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• W3019155769 abstract "•BMPs and osteogenic signals induce Pbrm1/PBAF expression in MSCs•Pbrm1/PBAF regulates Smad1/5/8 activation by remodeling chromatin at Bmpr/Tgfβr•Gain of BmprIβ in PBAF-deficient MSCs restores osteolineage priming•Pbrm1 loss further affects HSPC activity in a non-cell-autonomous mechanism Bone morphogenic protein (BMP)/transforming growth factor β (TGF-β) signaling determines mesenchymal-stromal-cell (MSC) osteolineage commitment and tissue identity. However, molecular integration of developmental signaling with MSC-intrinsic chromatin regulation remains incompletely understood. SWI/SNF-(BAF) is an ATP-dependent chromatin remodeler implicated in multi-cellular development. We show that BMPs and long-term osteogenic signals in MSCs selectively induce expression of polybromo BAF (PBAF) components Pbrm1, Arid2, and Brd7. Loss of Pbrm1/Arid2/Brd7 profoundly impairs osteolineage gene expression and osteogenesis without compromising adipogenesis. Pbrm1 loss attenuates MSC in vivo ossification. Mechanistically, Pbrm1/PBAF deficiency impairs Smad1/5/8 activation through locus-specific epi-genomic remodeling, involving Pbrm1 bromodomains, along with transcriptional downregulation of Bmpr/TgfβrII affecting BMP-early-responsive gene expression. Gain of function of BmprIβ, TgfβrII in PBAF-deficient MSCs partly restores Smad1/5/8 activation and osteogenesis. Pbrm1 loss further affects hematopoietic stem and progenitor activity through non-cell-autonomous regulation of microenvironment and niche-factor expression. Together, these findings reveal a link illustrating epi-genomic feedforward control of BMP/TGF-β signaling to transcriptional and cellular plasticity in the mesenchymal microenvironment and account for stromal-SWI/SNF in hematopoiesis. Bone morphogenic protein (BMP)/transforming growth factor β (TGF-β) signaling determines mesenchymal-stromal-cell (MSC) osteolineage commitment and tissue identity. However, molecular integration of developmental signaling with MSC-intrinsic chromatin regulation remains incompletely understood. SWI/SNF-(BAF) is an ATP-dependent chromatin remodeler implicated in multi-cellular development. We show that BMPs and long-term osteogenic signals in MSCs selectively induce expression of polybromo BAF (PBAF) components Pbrm1, Arid2, and Brd7. Loss of Pbrm1/Arid2/Brd7 profoundly impairs osteolineage gene expression and osteogenesis without compromising adipogenesis. Pbrm1 loss attenuates MSC in vivo ossification. Mechanistically, Pbrm1/PBAF deficiency impairs Smad1/5/8 activation through locus-specific epi-genomic remodeling, involving Pbrm1 bromodomains, along with transcriptional downregulation of Bmpr/TgfβrII affecting BMP-early-responsive gene expression. Gain of function of BmprIβ, TgfβrII in PBAF-deficient MSCs partly restores Smad1/5/8 activation and osteogenesis. Pbrm1 loss further affects hematopoietic stem and progenitor activity through non-cell-autonomous regulation of microenvironment and niche-factor expression. Together, these findings reveal a link illustrating epi-genomic feedforward control of BMP/TGF-β signaling to transcriptional and cellular plasticity in the mesenchymal microenvironment and account for stromal-SWI/SNF in hematopoiesis. Mesenchymal stromal cells (MSCs) constitute an integral component of the mammalian bone marrow microenvironment, regulating steady-state and stressed hematopoiesis (Baryawno et al., 2019Baryawno N. Przybylski D. Kowalczyk M.S. Kfoury Y. Severe N. Gustafsson K. Kokkaliaris K.D. Mercier F. Tabaka M. Hofree M. et al.A Cellular Taxonomy of the Bone Marrow Stroma in Homeostasis and Leukemia.Cell. 2019; 177: 1915-1932.e1916Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, Frenette et al., 2013Frenette P.S. Pinho S. Lucas D. Scheiermann C. 