Overall structure of the human cohesin-NIPBL-DNA complex solved by cryo-EM (24). The C2 and C3 clusters are indicated in the insets. (E) Fast SMT protocol (top) and 500 randomly selected single molecule trajectories for indicated species (bottom). Bound and diffusive fractions are indicated on the left of the respective panels. Scale bar is 4 μm. N_cells/N_tracks = 70/15,394 (WT), 142/82,691 (C1^mut), 76/46,879 (C2^mut), 75/27,535 (C1+C2^mut). (F) Intermediate SMT protocol captures the motion of bound NIPBL molecules (top). Representative trajectories of molecules in each of the four detected mobility states (bottom). Scale bar is 500 nm. N_cells/N_tracks/N_sub-tracks = 53/1,496/7,004 (WT), 65/5,769/19,632 (C1^mut), 69/4,398/22,317 (C2^mut), 67/1,746/7,729 (C1+C2^mut). (G) Population fractions for indicated mNIPBL species. Error bars = 95% confidence interval. (H) Differentially expressed genes upon NIPBL-KD rescued by 3xFLAG-Halo-mNIPBL-WT expression under basal conditions. (I) (Left) Heatmap of the 348 genes rescued by ectopic 3xFLAG-Halo-NIPBL-WT compared across the cell lines. (Right) Average expression bar plots for the genes in each cluster. See also, Figure S1, Videos S1–S2.

Figure 2:. Leu-rich clusters recruit diverse chromatin-associated proteins.

(A) (Left) Schematic representation of the proteomics experiments. (Right) Heatmap of abundance ratio of peptides detected in WT vs indicated NIPBL mutants. (B) MAU2 abundance detected in NIPBL-C1^mut and C2^mut relative to WT. p-value is reported from a one-way ANOVA. (C) NLRs for MAU2 against mNIPBL-C1^WT and mNIPBL-C2WT domains. Error bars denote standard deviation. ***p<0.001 (paired t-test). (D) Co-immunoprecipitation of MAU2 with NIPBL-WT, C1^mut, and C2^mut using the anti-FLAG antibody. (E) AlphaFold2-Multimer prediction of mNIPBL¹⁻¹²⁰ (yellow) with mMAU2 (silver). C1 is indicated in salmon. (F) Mass-spec log2(abundance ratio: C1^mut/C2^mut vs WT) of selected TFs. ****p<0.001 (one-way ANOVA). (G) Heatmap of gPCA interactions of NIPBL and MAU2 against a panel of TFs. See also, Figure S2.

Figure 3:. SR-LBDs interact with NIPBL through C2.

(A) NLRs of the NIPBL-C2^WT domain against indicated TFs. Green = positive and black = negative interactions. (B) Heatmap of interactions between NIPBL/MAU2 and NRs detected by gPCA. (C-E) NLRs for (C) NIPBL-C2^WT domain against the NRs that scored positively against NIPBL-WT; (D) GR against NIPBL-C2^WT/C2^mut domains; *p<0.05 (paired t-test); (E) NIPBL-C2^WT domain vs GR-WT, GR lacking its NTD (GR-ΔNTD), or its LBD (GR-ΔLBD), *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA and Tukey test for multiple comparisons). Error bars in panels A-E represent the standard deviation across measurements. (F-H) SPR sensorgrams for indicated human (h) SR-LBDs against human NIPBL-C2^WT (top) and C2^mut (bottom) peptides. (I) AlphaFold2-Multimer predictions for the interaction between hNIPBL-C2^WT peptide (salmon) and the LBDs of hGR, hAR, and hER. (J) Docking prediction for the hGR-LBD (blue) with the structured portion of hNIPBL (salmon). (K) Superposition of the pyDock prediction of the NIPBL-GR-LBD structure with the cryo-EM structure of the NIPBL-cohesin-DNA complex (24). See also, Figure S3.

Figure 4:. GR forms a ternary complex with NIPBL/MAU2 with implications for glucocorticoid signaling.

(A) gPCA results of MAU2 against GR-WT, GR-ΔNTD, and GR-ΔLBD. *p<0.05 (one-way ANOVA followed by Tukey test). Error bars denote the standard deviation. (B) AlphaFold2-Multimer prediction of the composite structure of mMAU2-mNIPBL¹⁻¹²⁰-mGR-LBD. (C) Triple-IP of FLAG-NIPBL, MAU2, and GR in cl15-WT cells. (D) Scatter plot of the top 50 genes whose Dex-response is affected upon NIPBL-KD. (E) PCA plot of the RNA-seq data across multiple conditions presented in Figures 1 and 4. See also, Figure S4.

Figure 5:. Possible models for a GR-NIPBL-MAU2 ternary complex.

(A) (Left) The glucocorticoid receptor ligand-binding domain (GR-LBD) interacts with the NIPBL¹⁻¹²⁰-MAU2 complex through helix 9. NIPBL¹⁻¹²⁰ and MAU2 interact through the C1 LxxLL motif cluster. (Right) The GR-LBD interacts with NIPBL-C2 through helix 12 in the AF-2 pocket. Key residues within C1 and C2 are indicated in the insets. (B) Possible configurations of the GR-NIPBL-MAU2 ternary complex: (Left) A single GR monomer can interact with both NIPBL-C2 through the AF-2 pocket and MAU2 through helix 9. (Middle and Right) Since the dimerization interface of GR is not occluded by either NIPBL or MAU2, a GR dimer can interact with NIPBL/MAU2 either through (Middle) a single GR molecule or (Right) with one GR molecule interacting with NIPBL-C2 and another one interacting with MAU2.