<br>多组学分析表明 FOXO4 参与鸡晶状体纤维细胞分化的遗传调控,Developmental Biology

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多组学分析表明 FOXO4 参与鸡晶状体纤维细胞分化的遗传调控
Developmental Biology ( IF 2.5 ) Pub Date : 2023-09-16 , DOI: 10.1016/j.ydbio.2023.09.005
Lisa Brennan ₁ , Joshua Disatham ₁ , A Sue Menko ₂ , Marc Kantorow ₁

Affiliation

鉴定新分化机制和途径的经典模型是晶状体，它由单层静止上皮细胞组成，这些细胞是成熟完全分化纤维细胞核心的祖细胞。晶状体上皮细胞分化为纤维细胞遵循一个协调程序，包括细胞周期退出、关键结构蛋白的表达和细胞器的标志性消除以实现透明度。尽管已确定多种机制和途径在晶状体分化中起关键作用，但控制晶状体分化的全部机制仍有待发现。先前的一项研究确定，特定的染色质可及性变化与必需晶状体纤维细胞基因的表达直接相关，这表明这些基因表达所需的转录因子的活性可以通过结合已识别的染色质区域来调节。对已鉴定的染色质可及区域的序列分析显示转录因子 FOXO4 结合序列的表达增强，表明 FOXO4 在这些基因表达中的直接作用。已知 FOXO4 可调节多种细胞过程，包括细胞对代谢和氧化应激的反应、细胞周期撤回和体内平衡，这表明 FOXO4 在晶状体细胞分化的调节中具有以前未确定的作用。为了进一步评估 FOXO4 的作用，我们采用多组学方法来分析全基因组 FOXO4 结合、关键基因的分化特异性表达和染色质可及性之间的关系。为了更好地鉴定活性启动子和增强子，我们还通过分析 H3K27ac 检查了组蛋白修饰。具体方法包括 CUT&RUN （FOXO4 结合和 H3K27ac 修饰）、RNA-seq （分化状态特异性基因表达）和 ATAC-seq （染色质可及性）。CUT&RUN 在晶状体纤维细胞基因组中鉴定了 20,966 个 FOXO4 结合位点和 33,921 个 H3K27ac 标记区域。RNA-seq 鉴定了 956 个与上皮细胞相比，纤维细胞中表达水平显著较高的基因（log2FC > 0.7， q < 0.05）和 2548 个表达水平显著较低的基因（log2FC < -0.7， q < 0.05）。综合分析确定了 1727 个分化状态特异性基因，这些基因与至少一个 FOXO4 结合位点最近，包括编码晶状体间隙连接（GJA1、GJA3）、晶状体结构蛋白（BFSP1、CRYBB1、ASL1）和晶状体透明度所需的基因（HSF4、NRCAM）。比较已发表的 ATAC-seq 数据中鉴定的 FOXO4 结合位点的多组学分析显示，染色质可及性与晶状体分化过程中 FOXO4 依赖性基因表达相关。结果为 FOXO4 在晶状体分化所需关键基因表达的调控中的重要要求提供了证据，并将染色质可及性和 H3K27ac 组蛋白修饰的表观遗传调控与 FOXO4 在晶状体纤维细胞分化过程中控制晶状体基因表达的功能联系起来。

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Multiomic analysis implicates FOXO4 in genetic regulation of chick lens fiber cell differentiation

A classic model for identification of novel differentiation mechanisms and pathways is the eye lens that consists of a monolayer of quiescent epithelial cells that are the progenitors of a core of mature fully differentiated fiber cells. The differentiation of lens epithelial cells into fiber cells follows a coordinated program involving cell cycle exit, expression of key structural proteins and the hallmark elimination of organelles to achieve transparency. Although multiple mechanisms and pathways have been identified to play key roles in lens differentiation, the entirety of mechanisms governing lens differentiation remain to be discovered. A previous study established that specific chromatin accessibility changes were directly associated with the expression of essential lens fiber cell genes, suggesting that the activity of transcription factors needed for expression of these genes could be regulated through binding access to the identified chromatin regions. Sequence analysis of the identified chromatin accessible regions revealed enhanced representation of the binding sequence for the transcription factor FOXO4 suggesting a direct role for FOXO4 in expression of these genes. FOXO4 is known to regulate a variety of cellular processes including cellular response to metabolic and oxidative stress, cell cycle withdrawal, and homeostasis, suggesting a previously unidentified role for FOXO4 in the regulation of lens cell differentiation. To further evaluate the role of FOXO4 we employed a multiomics approach to analyze the relationship between genome-wide FOXO4 binding, the differentiation-specific expression of key genes, and chromatin accessibility. To better identify active promoters and enhancers we also examined histone modification through analysis of H3K27ac. Specific methods included CUT&RUN (FOXO4 binding and H3K27ac modification), RNA-seq (differentiation state specific gene expression), and ATAC-seq (chromatin accessibility). CUT&RUN identified 20,966 FOXO4 binding sites and 33,921 H3K27ac marked regions across the lens fiber cell genome. RNA-seq identified 956 genes with significantly greater expression levels in fiber cells compared to epithelial cells (log2FC > 0.7, q < 0.05) and 2548 genes with significantly lower expression levels (log2FC < −0.7, q < 0.05). Integrated analysis identified 1727 differentiation-state specific genes that were nearest neighbors to at least one FOXO4 binding site, including genes encoding lens gap junctions (GJA1, GJA3), lens structural proteins (BFSP1, CRYBB1, ASL1), and genes required for lens transparency (HSF4, NRCAM). Multiomics analysis comparing the identified FOXO4 binding sites in published ATAC-seq data revealed that chromatin accessibility was associated with FOXO4-dependent gene expression during lens differentiation. The results provide evidence for an important requirement for FOXO4 in the regulated expression of key genes required for lens differentiation and link epigenetic regulation of chromatin accessibility and H3K27ac histone modification with the function of FOXO4 in controlling lens gene expression during lens fiber cell differentiation.

更新日期：2023-09-16

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