Mechanisms of transcriptional regulation by the histone variant H2A.Z

 描述（由申请人提供）：生物体适当调节发育、维持稳态和适当响应环境刺激的能力取决于其动态调节转录的能力。转录调控的一种形式涉及通过改变周围的染色质结构来改变DNA对转录机制的可及性，使用诸如将组蛋白变体并入核小体的机制。高度保守的组蛋白H2 A变体H2A.Z在不同的情况下充当转录的激活子或抑制子;然而，H2A.Z的相反作用背后的机制目前尚不清楚。在成熟的植物模型拟南芥中，需要H2A.Z来转录激活基因，如FLC，一种抑制从营养向生殖发育转变的发育开关基因。通过识别和表征拮抗H2A.Z在FLC转录激活中作用的因子，我们将更好地了解H2A.Z如何充当转录的正调节因子和负调节因子的机制。BRM是一种染色质重塑复合物亚基，BRM中的突变减轻了FLC转录激活中对H2A.Z的需求。我们假设H2A.Z通过使基因座的调节区的核小体不稳定来激活基因，如FLC，而转录抑制因子，如BRM，拮抗H2A.Z的功能，并通过稳定或滑动核小体以取代被H2A.Z取代的核小体来抑制转录。该项目的具体目标1将解决核小体稳定性或组成的变化是否可以解释在brm突变体或将H2A.Z掺入核小体中有缺陷的突变体中观察到的转录变化。不能将H2A.Z掺入FLC基因座处的核小体中的突变体提早开花，因为它们不激活FLC转录。在具体目标2中，我们将使用来自抑制早花表型并恢复FLC转录的正向遗传抑制筛选的突变体来检测拮抗FLC中H2A.Z功能的其他转录抑制因子。将使用全基因组测序绘制抑制突变图谱，并将鉴定和表征拮抗H2A.Z功能的致病基因。识别抑制因子将使我们能够完善我们的模型，了解为什么转录激活需要H2A.Z。该项目利用拟南芥中BRM和H2A.Z之间独特的遗传相互作用，通过确定特定的转录抑制因子如何拮抗H2A.Z功能来进一步了解H2A.Z功能。结果将解决我们的长期研究目标，了解如何纳入组蛋白变体调节转录调控的重要过程。这项工作将提供对转录调控机制的深入了解，由于H2A.Z与乳腺癌、膀胱癌和胰腺癌有关，它将为治疗转录过程失调的疾病（如这些癌症）提供潜在的治疗靶点。

 DESCRIPTION (provided by applicant): The ability of an organism to properly regulate development, maintain homeostasis and appropriately respond to environmental stimuli depends on its ability to dynamically regulate transcription. One form of transcriptional regulation involves changing how accessible DNA is to transcriptional machinery by making changes to the surrounding chromatin structure, using mechanisms such as incorporating histone variants into nucleosomes. The highly conserved histone H2A variant, H2A.Z, acts as either an activator or repressor of transcription in different contexts; however, the mechanisms behind the opposing roles of H2A.Z are presently unclear. In the well-established plant model Arabidopsis thaliana, H2A.Z is required to transcriptionally activate genes such as FLC, a developmental switch gene that represses the transition from vegetative to reproductive development. By identifying and characterizing factors that antagonize H2A.Z in its role in transcriptional activation of FLC, we will better understand the mechanisms of how H2A.Z acts as both a positive and negative regulator of transcription. Mutations in BRM, a chromatin remodeling complex subunit, alleviate the requirement for H2A.Z in transcriptional activation of FLC. We hypothesize that H2A.Z activates genes, such as FLC, by destabilizing nucleosomes at regulatory regions of a locus, while transcriptional repressors, such as BRM, antagonize the function of H2A.Z and inhibit transcription by stabilizing or sliding nucleosomes to replace those that were displaced by H2A.Z. Specific aim 1 of this project will address whether changes in nucleosome stability or composition can explain the changes in transcription observed in brm mutants or mutants defective in incorporating H2A.Z into nucleosomes. Mutants that cannot incorporate H2A.Z into nucleosomes at the FLC locus flower early because they do not activate FLC transcription. In specific aim 2, we will use mutants from a forward genetic suppressor screen that suppress the early flowering phenotype and restore FLC transcription to detect additional transcriptional repressors that antagonize H2A.Z function at FLC. Suppressor mutations will be mapped using whole genome sequencing and the causal genes that antagonize H2A.Z function will be identified and characterized. Identifying suppressors will allow us to refine our model about why H2A.Z is needed for transcriptional activation. This project takes advantage of a unique genetic interaction between BRM and H2A.Z, shown in Arabidopsis, in order to further understand H2A.Z function through determining how specific transcriptional repressors antagonize H2A.Z function. Results will address our long-term research goal of understanding how the incorporation of histone variants regulates the vital process of transcriptional regulation. This work will provide insight into transcriptional regulatin mechanisms and, since H2A.Z has been implicated in breast, bladder, and pancreatic cancer, it will provide potential targets for therapies to treat diseases in which transcriptional processes are misregulated, such as these cancers.