Deacetylation of FOXP1 by HDAC7 potentiates self-renewal of mesenchymal stem cells

Abstract

Background: Mesenchymal stem cells (MSCs) are widely used in a variety of tissue regeneration and clinical trials due to their multiple differentiation potency. However, it remains challenging to maintain their replicative capability during in vitro passaging while preventing their premature cellular senescence. Forkhead Box P1 (FOXP1), a FOX family transcription factor, has been revealed to regulate MSC cell fate commitment and self-renewal capacity in our previous study.

Methods: Mass spectra analysis was performed to identify acetylation sites in FOXP1 protein. Single and double knockout mice of FOXP1 and HDAC7 were generated and analyzed with bone marrow MSCs properties. Gene engineering in human embryonic stem cell (hESC)-derived MSCs was obtained to evaluate the impact of FOXP1 key modification on MSC self-renewal potency.

Results: FOXP1 is deacetylated and potentiated by histone deacetylase 7 (HDAC7) in MSCs. FOXP1 and HDAC7 cooperatively sustain bone marrow MSC self-renewal potency while attenuating their cellular senescence. A mutation within human FOXP1 at acetylation site (T176G) homologous to murine FOXP1 T172G profoundly augmented MSC expansion capacity during early passages.

Conclusion: These findings reveal a heretofore unanticipated mechanism by which deacetylation of FOXP1 potentiates self-renewal of MSC and protects them from cellular senescence. Acetylation of FOXP1 residue T172 as a critical modification underlying MSC proliferative capacity. We suggest that in vivo gene editing of FOXP1 may provide a novel avenue for manipulating MSC capability during large-scale expansion in clinical trials.

Keywords: Deacetylation; FOXP1; HDAC7; Mesenchymal stem cells; Self-renewal.

Fig. 1

Deacetylation of FOXP1 by HDAC7. a FOXP1 protein was enriched by immunoprecipitation (IP) from mesenchymal cells of human mesenchymal stem cells (MSCs) and was detected for acetylation by western blot with anti-Pan Acetyl-Lysine antibody (Ab). b A pcDNA-FOXP1-His plasmid was expressed in 293 T cells and FOXP1 was harvested via IP with anti-His Ab. Extensive acetylation was identified by mass spectrum analysis. c Mass spectral characterization of murine FOXP1 protein acetylation at amino acid T172. d Co-IP detection of the in vitro interaction of FOXP1 and HDAC7 in 293 T cells following their transfection with the indicated plasmids (representative of 3 independent experiments). e Co-IP detection of the in vivo interaction of FOXP1 and HDAC7 in bone marrow (BM) MSCs (representative of 3 independent experiments). f Immunostaining with anti-His (green) and anti-Flag (red) detects the co-localization of FOXP1 and HDAC7 within perinuclear region of C3H10T1/2 cells transfected with the indicated plasmids. Bar, 25 μm. g Deacetylation of FOXP1 by HDAC7 in 293 T cells. h Diagram depicting the expression subregions of FOXP1 protein. FOXP1-N: FOXP1 protein N-terminal(1-302aa); FOXP1-M: middle part (302-369aa), including zinc finger (ZF) and leucine zipper (LZ) domains; FOXP1-N/M: N-terminal and Middle part (1-369aa); FOXP1-C: C-terminal (369-673aa), containing the Forkhead (Fkh) domain [FOXP1-C(Fkh)]. Blue lines indicated the potential interaction domain between FOXP1 and HDAC7 protein. i–l Co-IP detection of the in vitro interaction of FOXP1-N/M and HDAC7-C in 293 T cells transfected with the indicated plasmids (representative of 3 independent experiments)

Fig. 2

HDAC7 facilitates FOXP1 protein stabilization. a The expression of FOXP1 protein increased as the dosage of HDAC7 increased. Different levels (0, 1 μg and 2 μg) of HDAC7 and FOXP1 (1 μg) expression plasmids were transfected into 293 T cells and then analyzed 24 h later. β-actin levels served as loading control here and in subsequent sub-figures. b The expression of FOXP1 increased following harvesting from 293 T cells post-transfection at the indicated times (12, 24, 36, 48 h) with the indicated plasmids (2 μg). c FOXP1 expression assessed in 293 T cells that were treated for 12 h with either Cycloheximide (CHX, an inhibitor of protein synthesis, 10 μg/ml), or Chloroquine (an inhibitor of lysosomal activity, 50 μM), or Bafilomycin A1 (an inhibitor of autophagy, 100 μM) and with MG132 (a proteasome inhibitor, 10 μM). d Stabilization of FOXP1 by HDAC7 is sensitive to HDAC inhibitor TSA. 293 T cells transfected with the indicated plasmids were treated with 40 nM TSA for 48 h, or 10 μM MG132 for 24 h prior harvesting of FOXP1 via its His tag. FOXP1 acetylation was assessed simultaneously (as described in the legend for Fig. 1). e HDAC7 reduces FOXP1 ubiquitination levels in 293 T cells transfected with the plasmids indicated on the figure. f HDAC7 facilitates FOXP1 accumulation both within the nucleus and the cytoplasm of 293 T cells transfected with the indicated plasmids. g Luciferase reporter assays demonstrate that FOXP1 transcriptional repression of p16 is enhanced by HDAC7. C3H/10T1/2 cells were co-transfected with p16-Luc, FOXP1-His, and/or HDAC7-Flag. Reporter activity is presented as relative luciferase units (RLU). Data shown are representative of 3 independent experiments). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, non-significant

