Fig 2: Targeting of HDAC4 and FOXP1 by miR-22 and constitutive acetylation of p53 may contribute to the overexpression of p21 and/or p27.a Diagram of the “seed” sequence for miR-22 targeting of HDAC4 and FOXP1. b Whole cell lysates prepared from S05 and 182R-6 cells were subjected to western blot analysis using antibodies to either HDAC4 or FOXP1. GAPDH served as a loading control. c 182R-6 cells transfected with either 40 nM miR-22 mimic or AllStars negative control siRNA or 50 nM miR-22 inhibitor or negative control were incubated for either 72 or 96 h. Whole cellular lysates were prepared and subjected to western blot analysis using the indicated antibodies. GAPDH served as a loading control. d 182R-6 cells transfected with either 40 nM miR-22 mimic or AllStars negative control siRNA or 50 nM miR-22 inhibitor or negative control were incubated for either 72 or 96 h. Whole cell lysates were prepared, and the immunoprecipitation and western blot analysis were performed as described in the “Methods” section

Fig 3: Expression of mineralization-associated HDAC, HDAC4 and HDAC5, in the dentin–pulp complex increased during human-DPC mineralization and after MMP13-loss. (A) Time-course human-DPC-culture indicates a progressive significant increase in both Hdac4 and 5 expressions during mineralization. (B) IHC-HDAC4 expression decreases in adults compared with developing teeth (a–f). HDAC4 demonstrated high expression at post-natal 3-day WT samples in the odontoblast layer (a, b) compared with unstained control (ai—inset). In adult samples, reduced expression of HDAC4 (c–f) was demonstrated in pulp, odontoblast (e, f), and predentin, with increased expression in mineralized dentin. Higher expression in dentin (d, f—black arrows) and odontoblast (f) was evident in Mmp13 −/− samples (black arrows). High HDAC4 expression in adult alveolar bone (c- black star; ci inset—white star) was evident. ai and di represent unstained controls of a and d, while ci is a 10× section of a different area of the same tooth illustrated in C. Scale bars = (a, ai, c, ci, d, di) 100 μm (original mag. ×10) and (b, e, f) 5 μm (original mag. ×63). (C) HDAC5 expression increased in Mmp13 −/− teeth. HDAC5 expression was low at 3-week in WT and MMP13KO teeth but high in bony and mineralized pulpal deposits evident in Mmp13 −/− samples [(b), small white arrow] and alveolar bone [(b), large white arrow]. Dentin–pulp HDAC5 expression increased at 6-week (c, d) and considerably at 3-month in the pulp, odontoblast, and predentin area of Mmp13 −/− samples (d–f, black arrows). (b–f) represent higher magnifications of the indicated area in (bi–fi), respectively. Scale bars = (a, bi) 50 μm (original mag. ×10), (b, ci, di, ei, fi) 20 µm (original mag. ×20), and (c, d) 10 μm (e, f) 5 μm (original mag. x 63). Abbreviations: Am, ameloblast; Bn, alveolar bone; Dn, dentin; Pp, pulp. Sections were examined from five mice at 3-day, six mice at 10-day postnatal and nine mice at 3 months for both genotypes. Hdac5 expression is significantly increased at 3-month in DPC cultures (D) from MMP13KO molars by RT-qPCR. (E) WB analysis of HDAC4 expression in proliferating mouse DPC cells was similar in MMP13KO and WT but decreased in WT differentiating DPC samples, while HDAC5 increased in differentiating samples and all KO samples supporting data in (Ca–f). HDAC6 expression level was similar in WT and MMP13KO but reduced in differentiating DPCs. All qRT-PCR and WB data shown are based on at least three independent biological samples carried out in triplicate. qRT-PCR data are shown as mean ± s.d., *p < 0.05 vs. control.

Fig 4: HDAC4 knockdown decreases TET2 expression. (A) Nomo-1 cells were transduced with siRNAs targeting indicated HDAC genes, and TET2 expression was determined by RT-qPCR. (B, C) HDAC4 knockdown using lentivirus to deliver shRNA to MDS-L and Nomo-1 cells. TET2 expression was determined by RT-qPCR (B) or Western blot (C). (D) Total BM and c-kit+ cells were harvested from HDAC4 WT or KO mice, and TET2 protein levels were determined by Western blot. (E) MDS-L and Nomo-1 cells were treated with 5 μM LMK-235 or 1 μM MS-275 for 24 hours, and TET2 expression was determined by RT-qPCR. (F) MDS-L cells treated with LMK-235 or MS-275 were analyzed by Western blotting for TET2 expression. H3K27Ac served as a positive control.

Fig 5: ZEB1 derepresses Itga1 transcription by regulating HDAC4 nuclear translocation.(A) Quantitative reverse transcription PCR analysis of Itga1 in 393P cells treated with 5-azacytidine-2′-deoxycytidine (5-Aza) or trichostatin A (TSA) alone or in combination. ***P < 0.001. (B and C) Quantitative reverse transcription PCR analysis of Itga1 in KP cells treated with or without TSA. E, epithelial cells; M, mesenchymal cells. **P < 0.01; ***P < 0.001. (D and E) Quantitative reverse transcription PCR analysis of Itga1 in 393P cells treated with indicated histone deacetylase (HDAC) inhibitors (D) or transfected with indicated siRNAs (E). **P < 0.002; ***P < 0.001. (F) Western blot analysis of HDAC4 (HD4), HDAC2 (HD2), CREB, and α-tubulin in indicated cell fractions. W, whole-cell lysate; C, cytoplasmic fraction; N, nuclear fraction. (G) Western blot analysis of phospho-HDAC4 (Ser246) (pHD4) and total HD4. (H) Confocal micrographs of Flag-tagged wild-type HD4 or mutant HD4 (S246/467/632A) (HD4SA) in 344SQ cells. Scale bar: 20 μm. (I) Western blot analysis of Itga1 and ectopic HD4 (FLAG) in 344SQ transfectants. (J) Luciferase reporter assay in 393P cells transfected with Itga1 promoter reporter (PItga1) and HD4SA expression vector or control vector (Vec). (K and L) PP2A activity in 393P_Vec and 393P_ZEB1 cells (K) and KP cell panel (L). (M) Quantitative reverse transcription PCR analysis of Itga1 in 393P cells treated with PP2A inhibitor LB-100 for 1 day. (N) Relative expression levels of PP2A subunits in 393P_Vec and 393P_ZEB1 cells quantified by RNA sequencing. *P < 0.05; **P < 0.01; ***P < 0.001. (O) Schema showing that ZEB1 depresses Itga1 expression by modulating HDAC4 nuclear accumulation through suppression of PP2A activity. Data are shown as the mean ± SEM from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. Two-tailed Student’s t test for 2-group comparisons; 1-way ANOVA test for multiple comparisons.