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
Mentions: LYAR has been shown to have zinc finger DNA-binding motifs (Supplementary Figure S3) (29). Since LYAR was able to bind the γ-globin promoter as demonstrated by ChIP assays, we asked if LYAR was able to bind DNA directly. To test this possibility, we selected LYAR-binding sequences using the CASTing (cyclic amplification and selection of targets) method (35,36). For this purpose, exogenously expressed HA-tagged LYAR from nuclei of K562 cells (Supplementary Figure S1B) was incubated with a pool of double-stranded oligonucleotides containing 26 nt of random core sequences flanked by primer sequences. DNA–protein complexes were immunoprecipitated with anti-HA antibody; the DNA was subsequently recovered, and subjected to 15–20 cycles of PCR amplification. The PCR products were then used in another round of selection and immunoprecipitation. After four such rounds of selection, the final PCR products were cloned, and 69 different clones were sequenced. The sequences obtained were then analyzed by aligning them as shown in Figure 2A and Supplementary Figure S5. We found that LYAR preferentially bound to a consensus motif containing GGTTAT (Figure 2B). Interestingly, the same nucleotide sequence occurs from position +26 to +32 on the γ-globin proximal promoter relative to the CAP site.
Affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China.