HAND1 and FOXF1 are mutually regulated and required for the expression of the MJH specific genes.
(A) Comparison of the genes affected by Hand1 KO or Foxf1 KO with cluster specific genes of E7.0 mesodermal cells. Dot plot showing that absolute (dot size) and relative (dot color) ratio of the cluster specific genes in WT, Hand1 KO or Foxf1 KO MES cells. >90% cluster specific genes were expressed in MES. Higher enrichment in C0-2 and lower enrichment in other clusters can be observed in Hand1 KO or Foxf1 KO MES cells. Bar plots showing the up- or down-regulated genes in Hand1 KO or Foxf1 KO MES cells. Lengths of bars representing the percentage of cluster specific genes which were up- or down-regulated. Dark colors indicating direct targets of Hand1 (left) and Foxf1 (right).
(B) Heatmap showing the transcriptomic similarity of MES cells and cell types of E7.0 mouse embryo. MES transcriptome data were generated using bulk RNA-seq. Cell-type specific transcriptomes of E7.0 mice were determined as the average of single-cell transcriptomes from each cell type. The gene set for comparison was defined as the collection of top 50 marker genes of each E7.0 cell type. Cosine similarity metric was used. Ant.PS, anterior primitive streak. Haem, haematoendothelial progenitors. Def.end, def.endoderm. Vis.end, visceral endoderm. Par.end, parietal endoderm. ExE.end, ExE endoderm. Surf.ect, surface ectoderm. ExE.ect, ExE ectoderm.
(C) Scatter plot showing gene expression FCs after Hand1 KO or Foxf1 KO. Dots representing genes with FC > 1.5 and adjusted P (Padj)-value < 1e-5 (Wald tests). Red and blue colors indicating genes co-activated/inhibited in Hand1 KO and Foxf1 KO cells. Correlation coefficient of FCs between Hand1 KO and Foxf1 KO is 0.74, P < 2.2e-16, t-test.
(D) Representative genome browser snapshots showing the localization of H3K27ac, H3K4me1, HAND1 and FOXF1 at the Hand1 and Foxf1 loci. The FOXF1-binding Hand1 enhancer (Hand1-eF) and the HAND1-binding Foxf1 enhancer (Foxf1-eH), for enhancer KO experiments, are highlighted.
(E-F) RT-qPCR showing that the reduction in RNA levels of the EEM marker genes after Hand1-eF KO can be rescued by HAND1 OE (e), and after Foxf1-eH KO can be rescued by FOXF1 OE.
(G) Cartoon illustrating target Foxf1-eH of by dCas9-VP64 and the genes in the vicinity of Foxf1-eH.
(H) RT-qPCR showing that the substantial increase in RNA levels of Foxf1 and the FOXF1 target genes, but not the Foxf1-eH neighbouring genes in Foxf1-eH CRISPRa cells. Data are the mean ± standard error of the mean from three independent experiments. Two-tailed unpaired Student’s t-test was performed.