Role of a Putative Histone Demethylase in the Ethylene Signaling Network

描述（申请人提供）：植物激素乙烯调节多种形态和生理反应，对植物生存很重要。本提案的目的是为了确定EIN6的作用，它在乙烯信号通路的一个未解决的部分中起作用。然而，在这些实验中提出的工具和方法将对植物生物学和其他许多领域产生影响。初步的序列分析表明，EIN6含有Jumonji结构域和锌指结构域，可能是一种染色质重塑酶。具体来说，EIN6包含一个与哺乳动物组蛋白去甲基化酶JARID1家族密切相关的Jumonji结构域，该家族最近被证明可以使组蛋白3赖氨酸4去甲基化（一种与活性转录相关的表观遗传标记）。组蛋白甲基化是一个特别感兴趣的领域，因为这种表观遗传修饰与癌症相关的细胞畸变有关。我们建议使用染色质免疫沉淀（ChIP）偶联靶DNA鉴定，通过Solexa测序技术作为一种在基因组水平上鉴定EIN6靶序列的无偏方法。我们提出的体外实验将确定EIN6是否具有去甲基化酶活性，这将是植物中组蛋白去甲基化酶的第一个例子，并为乙烯信号传导增加了一个有趣的调节水平。我们努力了解EIN6影响乙烯反应的机制，包括使用遗传方法鉴定与EIN6相互作用的蛋白质，以及在植物中进行遗传筛选以鉴定EIN6表型增强子。这些研究将使人们对乙烯信号转导的认识达到一个新的水平，并可能揭示新的信号成分。有了更完整的途径，我们可以开始将我们的知识应用于乙烯信号的农业和经济方面，包括水果成熟和对病原体攻击的反应。这里描述的具体目标集中在一个关键的植物突变体，是不敏感的关键生长激素，乙烯。确定这个先前未被表征的基因的作用将提高我们目前对植物中乙烯信号的理解。这是朝着我们将我们的知识应用于乙烯信号的农业和经济方面的长期目标迈出的重要一步，例如水果成熟和对病原体攻击的反应。这种感兴趣的蛋白质被预测为一种组蛋白去甲基化酶，这给我们的工作带来了一个有趣的旋转，因为这种类型的染色质修饰以前没有在植物中被描述过，并且与包括癌症在内的许多人类疾病有关。

DESCRIPTION (provided by applicant): The plant hormone ethylene regulates a variety of morphological and physiological responses important for plant survival. The aims of this proposal serve to define the role of EIN6, which functions in an unresolved section of the ethylene signaling pathway. However, tools and approaches proposed in these experiments will have implications for many areas of plant biology and beyond. Preliminary sequence analysis reveals that EIN6 contains Jumonji domains and zinc-finger domains, suggesting a role as a chromatin remodeling enzyme. Specifically, EIN6 contains a Jumonji domain that is closely related to the JARID1 family of mammalian histone demethylases, which have recently been shown to demethylate Histone 3 Lysine 4 (an epigenetic mark associated with active transcription). Histone methylation is an area of particular interest given that this epigenetic modification is connected to cellular aberrations associated with cancer. We propose the use of chromatin immunoprecipitation (ChIP) coupled to target DNA identification through Solexa sequencing technology as an unbiased approach to identify EIN6 target sequences at the genomic level. Our proposed in vitro assay will determine if EIN6 does possess demethylase activity, which would be the first example of a histone demethylase in plants and add an interesting level of regulation to ethylene signaling. Our efforts to understand the mechanism by which EIN6 influences the ethylene response includes using a genetic approach to identify proteins that interact with EIN6 and a genetic screen in planta to identify enhancers of the ein6 phenotype. These studies will bring a new level of understanding to ethylene signal transduction and may reveal novel signaling components. With a more complete view of the pathway we may begin to apply our knowledge to agricultural and economic aspects of ethylene signaling, including fruit ripening and response to pathogen attack. The Specific Aims described here are centered on a key plant mutant that is insensitive to the critical growth hormone, ethylene. Defining the role of this previously uncharacterized gene will improve our current understanding of ethylene signaling in plants. This is an important step towards our long-term goal of applying our knowledge to agricultural and economic aspects of ethylene signaling, such as fruit ripening and response to pathogen attack. This protein of interest is predicted to function as a histone demethylase, which puts an intriguing spin on our work as this type of chromatin modification has not been previously described in plants and is linked to many human diseases including cancer.