Fig 2: NCoR1 forms a complex with MEF2 and HDAC4 to repress the expression of hypertrophy-related genes in vivo AChIP analysis showing enrichment of NCoR1 in MEF2-binding regions on the promoters of Acta1 and Nppa in ventricular samples.BChIP analysis of enrichment of MEF2a on promoters of Acta1 and Nppa in ventricular samples. n = 3:3.C–FChIP analysis of enrichment of HDAC4 (C), HDAC5 (D), acetylated-Histone 4 (ac-H4) (E), and ac-H3 (F) on promoters of Acta1 and Nppa in ventricular samples. n = 4:4 for (C), n = 3:3 for (D, E, F).GChIP analysis of RNA polymerase II enrichment on the transcriptional regions of Acta1 and Nppa in ventricular samples. n = 3:3.HWorking model of NCoR1 function in cardiomyocytes. TSS indicates transcription start site.Data information: Data are presented as mean ± SEM. Student's t-test was used for statistical analysis.

Fig 3: EGCG reverses abnormal expression of ANP and BNP by inhibiting overexpression of HDAC5 and reversing hypoacetylation of H3K27ac in the heart of mice 12 W after TAC. (A) Expression of HDAC5 following treatment with different concentrations of EGCG. (B) Acetylation levels of H3K27ac. (C) Expression of HDAC5. (D) Reverse transcription-quantitative PCR analysis of mRNA levels of MEF2A. (E) Expression of MEF2A. (F) Co-IP using anti-HDAC5-protein G magnetic beads and immunoblotting with anti-H3K27ac, anti-MEF2A or anti-HDAC5 antibody. (G) Chromatin IP-PCR assay was used to identify the regulatory association between MEF2A, ANP and BNP. Input, positive control; IgG, negative control. Western blotting showing expression of (H) ANP and (I) BNP. *P<0.05 vs. Sham; #P<0.05 vs. TAC. n=6/group. EGCG, epigallocatechin gallate; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; TAC, transverse aortic constriction; HDAC5, histone deacetylase 5; H3K27ac, histone acetylated lysine 27 on histone H3; MEF2A, myocyte enhancer factor 2A; IP, immunoprecipitation; IB, immunoblot.

Fig 4: Six4 targets UTX to muscle-specific genes. (A) Proteins were immunoprecipitated from C2C12 nuclear extracts (24 h differentiation) using antibodies directed against UTX, Myog, Six4, or control IgG. Immunoprecipitated proteins, and 1/50th of input, were examined by western blot using the antibodies indicated. The left panel corresponds to an immunoprecipitation experiment, where the input and elution samples were processed together. The blot was cropped to remove lanes corresponding to flow-through fractions, and thus the input is separated from the elution fractions by a white bar but was run on the same gel and has the same exposure. Uncropped versions of the UTX and Six 4 analysis are shown in Supplementary Figure 6A. (B) Proteins were immunoprecipitated from nuclear extracts prepared from erythroleukemia cell line K562 using antibodies directed against Six4, Mef2, or control IgG. Immunoprecipitated proteins, and 1/10th of input, were examined by western blot using the antibodies indicated. The asterisk (*) marks a non-specific band observed from the UTX antibody in K562 cells. Mef2/IgG indicates that the two Mef2 bands observed in K562 cells co-migrated with IgG. To provide evidence that the stronger band observed in the Mef2 IP lane represents immunoprecipitated Mef2, we provide a shorter exposure of the blot in Supplementary Figure 6B where the slower migrating Mef2 band is distinguishable from the IgG. Note that all elution bands in this experiment migrate slightly higher than their counterpart in the input because the samples were eluted in a buffer containing urea. (C) Cross-linked chromatin from C2C12 cells (growing (0 h) or differentiated (24 h)) was subjected to ChIP analysis of Six4 binding. Immunopurified DNA was quantitated by qPCR using hydrolysis probes that recognize Myog (Region 3), CKm (Region 2), or IgH. (D) Western blot analysis of protein extracts isolated from cells infected with lentivirus expressing shRNA targeting Six4 or a scrambled control. Nuclear extracts prepared from cells infected with lentivirus expressing shRNA that target UTX or control (scrambled) were separated by SDS–PAGE and analysed by western blot using the antibodies indicated. Each panel represents two lanes run on the same gel that have been cropped due to space constraints. Uncropped blots are shown in Supplementary Figure 8. (E) RNA isolated from cells infected with lentivirus expressing Six4 targeting (or control) shRNA was reverse transcribed and subjected to qPCR analysis. Expression levels are expressed relative to DDX5. (F) Cross-linked chromatin from growing (-), or 24 h differentiated (+) C2C12 cells was subjected to ChIP analysis using antibodies directed against UTX. Immunopurified DNA was quantitated by qPCR using probes that recognize the promoter (Region 3) of Myog, or the upstream enhancer (Region 2) of CKm. (G) Native chromatin from growing (-), or 24 h differentiated (+) C2C12 cells was subject to ChIP analysis using antibodies directed against H3K27me3. Immunopurified DNA was quantitated by qPCR using probes that recognize the promoter (Region 3) of the Myog gene, or the upstream enhancer (Region 2) of CKm.

Fig 5: CLU interacted with MEF2A to upregulate COL15a1(A) Jaspar database was used to estimate the potential TFs that could bind to the promoter of COL15a1, and ChIP assay was performed to prove this in oeCLU and NC groups. (B) Luciferase reporter assay was performed in shMEF2A cells to detect the luciferase activity. The mRNA expression of COL15a1 was then explored with qRT-PCR in shMEF2A, oeCLU, and oeCLU + shMEF2A groups. (C) The mutated promoter of COL15a1 was constructed. The luciferase activity of MEF2A was then detected in WT and mutant groups with cells transfected by shCLU, shMEF2A, oeCLU + shMEF2A, and NC. (D) Immunoprecipitation assay was performed with total, nucleus, and cytoplasm protein respectively to study the directly interaction between CLU and MEF2A. (E) Co-localized IF staining displayed the distribution of CLU and MEF2A. (F) The analysis of COL15a1 expression in seminoma based on UALCAN database. (G) The mechanism of CLU in inhibiting testicular seminoma metastasis.