Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Theranostics. 2022 Jan 1;12(2):530-541. doi: 10.7150/thno.62760. eCollection 2022.
Histone H4 lysine16 acetylation (H4K16Ac) modulates chromatin structure by serving as a switch from a repressive to a transcriptionally active state. This euchromatin mark is associated with active transcription. In this study, we investigated the effects of H4K16Ac on the expression of pro-fibrotic genes in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and in an aging murine model of lung fibrosis. The lung tissues and fibroblasts from human IPF/non-IPF donors and from aged mice with/without bleomycin induced lung fibrosis were used in this study. The H4K16Ac levels were examined by immunohistochemistry or western blots. RNA silencing of H4K16Ac acetyltransferase Mof was used to reduce H4K16Ac levels in IPF fibroblasts. The effects of reduced H4K16Ac on pro-fibrotic gene expression were examined by western blots and real-time PCR. The association of H4K16Ac with these genes' promoter region were evaluated by ChIP assays. The gene expression profile in siRNA Mof transfected IPF cells were determined by RNA-Seq. The impact of H4K16Ac levels on lung fibrosis was evaluated in an aging murine model. Aged mice with bleomycin induced lung fibrosis showed increased H4K16Ac levels. Human lung fibroblasts with siRNA Mof silencing demonstrated reduced H4K16Ac, and significantly down-regulated profibrotic genes, such as α-smooth muscle actin (α-SMA), collagen I, Nox4, and survivin. ChIP assays confirmed the associations of these pro-fibrotic genes' promoter region with H4K16Ac, while in siRNA Mof transfected cells the promoter/H4K16Ac associations were depleted. RNA-seq data demonstrated that Mof knockdown altered gene expression and cellular pathways, including cell damage and repair. In the aging mice model of persistent lung fibrosis, 18-month old mice given intra-nasal siRNA Mof from week 3 to 6 following bleomycin injury showed improved lung architecture, decreased total hydroxyproline content and lower levels of H4K16Ac. These results indicate a critical epigenetic regulatory role for histone H4K16Ac in the pathogenesis of pulmonary fibrosis, which will aid in the development of novel therapeutic strategies for age-related diseases such as IPF.
组蛋白 H4 赖氨酸 16 乙酰化 (H4K16Ac) 通过充当从抑制状态到转录激活状态的开关来调节染色质结构。这种常染色质标记与活跃的转录有关。在这项研究中，我们研究了 H4K16Ac 对特发性肺纤维化 (IPF) 患者的肺成纤维细胞和衰老小鼠肺纤维化模型中促纤维化基因表达的影响。 本研究使用了来自特发性肺纤维化/非特发性肺纤维化供体的肺组织和肺成纤维细胞以及接受博来霉素诱导肺纤维化的老年小鼠。通过免疫组织化学或 Western blot 检测 H4K16Ac 水平。使用 H4K16Ac 乙酰转移酶 Mof 的 RNA 沉默来降低 IPF 成纤维细胞中的 H4K16Ac 水平。通过 Western blot 和实时 PCR 检测降低 H4K16Ac 对促纤维化基因表达的影响。通过 ChIP 测定评估 H4K16Ac 与这些基因启动子区域的关联。通过 RNA-Seq 确定转染 siRNA Mof 的 IPF 细胞中的基因表达谱。通过评估衰老小鼠模型中的 H4K16Ac 水平来评估其对肺纤维化的影响。 接受博来霉素诱导肺纤维化的老年小鼠表现出 H4K16Ac 水平升高。用 siRNA Mof 沉默的人肺成纤维细胞显示出 H4K16Ac 减少，并且显著下调了促纤维化基因，如α-平滑肌肌动蛋白 (α-SMA)、胶原蛋白 I、Nox4 和 survivin。ChIP 测定证实了这些促纤维化基因启动子区域与 H4K16Ac 的关联，而在转染 siRNA Mof 的细胞中，启动子/H4K16Ac 关联被耗尽。RNA-seq 数据表明，Mof 敲低改变了基因表达和细胞途径，包括细胞损伤和修复。在持续肺纤维化的衰老小鼠模型中，3 至 6 周龄接受博来霉素损伤后给予鼻腔内 siRNA Mof 的 18 个月大的小鼠显示出改善的肺结构、总羟脯氨酸含量降低和 H4K16Ac 水平降低。 这些结果表明组蛋白 H4K16Ac 在肺纤维化发病机制中具有关键的表观遗传调节作用，这将有助于为特发性肺纤维化等与年龄相关的疾病开发新的治疗策略。