Fig. 3

Knockout of FOXP1/HDAC7 exacerbates bone mass loss. a Representative dorsal view of 3-month-old Foxp1^fl/fl, Foxp1_Prx1^∆/∆, HDAC7^+/−and Foxp1_Prx1^∆/∆;HDAC7^+/− double KO mice. b Western blot detection of FOXP1 and HDAC7 within BM MSCs of mice at 3 months of age. c qPCR validation of mRNA level of FOXP1 and HDAC7 in MSCs from mice at 3 months of age. Data shown are representative of 3 independent assays. d 3D view of cortical and trabecular bone structure by µCT analysis of femur bones of mice at 3 months of age. e–f Quantification of cortical and trabecular bone properties by µCT analysis (n = 6). Abbreviations: BV/TV, bone volume/tissue volume; BMD, bone mineral density; Tb. N., trabecular bone number; Tb. Sp., trabecular bone spacing; Tb. Th., trabecular bone thickness; BS, bone surface; Cb. Th., cortical bone thickness. Two-tailed Student's t tests for comparisons between two groups. Each group consists of 9 mice of each phenotype. g Crystal purple staining of CFU-F colonies of MSCs from 3-month-old mice (n = 3) of the indicated genotypes.*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, non-significant

Fig. 4

FOXP1/HDAC7 deficiency impairs the self-renewal potency of MSCs a Population doubling curve of BM MSCs of Foxp1^fl/fl, Foxp1_Prx1^∆/∆, HDAC7^+/−, and Foxp1_Prx1^∆/∆;HDAC7^+/− 3-month-old transgenic mice. Each group consists of 3 mice of each phenotype. b SA-β-gal staining (left panel) and qPCR analysis (right panel) for cellular senescence markers at passage 5 of MSCs in transgenic mice of each genotypes (n = 3) indicated in panel A. Bar, 200 μm. c Immunofluorescence of LAP2 (red) and DAPI (blue) for passage 5 MSCs from transgenic mice (n = 3) of genotype indicated in (a). Right panel shows the quantification of LAP2-positive cells. Bar, 100 μm. d Immunofluorescence of γH2AX (Green) and DAPI (blue) for passage 5 MSCs from knockout mice (n = 3) of each genotype indicated in (a). Right panel shows the quantification of γH2AX-positive cells. Bar, 100 μm. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, non-significant. e Detection of LAP2 and P16 protein expression in passage-5 MSCs from bone marrows of Foxp1^fl/fl, Foxp1_Prx1^Δ/Δ, HDAC7^+/−, and Foxp1_Prx1^Δ/Δ;HDAC7^+/− mice (n = 3)

Fig. 5

FOXP1/HDAC7 retroviral-mediated overexpression enhances the expansive potency of hMSCs. a Population doubling curves of hMSCs infected with retroviral pMSCV-FOXP1 and/or pMSCV-HDAC7. Human (h)MSCs were infected with pMSCV empty vector as a control. Data shown are representative of 3 independent replications. b qPCR analysis of p16/p21 expression in hMSCs overexpressing FOXP1/HADC7-. n = 3. Data shown are representative of 3 independent replications. c SA-β-gal staining at passage 5 of FOXP1/HDAC7 retroviral overexpressing hMSCs. Right panel, quantification of percentage of SA-β-gal-positive cells. Bar, 200 μm. d Immunofluorescence of LAP2 (red) and DAPI (blue) of passage 5 MSCs from mice (n = 3) as indicated. Right panel, quantification of LAP2-positive cells. Bar, 100 μm. e Immunofluorescence of γH2AX (Green) and DAPI (blue) for passage 5 MSCs from mice (n = 3) as indicated. Right panel, quantification of γH2AX-positive cells. Bar, 100 μm. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, non-significant

Fig. 6

hFOXP1^T176G mutation potentiates the replicative capacity of hMSCs. a A murine FOXP1-T172G substitution mutant transfected into 293 T cells is enriched in global acetylation relative to FOXP1 wild type (WT) control. Proteins were pulled down via their His-tags by anti-His, fractionated on SDS-PAGE and then blotted with either pan anti-Acetyl-Lysine antibody (Ace) or for loading control antibodies specific for HA tag or for β-actin. b The ubiquitination levels of FOXP1-T172G were increased as compared to FOXP1 controls following transfection and fractionation in 293 T cells as described in panel A. c Schematic diagram depicting the strategy employed for hFOXP1^T176G engineering in human Embryonic Stem Cells (hESCs) via transfection with episomal Cas9n/sgRNA and donor vectors. d Validation of hFOXP1^T176G-engineered hESCs by Sanger sequencing. The mutated leucine to glycine codon (GGC) is boxed in red (lower panel) relative to the wild type codon (ACC) shown in the left panel as black. e Crystal purple staining identifies CFU-F-positive colonies of hMSCs at passages P3 and P10. hMSCs shown were directly induced from hFOXP1^T176G-engineered hESCs. Bar, 100 μm. f Population doubling curve of hFOXP1^T176G-engineered hMSCs. Results shown are representative of 3 independent measurements. g Q-PCR analysis of cellular senescence as indicated by levels of cell cycle (p16, p21, p27) and tumor repressor (p53, Rb) transcript levels within hMSCs at P15. h SA-β-gal staining identifies cellular senescence of hFOXP1^T176G-engineered hMSCs at P10 and P15. Data are representative of 3 independent measurements. Bar, 100 μm. i DNA damage as measured in hMSCs at P15 by immunofluorescence of Lamina-associated polypeptide 2 (LAP2) (left panel). Right panel, quantification of γH2AX-positive cells. n = 3. Bar, 100 μm. j Immunofluorescence of γH2AX in hMSCs at P15. Right panel is quantification of γH2AX-positive cells. Results shown are representative of 3 independent measurements. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, non-significant. Bar, 100 μm. k Schematic diagram showing that deacetylation of acetylation marks (blue dots) from FOXP1 by HDAC7 potentiates its effect on promoting self-renewal (indicated by the red arrow) of MSCs