(E-166) Elevated Rho Signaling Downstream of the R206H ACVR1 Mutation in FOP Alters Chromatin Organization

Introduction:: Fibrodysplasia ossificans progressiva (FOP) is a rare musculoskeletal disorder caused by a gain-of-function mutation (R206H; c.617 G > A) in the bone morphogenetic protein (BMP) type I receptor ACVR1 [1]. As a result, enhanced activity of ACVR1 increases downstream BMP pathway signaling, and induces the formation of extraskeletal bone, or heterotopic ossification (HO), in soft connective tissues [1]. There are currently no treatments that are effective in preventing this HO. Signaling pathways, including the BMP pathway, regulate cell function through transcriptional regulation mediated in part by chromatin reorganization. Elucidating the molecular mechanisms of altered signaling in FOP that perturbs spatio-temporal genome organization is crucial for developing novel therapeutics. However, how the R206H mutation impacts chromatin organization and the molecular mechanisms by which this occurs have not been examined. To this end, using super-resolution stochastic optical reconstruction microscopy (STORM) and computational analyses, we examined how the FOP ACVR1 R206H mutation in mouse embryonic fibroblasts (MEFs) alters nanoscale histone organization and assessed how treatment with BMP ligands regulates histone organization. Further, given that we had previously identified altered mechanobiological signaling (increases in cell contractility and misinterpretation of substrate rigidity via Rho/ROCK) in FOP cells [1], we investigated whether blockade of this pathway using clinically used small molecule inhibitors could restore chromatin reorganization in Acvr1^R206H/+ (FOP) cells.


Materials and Methods:: Acvr1^+/+ (wild-type, WT) and Acvr1^R206H/+ (FOP) primary MEFs were isolated and cultured in basal growth media (BM) [1]. Study 1: To investigate how the FOP mutation alters nanoscale chromatin organization, STORM imaging (Nanoimager, ONI) was carried out. WT and FOP MEFs were fixed, immunostained for histone-H2B (H2B), and incubated with secondary antibodies custom labeled with activator-reporter dye pairs [2, 3]. Then, images were analyzed using the Nanoimager software (ONI) and custom Voronoi-based clustering analysis in MATLAB [2, 3]. The chromatin condensation parameter was determined by normalizing the Voronoi density of H2B to the mean Voronoi polygon density across the nucleus [2, 3]. Study 2: To explore how activated BMP signaling differentially regulates chromatin organization, WT or FOP MEFs (passage 1) were treated with recombinant BMP4 (15 ng/mL or 30 ng/mL) in serum-free BM overnight and STORM data was obtained for each ligand concentration. In addition, STORM data was obtained for WT and FOP MEFs in the presence of low-dose BMP4 (15 ng/mL) at various time points (0.5hr, 1hr, 3hr, 12hr) to evaluate the differential sensitivity to BMP4 ligand. Study 3: To gain additional insight into the crosstalk between the BMP and the Rho/ROCK mechano-signaling pathways in disease-mediated chromatin reorganization, WT MEFs were treated with Lysophosphatidic acid (LPA), a Rho activator, for 1 hour, while FOP MEFs were treated with Fasudil (FD), a ROCK inhibitor, for 0.5 hours. STORM images were obtained. Statistical analyses were performed using a student’s t-test or ANOVA with Tukey’s post hoc testing.


Results, Conclusions, and Discussions:: We successfully imaged the organization of histone H2B within the nucleus of MEFs isolated from Acvr1^R206H/+ (FOP) mice using STORM (Fig. 1). STORM analysis revealed condensed nano-sized H2B clusters in WT MEFs (WT, Fig. 1A). In FOP MEFs, less condensed H2B clusters were observed (FOP, Fig. 1A). Through quantification, we found no differences in the total number of H2B localization per nuclear area (Fig. 1B) while the Acvr1^R206H mutation significantly decreased the chromatin condensation parameter, indicating an increase in chromatin decondensation with the mutation (Fig. 1C). When WT or FOP MEFs were treated with BMP4, cells responded differently to the treatments based on genotype. In particular, a high dose of BMP4 (30 ng/mL) decreased chromatin condensation in WT MEFs, while it significantly increased chromatin condensation in FOP MEFs (Fig. 2A). In addition, we examined the time-dependent response of WT and FOP MEFs to BMP4 (15 ng/mL) treatment. Interestingly, FOP MEFs showed a more rapid and greater magnitude change in chromatin condensation compared to WT MEFs, consistent with FOP MEFs being more sensitive to BMP4 (Fig. 2B). When we treated WT MEFs with the Rho activator, LPA (to increase cellular contractility), chromatin condensation in WT MEFs became similar to that of FOP cells (Fig. 3A, B). On the other hand, the addition of Fasudil (FD, a Rho kinase inhibitor) returned the nanoscale chromatin condensation level in FOP MEFs to that of WT MEFs (Fig. 3B). Taken together, MEFs with the FOP mutation Acvr1^R206H have less condensed chromatin and are more sensitive to BMP4 ligands. We also found that the Rho/ROCK pathway contributes to this process. These data support crosstalk between the BMP pathway and Rho/ROCK mechano-signaling pathway in disease-mediated chromatin reorganization that potentially alters the transcriptional activations in FOP cells. Of note, chromatin condensation levels in FOP MEFs were rescued with FD, presenting a possible therapeutic target for the treatment of FOP. Ongoing studies are focused on further exploring the crosstalk between BMP signaling pathway and mechano-signaling pathway and identifying novel epigenetic mechanisms of FOP pathology that alter chromatin organization and phenotype, providing critical information to support new therapies in FOP.


Acknowledgements (Optional): : This work is supported by NIH K01 AR077087, NSF CMMI 154857, NIH R01 AR071399, and the International FOP Association.

References (Optional): : [1] Stanley+ 2019, JBMR. [2] Heo+ 2022, Nature BME; [3] Otterstrom+ 2019, Nucleic Acids Res.