Epigenic Control of ENaC Transcription and Sodium Transport

描述(由申请人提供)：该项目的主要目标是以ENaC(上皮钠通道)为模型，确定在mIMCD3细胞和小鼠肾脏中控制钠稳态的新的表观遗传机制。ENaC突变与利德尔综合征和PHA-1(假性低醛固酮增多症1型)的相关性以及醛固酮对ENaC的紧密而复杂的调节表明ENaC在钠平衡和血压调节中的重要性。醛固酮是钠吸收的主要调节剂，主要通过在多个水平上控制ENaC功能发挥作用，包括转录和细胞表面表达。在经典模型中，醛固酮通过激活两种不同但相似的核受体(核受体)来增强转录：MR和GR(盐皮质激素和糖皮质激素受体)。小鼠MR的破坏导致PHA-1和动物在出生后第10天左右死亡。最近，我们报道了另一种信号通路，通过一系列事件，包括组蛋白H3 K79甲基转移酶Dot1a(端粒沉默干扰物1)和可能的转录因子AF9(9号染色体上的ALL1融合基因)的减少，SGK1(血清和糖皮质激素调节的激酶)介导的AF9的磷酸化，从而下调Dot1a-AF9的相互作用，从而下调Dot1a-AF9的相互作用，并在mIMCD3细胞的ENaC1启动子上靶向组蛋白H3 K79的低甲基化。尽管有这些发现，但NR/醛固酮和Dot1a/AF9复合体之间可能的拮抗作用仍未解决；醛固酮刺激的ENaC转录和ENaC活性之间的相关性仍未很好地确定；导致PHA-1的MR干扰所产生的分子缺陷仍不清楚。在这项提案中，我们打算填补这些空白。在目标1中，我们将验证一种假设，即NR-醛固酮和Dot1a-AF9复合体通过MR-AF9和/或GR-AF9相互作用来动态控制mIMCD3细胞中ENaC1的转录，从而相互抑制对手的DNA结合活性：目标2将验证ENaC转录变化转化为mIMCD3细胞中ENaC活性变化的假设；而Aim 3将对MR-/-小鼠进行更全面的研究，以检验MR缺乏对我们新的醛固酮网络中的组件和其他ENaC调节因子的调节被解除的假设，这有助于PHA-1的发展，并证实这两个复合体对ENaC转录和活性的相互拮抗作用，如目标1和2所定义的。因此，拟议的研究将1)整合醛固酮作用的两种模式：激活NR和解除Dot1a-AF9介导的抑制；2)将醛固酮作用与染色质介导的ENaC转录激活、增强的ENaC活性和Na运输联系起来；以及3)揭示PHA-1发展的分子机制。 公共卫生相关性 钠平衡对血压控制很重要，主要是通过类固醇激素醛固酮对ENaC的分子作用实现的，ENaC是一种离子通道，将Na通过细胞膜输送到细胞内。这项拟议的研究旨在利用一个现有的基因工程小鼠模型，确定将醛固酮作用与染色质修饰、ENaC转录激活和增强小鼠肾脏钠摄取联系起来的新机制。

DESCRIPTION (provided by applicant): The broad goal of this project is to define novel epigenetic mechanisms controlling Na+ homeostasis in mIMCD3 cells and in mouse kidney, with ENaC (epithelial sodium channel) as the model. The association of ENaC mutations with Liddle's syndrome and PHA-1 (pseudohypoaldosteronism type 1) as well as the tight and complex regulation of ENaC by aldosterone indicates the importance of ENaC in the regulation of Na+ balance and blood pressure. Aldosterone is a major regulator of Na+ absorption and acts primarily by controlling ENaC function at multiple levels, including transcription and cell surface expression. In the classical model, aldosterone enhances transcription by activating two distinct but similar types of NR (nuclear receptors): MR and GR (mineralocorticoid and glucocorticoid receptors). MR disruption in mouse leads to PHA-1 and animal death around day 10 after birth. Recently, we reported an alternative signaling pathway that couples aldosterone action to chromatin modifications and ENaC1 transcriptional activation through a series of events including reduction of histone H3 K79 methyltransferase Dot1a (disruptor of telomeric silencing 1) and putative transcription factor AF9 (ALL1-fused gene from chromosome 9), SGK1 (serum and glucocorticoid regulated kinase)-mediated phosphorylation of AF9 and thus downregulation of Dot1a-AF9 interaction, and targeted histone H3 K79 hypomethylation at the ENaC1 promoter in mIMCD3 cells. Despite these findings, the putative antagonism between the NR/aldosterone and Dot1a/AF9 complexes remains unaddressed; the correlation between aldosterone-stimulated ENaC transcription and ENaC activity is still not well defined; and the molecular defects derived from MR disruption that result in PHA-1 remain elusive. In this proposal, we intend to fill these gaps. In Aim 1, we will test the hypothesis that the NR-aldosterone and Dot1a-AF9 complexes mutually inhibit the DNA binding activity of their opponent through MR-AF9 and/or GR-AF9 interactions to dynamically control ENaC1 transcription in mIMCD3 cells; Aim 2 will test the hypothesis that changes in ENaC transcription translate into changes in ENaC activity in mIMCD3 cells; and Aim 3 will examine MR-/- mice more fully to test the hypothesis that the components in our new aldosterone network and other ENaC regulatory factors are deregulated by MR deficiency, which contributes to the development of PHA-1, and to confirm the mutual antagonism of the two complexes on ENaC transcription and activity as defined in Aim 1 and 2. Therefore, the proposed studies will 1) integrate the two modes of aldosterone action: activation of NR and relief of Dot1a-AF9-mediated repression; 2) link aldosterone action to chromatin-mediated ENaC transcriptional activation, enhanced ENaC activity and Na+ transport; and 3) shed new light on molecular mechanisms of PHA-1 development. PUBLIC HEALTH RELEVANCE Na+ balance is important for blood pressure control and is primarily achieved by the molecular action of the steroid hormone aldosterone on ENaC, an ion channel that transports Na+ through the cell membrane into the cells. The proposed studies are aimed to define novel mechanisms linking aldosterone action to chromatin modifications, transcriptional activation of ENaC, and enhanced Na+ uptake in mouse kidney, using one existing genetically engineered mouse model.