Mechanisms of transcriptional regulation by the histone variant H2A.Z

 描述(申请人提供)：生物体适当地调节发育、维持动态平衡和适当地对环境刺激作出反应的能力取决于其动态调节转录的能力。一种形式的转录调控涉及通过改变周围的染色质结构，例如将组蛋白变体掺入核小体，来改变DNA对转录机器的可及性。高度保守的组蛋白H_2A变异体H_2A.Z在不同的环境中既可以作为转录的激活因子，也可以作为转录的抑制因子；然而，H_2A_Z相反作用背后的机制目前尚不清楚。在成熟的拟南芥植物模型中，H2A.Z需要转录激活FLC等基因，FLC是一种发育开关基因，可以抑制从营养发育到生殖发育的转变。通过确定和表征在转录激活FLC中拮抗H2A.Z的因素，我们将更好地理解H2A.Z作为转录正负调控因子的机制。BRM是一种染色质重塑复合亚基，它的突变降低了FLC转录激活中对H2A.Z的需求。我们假设，H2A.Z通过破坏基因座调控区域的核小体的稳定来激活基因，如FLC，而转录抑制因子，如BRM，通过稳定或滑动核小体来取代被H2A.Z取代的核小体来拮抗H2A.Z的功能并抑制转录。本项目的特定目标1将解决核小体稳定性或组成的变化是否能解释在BRM突变体或将H2A.Z结合到核小体缺陷的突变体中观察到的转录变化。不能将H2A.Z结合到FLC基因座的核小体中的突变体很早就开花了，因为它们不激活FLC转录。在特定的目标2中，我们将使用来自正向遗传抑制因子筛选的突变体来抑制早花表型并恢复FLC转录，以检测额外的转录抑制因子，这些转录抑制因子在FLC中拮抗H2A.Z功能。将使用全基因组测序来绘制抑制突变的图谱，并将识别和表征拮抗H2A.Z功能的原因基因。识别抑制子将使我们能够改进我们的模型，解释为什么转录激活需要H2A.Z。本项目利用BRM和H2 A.Z之间独特的遗传相互作用，在拟南芥中显示，以便通过确定特定的转录抑制因子如何拮抗H2 A.Z功能来进一步了解H2 A.Z的功能。结果将解决我们的长期研究目标，即了解组蛋白变体的加入如何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洞察，由于H2A.Z已与乳腺癌、膀胱癌和胰腺癌有关，它将为治疗转录过程调控错误的疾病提供潜在的靶点，如这些癌症。

 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.