A transcriptome and chromatin landscape analysis of vitreous-induced lens fiber cell differentiation in mouse lens epithelial explants (RNA-Seq). A transcriptome and chromatin landscape analysis of vitreous-induced lens fiber cell differentiation in mouse lens epithelial explants (RNA-Seq)

Lens epithelial explants consist of lens epithelial cells (P8 FVB/N mice) grown in vitro on their native basement membrane, the lens capsule. For decades, biologists have used lens epithelial explants to study lens fiber cell differentiation. However, the global change in the accessibility of the chromatin and transcriptome during the process of explanting and culture is unknown. Therefore, P8 FVB/N lens epithelial explants cultured in either unsupplemented media or media containing 50% bovine vitreous humor for one or five days were collected. Chromatin and RNA was collected for ATAC-sequencing and RNA-sequencing respectively. Differentially accessible regions and differentially expressed genes were identified for each condition to provide a genome wide view of chromatin architecture and gene expression during fiber cell differentiation in vitro. Vitreous humor generally increased chromatin accessibility in promoter regions of genes associated with fiber differentiation and immune response, and this was associated with increased transcript levels for these genes. In contrast, vitreous had relatively little effect on the accessibility of most of the genes highly expressed in the lens epithelium despite dramatic reductions in the transcript levels of these genes. Overall design: Comparative gene expression profiling analysis of RNA-seq data for lens epithelial explants with different treatments (Culture Media (CM) and Differentiation Media (DM)) and different time points (Immediately after the lens epithelium collection, 24 hrs in culture media (D0), after an additional day in CM (D1_CM) or DM (D1_DM), after an additional (relative to Day 0) 5 days in CM (D5_CM) or DM (D5_ DM). Biological replicates were collected in triplicate.

晶状体上皮外植体（lens epithelial explants）指在其天然基底膜——晶状体囊（lens capsule）上体外培养的P8代FVB/N小鼠晶状体上皮细胞（lens epithelial cells）。数十年来，生物学家一直利用该模型研究晶状体纤维细胞分化，但在外植与培养过程中，染色质可及性与转录组的全局变化尚未明确。为此，研究人员收集了分别在无补充培养基或含50%牛玻璃体（bovine vitreous humor）的培养基中培养1天或5天的P8 FVB/N小鼠晶状体上皮外植体，分别提取染色质与RNA，用于ATAC测序（ATAC-sequencing）与RNA测序（RNA-sequencing）。通过鉴定各实验条件下的差异可及区域与差异表达基因，本研究获得了体外纤维细胞分化过程中染色质结构与基因表达的全基因组视图。结果显示，牛玻璃体通常可提升与纤维分化及免疫应答相关基因启动子区域的染色质可及性，这与这类基因的转录水平上调相一致。与之相对，尽管晶状体上皮中高表达基因的转录水平出现显著下调，但牛玻璃体对这类基因的可及性影响相对微弱。总体实验设计：本研究对不同处理组（基础培养基（CM）与分化培养基（DM））、不同时间点的晶状体上皮外植体的RNA-seq数据开展比较基因表达谱分析，时间点涵盖：晶状体上皮收集后即刻、基础培养基中培养24小时（D0）、在基础培养基（CM）中继续培养1天（D1_CM）或分化培养基（DM）中继续培养1天（D1_DM）、相较于第0天在CM中继续培养5天（D5_CM）或DM中继续培养5天（D5_DM）。所有实验均设置三次生物学重复。