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Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment.Cell. 2007; 131: 324-336Abstract Full Text Full Text PDF PubMed Scopus (1618) Google Scholar, Spiegel et al., 2008Spiegel A. Kalinkovich A. Shivtiel S. Kollet O. Lapidot T. Stem cell regulation via dynamic interactions of the nervous and immune systems with the microenvironment.Cell Stem Cell. 2008; 3: 484-492Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). MSCs are multi-potent in nature and differentiate into osteoblasts, adipocytes, and chondrocytes in response to diverse signaling cues (Bianco et al., 2008Bianco P. Robey P.G. Simmons P.J. Mesenchymal stem cells: revisiting history, concepts, and assays.Cell Stem Cell. 2008; 2: 313-319Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar, Gronthos et al., 1994Gronthos S. Graves S.E. Ohta S. Simmons P.J. The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors.Blood. 1994; 84: 4164-4173Crossref PubMed Google Scholar, Méndez-Ferrer et al., 2010Méndez-Ferrer S. Michurina T.V. Ferraro F. Mazloom A.R. Macarthur B.D. Lira S.A. Scadden D.T. Ma’ayan A. Enikolopov G.N. Frenette P.S. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.Nature. 2010; 466: 829-834Crossref PubMed Scopus (2323) Google Scholar). Bone morphogenic proteins (BMPs) belong to the transforming growth factor β (TGF-β) superfamily of proteins that critically regulate osteolineage differentiation of MSCs by regulating downstream Smad signaling and osteogenic gene expression, including Alpl, Runx2, and Osx (Attisano and Wrana, 2002Attisano L. Wrana J.L. Signal transduction by the TGF-beta superfamily.Science. 2002; 296: 1646-1647Crossref PubMed Scopus (1094) Google Scholar, Chen et al., 2004Chen D. Zhao M. Mundy G.R. Bone morphogenetic proteins.Growth Factors. 2004; 22: 233-241Crossref PubMed Scopus (1646) Google Scholar, Goldman et al., 2009Goldman D.C. Bailey A.S. Pfaffle D.L. Al Masri A. Christian J.L. Fleming W.H. BMP4 regulates the hematopoietic stem cell niche.Blood. 2009; 114: 4393-4401Crossref PubMed Scopus (86) Google Scholar, Hassan et al., 2004Hassan M.Q. Javed A. Morasso M.I. Karlin J. Montecino M. van Wijnen A.J. Stein G.S. Stein J.L. Lian J.B. Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene.Mol. Cell. Biol. 2004; 24: 9248-9261Crossref PubMed Scopus (227) Google Scholar, Massagué et al., 2000Massagué J. Blain S.W. Lo R.S. TGFbeta signaling in growth control, cancer, and heritable disorders.Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2018) Google Scholar, Massagué et al., 2005Massagué J. Seoane J. Wotton D. Smad transcription factors.Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1845) Google Scholar, Medici et al., 2010Medici D. Shore E.M. Lounev V.Y. Kaplan F.S. Kalluri R. Olsen B.R. Conversion of vascular endothelial cells into multipotent stem-like cells.Nat. Med. 2010; 16: 1400-1406Crossref PubMed Scopus (528) Google Scholar). Activated Smads and Runx2 cooperate to orchestrate downstream gene expression required for osteoblast differentiation (Massagué et al., 2005Massagué J. Seoane J. Wotton D. Smad transcription factors.Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1845) Google Scholar, Phimphilai et al., 2006Phimphilai M. Zhao Z. Boules H. Roca H. Franceschi R.T. BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype..J. Bone Miner. Res. 2006; 21: 637-646Crossref PubMed Scopus (278) Google Scholar). In the last decades, although significant efforts have been made to determine BMP-signal-dependent osteoblast differentiation, molecular synchronization of BMP/Smad signal transduction with chromatin remodelling, transcriptional activation, and MSC osteogenic fate commitment remain inadequately understood. Therefore, identification and molecular characterization of regulatory nodes that essentially connect tissue microenvironment signals with chromatin architecture is an important aspect of multi-cellular development and regenerative medicine. SWI/SNF (BAF) is an evolutionarily conserved, multi-subunit ATP-dependent chromatin remodeling complex that plays important roles in pluripotency, cellular reprogramming, and lineage differentiation (de la Serna et al., 2006de la Serna I.L. Ohkawa Y. Imbalzano A.N. Chromatin remodelling in mammalian differentiation: lessons from ATP-dependent remodellers.Nat. Rev. Genet. 2006; 7: 461-473Crossref PubMed Scopus (302) Google Scholar, Ho et al., 2009Ho L. Jothi R. Ronan J.L. Cui K. Zhao K. Crabtree G.R. An embryonic stem cell chromatin remodeling complex, esBAF, is an essential component of the core pluripotency transcriptional network.Proc. Natl. Acad. Sci. USA. 2009; 106: 5187-5191Crossref PubMed Scopus (314) Google Scholar, Lessard et al., 2007Lessard J. Wu J.I. Ranish J.A. Wan M. Winslow M.M. Staahl B.T. Wu H. Aebersold R. Graef I.A. Crabtree G.R. An essential switch in subunit composition of a chromatin remodeling complex during neural development.Neuron. 2007; 55: 201-215Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, Wilson and Roberts, 2011Wilson B.G. Roberts C.W. SWI/SNF nucleosome remodellers and cancer.Nat. Rev. Cancer. 2011; 11: 481-492Crossref PubMed Scopus (846) Google Scholar). SWI/SNF activity has been linked with enhancer function in gene expression regulation (Nakayama et al., 2017Nakayama R.T. Pulice J.L. Valencia A.M. McBride M.J. McKenzie Z.M. Gillespie M.A. Ku W.L. Teng M. Cui K. Williams R.T. et al.SMARCB1 is required for widespread BAF complex-mediated activation of enhancers and bivalent promoters.Nat. Genet. 2017; 49: 1613-1623Crossref PubMed Scopus (111) Google Scholar, Wang et al., 2017Wang X. Lee R.S. Alver B.H. Haswell J.R. Wang S. Mieczkowski J. Drier Y. Gillespie S.M. Archer T.C. Wu J.N. et al.SMARCB1-mediated SWI/SNF complex function is essential for enhancer regulation.Nat. Genet. 2017; 49: 289-295Crossref PubMed Scopus (161) Google Scholar). Several reports have suggested that two structurally similar but functionally distinct SWI/SNF complexes exist in the mammalian system, namely classical BAF and polybromo BAF (PBAF) complex (Hodges et al., 2016Hodges C. Kirkland J.G. Crabtree G.R. The Many Roles of BAF (mSWI/SNF) and PBAF Complexes in Cancer.Cold Spring Harb. Perspect. Med. 2016; 6: a026930Crossref PubMed Scopus (186) Google Scholar). BAF and PBAF complexes have overlapping subunit composition, including ATPase subunit Smarca4 (Brg1) and core component Smarcc1 (Baf155) (Flowers et al., 2009Flowers S. Nagl Jr., N.G. Beck Jr., G.R. Moran E. Antagonistic roles for BRM and BRG1 SWI/SNF complexes in differentiation.J. Biol. Chem. 2009; 284: 10067-10075Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). However, the presence of Pbrm1, Arid2, and Brd7 proteins, containing multiple bromodomains important for transcriptional activation by recognizing acetylated lysine residues within histone tails in a context-dependent manner, distinguishes PBAF from the classical BAF complex (Hodges et al., 2016Hodges C. Kirkland J.G. Crabtree G.R. The Many Roles of BAF (mSWI/SNF) and PBAF Complexes in Cancer.Cold Spring Harb. Perspect. Med. 2016; 6: a026930Crossref PubMed Scopus (186) Google Scholar, Wang et al., 2004Wang Z. Zhai W. Richardson J.A. Olson E.N. Meneses J.J. Firpo M.T. Kang C. Skarnes W.C. Tjian R. Polybromo protein BAF180 functions in mammalian cardiac chamber maturation.Genes Dev. 2004; 18: 3106-3116Crossref PubMed Scopus (137) Google Scholar, Yan et al., 2005Yan Z. Cui K. Murray D.M. Ling C. Xue Y. Gerstein A. Parsons R. Zhao K. Wang W. PBAF chromatin-remodeling complex requires a novel specificity subunit, BAF200, to regulate expression of selective interferon-responsive genes.Genes Dev. 2005; 19: 1662-1667Crossref PubMed Scopus (184) Google Scholar). Unlike PBAFs, the canonical BAF complex expresses Arid1a (Baf250a) or Arid1b (Baf250b) subunits. In support of an epi-genomic connection to MSC fate determination, a recent study has revealed that H3K9me3 and H3K27m3 demethylases, Kdm4b and Kdm6b, respectively, can promote osteolineage differentiation of human MSCs (Fu et al., 2016Fu G. Ren A. Qiu Y. Zhang Y. Epigenetic Regulation of Osteogenic Differentiation of Mesenchymal Stem Cells.Curr. Stem Cell Res. Ther. 2016; 11: 235-246Crossref PubMed Scopus (21) Google Scholar, Ye et al., 2012Ye L. Fan Z. Yu B. Chang J. Al Hezaimi K. Zhou X. Park N.H. Wang C.Y. Histone demethylases KDM4B and KDM6B promotes osteogenic differentiation of human MSCs.Cell Stem Cell. 2012; 11: 50-61Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Enhancer activation in MSCs has been linked with osteogenesis (Agrawal Singh et al., 2019Agrawal Singh S. Lerdrup M. Gomes A.R. van de Werken H.J. Vilstrup Johansen J. Andersson R. Sandelin A. Helin K. Hansen K. PLZF targets developmental enhancers for activation during osteogenic differentiation of human mesenchymal stem cells.eLife. 2019; 8: e40364Crossref PubMed Scopus (21) Google Scholar). Polycomb group proteins were also implicated in MSC fate mapping and skeletal development (Hemming et al., 2017Hemming S. Cakouros D. Codrington J. Vandyke K. Arthur A. Zannettino A. Gronthos S. EZH2 deletion in early mesenchyme compromises postnatal bone microarchitecture and structural integrity and accelerates remodeling.FASEB J. 2017; 31: 1011-1027Crossref PubMed Scopus (39) Google Scholar). In addition, previous reports indicated Smarca4-mediated regulation of Alpl in skeletal gene expression (Flowers et al., 2014Flowers S. Patel P.J. Gleicher S. Amer K. Himelman E. Goel S. Moran E. p107-Dependent recruitment of SWI/SNF to the alkaline phosphatase promoter during osteoblast differentiation.Bone. 2014; 69: 47-54Crossref PubMed Scopus (6) Google Scholar, Young et al., 2005Young D.W. Pratap J. Javed A. Weiner B. Ohkawa Y. van Wijnen A. Montecino M. Stein G.S. Stein J.L. Imbalzano A.N. Lian J.B. SWI/SNF chromatin remodeling complex is obligatory for BMP2-induced, Runx2-dependent skeletal gene expression that controls osteoblast differentiation.J. Cell. Biochem. 2005; 94: 720-730Crossref PubMed Scopus (81) Google Scholar). Smarca4 was also shown to regulate MSC senescence (Alessio et al., 2010Alessio N. Squillaro T. Cipollaro M. Bagella L. Giordano A. Galderisi U. The BRG1 ATPase of chromatin remodeling complexes is involved in modulation of mesenchymal stem cell senescence through RB-P53 pathways.Oncogene. 2010; 29: 5452-5463Crossref PubMed Scopus (39) Google Scholar, Napolitano et al., 2007Napolitano M.A. Cipollaro M. Cascino A. Melone M.A. Giordano A. Galderisi U. Brg1 chromatin remodeling factor is involved in cell growth arrest, apoptosis and senescence of rat mesenchymal stem cells.J. Cell Sci. 2007; 120: 2904-2911Crossref PubMed Scopus (45) Google Scholar). Importantly, Arid1a was shown to regulate pool size of fetal liver hematopoietic stem cells (HSCs) in a non-cell-autonomous mechanism, where Arid1a mutant stromal cells demonstrated hematopoiesis-supporting potential (Krosl et al., 2010Krosl J. Mamo A. Chagraoui J. Wilhelm B.T. Girard S. Louis I. Lessard J. Perreault C. Sauvageau G. A mutant allele of the Swi/Snf member BAF250a determines the pool size of fetal liver hemopoietic stem cell populations.Blood. 