Fig 1: JMJD1A mediates iron-dependent demethylation of H3K9me2 in the Pparg region. (A) Pparg mRNA levels on Day 2 in a series of 3T3-L1 cell lines stably expressing either sh-Jmjd1a, sh-Jmjd2b, sh-Jhdm1d, sh-Jmjd2c, sh-Jmjd2d, sh-Utx, sh-Phf2, sh-Jmjd2a, or sh-Jmjd3 were determined by qPCR (n = 3 biological replicates). The mRNA levels normalized to the Cyclophilin B level in the cell lines stably expressing an shRNA for each target enzyme (shRNA #1 or shRNA #2) is shown as a ratio to the level of the corresponding control cell line (sh-Ctrl). Data are shown as the mean ± s.e.m. The two-tailed Student's t-test was performed for the cell lines expressing either sh-Jmjd2c, sh-Jmjd2d, or sh-Phf2. One-way ANOVA followed by the Dunnett test was performed for the other cell lines. *P < 0.05. (B) Each knockdown line was retrovirally transduced with the corresponding enzyme with or without the indicated mutation in the iron-binding site. Pparg mRNA levels on Day 2 were measured by qPCR (n = 3 biological replicates). The full-length mouse sequence of the corresponding enzyme gene was used for overexpression, except for the partial sequence encoding amino acids (a.a.) 71 to 940 of JHDM1D and the human version for UTX. Data are shown as the mean ± s.e.m. One-way ANOVA followed by the Tukey-Kramer test was performed for statistical analysis. *P < 0.05. (C) ChIP-qPCR analysis of H3K9me2 on Pparg and Actb genes in JMJD1A-KD cells (sh-Jmjd1a #1) and its control cell line (sh-Ctrl) on Day 2 (mean ± s.e.m. of three biological replicates). ChIP signals were presented as a percentage of input DNA. The two-tailed Student's t-test was performed for statistical analysis. *P < 0.05, ***P < 0.001, ****P < 0.0001, N.S., not significant. (D) ORO staining was performed on Day 8 in JMJD1A-KD cells (sh-Jmjd1a #1), or the JMJD1A-KD cells (sh-Jmjd1a #1) that stably express mouse JMJD1A harboring an shRNA-resistant mutation with [JMJD1A (H1122A-shR)] or without [JMJD1A (WT-shR)] additional mutations in the iron-binding site. (E) JMJD1A-KD cells (sh-Jmjd1a #1) overexpressing either lacZ [Ctrl (lacZ)], JMJD1A (WT-shR), or JMJD1A (H1122A-shR) were induced to differentiate, and mRNA levels of Jmjd1a and Pparg on Day 2 were measured by qPCR (n = 3 biological replicates). Data are shown as the mean ± s.e.m. One-way ANOVA followed by the Tukey-Kramer test was performed for statistical analysis. *P < 0.05. (F) ChIP-qPCR analysis of H3K9me2 on Pparg and Actb genes. JMJD1A-KD cells (sh-Jmjd1a #1) overexpressing either lacZ [Ctrl (lacZ)], JMJD1A (WT-shR), or JMJD1A (H1122A-shR) were differentiated with 100 µM DFO [DFO(+)] (bottom) or vehicle [DFO(-)] (top). ChIP-qPCR was performed on Day 2 (mean ± s.e.m. of three biological replicates). One-way ANOVA followed by the Tukey-Kramer test was performed for statistical analysis. *P < 0.05; N.S.: not significant. (G) JMJD1A-KD cells (sh-Jmjd1a #1) overexpressing either lacZ [Ctrl (lacZ)], JMJD1A (WT-shR), or JMJD1A (H1122A-shR) were induced to differentiate, and protein levels of PPAR? and ß-actin on Day 2 of differentiation were determined by immunoblot analysis using whole cell extracts. Uncropped images are shown in Supplementary Figure S10. (H) Adipocyte differentiation of 3T3-L1 cells was induced with or without treatment of 100 µM DFO for the first 2 days, and mRNA levels of Jmjd1a during adipocyte differentiation were analyzed by RNA-seq, as described in Figure 2D. Data are presented in TPM and circles indicate two biological replicates.
Fig 2: Crucial role of iron in epigenetic rewriting during adipocyte differentiation mediated by JMJD1A and TET2 activity.
Fig 3: Iron regulates the H3K9me2 demethylase activity of JMJD1A. (A) Histone demethylase activity of JMJD1A was determined by the HTRF demethylation assay using the recombinant JMJD1A protein and its substrate (biotinylated-H3K9me1 or biotinylated-H3K9me2). The vertical axis represents DF%: DF% = ([665 nm/620 nm of Enzyme (+) condition] / [665 nm/620 nm of Enzyme (-) condition] -1) ×100). Data are shown as the mean ± SD of 3 technical replicates. (B) Cells transfected with pCAG-HA-Jmjd1a were cultured in the presence or absence of 100 µM DFO for 2 days, and immunostained with anti-H3K9me2 and anti-HA antibodies. Counterstaining was performed using Hoechst. Dashed circles indicate HA-JMJD1A-positive cells. Scale bar, 50 µm. (C) JMJD1A activity was determined by the in vitro demethylation assay in the presence or absence of DFO. Flag-Twin-Strep-tagged-WT-JMJD1A or Flag-Twin-Strep-tagged-H1122A-JMJD1A were affinity purified from 3T3-L1 preadipocytes overexpressing the respective proteins. Acquired purified WT-JMJD1A or H1122A-JMJD1A was reacted with the synthesized H3K9me2 peptide in a buffer (50 mM HEPES–KOH, pH 7.5, 1 mM a-KG, 2 mM ascorbic acid, and 70 µM ferrous ammonium sulfate) with or without 100 µM DFO (top). Flag-Twin-Strep-tagged-WT-JMJD1A WT or Flag-Twin-Strep-tagged-H1122A-JMJD1A was affinity purified from 3T3-L1 cells induced to differentiate with or without 100 µM DFO for 2 days. The resultant purified WT-JMJD1A or H1122A-JMJD1A was reacted with the synthesized H3K9me2 peptide in an iron-free buffer (50 mM HEPES–KOH, pH 7.5, 1 mM a-KG, and 2 mM ascorbic acid) (bottom). Protein levels were determined by performing immunoblot analysis using anti-H3K9me2 and anti-FLAG. Uncropped images are shown in Supplementary Figure S10. (D) Eight-week-old male mice were intraperitoneally injected with DFO (100 mg/kg BW/day) for 2 weeks under a high-fat, high-cholesterol diet, and H3K9me2 levels in epididymal WAT were determined by ChIP-qPCR (mean ± s.e.m. of three biological replicates). ChIP signals were presented as a percentage of input DNA. The two-tailed Student t-test was performed for statistical analysis. *P < 0.05, **P < 0.01
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