Fig. 9: Breed-specific 19-bp duplication affects NORFA transcription by altering NFIX recruitment.

a Polymorphisms of the NORFA gene in Erhualian (n = 106), Landrace (n = 94), and Yorkshire (n = 144) sows. Schematic showing the NORFA gene and the location of 5 SNPs and a 19-bp duplication. Genotype and allele frequency were calculated and are represented with columns and pie diagrams, respectively. b The luciferase activities of NORFA promoter containing 19-bp duplication alleles were detected. c, d The copy number (c) and expression levels (d) of NORFA in granulosa cells (different sizes) from A1/A1 and A2/A2 genotype sows (n = 3) were detected by qRT-PCR. e NFIX protein levels in granulosa cells after overexpression or knockdown of NFIX were detected by western blotting. f The expression levels of NORFA in granulosa cells after NFIX was overexpressed or silenced were measured by qRT-PCR. g The effects of NFIX on the luciferase activities of NORFA promoter containing 19-bp duplication alleles. FC indicates fold change. h Analysis of NFIX-binding motif by Jaspar prediction tools (upper) and ChIP-seq data (lower). i ChIP-qPCR assay. The binding enrichment of NFIX on NORFA promoter containing 19-bp duplication alleles were detected by ChIP-qPCR assay. FC means fold change. Beads indicates no antibody, IgG was used as negative control, and the enrichment was normalized to 0.2 input. Data in b–g and i are represented as mean ± S.E.M. with three independent experiments. P values were calculated by a two-tailed Student’s t test. *P < 0.05 and **P < 0.01.