P.E95K mutation in Indian hedgehog causing brachydactyly type A1 impairs IHH/Gli1 downstream transcriptional regulation

Lu Shen,Likuan Xiong,Xuhan Yang,Shengying Qin,Gang Ma,Xi Wu,Lei Cai,Chunling Wan,Yunfeng Ruan,Xueli Gong,Lin He,Chao Yang,Yuyu Xiong,Ye Shi

doi:10.1186/s12863-018-0697-5

Abstract

BackgroundBrachydactyly type A1 (BDA1, OMIM 112500) is a rare inherited malformation characterized primarily by shortness or absence of middle bones of fingers and toes. It is the first recorded disorder of the autosomal dominant Mendelian trait. Indian hedgehog (IHH) gene is closely associated with BDA1, which was firstly mapped and identified in Chinese families in 2000. Previous studies have demonstrated that BDA1-related mutant IHH proteins affected interactions with its receptors and impaired IHH signaling. However, how the altered signaling pathway affects downstream transcriptional regulation remains unclear.ResultsBased on the mouse C3H10T1/2 cell model for IHH signaling activation, two recombinant human IHH-N proteins, including a wild type protein (WT, amino acid residues 28–202) and a mutant protein (MT, p.E95k), were analyzed. We identified 347, 47 and 4 Gli1 binding sites in the corresponding WT, MT and control group by chromatin immunoprecipitation and the overlapping of these three sets was poor. The putative cis regulated genes in WT group were enriched in sensory perception and G-protein coupled receptor-signaling pathway. On the other hand, putative cis regulated genes were enriched in Runx2-related pathways in MT group. Differentially expressed genes in WT and MT groups indicated that the alteration of mutant IHH signaling involved cell-cell signaling and cellular migration. Cellular assay of migration and proliferation validated that the mutant IHH signaling impaired these two cellular functions.ConclusionsIn this study, we performed integrated genome-wide analyses to characterize differences of IHH/Gli1 downstream regulation between wild type IHH signaling and the E95K mutant signaling. Based on the cell model, our results demonstrated that the E95K mutant signaling altered Gli1-DNA binding pattern, impaired downstream gene expressions, and leaded to weakened cellular proliferation and migration. This study may help to deepen the understanding of pathogenesis of BDA1 and the role of IHH signaling in chondrogenesis.

Highlights

Type A1 (BDA1, OMIM 112500) is a rare inherited malformation characterized primarily by shortness or absence of middle bones of fingers and toes
Activation of Indian hedgehog (IHH) signaling pathway in C3H10T1/2 cells induced by Indian Hedghog N-terminus protein (IhhN) proteins According to previous brachydactyly type A1 (BDA1) cell model [19], after incubation of C3H10T1/2 cells with 750-nM IhhN protein for 48 h, targets of activated IHH signaling, such as Gli1, Ptch1 and Ihh, were examined using quantitative real time PCR in two groups (Fig. 1)
These results suggested that E95K mutant IHH weakened the activation of IHH signaling

Summary

Introduction

Type A1 (BDA1, OMIM 112500) is a rare inherited malformation characterized primarily by shortness or absence of middle bones of fingers and toes. It is the first recorded disorder of the autosomal dominant Mendelian trait. Previous studies have demonstrated that BDA1-related mutant IHH proteins affected interactions with its receptors and impaired IHH signaling. How the altered signaling pathway affects downstream transcriptional regulation remains unclear. Gli-mediated regulation of IHH signaling in vertebrates controls downstream gene expressions during the cartilage development [11]. How the BDA1-related mutant IHH signaling changes Gli-mediated transcriptional regulation remains unclear

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