Regulatory role of PI3K signaling pathways in lens differentiation and function

PI3K信号通路在晶状体分化和功能中的调节作用

• 批准号: 10580706
• 负责人: Marc Kantorow
• 金额: $ 51.83万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10580706
• 关键词: AKT Signaling Pathway; AKT inhibition; ATAC-seq; Anterior; Autophagocytosis; Binding; Binding Sites; Biological Assay; Catalytic Domain; Cell Differentiation process; Cell Nucleus; Cell physiology; Cells; Chick Embryo; Chromatin; Complex; Consensus; Crystallins; Cytoplasm; DNA; DNA Binding; Development; Down-Regulation; EPHA2 gene; Epithelial Cells; Epithelium; Event; Excision; Exhibits; FYCO1 gene; Family; Funding; Gene Expression; Gene Expression Profile; Genes; Genome; Golgi Apparatus; Grant; High-Throughput RNA Sequencing; Image; Individual; Investigation; Lens Fiber; Lens development; Light; Link; MLLT7 gene; Maps; Microdissection; Mitochondria; Modeling; Nuclear; Nuclear Translocation; Nucleic Acid Regulatory Sequences; Organ; Organelles; PI3K/AKT; PIK3CG gene; Pathway interactions; Pattern; Phenotype; Phosphorylation; Play; Process; Regulation; Retina; Role; Signal Induction; Signal Pathway; Signal Transduction; Tissues; Undifferentiated; cell type; fiber cell; gene induction; genome-wide; lens; lens induction; lens transparency; programs; spatiotemporal; transcription factor; transcriptome sequencing

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.

使用ATACseq和RNAseq的组合方法，我们的研究提供了第一个证据，表明 在染色质可及性是至关重要的分化状态特异性表达的各种基因 对于从透镜上皮细胞到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位点可能调节广泛的透镜基因，最重要的是 FOXO 4。在透镜细胞分化中具有必要作用的基因，其包含共有DNA结合序列 FOXO 4的主要基因包括EPHA 2、NrCAM、δ-晶状体蛋白、Notch 1和FYCO 1。FOXO家族，包括FOXO 4， 由PI 3 K/Akt信号通路调节。PI 3 K/Akt对FOXO 4的磷酸化将其隔离在 细胞质和PI 3 K/Akt信号的抑制是FOXO 4输入到细胞核所必需的， 调节基因表达。FOXO 4的功能从未在透镜中检查过。我们建议 确定PI 3 K/AKT对透镜纤维细胞FOXO 4依赖性基因表达的调控作用 分化这将通过以下方式实现：1）确定细胞核增殖对PI 3 K/Akt抑制的需求， FOXO 4在透镜上皮细胞向纤维细胞转变过程中的易位; 2）建立 PI 3 K/Akt抑制和含有FOXO 4结合的透镜分化特异性基因的表达 3）证明FOXO 4与不同的染色质可及DNA调控区的结合， 使用靶向-CHIP测定对透镜分化至关重要的基因;和4）建立光谱、范围和 透镜细胞分化过程中FOXO 4调控基因的空间表达模式。我们还发现 PI 3 K/Akt信号传导轴在调节去除细胞凋亡的时间和机制中起着重要作用。 线粒体，ER和高尔基体从中央光路和多个PI 3 K下游信号通路 是消除细胞核以形成透镜无细胞器区（OFZ）的过程所必需的。这个过程是 这是形成能够将光图像聚焦在视网膜上的成熟透镜所需要的。我们将探讨PI 3 K如何 信号通路调节细胞核和其他细胞器的消除以形成无细胞器的透镜 区域的研究旨在1）确定单个PI 3 K p110催化亚基在调节 2）证实在抑制PI 3 K/Akt信号传导后诱导自噬 轴负责从发育中的透镜中去除线粒体、ER和高尔基体; 3）确定 不同PI 3 K信号通路的失活与所需机制靶点的激活之间的联系 消除细胞核以形成OFZ;以及4）研究在细胞核中Akt活性环与细胞核中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。