2010; 116: 1678-1684Crossref PubMed Scopus (35) Google Scholar). In this study, we have identified that short-term BMP/Smad signaling as well as long-term osteogenic signals, in established murine MSCs and in human normal bone-marrow-derived primary MSCs, cause significant and specific induction in the expression of Pbrm1, Arid2, and Brd7. We demonstrate that deficiency of Pbrm1/PBAFs profoundly impairs BMP-dependent Smad1/5/8 activation, osteogenic gene expression, and osteolineage differentiation. Interestingly, Pbrm1-deficient MSCs also showed a defective in vivo ossification. Pbrm1/PBAF loss did not affect MSC proliferation and survival and adipogenic differentiation potential. Mechanistically, Pbrm1/PBAF-loss-associated impaired Smad1/5/8 activation was due to an altered Pbrm1 bromodomain function, chromatin accessibility, and expression of Bmp/Tgfβ receptor genes. Gain of BmprIβ/TgfβrII expression partly restored BMP-2-dependent Smad1/5/8 activation and promoted osteogenic gene expression in PBAF-deficient MSCs. Furthermore, we provide evidence that the loss of Pbrm1 impairs the expression of critical hematopoietic microenvironment/niche factors, which results in a defective non-cell autonomous support of hematopoietic stem and progenitor cell (HSPC) activity. Overall, these results reveal a feedforward regulation between BMP/Smad and PBAFs and posit critical roles of Pbrm1/PBAF in integrating BMP/Smad signaling and osteogenic gene expression in mammalian MSC lineage commitment. BMP signaling plays a crucial role in osteogenic differentiation of MSCs. OP9 cells have been considered as a bona fide murine MSC line that possesses osteogenic, adipogenic, and chondrogenic differentiation potential (Gao et al., 2010Gao J. Yan X.L. Li R. Liu Y. He W. Sun S. Zhang Y. Liu B. Xiong J. Mao N. Characterization of OP9 as authentic mesenchymal stem cell line.J. Genet. Genomics. 2010; 37: 475-482Crossref PubMed Scopus (51) Google Scholar, Lane et al., 2012Lane S.W. De Vita S. Alexander K.A. Karaman R. Milsom M.D. Dorrance A.M. Purdon A. Louis L. Bouxsein M.L. Williams D.A. Rac signaling in osteoblastic cells is required for normal bone development but is dispensable for hematopoietic development.Blood. 2012; 119: 736-744Crossref PubMed Scopus (18) Google Scholar). Using OP9 cells as a model system to investigate osteogenic differentiation of MSCs, we sought to determine the role of the SWI/SNF (BAF) chromatin remodeler in osteogenesis. Gene expression analysis identified a significant induction in Pbrm1 (Baf180), along with classical osteogenic markers, including Alpl and transcription factors Osx and Runx2, in OP9 cells treated with BMP-2 in a time-dependent manner (Figure 1A; Figures S1A and S1B). In addition, there was an increase in the expression of Arid2 (Baf200) and Brd7, two other important subunits of the mammalian polybromo BAF (PBAF) complex in BMP-2-treated cells (Figure 1A; Figures S1A and S1B). Similar to OP9 cells, BMP-4/7 treatment also induced PBRM1, ARID2, and BRD7 expression in human HS-5 cells as well as in human normal bone-marrow-derived primary MSCs (Figures 1B and 1C; Figures S1C and S1D). In agreement with their transcriptional upregulation, BMP signaling also induced the expression of PBAF proteins (Figures 1D and 1E; Figure S1E). We did not observe appreciable changes in the expression of the remaining SWI/SNF subunits (Figure 1C; Figures S1A and S1C). BMPs regulate downstream gene expression through Smad signaling (Attisano and Wrana, 2002Attisano L. Wrana J.L. Signal transduction by the TGF-beta superfamily.Science. 