Zeng Q, Wan Y, Zhu P, Zhao M, Jiang F, Chen J, Tang M, Zhu X, Li Y, Zha H, Wang Y, Hu M, Mo X, Zhang Y, Chen Y, Chen Y, Ye X, Bodmer R, Ocorr K, Jiang Z, Zhuang J, Yuan W, Wu X
The Center for Heart Development, State Key Laboratory of Development Biology, Key Laboratory of MOE for Development Biology and Protein Chemistry, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
Cardiovascular Surgery, Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510100, China.
Curr Mol Med. 2018 Mar 9;17(7):509-517. doi: 10.2174/1566524018666180212145714.
The basic helix-loop-helix (bHLH) protein families are a large class of transcription factors, which are associated with cell proliferation, tissue differentiation, and other important development processes. We reported that the Nuclear localized protein-1 (Nulp1) might act as a novel bHLH transcriptional factor to mediate cellular functions. However, its role in development in vivo remains unknown.
Nulp1 (dNulp1) mutants are generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal. Expression of Wg target genes are analyzed by qRT-PCR. We use the Top-Flash luciferase reporter assay to response to Wg signaling.
Here we show that Drosophila Nulp1 (dNulp1) mutants, generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal, are partially homozygous lethal and the rare escapers have bent femurs, which are similar to the major manifestation of congenital bent-bone dysplasia in human Stuve- Weidemann syndrome. The fly phenotype can be rescued by dNulp1 over-expression, indicating that dNulp1 is essential for fly femur development and survival. Moreover, dNulp1 overexpression suppresses the notch wing phenotype caused by the overexpression of sgg/GSK3β, an inhibitor of the canonical Wnt cascade. Furthermore, qRT-PCR analyses show that seven target genes positively regulated by Wg signaling pathway are down-regulated in response to dNulp1 knockout, while two negatively regulated Wg targets are up-regulated in dNulp1 mutants. Finally, dNulp1 overexpression significantly activates the Top-Flash Wnt signaling reporter.
We conclude that bHLH protein dNulp1 is essential for femur development and survival in Drosophila by acting as a positive cofactor in Wnt/Wingless signaling.
碱性螺旋-环-螺旋（bHLH）蛋白家族是一大类转录因子，与细胞增殖、组织分化及其他重要的发育过程相关。我们曾报道核定位蛋白-1（Nulp1）可能作为一种新型bHLH转录因子介导细胞功能。然而，其在体内发育中的作用仍不清楚。
通过CRISPR/Cas9靶向其C末端未知功能结构域（DUF654）来产生Nulp1（dNulp1）突变体。采用qRT-PCR分析Wg靶基因的表达。我们使用Top-Flash荧光素酶报告基因检测法来响应Wg信号。
在此我们表明，通过CRISPR/Cas9靶向其C末端未知功能结构域（DUF654）产生的果蝇Nulp1（dNulp1）突变体部分纯合致死，罕见的存活者有股骨弯曲，这与人Stuve-Weidemann综合征中先天性弯骨发育不良的主要表现相似。果蝇的这种表型可通过dNulp1过表达得到挽救，表明dNulp1对果蝇股骨发育和存活至关重要。此外，dNulp1过表达可抑制由经典Wnt级联反应抑制剂sgg/GSK3β过表达引起的缺刻翅表型。此外，qRT-PCR分析表明，Wg信号通路正向调控的7个靶基因在dNulp1敲除后下调，而2个受Wg负调控的靶基因在dNulp1突变体中上调。最后，dNulp1过表达显著激活Top-Flash Wnt信号报告基因。
我们得出结论，bHLH蛋白dNulp1通过作为Wnt/Wingless信号通路中的正向辅助因子，对果蝇股骨发育和存活至关重要。
