Figure 1

Biochemical analysis of the ET domain of BRD4 binding to JMJD6. (A) 2D ¹⁵N-HSQC spectra of the ET domain in the free form (black) and in complex with the JMJD6 peptide (red). The protein concentration was 0.05 mM and the molar ratio of the protein to peptide was 1:10. (B) ITC measurement of the ET domain titrated by the JMJD6 peptide. (C) Thermal stability of the full-length JMJD6 determined by differential scanning fluorimetry. The averaged fluorescent intensities were normalized using T _m and a obtained by fitting thermal shift data to a theoretical equation (see Experimental procedures). The profiles in black, green, red and blue were obtained from free JMJD6, JMJD6/ET, JMJD6/ssRNA and JMJD6/ET/ssRNA, respectively. The standard deviations of the profiles were calculated from three independent experiments. In control experiments, samples of only ET, ssRNA, dsRNA, ssDNA or dsDNA without JMJD6 had no fluorescent intensity. (D) Thermal stability chart of JMJD6 with/without ET, ssRNA, dsRNA, ssDNA and dsDNA. T _m is presented as mean ± SEM (n = 3). P-values calculated by the t-test indicate a statistically significant difference. (E) HEK293T cells were transiently transfected with Flag-BRD4WT (Flag-BRD4ΔET or Flag-BRD4 ET mutant) and His-JMJD6 (1–403), and cell lysates were harvested 48 h post-transfection. Proteins were separated by SDS-PAGE and immunoblotted with antibodies specific to Flag, NSD3, JMJD6 and H4ac. (F) Harvested cell lysates of double-transfected Flag-BRD4WT and His-JMJD6 were added different amounts of ssRNA and followed through the same procedures for Western blotting analysis. At 1 μM ssRNA, the binding of JMJD6 to BRD4 appears much stronger. (G) Harvested cell lysates were added 1 μM ssRNA, dsDNA or ssDNA and followed through the same procedures for Western blotting analysis.