2002; 296: 1646-1647Crossref PubMed Scopus (1094) Google Scholar, Massagué et al., 2005Massagué J. Seoane J. Wotton D. Smad transcription factors.Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1845) Google Scholar). We asked whether BMP-driven induction in PBAFs is dependent on Smad activation. Pharmacological inhibition of Smad1/5/8 phosphorylation attenuated PBAF transcriptional induction (Figure 1F; Figure S2A). In agreement with this result, silencing of Smad1 also impaired BMP-2-driven transcriptional induction of Pbrm1 and Alpl, suggesting BMP/Smad-dependent regulation of PBAF gene expression (Figure S2B). Mechanistically, BMP-2-mediated Pbrm1 induction in MSCs was associated with chromatin occupancy of active transcription marks p300 and H3K27ac along with an increased p-Smad1/5/8 level at the Pbrm1 transcription start site (TSS; −200 bp through +100 bp) (Figure S2C). Consistent with these findings, there was a significant increase in PBAF gene expression in OP9 cells and also in other human MSCs that were differentiated over 21 days in the presence of osteogenic conditioned media (Figures 1G and 1H; Figure S2D). Together, these results indicate that the expression of Pbrm1, along with other components of PBAF machinery, is associated with BMP signaling and mammalian mesenchymal stromal osteogenic commitment. Next, we investigated whether stromal loss of Pbrm1 affects MSC osteogenic commitment. OP9 cells were transduced at a multiplicity of infection (MOI) of 5, with lentivirus particles carrying two different short hairpin RNA (shRNA) constructs targeting against Pbrm1 (sh-Pbrm1_B and sh-Pbrm1_C) or sh-Control and co-expressing GFP. After 48 h of transduction, GFP-expressing cells were sorted using flow cytometry and expanded subsequently (Figure 2A). Interestingly, there were dramatic impairments in the induction of Alpl, Osx, and Runx2 in response to BMP-2 signaling in Pbrm1-deficient MSCs compared to the control (Figure 2B; Figure S3A). Pbrm1 loss also attenuated the expression induction of Dlx3 and Dlx5, late-responsive osteogenic genes; Bglap (Osteocalcin) and Bsp1 (Osteopontin); along with H3K9me3/H3K27me3 demethylases Kdm4b and Kdm6b (Figure S3A). Long-term osteogenic differentiation assays further indicated that Pbrm1 loss in OP9 cells resulted in a significant reduction in osteogenic gene expression and osteogenesis in vitro (Figures 2C and 2D; Figure S3B). Similarly, PBRM1 loss also impaired BMP-4/7-induced osteogenic signaling in HS-5 and human primary MSCs (Figures 2E and 2F; Figures S3C and S4A). Conversely, gain of function of Pbrm1 in wild-type MSCs upregulated BMP-2-induced osteogenic gene expression (Figure S4B). We did not observe any change in the proliferation or survival of Pbrm1-deficient MSCs (Figures S4C and S4D). Sucrose density gradient centrifugation analysis also revealed that the absence of Pbrm1 did not impart any overall change in the nuclear SWI/SNF complex integrity under steady-state conditions (Figure S4E). To further understand the physiological significance of Pbrm1 deficiency in osteoblast differentiation, wild-type and Pbrm1-deficient OP9 cells together with hydroxyapatite/tri-calcium phosphate particles were transplanted subcutaneously into C57/BL6 mice and analyzed for in vivo ossification. Interestingly, 12-weeks post-transplantation image analysis of