Fig. 7: XCP directly modulates the demethylase activity of PHF8.
A Diagram showing the flow of in vitro demethylase reactions using recombinant FLAG-PHF8, His6-XCP and H3K9me2 peptide (left). Dot bot showing the demethylase activity of PHF8 on H3K9me2 peptide in the presence or absence of XCP (right). Total Histone H3 was used as a loading control for dot blot. B RT-qPCR (top) and Western blot (bottom) showing dox-inducible ectopic expression of full length lncRNA1456 in its wild-type form (Wt), which expresses the XCP peptide, or an ATG mutant (Mut) version which abolishes XCP peptide production. For RT-qPCR, RNA levels were quantified by normalizing to housekeeping gene RPL19 mRNA. Each bar represents the mean + SEM; n = 3. For Western blot, β-tubulin was used a loading control. C Bar graphs showing global changes in H3K9 methylation modifications assayed by mass spectrometry of MCF-7 histones in the absence (top) or presence (bottom) of neighboring H3K14ac. Each bar represents the mean ± SEM; n = 3. Significance calculated by 1-sided Welch’s t test. D Western blot analysis of MCF-7 histone preparation used for mass spectrometry analysis (C), corroborating levels of H3K9me2. Total Histone H3 was used as a loading control. E Diagram summarizing model: XCP, encoded by lncRNA1456, interacts with PHF8, a histone demethylase, and directly regulates its activity to modulate gene expression. Diagram generated using BioRender. [See also Fig. S7].
