Long non-coding RNA lncMGC mediates the expression of TGF-β-induced genes in renal cells via nucleosome remodelers.
Long non-coding RNA lncMGC mediates the expression of TGF-β-induced genes in renal cells via nucleosome remodelers.
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DOI：
10.3389/fmolb.2023.1204124
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发表时间：
2023
影响因子：
5
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背景：微小RNAs(MiRNAs)和长非编码RNAs(LncRNAs)在糖尿病肾病(DKD)中起关键作用。转化生长因子-β(转化生长因子-β)调节miR-379miRNAs及其宿主转录产物-mGc的表达，使其在糖尿病小鼠肾小球中增多，促进早期糖尿病肾病的发生。然而，lncMGC的生化功能尚不清楚。在这里，我们通过体外转录的LncMGC RNA下拉和质谱仪鉴定了LncMGC相互作用的蛋白质。我们还通过CRISPR-Cas9编辑创建了lncMGC基因敲除(KO)小鼠，并使用来自KO小鼠的原代小鼠系膜细胞(MMC)来检测lncMGC对DKD相关基因表达、启动子组蛋白修饰的变化以及染色质重塑的影响。方法：体外转录的LncMGC RNA与HK2细胞(人肾细胞系)的裂解物混合。用质谱仪鉴定与lncMGC相互作用的蛋白质。用RNA免疫沉淀和定量聚合酶链式反应对候选蛋白进行鉴定。将Cas9和GUIDE RNA注射到小鼠卵子中，建立IncMGC-KO小鼠。用转化生长因子-β处理野生型(WT)和LncMGC-KO MMCs，通过RNA-seq和qPCRRNA表达和组蛋白修饰(染色质免疫沉淀)和染色质重构/开放染色质(转座酶可及染色质测序分析，atac-seq)进行检测。结果：几种核小体重塑因子包括SMARCA5和SMARCC2被质谱学鉴定为lncMGC相互作用蛋白，并经RNA免疫沉淀-定量聚合酶链式反应证实。LncMGC-KO小鼠MMC未见基础表达或转化生长因子-β诱导表达。在经转化生长因子-β处理的WT MMCs中，组蛋白H3K27乙酰化和SMARCA5在lncMGC启动子上的浓集增加，而在LncMGC-KO MMCs中显著减少。在转化生长因子-β处理的情况下，lncMGC启动子区域的ATAC峰和许多其他DKD相关基因包括COL4A3和COL4A4显著低于WT MMCs。在ATAC峰中富含锌指(ZF)、ADARD和SMAD基序。在lncMGC基因中还发现了ZF和干旱位点。结论：LncMGC RNA与多种核小体重塑因子相互作用，促进染色质松弛，增强LncMGC自身及其他促纤维化基因的表达。LncMGC/核小体重构体复合体促进部位特异性染色质的可及性，以增强靶肾细胞中的DKD相关基因。
Background: MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) play key roles in diabetic kidney disease (DKD). The miR-379 megacluster of miRNAs and its host transcript lnc-megacluster (lncMGC) are regulated by transforming growth factor-β (TGF-β), increased in the glomeruli of diabetic mice, and promote features of early DKD. However, biochemical functions of lncMGC are unknown. Here, we identified lncMGC-interacting proteins by in vitro-transcribed lncMGC RNA pull down followed by mass spectrometry. We also created lncMGC-knockout (KO) mice by CRISPR-Cas9 editing and used primary mouse mesangial cells (MMCs) from the KO mice to examine the effects of lncMGC on the gene expression related to DKD, changes in promoter histone modifications, and chromatin remodeling. Methods: In vitro-transcribed lncMGC RNA was mixed with lysates from HK2 cells (human kidney cell line). lncMGC-interacting proteins were identified by mass spectrometry. Candidate proteins were confirmed by RNA immunoprecipitation followed by qPCR. Cas9 and guide RNAs were injected into mouse eggs to create lncMGC-KO mice. Wild-type (WT) and lncMGC-KO MMCs were treated with TGF-β, and RNA expression (by RNA-seq and qPCR) and histone modifications (by chromatin immunoprecipitation) and chromatin remodeling/open chromatin (by Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) were examined. Results: Several nucleosome remodeling factors including SMARCA5 and SMARCC2 were identified as lncMGC-interacting proteins by mass spectrometry, and confirmed by RNA immunoprecipitation–qPCR. MMCs from lncMGC-KO mice showed no basal or TGF-β-induced expression of lncMGC. Enrichment of histone H3K27 acetylation and SMARCA5 at the lncMGC promoter was increased in TGF-β-treated WT MMCs but significantly reduced in lncMGC-KO MMCs. ATAC peaks at the lncMGC promoter region and many other DKD-related loci including Col4a3 and Col4a4 were significantly lower in lncMGC-KO MMCs compared to WT MMCs in the TGF-β-treated condition. Zinc finger (ZF), ARID, and SMAD motifs were enriched in ATAC peaks. ZF and ARID sites were also found in the lncMGC gene. Conclusion: lncMGC RNA interacts with several nucleosome remodeling factors to promote chromatin relaxation and enhance the expression of lncMGC itself and other genes including pro-fibrotic genes. The lncMGC/nucleosome remodeler complex promotes site-specific chromatin accessibility to enhance DKD-related genes in target kidney cells.