Fig. 5: Multi-omics analysis reveals MSX2 as the downstream target of ZNF124.
A Schematic summary for analysis of ZNF124 target genes. B, C Distribution of ZNF124-binding peaks showed strong enrichment within 1 kb upstream of the transcription start sites (TSS). D The distribution of ZNF124-binding sites within different gene regions. E The consensus sequences of ZNF124-binding sites detected by HOMER motif analysis with CUT&Tag data. The triangle arrows indicate the shared motif between de novo and known groups. F Volcano plots displaying the differentially expressed genes (DEGs) between Gm20541f/f and Gm20541SKO retinas and their statistical significance. Red represents up-regulated genes, blue represents down-regulated genes. G Venn diagram of DEGs identified by RNA-seq analysis along with ZNF124 binding genes by CUT&Tag and CHIP-exo data. Three genes were selected according to the overlaps. The RNA-seq and CUT&Tag data was acquired from present study, while the CHIP-exo data were obtained from GEO datasets (GSE78099), which was conducted in published paper. H RNA-seq results show that the filtered 3 genes are downregulated in Gm20541SKO retinas. I Integrative Genomics Viewer (IGV) tracks displaying the distribution of ZNF124-binding peaks among the filtered genes according to CUT&Tag data. J The lower diagram indicates the location of the binding site of ZNF124 in the MSX2 gene. The upper panel shows the ChIP-qPCR data of ZNF124 binding to the MSX2 promoter using primers as indicated. The HEK293T cells were transfected with the empty vector (used as negative control) or pcDNA3.1-ZNF124-Flag plasmid. Results are presented as the percentage of ChIP/Input (n = 4 technical replicates). K The upper panel shows the typical consensus sequences of ZNF124-binding sites. The lower panel illustrates the sequences of ZNF124-binding sites aligned to the promoter region of the MSX2 gene. The ZNF124-binding sites for ZNF124 are indicated in red letters. The green line refers to the promoter region of MSX2 gene. L Schematic diagram of the construction of the pGL luciferase reporter plasmid. The pRL-TK-hRluc plasmid was used as the baseline control. “WT” indicates the aligned ZNF124-binding sites in the MSX2 promoter region, and “Mut” refers to the mutant version of the sequences. M The luciferase assay shows that ZNF124 overexpression causes an increase in fluorescence in the wild-type MSX2 promoter group but not in the mutant group. Data were normalized with Renilla luciferase values used as an internal control. N RT-qPCR verified the RNA-seq results for the indicated genes (n = 5 technical replicates). O Western blot analysis of the expression of MSX2 in Gm20541f/f and Gm20541SKO retinas. GAPDH was used as a loading control. O Quantitative analysis of the expression levels of each subunit, with the statistical comparison between groups (n = 4 technical replicates). ***P < 0.001. The data are represented as means ± SEM.