the recipient mice revealed bone-like structure formation only for the wild-type group, but no appreciable ossification was observed in mice transplanted with Pbrm1-deficient cells. Histopathological analysis of the implanted scaffolds further confirmed a significant reduction in the formation of mineralized bony tissues in Pbrm1-deficient MSCs compared to the control (Figure 2G). Homeostatic balance between MSC lineage commitment into mature osteoblasts, adipocytes, or chondrocytes is considered as a central feature of bone formation. We, therefore, asked whether the loss of Pbrm1-associated defective osteogenesis was at the expense of an enhanced adipogenic or chondrogenic differentiation. However, Pbrm1 deficiency did not influence adipocyte differentiation or alter the expression of key adipogenic transcription factor Pparγ (Figures S5A and S5B). Although Pbrm1 loss did not apparently affect chrondrogenic differentiation, there was an impaired transcriptional induction of Sox9 in Pbrm1-deficient MSCs (Figures S5C and S5D). Together, these results suggest that Pbrm1 plays an important role in regulating BMP signaling and osteogenic fate determination. Next, we investigated whether the loss of two other PBAF components, namely Arid2 and Brd7, could exert a similar effect in MSC osteolineage differentiation. OP9 cells were transduced with lentivirus particles at a MOI of 2.5 for each construct expressing sgRNAs targeting Arid2 or Brd7 and co-expressing Cas9 and GFP (Figure 2A). Similar to Pbrm1 deficiency, CRISPR-Cas-driven loss of Arid2 or Brd7 also impaired induction in osteogenic gene expression in response to BMP-2 signaling and attenuated long-term osteogenic differentiation (Figures 2H–2K; Figures S5E and S5F). Loss of Arid2 or Brd7 had no effect on proliferation or survival of MSCs (Figures S4C and S4D). In addition, similar to Pbrm1 loss, a deficiency of Arid2 or Brd7 did not affect adipocyte differentiation (Figures S5A and S5B). Silencing of classical BAF-restricted components Arid1a (Baf250a) and Arid1b (Baf250b) impaired BMP-2-mediated Alpl induction and MSC long-term osteogenic commitment (Figures S5G–S5J). Together, these results highlight the critical function of the PBAF complex in regulating osteogenesis. We further interrogated whether the loss of each subunit of PBAF can result in a compensatory increase in expression of the remaining components. Indeed, there was a significant increase in Pbrm1 gene as well as protein expression in Arid2- or Brd7-deficient stromal cells (Figure 2A; Figure S5K). However, expression of Arid2 and Brd7 were similarly maintained in Pbrm1-defcient MSCs compared to control (Figure S5K). In agreement with these findings, additional silencing of Pbrm1 in Arid2-deficient cells resulted in a compound loss of Alpl induction (Figure S5L). Together, these results indicate that Pbrm1 and PBAF play important roles in determining BMP signaling-induced osteogenic gene expression and MSC osteogenic lineage commitment. Having identified Pbrm1 as an important molecular regulator of osteogenic gene expression and osteoblast differentiation, we investigated the effects of Pbrm1 loss in BMP-2/TGFβ downstream Smad signal transduction pathways. Deficiency of Pbrm1 in MSCs resulted in a significant reduction in BMP-2-induced Smad1/5/8 phosphorylation (Figure 3A; Figure S6A). Similarly, a loss of Arid2 or Brd7 also diminished Smad1/5/8 activation (Figure 3A). There was also a reduction in Erk1/2 activation in Pbrm1-deficient, but not Arid2/Brd7-deficient, MSCs in response to BMP-2/TGF-β1 signaling (Figures 3A and 3B). In addition, Pbrm1/PBAF loss further impaired TGF-β1-driven