Epigenetic control of type 2 and 3 deiodinases in myogenesis: role of Lysine-specific Demethylase enzyme and FoxO3

Abstract

The proliferation and differentiation of muscle precursor cells require myogenic regulatory factors and chromatin modifiers whose concerted action dynamically regulates access to DNA and allows reprogramming of cells towards terminal differentiation. Type 2 deiodinase (D2), the thyroid hormone (TH)-activating enzyme, is sharply upregulated during myoblast differentiation, whereas type 3 deiodinase (D3), the TH-inactivating enzyme, is downregulated. The molecular determinants controlling synchronized D2 and D3 expression in muscle differentiation are completely unknown. Here, we report that the histone H3 demethylating enzyme (LSD-1) is essential for transcriptional induction of D2 and repression of D3. LSD-1 relieves the repressive marks (H3-K9me2-3) on the Dio2 promoter and the activation marks (H3-K4me2-3) on the Dio3 promoter. LSD-1 silencing impairs the D2 surge in skeletal muscle differentiation while inducing D3 expression thereby leading to a global decrease in intracellular TH production. Furthermore, endogenous LSD-1 interacts with FoxO3a, and abrogation of FoxO3-DNA binding compromises the ability of LSD-1 to induce D2. Our data reveal a novel epigenetic control of reciprocal deiodinases expression and provide a molecular mechanism by which LSD-1, through the opposite regulation of D2 and D3 expression, acts as a molecular switch that dynamically finely tunes the cellular needs of active TH during myogenesis.

Figure 1.

Deacetylase inhibitors upregulate D2 expression and thyroid hormone concentration. (A) Proliferating C2C12 cells were treated with different deacetylase inhibitors (see ‘Materials and Methods’ section) for 24 h, and harvested for RT-PCR analysis of D2 mRNA expression. Cyclophillin A was used as internal control. Normalized copies of D2 in untreated cells (CTR) were set as 1. (B) C2C12 cells were transfected with a 1300 bp mouse Dio2 5′-flanking region driving the Luciferase construct (Dio2-Luc) and CMV-Renilla as internal control and treated with different deacetylase inhibitors for 24 h. The results are shown as means ± SD of the LUC/Renilla ratios from at least three separate experiments, performed in duplicate. (C) C2C12 cells were transfected with TRE3TK-Luc construct and CMV-Renilla as internal control and treated with different deacetylase inhibitors for 24 and 48 h. The results are shown as means ± SD of the LUC/Renilla ratios from at least three separate experiments, performed in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

Dio2 promoter is regulated by histone acetylation and methylation at specific lysine residues. (A) Top: Schematic diagram depicts Dio2 promoter with TATA box and binding sites for different transcription factors. Amplified region in the ChIP assayes is underlined. Bottom: The histone H3 acetylation profile of the Dio2 promoter was evaluated by ChIP analysis in proliferating and differentiated muscle cells. mpcs were grown in proliferating or differentiating conditions (see ‘Materials and Methods’ section), and chromatin was immunoprecipitated using the H3-pan-acetylated antibody. The amount of precipitated chromatin was calculated relative to the total input chromatin and expressed as percentage of the total. (B) ChIP assays were carried out to evaluate the in vivo methylation status of histone H3 on the Dio2 promoter. Chromatin extracted from proliferating and differentiated mpcs cells was immunoprecipitated using the indicated antibodies for detection of H3-K4 methylation. The amount of precipitated chromatin was calculated relative to the total input chromatin and expressed as percentage of the total. (C) ChIP analysis of H3-K9 mono-, di- and tri-methylated lysines on Dio2 promoter were performed as indicated above. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

