The Role and Mechanisms of Action of PML II on Regulating Gene Expression

PML II调节基因表达的作用及机制

• 批准号: 1643098
• 金额: --
• 依托单位: University of Warwick
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1643098
• 关键词: Role; Mechanisms; Action; PML; II

Promyelocytic leukaemia protein (PML), initially identified in acute promyelocytic leukaemia (APL) cancer patients, is a pleiotropic protein implicated in a range of cellular functions such as regulating transcription, defence against viral infection and control of apoptosis and cell growth. The PML gene consists of nine exons giving rise to several different splice-variant transcripts. The isoforms are divided into seven groups, PML I - VII, and all contain an identical N-terminal region encoded by exons 1 to 6 including a RING-finger, two B-box and a Coiled-Coil motif. The differences in isoform structure leads to variation in isoform activity. PML nuclear bodies (NBs) are organised by the PML protein and recruit numerous partner proteins to regulate cellular activities.PML-II has been shown to be required for efficient interferon response gene expression, and transcription and modification of chromatin architecture are two functions controlled by PML NBs. This project aims to understand how PML-II functions in the IFN response, focusing on events at the IFNBeta promoter. In uninfected cells, the IFNBeta gene is constitutively repressed, however, upon viral infection the gene is transiently activated. ChIP assays have shown there is a direct correlation between acetylation at histone H4 and the transcriptional activity of the IFNBeta promoter region. While repressed, the IFNBeta promoter region was hypoacetylated, silencing the IFNBeta genes. Upon inhibition of histone deacetylase, hyperacetylation of histone H4 occurs at the IFNBeta promoter leading to increased IFN levels, conferring the antiviral state. Other modifications found in the IFNBeta promoter site, such as methylations on a subset of lysine residues on histones H3 and H4 occur during activation of gene transcription. I will investigate these modifications at the IFNBeta gene to look for markers of transcription activation, such as histone 3 lysine 4 trimethylation (H3K4me3) and modifications for repression of transcription such as histone 3 lysine 27 trimethylation (H3K27me3). The balance between H3K4me3 verses H3K27me3 may indicate the transcriptional activity at the genes of interest. By looking at differences that arise when PMLII has been reduced, we will identify functions associated with histone modifications that involve PML. Class switching of whole histones also occurs during activation of gene transcription. Eukaryotic DNA is packed into nucleosomes composed of 4 different histone proteins: H2A, H2B, H3 and H4. There are 5 variants of H3 histone protein, the main two being H3.1 and H3.3. H3.1 is enriched in regions of transcriptional repression and coincides with H3K27me3 sites. However, at regions with high transcriptional activity, H3.3 is more abundant. There is a suggested role for the PML protein in class switching from H3.1 to H3.3 via its interactions with death-associated protein 6 (DAXX) at SUMOylated sites on PML in PML NBs. PML opposes H3.3 histone deposition by DAXX and loss of PML alters chromatin composition, shifting histone modifications to repressive H3K27me3 markers and increased H3.3 deposition. The exchange of H3.1 to H3.3 at the IFN-Beta promoter is something I will investigate in wildtype and knockdown models of PMLII.

早幼粒细胞白血病蛋白（PML）最初在急性早幼粒细胞白血病（APL）癌症患者中鉴定，是一种涉及一系列细胞功能的多效性蛋白，例如调节转录、防御病毒感染以及控制细胞凋亡和细胞生长。PML基因由九个外显子组成，产生几种不同的剪接变体转录本。亚型分为7组，PML I-VII，并且都含有由外显子1至6编码的相同的N-末端区域，包括环指、两个B-盒和卷曲螺旋基序。异构体结构的差异导致异构体活性的变化。PML核体（NB）由PML蛋白组织并募集许多伴侣蛋白来调节细胞活性，PML-II已被证明是有效的干扰素应答基因表达所必需的，并且转录和染色质结构的修饰是由PML NB控制的两个功能。该项目旨在了解PML-II在IFN应答中的功能，重点关注IFN β启动子的事件。在未感染的细胞中，IFN β基因被组成性抑制，然而，在病毒感染时，该基因被瞬时激活。ChIP分析显示组蛋白H4的乙酰化与IFN β启动子区域的转录活性之间存在直接相关性。当抑制时，IFN β启动子区域低乙酰化，使IFN β基因沉默。在抑制组蛋白脱乙酰酶后，组蛋白H4在IFN β启动子处发生超乙酰化，导致IFN水平增加，从而赋予抗病毒状态。在IFN β启动子位点发现的其他修饰，如组蛋白H3和H4上赖氨酸残基亚组的甲基化，发生在基因转录激活期间。我将研究IFN β基因的这些修饰，以寻找转录激活的标志物，如组蛋白3赖氨酸4三甲基化（H3 K4 me 3）和抑制转录的修饰，如组蛋白3赖氨酸27三甲基化（H3 K27 me 3）。H3 K4 me 3与H3 K27 me 3之间的平衡可以指示在感兴趣的基因处的转录活性。通过观察PMLII减少时出现的差异，我们将确定与涉及PML的组蛋白修饰相关的功能。整个组蛋白的类别转换也发生在基因转录的激活过程中。真核DNA被包装在由4种不同的组蛋白组成的核小体中：H2 A，H2 B，H3和H4。H3组蛋白有5种变体，主要的两种是H3.1和H3.3。H3.1在转录抑制区域富集，并且与H3 K27 me 3位点一致。然而，在具有高转录活性的区域，H3.3更丰富。在PML NB中，PML蛋白通过与死亡相关蛋白6（DAXX）在PML上SUMO化位点的相互作用，在从H3.1到H3.3的类别转换中发挥作用。PML通过DAXX对抗H3.3组蛋白沉积，并且PML的丧失改变染色质组成，将组蛋白修饰转移到抑制性H3 K27 me 3标记物并增加H3.3沉积。在IFN-β启动子处H3.1到H3.3的交换是我将在PMLII的野生型和敲低模型中研究的。