Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance

Abstract

Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5'-triphosphate)-driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine-dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes.

Keywords: BAF complex; BRG; BRM; PB1; chemical probe; chromatin remodelling,embryonic stem cells; epigenetics; trophoblast stem cells.

Fig. 1. Characterization of the interaction of SA with family VIII bromodomains.

(A) Selectivity of SA assessed using temperature shift (ΔT_m) assays. Screened targets are labeled in the phylogenetic tree based on the BRD family structure–based alignment (11). (B) Isothermal titration data of the interaction of SA with PB1(5). The raw binding heats for each injection as well as the normalized binding enthalpies (inset) are shown. (C). Sequence alignment of a ZA-loop segment of bromodomains of family VIII as well as BRD4(1) and CBP (CREB-binding protein) (left panel) and structural overview of the SA/PB1(5) complex. Hydrogen bonds to the conserved asparagine (N⁷⁰⁷) and tyrosine (Y⁶⁶⁴) are shown as dotted green lines. (D) Binding mode of a typical acetyl lysine mimetic fragment (N-methyl-2-pyrrolidone) in PB1(5) (left panel) and SA (right panel). Water molecules are shown as solid spheres. Waters present in the PB1(5) complex are superimposed onto the SA cocrystal structure to demonstrate the displacement of four structural waters by the ligand. (E) Superposition of apo-PB1(5) (PDB ID: 3G0J) and apo-BRD4(1) (PDB ID: 2oss); blue spheres represent waters from the PB1(5)/NMP structure. There is a missing water indicated with a green arrow for PB1(5) structures.

Fig. 2. Selectivity, potency, and cocrystal structure of PFI-3 with BRG1.

(A) Chemical structure of PFI-3. (B) Selectivity screening data of PFI-3 using temperature shift assay. The temperature shifts are mapped onto the phylogenetic tree using red spheres as indicated in the figure. (C) ITC data measured for PFI-3 using the two BRM bromodomain isoforms (see fig. S1) BRG1 and PB1(5). Raw injection heats as well as normalized binding enthalpies and the corresponding nonlinear least squares fits (inset) are shown. (D) 2F_o − F_c omit electron density map contoured at 1.5 σ around the inhibitor at 2. (E) Details of the interaction of PFI-3 with the BRG1 bromodomain. (F) Surface representation of the BRG1 acetyl lysine binding site in complex with PFI-3.

Fig. 3. FRAP experiments and loss of stemness, and skewed differentiation in embryonic stem cells (ESCs).

(A) Influence of PFI-3 or the inactive PFI-3oMet on half recovery times of U2OS cells transfected with wild-type (WT) full-length GFP-BRM or the N1464F mutant construct. Cells were treated with 2.5 μM SAHA (shown by “*”) to increase the assay window. (B) Time dependence of fluorescence recovery in the bleached area of cells expressing WT or mutant GFP-BRM with the corresponding treatment as in (A). All PFI-3–treated samples showed a P < 0.0001 compared to WT treated with SAHA. Curves represent averaged data of at least 10 replicates. (C) Phase-contrast microscopy images of ESCs treated with the negative control substance PFI-3oMet or PFI-3. (D) qRT-PCR analyses of the transcription levels for the indicated stemness genes after treatment for 1 or 8 days. (E) mRNA levels of epiblast differentiation genes, representative of endoderm (Gata4), mesoderm (T), and ectoderm (Nes) cultured for 1 or 4 days. The stemness marker Oct4 was used as differentiation control. (D and E) mRNA levels normalized to Gapdh, Hprt, and Ppia are represented as means + SEM relative to the expression in ESCs treated with control compound for 24 hours. Experiments were independently repeated at least three times in triplicate. *P < 0.05; ***P < 0.001. ESCs were treated with the negative control substance PFI-3oMet or PFI-3 inhibitor in the presence (C and D) or absence (E) of leukemia inhibitory factor (LIF) in stemness or differentiation conditions, respectively. Red bars represent PFI-3–treated cells, and white bars represent the corresponding control experiments.

Fig. 4. TSC differentiation is affected by PFI-3 inhibition.

Comparison of gene expression in TSCs treated with PFI-3 or control substance during stemness and differentiation. (A to C) Differentially transcribed genes are displayed normalized and clustered to the number of reads shown as (A) tile array and (B and C) dot blots. (D) Quantification of stemness (Eomes) and differentiation markers (Prl3d1, Tpbpa) expression by RT-PCR. mRNA levels were normalized to Gapdh, Hprt, and Ppia, and values represent the means + SEM relative to the expression in TSCs treated with control compound at day 0. Experiments were independently repeated at least three times in triplicate. ***P < 0.001.