activation of Smad1/5/8 and Smad2/3 signaling pathways (Figure 3B; Figure S6B). Considering the transcriptional regulatory role of SWI/SNF in mammalian cells, we argued that impaired activation of Smad1/5/8 may be secondary to the corresponding reduction in Smad1/5/8 gene expression. However, there was no change in the expression of Smad transcripts or their protein levels in Pbrm1/PBAF-deficient stromal cells (Figures 3A and 3B; Figures S6B and S6C). To understand the mechanism of Pbrm1-dependent Smad1/5/8 activation, we performed RNA sequencing (RNA-seq) analysis of Pbrm1-deficient and control OP9 cells (Figure 3C; Data S2). Interestingly, differential gene expression analysis identified significant downregulation of BmprII, BmprIβ, and TgfβrII transcripts in Pbrm1 loss of function cells under basal conditions (Figure 3C; Figure S6D). Expression of the Bmpr/Tgfβr genes was also attenuated in Arid2- or Brd7-deficient stromal cells (Figure S6E). Unlike control MSCs, Pbrm1-deficient stromal cells did not show an induction of BmprII, BmprIβ, and TgfβrII expression (Figures 3D and 3E; Figure S6F). In addition, a similar impairment in the induction of BMPR pathway genes was also observed in PBRM1/PBAF-deficient HS-5 and human primary MSCs (Figure S7A). We then investigated whether reintroduction of Pbrm1 function in PBAF-deficient MSCs would restore Bmpr/Tgfβr gene expression. To this end, we used doxycycline-inducible lentiviral constructs overexpressing human BAF180 (PBRM1), which was resistant to shRNA sequences designed against murine Pbrm1 (Figure 4A) (Gao et al., 2017Gao W. Li W. Xiao T. Liu X.S. Kaelin Jr., W.G. Inactivation of the PBRM1 tumor suppressor gene amplifies the HIF-response in VHL−/− clear cell renal carcinoma.Proc. Natl. Acad. Sci. USA. 2017; 114: 1027-1032Crossref PubMed Scopus (73) Google Scholar). Interestingly, gain of function of full-length BAF180 (BAF180WT) caused an induction of BmprIβ and BmprII gene expression in Pbrm1-deficient cells (Figures 4B–4E). Transcriptional activation was accompanied with restoration of BmprIβ/BmprII protein expression (Figures 4B–4E). The presence of six consecutive bromodomains in BAF180 confers its transcriptional activation property by recognizing and interacting with acetylated histones. We, therefore, interrogated whether bromodomain-deleted BAF180 mutants could affect Bmpr gene expression. Indeed, doxycycline-inducible overexpression of bromodomain-deleted loss of function BAF180 mutants (BAF180Δ2BD and BAF180ΔBD) impaired the induction of BmprIβ/BmprII (Figures 4B–4E). The absence of all six bromodomains of BAF180 had a profound inhibitory effect in Bmpr transcriptional induction in comparison to the deletion of two bromodomains (Figures 4B–4E). In addition, we had also overexpressed a BAF180Q1298 nonsense mutant, which is commonly associated with renal carcinoma (Gao et al., 2017Gao W. Li W. Xiao T. Liu X.S. Kaelin Jr., W.G. Inactivation of the PBRM1 tumor suppressor gene amplifies the HIF-response in VHL−/− clear cell renal carcinoma.Proc. Natl. Acad. Sci. USA. 2017; 114: 1027-1032Crossref PubMed Scopus (73) Google Scholar), resulting i" @default.
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• W3019155769 date "2020-04-01" @default.
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• W3019155769 title "Pbrm1 Steers Mesenchymal Stromal Cell Osteolineage Differentiation by Integrating PBAF-Dependent Chromatin Remodeling and BMP/TGF-β Signaling" @default.
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• W3019155769 doi "https://doi.org/10.1016/j.celrep.2020.107570" @default.
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