Changes in DNA Methylation Hallmark Alterations In Chromatin Accessibility And Gene Expression For Eye Lens Differentiation [RNA-seq]

Methylation at cytosines (mCG) is a well-known regulator of gene expression but its requirements for cellular differentiation have yet to be fully elucidated. A well-studied cellular differentiation model system is the eye lens, consisting of a single anterior layer of epithelial cells that migrate laterally and differentiate into a core of fiber cells. Here, we explore the genome-wide relationships between mCG methylation, chromatin accessibility and gene expression during differentiation of eye lens epithelial cells into fiber cells. Whole genome bisulfite sequencing identified 7621 genomic loci exhibiting significant differences in mCG levels between lens epithelial and fiber cells. Changes in mCG levels were inversely correlated with the differentiation state-specific expression of 1285 genes preferentially expressed in either lens fiber or lens epithelial cells (Pearson correlation r = -0.37, p < 1x10-42). mCG levels were inversely correlated with chromatin accessibility determined by Assay for transposase-accessible sequencing (ATAC-seq) (Pearson correlation r = -0.86, p < 1x10-300). Many of the genes exhibiting altered regions of DNA methylation, chromatin accessibility and gene expression levels in fiber cells relative to epithelial cells are associated with lens fiber cell structure, homeostasis and transparency. These include lens crystallins (CRYBA4, CRYBB1, CRYGN, CRYBB2), lens beaded filament proteins (BFSP1, BFSP2), transcription factors (HSF4, SOX2, HIF1A), and Notch signaling pathway members (NOTCH1, NOTCH2, HEY1, HES5). Analysis of regions exhibiting cell-type specific alterations in DNA methylation revealed an overrepresentation of consensus sequences of multiple transcription factors known to play key roles in lens cell differentiation including HIF1A, SOX2, and the MAF family of transcription factors. Collectively, these results link DNA methylation with control of chromatin accessibility and gene expression changes required for eye lens differentiation. The results also point to a role for DNA methylation in the regulation of transcription factors previously identified to be important for lens cell differentiation. Overall design: Examination of differentially methylated mCG regions between 3 biological replicates of pooled lens epithelial cells and 3 biological replicates of pooled lens fiber cells obtained from embryonic day 13 chick lenses and correlating with differentially expressed genes.

胞嘧啶甲基化（mCG）是公认的基因表达调控因子，但其介导细胞分化的具体调控需求尚未完全阐明。眼晶状体是被广泛研究的细胞分化模型系统，其由单层前端上皮细胞组成，这些上皮细胞可侧向迁移并分化为晶状体纤维细胞构成的核心区域。本研究旨在探究眼晶状体上皮细胞向纤维细胞分化过程中，全基因组范围内mCG甲基化、染色质开放性与基因表达之间的关联。全基因组亚硫酸氢盐测序（Whole Genome Bisulfite Sequencing）共鉴定出7621个基因组位点，其mCG水平在晶状体上皮细胞与纤维细胞间存在显著差异。mCG水平的变化与1285个优先在晶状体纤维细胞或上皮细胞中表达的分化状态特异性基因的表达呈负相关（皮尔逊相关系数r=-0.37，p<1×10^-42）。mCG水平与通过转座酶可及性测序（Assay for Transposase-Accessible Chromatin Sequencing, ATAC-seq）测定的染色质开放性呈负相关（皮尔逊相关系数r=-0.86，p<1×10^-300）。相较于上皮细胞，纤维细胞中存在DNA甲基化、染色质开放性及基因表达水平改变区域的诸多基因，均与晶状体纤维细胞的结构、稳态及透明度相关。此类基因包括晶状体蛋白（CRYBA4、CRYBB1、CRYGN、CRYBB2）、晶状体串珠状丝蛋白（BFSP1、BFSP2）、转录因子（HSF4、SOX2、HIF1A）以及Notch信号通路成员（NOTCH1、NOTCH2、HEY1、HES5）。对细胞类型特异性DNA甲基化改变区域的分析显示，多个已知在晶状体细胞分化中发挥关键作用的转录因子的保守结合序列出现显著富集，其中包括HIF1A、SOX2以及MAF家族转录因子。综上，本研究结果将DNA甲基化与眼晶状体分化所需的染色质开放性调控及基因表达变化建立了关联。该研究结果同时表明，DNA甲基化可调控此前已被证实对晶状体细胞分化至关重要的转录因子。实验整体设计：对13日龄鸡胚晶状体中分离得到的3份混合晶状体上皮细胞生物学重复样本与3份混合晶状体纤维细胞生物学重复样本之间的差异mCG甲基化区域进行检测，并将其与差异表达基因进行关联分析。