Bhargavan Biju, Chhunchha Bhavana, Kubo Eri, Singh Dhirendra P
Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE, USA.
Department of Ophthalmology, Kanazawa Medical University, Ishikawa, 9200293, Japan.
Cell Death Discov. 2024 Jun 22;10(1):296. doi: 10.1038/s41420-024-02076-2.
The physiological quantum of stress-inducible transcriptional protein, Lens Epithelium-Derived Growth Factor (LEDGF), is vital for the maintenance of cellular physiology. Erratic epigenetic reprogramming in response to oxidative stress or with advancing age is found to be a major cause in the gene silencing, leading to pathobiologies. Using aging human (h) eye lens/lens epithelial cells (LECs) coupled with redox-active Peroxiredoxin 6 (Prdx6)-deficient (Prdx6) mLECs as model systems, herein, we showed that in aging/oxidative stress, the human LEDGF gene was regulated by unique methylation patterns of CGs nucleotides within and around the Sp1 binding site(s) of CpG island of the LEDGF promoter (-170 to -27nts). The process caused the repression of LEDGF and its target, Hsp27, resulting in reactive oxygen species (ROS) amplification and cellular insults. This phenomenon was opposed to the unmethylated promoter in LECs. Clinically, we observed that the loss of LEDGF in the Prdx6 mLECs or aging lenses/LECs, correlating with increased expression of DNMT1, DNMT3a, and DNMT3b along with the methyl CpG binding protein 2 (MeCP2). Upon oxidative stress, the expression of these molecules was increased with the dramatic reduction in LEDGF expression. While demethylating agent, 5-Aza deoxycytidine (5-AzaC) transposed the aberrant methylation status, and revived LEDGF and Hsp27 expression. Mechanistically, the chloramphenicol acetyltransferase (CAT) reporter gene driven by the LEDGF promoter (-170/ + 35) and ChIP assays uncovered that 5-AzaC acted on GC/Sp1 sites to release LEDGF transcription. The data argued, for the first time, that de novo methylation of CGs around and within Sp1 sites of the CpG island directly disrupted Sp1 activity, which ensued in LEDGF repression and its biological functions. The findings should improve our understanding of cellular insults-associated with aberrant DNMTs-mediated LEDGF's activity, and can offer strategies for therapeutic intervention to halt aging/oxidative stress-induced abnormalities.
应激诱导转录蛋白晶状体上皮衍生生长因子（LEDGF）的生理量对于维持细胞生理至关重要。研究发现，响应氧化应激或随着年龄增长而发生的不稳定表观遗传重编程是基因沉默的主要原因，进而导致病理生物学变化。本文以衰老的人眼晶状体/晶状体上皮细胞（LECs）以及缺乏氧化还原活性过氧化物酶6（Prdx6）的小鼠晶状体上皮细胞（mLECs）作为模型系统，表明在衰老/氧化应激状态下，人LEDGF基因受LEDGF启动子（-170至-27 nt）CpG岛的Sp1结合位点及其周围CG核苷酸独特甲基化模式的调控。这一过程导致LEDGF及其靶标Hsp27受到抑制，从而导致活性氧（ROS）扩增和细胞损伤。这种现象与LECs中未甲基化的启动子情况相反。临床上，我们观察到Prdx6 mLECs或衰老晶状体/LECs中LEDGF的缺失，与DNA甲基转移酶1（DNMT1）、DNA甲基转移酶3a（DNMT3a）和DNA甲基转移酶3b（DNMT3b）以及甲基CpG结合蛋白2（MeCP2）表达增加相关。在氧化应激时，这些分子的表达增加，同时LEDGF表达显著降低。而脱甲基剂5-氮杂脱氧胞苷（5-AzaC）可逆转异常甲基化状态，恢复LEDGF和Hsp27的表达。从机制上讲，由LEDGF启动子（-170 / +35）驱动的氯霉素乙酰转移酶（CAT）报告基因以及染色质免疫沉淀（ChIP）分析表明，5-AzaC作用于GC/Sp1位点以释放LEDGF转录。这些数据首次表明，CpG岛Sp1位点周围及内部CG的从头甲基化直接破坏了Sp1活性，进而导致LEDGF受到抑制及其生物学功能丧失。这些发现应能增进我们对与异常DNMT介导的LEDGF活性相关的细胞损伤的理解，并可为治疗干预提供策略，以阻止衰老/氧化应激诱导的异常情况。
