Human fetal globin gene expression is regulated by LYAR.
Bottom Line: We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent.We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells.Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene.
Affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China.Show MeSH
Related in: MedlinePlus
Mentions: In previous studies, we have shown that PRMT5 interacts with DNMT3A, thereby coupling DNA methylation and histone arginine methylation on the human γ-promoter to silence γ-globin gene expression (27). To identify other potential proteins interacting with PRMT5, we performed immunoprecipitation experiments using anti-Flag antibody conjugated to sepharose beads to precipitate PRMT5 and associated proteins from cellular extracts of K562 cells stably overexpressing Flag-tagged PRMT5. Proteins were eluted with 3× Flag peptide and subjected to mass spectrometry analysis. We found that, in addition to previously identified PRMT5-interaction partner proteins, such as MEP50 and Nucleolin (33,34), peptides corresponding to a new protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) were also present (Figure 1A) (29). To confirm the interaction between LYAR and PRMT5, endogenous proteins from K562 cells were analyzed by immunoprecipitation and western blot. We found that PRMT5 was co-immunoprecipitated with LYAR protein from K562 cellular extract by anti-LYAR antibody (Figure 1B). Furthermore, GST pull-down assays demonstrated that these proteins interacted directly (Figure 1C), and their interaction was mediated by amino acids 154–239 of LYAR containing a stretch of mostly charged residues (Figure 1D and boxed sequence in Supplementary Figure S3). Immunofluorescence staining demonstrated that LYAR co-localized with PRMT5 in the nucleus (Figure 1E). As PRMT5 was previously demonstrated to bind the γ-globin promoter, we determined if LYAR also bound the γ-globin promoter. ChIP-reChIP experiments showed that LYAR and PRMT5 together bound the γ globin promoter, but did not bind the MyoD promoter (Figure 1F). Furthermore, enriched binding of LYAR occurred around the proximal promoter and the first exon region of the γ-globin gene (Supplementary Figure S4).
Affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China.