LSD-1 increases the expression of D2 in muscle cells. (A) LSD-1 mRNA levels were analysed by RT-PCR in proliferating and differentiated mpcs cells. (B) Western blot analysis of LSD-1, MyoD and myogenin levels in proliferating and differentiated mpcs. (C) D2 mRNA levels were measured by RT-PCR in the same cells as in (B). (D) C2C12 cells were transiently transfected with LSD-1-expressing plasmid or an siRNA for specific LSD-1 silencing, and D2 mRNA levels were measured by RT-PCR. Effective LSD-1 expression and silencing were evaluated by western blot (Top). (E) D2 mRNA was measured in proliferating and differentiated C2C12 cells treated with 3 mM pargilin for 24 h. (F) TH concentration was evaluated in proliferating C2C12 cells on pargilin treatment for 24 and 48 h by co-transfecting the T3TRETK-Luc promoter and CMV-Renilla as internal control. (G) Effect of LSD-1, LSD-1 m and LSD-1Δ on D2 mRNA levels in transiently transfected C2C12 cells. (H) mpcs were transfected with Flag-tagged LSD-1 plasmid, and chromatin extracted from proliferating and differentiated cells was immunoprecipitated using the anti-Ha antibody. RT-PCR was carried out with oligos amplifying Dio2 promoter region on purified DNA to evaluate the amount of LSD-1 bound fragments. The amount of precipitated chromatin was calculated relative to the total input chromatin and expressed as percentage of the total. The results are shown as means ± SD of the LUC/Renilla ratios from at least three separate experiments, performed in duplicate. *P < 0.05, **P < 0.01.

Figure 4.

LSD-1 inversely regulates D3 expression. (A) D3 mRNA and protein levels in C2C12 cells transiently transfected with LSD-1-expressing plasmid or an siRNA for specific LSD-1 silencing (iLSD-1). (B) mpcs were transfected with Flag-tagged LSD-1 plasmid, and chromatin extracted from proliferating and differentiated cells was immunoprecipitated using the anti-Ha antibody. RT-PCR was carried out with oligos amplifying Dio3 promoter region on purified DNA to evaluate the amount of LSD-1 bound fragments. The amount of precipitated chromatin was calculated relative to the total input chromatin and expressed as percentage of the total. (C) ChIP assays were carried out to evaluate the in vivo methylation status of histone H3 on the Dio3 promoter. Chromatin extracted from proliferating and differentiated mpcs was immunoprecipitated using the indicated antibodies. The amount of precipitated chromatin was calculated relative to the total input chromatin and expressed as percentage of the total. *P < 0.05, **P < 0.01. (D–G) ChIP assays were carried out to evaluate the K4 and K9 methylation status of histone H3 on the Dio2 and Dio3 promoter. Chromatin extracted from proliferating and differentiated mpcs, transiently transfected with control siRNA (iCTR) or specific LSD-1-targeting siRNA (iLSD-1), was immunoprecipitated using the indicated antibodies for detection of H3-K4 or H3-K9 methylation. The amount of precipitated chromatin was calculated relative to the total input chromatin and expressed as percentage of the total. **P < 0.01, ***P < 0.001.

Figure 5.

LSD-1 interacts with FoxO3 on Dio2 promoter region. (A) Co-IP assay of transfected Flag-LSD-1 and Ha-FoxO3 proteins. Extracts from transiently transfected HEK-293 cells were immunoprecipitated with anti-Flag antibody, and anti-Ha antibody for reverse Co-IP. The western blot was decoded using anti-Ha and anti-Flag antibodies, respectively. Inputs were controlled for FoxO3 and LSD-1 expression by performing anti-Ha and anti-Flag immunoblot. (B) Co-IP assay on endogenous FoxO3 and LSD-1 proteins. Two milligrams of total extracts from proliferating and differentiated mpcs were subjected to immunoprecipitation with FoxO3 antibody or a negative antibody as control, and the western blot was decoded using the anti-LSD-1 antibody. (C) Re-ChIP experiments were performed to verify the binding of FoxO3/LSD-1 complex to Dio2 and Dio3 promoters. Chromatin extracted from proliferating and differentiated mpcs was immunoprecipitated using the indicated antibodies. The amount of precipitated chromatin was calculated relative to the total input chromatin and expressed as percentage of the total.

Figure 6.

FoxO3/LSD-1 complex regulates D2 expression. (A) mpcs were transfected with CMV-Flag, Flag-LSD-1 and Ha-FoxO3 or Ha-FoxO3 dn plasmids, and ChIP assay was performed using the anti-Flag antibody to verify effective LSD-1 binding to the Dio2 promoter. (B) D2 mRNA was measured by RT-PCR from transiently transfected C2C12 cells with the indicated expression vectors. (C) Differentiation markers Myogenin and MHC were measured by western blot analysis in mpcs transiently transfected with the indicated plasmids or siRNA constructs. (D) RT-PCR was carried out on the same cells to measure mRNA levels of Myogenin and muscle creatin kinase (CKM) levels. (E) Schematic diagram illustrating the LSD-1-FoxO3-regulated remodelling of chromatin in the Dio2 promoter region in proliferating and differentiated muscle cells.