Department of Genetics, Microbiology and Statistics, Faculty of Biology, CIBERER, IBUB, IRSJD, Universitat de Barcelona, 08028 Barcelona, Spain.
Musculoskeletal Research Group, Centro de Investigación Biomédica en Red en Fragilidad y Envejecimiento Saludable (CIBERFES), ISCIII, IMIM (Hospital del Mar Medical Research Institute), 08003 Barcelona, Spain.
Int J Mol Sci. 2021 Jan 6;22(2):489. doi: 10.3390/ijms22020489.
encodes the sclerostin protein, which acts as a key extracellular inhibitor of the canonical Wnt pathway in bone, playing a crucial role in skeletal development and bone homeostasis. The objective of this work was to assess the functionality of two variants previously identified (the rare variant rs570754792 and the missense variant p.Val10Ile) and to investigate the physical interactors of the proximal promoter region in bone cells. Through a promoter luciferase reporter assay we show that the minor allele of rs570754792, a variant located in the extended TATA box motif, displays a significant decrease in promoter activity. Likewise, through western blot studies of extracellular and intracellular sclerostin, we observe a reduced expression of the p.Val10Ile mutant protein. Finally, using a circular chromosome conformation capture assay (4C-seq) in 3 bone cell types (MSC, hFOB, Saos-2), we have detected physical interactions between the proximal promoter and the enhancer, several additional enhancers located between and and a distant region containing exon 18 of . In conclusion, presents functional regulatory and missense variants that affect its expression and displays physical contacts with far reaching genomic sequences, which may play a role in its regulation within bone cells.
编码骨硬化蛋白（sclerostin），该蛋白是骨中经典 Wnt 通路的关键细胞外抑制剂，在骨骼发育和骨稳态中发挥着重要作用。本研究旨在评估先前鉴定的两种变体（罕见变体 rs570754792 和错义变体 p.Val10Ile）的功能，并研究骨细胞中近端启动子区域的物理相互作用体。通过启动子荧光素酶报告基因检测，我们发现位于扩展 TATA 盒基序中的 rs570754792 等位基因的次要等位基因，其启动子活性显著降低。同样，通过对外源和胞内骨硬化蛋白的 Western blot 研究，我们观察到 p.Val10Ile 突变蛋白的表达减少。最后，我们在 3 种骨细胞类型（MSC、hFOB、Saos-2）中使用环状染色体构象捕获分析（4C-seq），检测到近端启动子与增强子之间的物理相互作用，以及位于 和 之间的几个额外增强子，以及包含外显子 18 的远端区域。总之， 表现出功能性调节和错义变体，影响其表达，并与远距离基因组序列发生物理接触，这可能在骨细胞中发挥其调控作用。
