Convergent biological pathways underlying the Kallmann syndrome-linked genes Hs6st1 and Fgfr1

Abstract

Kallmann syndrome (KS) is a congenital disorder characterized by idiopathic hypogonadotropic hypogonadism and olfactory dysfunction. KS is linked to variants in >34 genes, which are scattered across the human genome and show disparate biological functions. Although the genetic basis of KS is well studied, the mechanisms by which disruptions of these diverse genes cause the same outcome of KS are not fully understood. Here we show that disruptions of KS-linked genes affect the same biological processes, indicating convergent molecular mechanisms underlying KS. We carried out machine learning-based predictions and found that KS-linked mutations in heparan sulfate 6-O-sulfotransferase 1 (HS6ST1) are likely loss-of-function mutations. We next disrupted Hs6st1 and another KS-linked gene, fibroblast growth factor receptor 1 (Fgfr1), in mouse neuronal cells and measured transcriptome changes using RNA sequencing. We found that disruptions of Hs6st1 and Fgfr1 altered genes in the same biological processes, including the upregulation of genes in extracellular pathways and the downregulation of genes in chromatin pathways. Moreover, we performed genomics and bioinformatics analyses and found that Hs6st1 and Fgfr1 regulate gene transcription likely via the transcription factor Sox9/Sox10 and the chromatin regulator Chd7, which are also associated with KS. Together, our results demonstrate how different KS-linked genes work coordinately in a convergent signaling pathway to regulate the same biological processes, thus providing new insights into KS.

Figure 1

KS-linked mutations in HS6ST1 affect the protein structure. (A) Diagram of the human HS6ST1 protein and the six KS-linked mutations. (B) Three-dimensional protein structure of human HS6ST1 predicted using the AlphaFold. (C) The hydrophobicity and electrostatic potential of the WT and two mutant HS6ST1 proteins. Yellow arrows indicate the loci of property changes at the protein surface.

Figure 2

Hs6st1 knockdown alters the transcriptome. (A) Diagram of the experiment design. (B) qPCR results showing the relative mRNA levels (2^-ΔΔCt values) of Hs6st1 in the control (Ctrl) and Hs6st1 knockdown (KD) samples. Ctrl, n = 3; KD, n = 3. ^***P-value < 0.001, one-tailed t-test. (C) Principal component (PC) analysis results. (D) Volcano plot showing the fold change and FDR. Red dots represent the 740 upregulated genes, and green dots represent the 696 downregulated genes. (E) qPCR results showing the relative mRNA levels (2^-ΔΔCt values) of six genes in the independent experiments of Hs6st1 knockdown. Ctrl, n = 2; KD, n = 8. ^*P-value < 0.05; ^**P-value < 0.01; ^***P-value < 0.001; one-tailed t-test.

Figure 3

Hs6st1 knockdown promotes genes in the extracellular pathways and inhibits genes in the chromatin pathways. (A) Gene Ontology pathways enriched by the upregulated genes upon Hs6st1 knockdown. (B) Gene Ontology pathways enriched by the downregulated genes upon Hs6st1 knockdown. (C) GSEA results showing the upregulation of the ECM glycoproteins pathway and the downregulation of the covalent chromatin modification pathway.

Figure 4

Fgfr1 knockdown alters the transcriptome. (A) Diagram of the experiment design. (B) qPCR results showing the relative mRNA levels (2^-ΔΔCt values) of Fgfr1 in control (Ctrl) and Fgfr1 knockdown (KD) samples. Ctrl, n = 3; KD, n = 5. ^***P-value < 0.001, one-tailed t-test. (C) Principal component (PC) analysis results. (D) Volcano plot showing the fold change and FDR. Red dots represent the 2526 upregulated genes, and green dots represent the 2278 downregulated genes. (E) qPCR results showing the relative mRNA levels (2^−ΔΔCt values) of six genes in the independent experiments of Fgfr1 knockdown. Ctrl, n = 2; KD, n = 7. ^*P-value < 0.05; ^**P-value < 0.01; ^***P-value < 0.001; one-tailed t-test. (F) Gene Ontology pathways enriched by the upregulated genes upon Fgfr1 knockdown. (G) Gene Ontology pathways enriched by the downregulated genes upon Fgfr1 knockdown. (H) GSEA results showing the upregulation of the ECM binding pathway and the downregulation of the mRNA splicing pathway.

Figure 5

A group of 1052 genes are regulated by both Hs6st1 and Fgfr1. (A) Comparison of the differentially expressed genes (DEGs) between the Hs6st1 knockdown (KD) and Fgfr1 KD. Note, 1052 DEGs were overlapped. (B) Heatmap showing the expression changes of the 1052 overlapping DEGs in the two KD experiments. (C) Scatter plot showing the fold changes of the 1052 overlapping DEGs in the two KD experiments. (D) Gene Ontology pathways enriched by the 492 upregulated overlapping DEGs. (E) Gene Ontology pathways enriched by the 557 downregulated overlapping DEGs.

Figure 6

Hs6st1 and Fgfr1 regulate gene transcription likely via the transcription factor Sox9. (A) The pipeline of motif analysis. (B) The top five identified motifs. (C) Expression profiles of Sox9 and Sox10 upon Hs6st1 knockdown (siHs6st1) and Fgfr1 knockdown (siFgfr1). ^*P-value < 0.05; ^***P-value < 0.001; ns, P-value > 0.05; one-tailed t-test. (D) Heatmaps of ChIP-seq profiles of Sox9, H3K4me3 and H3K27ac at the promoter regions of the 1052 overlapping DEGs. TSS, transcription start site. (E) Sox9 ChIP-seq profiles at the promoter regions of the 1052 overlapping DEGs and the 1000 control genes (Ctrl). (F) Boxplot of Sox9 ChIP-seq sequencing reads at the promoter regions. ^**P-value = 0.001; one-tailed t-test. (G) Snapshots of ChIP-seq profiles at four DEGs.

Figure 7

A convergent signaling pathway underlying the KS-linked genes. (A) Expression profiles of the chromatin regulator Chd7 upon the two knockdown experiments. ^**P-value < 0.01; one-tailed t-test. (B) Heatmap of Chd7 ChIP-seq profiles at the promoter regions of the 1052 overlapping DEGs. (C) Chd7 ChIP-seq profiles at the promoter regions of the 1052 overlapping DEGs and the 1000 control genes (Ctrl). (D) Proposed model of the convergent pathway underlying the KS-linked genes Hs6st1, Fgf, Fgfr1, Sox10 and Chd7. HS, heparan sulfate. HSPG, heparan sulfate proteoglycan.