Fig. 6. CircTmem241 recruits Ash1l onto Elk3 promoter to initiate its transcription.
a Immunoprecipitation assay was performed using BM cells from WT mice with anti-Nono antibody or IgG. Eluted fractions were resolved by SDS-PAGE, followed by silver staining and mass spectrometry. b BM cell lysates were incubated with anti-Nono antibody or IgG, followed by western blotting. c RNA-pulldown assay using biotin-labeled circTmem241 transcripts with lysates from Nono+/+ and Nono−/− bone marrow cells. d Interaction between circTmem241 and Ash1l was measured by in vitro binding assay. e DNA FISH showed that Elk3 promoter co-localized with Nono and circTmem241. Scale bar, 2 μm. f BM cells from circTmem241+/+ and circTmem241−/− mice were lysed and treated with 1% formaldehyde for cross-linking. Anti-Nono antibody was incubated with treated lysates for ChIP assays, followed by size fractionation with sucrose gradient ultracentrifugation. Eluate gradients were examined by Western blotting and PCR assay. g Enrichment of Ash1l on Elk3 gene promoter was analyzed by ChIP assay with anti-Ash1l antibody. n = 3 for each group. h, i Enrichment of indicated histone modifications on Elk3 promoter was analyzed by ChIP assay. n = 3 for each group. j Enrichment of H3K4me3 on the Elk3 promoter in indicated ILC progenitor cells (Lin−CD127+c-KitintSca-1intα4β7+) was examined. n = 3 for each group. k WT, Nono-deficient, or Nono- and circTmem241-deficient ILCPs were subjected to nuclear run-on assay, followed by RT-PCR analysis. n = 3 for each group. l, m Relative mRNA l and protein m levels of Elk3 in indicated ILCPs were analyzed. n = 3 for each group in l. ***P < 0.001. Data were analyzed by an unpaired two-side Student’s t test and shown as means ± SD. Data are representative of at least three independent experiments. Source data are provided as a Source Data file.