Center for Comparative Biomedicine, Key laboratory of Systems Biomedicine (Ministry of Education), Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China.
Yi Chuan. 2022 Aug 20;44(8):695-764. doi: 10.16288/j.yczz.22-160.
The regulation of target genes by distal enhancers usually determines the fate and function of cells. Active enhancers in specific regions of chromatin may transcribe bidirectionally to produce long non-coding enhancer RNA (eRNA) to regulate gene expression. We recently found that an antisense enhancer eRNA PEARL (Pcdh eRNA associated with R-loop formation) regulates gene expression of members of the Pcdhα cluster via R-loop formation. To further explore the biological function of eRNA, we performed additional genetic and molecular experiments such as CRISPR (clustered regularly interspaced short palindromic repeats) DNA-fragment editing, RT-PCR, and qPCR. First, we performed expression analyses of the HS5-1 eRNA PEARL and found that it was expressed in a tissue-specific manner. In addition, upon CRISPR DNA-fragment deletion or inversion of the CTCF sites in the HS5-1 enhancer region, the expression of eRNA PEARL was reduced to 2%-10% and the expression of Pcdhα gene cluster was also reduced to 13%-68% of the original levels. Finally, deletion of the bidirectional transcription start site (TSS) of HS5-1 eRNA or inversion of TSS of the eRNA PEARL resulted in approximately 60% or 40% decrease of levels of Pcdhα gene expression. In summary, these data suggested a functional role of the HS5-1 eRNA in gene regulation of the Pcdhα cluster, providing a new direction for future researches on the regulatory mechanisms of clustered Pcdh gene expression in the brain.
远端增强子对靶基因的调控通常决定细胞的命运和功能。染色质特定区域的活性增强子可能双向转录产生长非编码增强子 RNA（eRNA），以调节基因表达。我们最近发现，反义增强子 eRNA PEARL（与 R 环形成相关的 Pcdh 增强子 RNA）通过 R 环形成调节 Pcdhα 簇成员的基因表达。为了进一步探索 eRNA 的生物学功能，我们进行了额外的遗传和分子实验，如 CRISPR（成簇规律间隔短回文重复）DNA 片段编辑、RT-PCR 和 qPCR。首先，我们对 HS5-1 eRNA PEARL 的表达进行了分析，发现它具有组织特异性表达。此外，在 CRISPR DNA 片段缺失或 CTCF 位点在 HS5-1 增强子区域的反转后，eRNA PEARL 的表达降低到原来水平的 2%-10%，Pcdhα 基因簇的表达也降低到原来水平的 13%-68%。最后，HS5-1 eRNA 的双向转录起始位点（TSS）缺失或 eRNA PEARL 的 TSS 反转导致 Pcdhα 基因表达水平降低约 60%或 40%。综上所述，这些数据表明 HS5-1 eRNA 在 Pcdhα 簇基因调控中发挥了功能作用，为未来研究大脑中聚类 Pcdh 基因表达的调控机制提供了新的方向。
