Regulatory role of PI3K signaling pathways in lens differentiation and function

使用ATACseq和RNAseq相结合的方法，我们的研究提供了变化的第一个证据 在染色质中，可及性对多种基因的分化状态特异性表达至关重要 对于晶状体上皮细胞向纤维细胞的转变至关重要。这些研究还确定了关键的DNA调控 区域和转录因子结合部位可能调节广泛的晶状体基因，最重要的是 FOXO4.含有共同DNA结合序列的在晶状体细胞分化中起必要作用的基因 对于FOXO4，包括EphA2、NrCAM、δ-晶体蛋白、Notch1和FYCO1。FOXO系列，包括FOXO4，是 受PI3K/Akt信号通路调控。PI3K/Akt对FOXO4的磷酸化将其隔离在细胞内 FOXO4进入细胞核需要细胞质和PI3K/Akt信号的抑制 调控基因表达。FOXO4的功能从未在晶状体中被检测过。我们建议 PI3K/AKT对晶状体纤维细胞FOXO4依赖基因表达的调控作用 差异化。这将通过1)确定对核的PI3K/Akt抑制的要求来实现 FOXO4在晶状体上皮细胞向纤维细胞转变过程中的移位；2)建立晶状体上皮细胞与纤维细胞之间的联系 PI3K/Akt抑制与含FOXO4结合的晶状体分化特异性基因的表达 序列；3)展示了FOXO4与不同的染色质可及DNA调节区的结合 使用靶向芯片分析对晶状体分化至关重要的基因；以及4)建立光谱、范围和 晶状体细胞分化过程中FOXO4调控基因的空间表达模式我们还发现 PI3K/Akt信号轴在调节去除 线粒体、内质网和高尔基体来自中央光路和多个PI3K下游信号通路 是消除核形成晶状体细胞器自由区(OFZ)的过程所必需的。这个过程是 需要创造一种成熟的透镜，能够将光线图像聚焦到视网膜上。我们将探索PI3K是如何 信号通路调节细胞核和其他细胞器的消除以形成晶状体细胞器游离 旨在1)确定单个PI3K p110催化亚基在调控中的功能 OFZ的形成；2)证实抑制PI3K/Akt信号后自噬的诱导 Axis负责从正在发育的晶状体中移除线粒体、内质网和高尔基体；3)确定 不同PI3K信号通路失活与所需机械性靶点激活之间的联系 消除原子核以形成OFZ；以及4)研究Akt活性环之间的潜在联系 皮质和核纤维细胞的边界以及OFZ外边界的调节。

Using a combined ATACseq and RNAseq approach our studies have provided the first evidence that changes in chromatin accessibility are crucial to the differentiation state-specific expression of a wide variety genes essential to the transition from lens epithelial to fiber cells. These studies also identified key DNA regulatory regions and transcription factor binding sites likely to regulate a wide-range of lens genes, most importantly FOXO4. Genes with requisite roles in lens cell differentiation that contain a consensus DNA binding sequence for FOXO4 include EPHA2, NrCAM, δ-crystallin, Notch1 and FYCO1. The FOXO family, including FOXO4, is regulated by the PI3K/Akt signaling pathway. Phosphorylation of FOXO4 by PI3K/Akt sequesters it in the cytoplasm and suppression of PI3K/Akt signaling is required for FOXO4 import to the nucleus for its role in regulating gene expression. The function of FOXO4 has never been examined in the lens. We propose to establish the role of PI3K/AKT regulation of FOXO4-dependent gene expression in lens fiber cell differentiation. This will be accomplished by 1) identifying the requirement for PI3K/Akt inhibition for the nuclear translocation of FOXO4 in the transition of lens epithelial to fiber cells; 2) establishing the link between PI3K/Akt inhibition and the expression of lens differentiation-specific genes containing FOXO4 binding sequences; 3) demonstrating the binding of FOXO4 to distinct chromatin accessible DNA regulatory regions in genes crucial to lens differentiation using targeted-CHIP assays; and 4) establishing the spectrum, range and spatial expression patterns of genes regulated by FOXO4 during lens cell differentiation. We also discovered that the PI3K/Akt signaling axis plays an essential role in regulating the timing and mechanism that removes mitochondria, ER and Golgi from the central light path and that multiple PI3K-downstream signaling pathways are required for the process of eliminating nuclei to form the lens Organelle Free Zone (OFZ). This process is required to create a mature lens capable of focusing light images on the retina. We will explore how PI3K signaling pathways regulate the elimination of nuclei and other organelles to form the lens Organelle Free Zone in studies aimed at 1) identifying the functions of individual PI3K p110 catalytic subunits in regulating formation of the OFZ; 2) confirming that the induction of autophagy following inhibition of the PI3K/Akt signaling axis is responsible for the removal of mitochondria, ER and Golgi from the developing lens; 3) determining the link between inactivation of different PI3K signaling pathways and activation of the mechanistic targets required to eliminate nuclei to form the OFZ; and 4) investigating the potential link between the ring of Akt activity at the border of cortical and nuclear fiber cells and the regulation of the outer boundary of the OFZ.