School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia.
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia.
Biochim Biophys Acta Mol Cell Biol Lipids. 2017 Jul;1862(7):647-657. doi: 10.1016/j.bbalip.2017.03.009. Epub 2017 Mar 23.
The two control points of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and squalene monooxygenase (SQLE) are known targets of the transcription factor sterol-regulatory element binding protein-2 (SREBP-2). Yet the location of the sterol-regulatory elements (SREs) and cofactor binding sites, nuclear factor-Y (NF-Y) and specificity protein 1 (Sp1), have not been satisfactorily mapped in the human SQLE promoter, or at all in the human HMGCR promoter.
We used luciferase reporter assays to screen the sterol-responsiveness of a library of predicted SRE, Sp1 and NF-Y site mutants and hence identify bone fide binding sites. We confirmed SREs via an electrophoretic mobility shift assay (EMSA) and ChIP-PCR.
We identified two SREs in close proximity in both the human HMGCR and SQLE promoters, as well as one NF-Y site in HMGCR and two in SQLE. In addition, we found that HMGCR expression is highly activated only when SREBP-2 levels are very high, in contrast to the low density lipoprotein receptor (LDLR), a result reflected in mouse models used in other studies.
Both HMGCR and SQLE promoters have two SREs that may act as a homing region to attract a single SREBP-2 homodimer, with HMGCR being activated only when there is absolute need for cholesterol synthesis. This ensures preferential uptake of exogenous cholesterol via LDLR, thereby conserving energy.
We provide the first comprehensive investigation of SREs and NF-Ys in the human HMGCR and SQLE promoters, increasing our fundamental understanding of the transcriptional regulation of cholesterol synthesis.
胆固醇合成的两个控制点，3-羟-3-甲基戊二酰基辅酶 A 还原酶（HMGCR）和角鲨烯单加氧酶（SQLE）是转录因子固醇调节元件结合蛋白-2（SREBP-2）的已知靶点。然而，固醇调节元件（SREs）和辅助因子结合位点，核因子-Y（NF-Y）和特异性蛋白 1（Sp1）的位置在人类 SQLE 启动子中尚未得到令人满意的映射，或者在人类 HMGCR 启动子中根本没有映射。
我们使用荧光素酶报告基因检测筛选了预测的 SRE、Sp1 和 NF-Y 位点突变体文库的固醇反应性，从而鉴定了真正的结合位点。我们通过电泳迁移率变动分析（EMSA）和 ChIP-PCR 来确认 SREs。
我们在人类 HMGCR 和 SQLE 启动子中都发现了两个紧密相邻的 SRE，在 HMGCR 中有一个 NF-Y 位点，在 SQLE 中有两个。此外，我们发现 HMGCR 的表达只有在 SREBP-2 水平非常高时才会被高度激活，与低密度脂蛋白受体（LDLR）形成鲜明对比，这一结果反映了其他研究中使用的小鼠模型。
HMGCR 和 SQLE 启动子都有两个 SREs，它们可能作为一个归巢区域来吸引单个 SREBP-2 同源二聚体，只有在绝对需要胆固醇合成时，HMGCR 才会被激活。这确保了通过 LDLR 优先摄取外源性胆固醇，从而节省能量。
我们首次对人类 HMGCR 和 SQLE 启动子中的 SREs 和 NF-Ys 进行了全面调查，增加了我们对胆固醇合成转录调控的基本理解。
