Gene-regulatory networks in podocytes in health and disease

足细胞是维持肾脏过滤功能的关键。足细胞的损伤导致尿蛋白排泄，这反过来又导致肾脏疾病的进展。在对损伤的反应中，足细胞通过改变其基因表达模式和激活向细胞核发出信号的途径来采用涉及细胞核的机制。然而，尽管对遗传性足细胞疾病由核蛋白如WT 1突变引起的认识由来已久，但对足细胞中的这种核机制还没有很好的理解。在这种情况下，转录因子（TF）WT1起着重要的作用，因为它是足细胞发育和维持不可或缺的。因此，我们以前的特点是WT1依赖的足细胞基因调控网络在小鼠体内。在这个网络中，WT1作为先驱TF，并与其他足细胞TF的基因如Mafb和Lmx1b结合。此外，在足细胞疾病模型中，与足细胞损伤途径相关的基因被WT1差异性地结合。基于这些发现，我们现在的目标是测试的假设，在健康的足细胞，WT1编排进一步的足细胞TF的结合，这反过来又承担特定功能的足细胞生理。为此，我们将采用染色质免疫沉淀（ChIPseq）来表征与足细胞相关的各种TF在全基因组中的结合位点。通过RNA测序（RNAseq）对小鼠足细胞进行基因表达分析将补充这一数据，并确定TF对定义足细胞组分的特异性基因调控作用。通过生物信息学分析将这些数据集与现有的WT1网络整合，将允许详细了解TF在足细胞维护中的功能以及遗传性足细胞疾病的发病机制。在第二种方法中，我们的目标是研究WT1和生理足细胞TF网络如何与足细胞损伤后激活的通路的核效应物相互作用。为此，我们将使用遗传和毒性足细胞损伤的小鼠模型，并通过ChIPseq在足细胞疾病的早期和晚期评估效应子和WT1结合位点。同时，我们将通过RNAseq评估足细胞基因表达随疾病的变化。所有数据集的生物信息学分析将使我们能够识别生理和病理基因调控网络之间的依赖关系，并在系统生物学方法中在核水平上表征疾病阶段特异性足细胞反应。我们期望获得显着的洞察足细胞损伤后的基因调控网络的重编程WT1以及进一步TF相关的足细胞维护。此外，我们将确定这种重编程对足细胞信号通路的影响，并确定其在足细胞损伤中的作用。因此，这些发现将阐明足细胞疾病的既定和新机制。

Podocytes are key to maintaining the filtration function of our kidneys. Damage to podocytes results in urinary protein excretion, which in turn leads to progression of kidney disease. In response to damage, podocytes employ mechanisms involving the cell nuclei by changing their gene expression pattern and by activating pathways that signal to the nucleus. However, despite longstanding knowledge of hereditary podocyte disease caused by mutations in nuclear proteins such as WT1, such nuclear mechanisms in podocytes are not well understood. In this context, the transcription factor (TF) WT1 plays an important role as it is indispensable to podocyte development and maintenance. Thus, we have previously characterized the WT1-dependent podocyte gene regulatory network in mice in vivo. In this network, WT1 acts as a pioneering TF and is bound to the genes of further podocyte TF such as Mafb and Lmx1b. Furthermore, in a podocyte disease model, genes relevant to podocyte damage pathways are differentially bound by WT1. Based on these findings, we now aim to test the hypothesis that in healthy podocytes, WT1 orchestrates the binding of further podocyte TFs, which in turn assume specific functions in podocyte physiology. To this end, we will employ chromatin immunoprecipitation (ChIPseq) to characterize binding sites various TFs relevant to podocytes in genome wide fashion. Gene expression analysis of mouse podocytes by RNA sequencing (RNAseq) will complement this data and identify TF-specific gene regulatory effects on defined podocyte components. The integration of these datasets with the existing WT1 network by bioinformatic analyses will allow for detailed insight into the function of TFs in podocyte maintenance as well as pathogenetic mechanisms in hereditary podocyte disease. In a second approach, we aim to investigate how WT1 and the physiologic podocyte TF network interact with the nuclear effectors of pathways activated upon podocyte damage. To this end, we will use mouse models of genetic and toxic podocyte damage and assess effector and WT1 binding sites by ChIPseq at an early and late stage of podocyte disease. In parallel, we will assess podocyte gene expression changes with disease by RNAseq. Bioinformatic analyses of all datasets will the enable us to identify dependencies between physiologic and pathologic gene regulatory networks, and to characterize a disease stage specific podocyte response on a nuclear level in a systems biology approach. We anticipate gaining significant insight into reprogramming of gene regulatory networks upon podocyte damage by both WT1 as well as further TFs relevant to podocyte maintenance. Furthermore, we will identify the effects of such reprogramming focusing on podocyte signaling pathways with an established role in podocyte damage. These findings will therefore shed light on established and novel mechanisms in podocyte disease.