Extended Data Fig. 4: NFIL3 binds to the –165 kb Zeb2 enhancer.

a, Representative flow plots showing DC potential of Hoxb8 cell line. The Hoxb8 cells were washed twice with cold PBS to remove residual β-estradiol and cytokines before standard Flt3L culture for 7 days. b, c, Frequency of cDC1 and representative flow plots showing cDCs differentiated from Hoxb8 cells retrovirally expressing NFIL3. Data are pooled from two independent experiments (n = 6 for each cell line). d, Nfil3 transcripts, measured by RT-qPCR, in GFP-NFIL3^– CDP, GFP-NFIL3⁺ CDP, and Hoxb8 cell lines retrovirally expressing NFIL3 used for CUT&RUN and ChIP-seq experiments. Data are pooled from three independent experiments for GFP-NFIL3^– CDP, GFP-NFIL3⁺ CDP, and two independent experiments for Hoxb8 cell lines. e, Rank of the NFIL3 CUT&RUN and ChIP-seq peaks at the –165 kb Zeb2 enhancer. f, Alignment of human, genome draft hg19, and mouse, genome draft mm10, for the –165 kb Zeb2 enhancer regions. Red boxes indicate NFIL3 binding sites. g, EMSA showing NFIL3 binding at –165 kb Zeb2 enhancer. A 37 bp ³²P-labelled DNA probe encompassing the NFIL3 binding site 1 from the –165 kb Zeb2 enhancer was synthesized as site 1 probe. A 40 bp ³²P-labelled DNA probe containing an optimized NFIL3 motif and flanking sequence of the NFIL3 binding site 3 from the –165 kb Zeb2 enhancer was synthesized as N > C probe. NFIL3 binding specificity was demonstrated by competition with non-radioactively labeled probes and supershift with anti-NFIL3 antibody. The competitor 1 indicates site 1, and the competitor 1mut has the same sequence as site 1 except that the NFIL3 binding site 1 was mutated. Data shown are one of two similar experiments. For gel source data, see Supplementary Figure 1. Mean ± s.d.

Source Data