Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan.
Division of Biology, Department of Biological Sciences, School of Science, Hokkaido University, Sapporo 060-0810, Japan.
J Reprod Dev. 2020 Oct 13;66(5):435-444. doi: 10.1262/jrd.2019-161. Epub 2020 Jun 5.
Specific gene expression in granulosa cells is key for the function of ovary, but the molecular mechanism of transcriptional activation is not well studied. Here we investigated the regulatory mechanism of the mouse stearoyl-CoA desaturase 2 (Scd2) gene encoding an enzyme for lipid metabolism. Northern blot and in situ hybridization indicated that the mouse Scd2 mRNA was highly expressed in ovarian granulosa cells. We found four conserved noncoding sequences (CNSs) and two long noncoding RNAs (lncRNAs) transcribed from regions upstream of the Scd2 gene as candidates of regulatory elements/factors. These lncRNAs were predominantly transcribed in the opposite direction to Scd2 and localized in nuclei and showed the correlation with Scd2 expression, raising the possibility of their transcriptional regulatory roles. Indeed, knockdown of both lncRNAs, lncRNA-sc1 and lncRNA-sc2, significantly decreased the Scd2 mRNA level in primary granulosa cells. Then, we investigated the histone modification pattern at this locus by a chromatin immunoprecipitation assay, and two CNSs, CNS1 and CNS2, were found to be marked with high levels of histone H3K9/K27 acetylation in primary granulosa cells. By a reporter gene assay, both CNS1 and CNS2 interdependently exhibited enhancer activity for the Scd2 promoter in primary granulosa cells. These data suggest that the mouse Scd2 gene is activated by two lncRNAs and interdependent enhancers in ovarian granulosa cells, which provides a new insight into transcriptional activation in granulosa cells.
颗粒细胞中特定基因的表达是卵巢功能的关键，但转录激活的分子机制尚未得到很好的研究。在这里，我们研究了编码脂质代谢酶的小鼠硬脂酰辅酶 A 去饱和酶 2 (Scd2) 基因的调控机制。Northern blot 和原位杂交表明，小鼠 Scd2 mRNA 在卵巢颗粒细胞中高度表达。我们发现了四个保守的非编码序列 (CNSs) 和两个长非编码 RNA (lncRNA)，它们从 Scd2 基因的上游区域转录，作为候选的调节元件/因子。这些 lncRNA 主要以与 Scd2 相反的方向转录，并定位于核内，与 Scd2 表达相关，提示它们具有转录调节作用。事实上，两种 lncRNA（lncRNA-sc1 和 lncRNA-sc2）的敲低均显著降低了原代颗粒细胞中 Scd2 mRNA 的水平。然后，我们通过染色质免疫沉淀试验研究了该基因座的组蛋白修饰模式，发现两个 CNSs（CNS1 和 CNS2）在原代颗粒细胞中标记有高水平的组蛋白 H3K9/K27 乙酰化。通过报告基因试验，CNS1 和 CNS2 相互依赖地在原代颗粒细胞中表现出 Scd2 启动子的增强子活性。这些数据表明，小鼠 Scd2 基因在卵巢颗粒细胞中被两种 lncRNA 和相互依赖的增强子激活，为颗粒细胞中的转录激活提供了新的见解。
