Finding the Balance: Repression of Plant Gene Expression

所有的生命在很大程度上取决于有机体设定各自基因表达水平的能力。教科书模型声称，基因调控是由转录因子(TF)控制的；转录因子是将基因组的特定区域绑定在一起，以开启或关闭目标基因表达的蛋白质。转录因子提供特异性，识别要调控的正确基因，但它们招募其他蛋白质来实际设定基因表达水平。许多转录因子通过招募被称为辅抑制子的蛋白质来关闭基因表达。辅阻遏子不能结合DNA，但一旦被特定的转运蛋白带到靶点，它们就可以关闭目标基因的表达。最近的研究反对这种简单化的模型，而是支持一个更微妙的模型，涉及到激活和抑制之间的调节平衡，以提供适当的表达水平。矛盾的是，在动物身上，抑制因子被发现是既需要抑制又需要激活的复合体的一部分。Gro/Tup1家族是一类重要的动植物辅阻遏子。在植物中，这些辅阻遏子的主要组称为TPL/TPR。TPL/TPR参与多种不同的植物过程，是植物正常生长发育的绝对基础。尽管这些调节剂是如此关键，但人们对它们的作用模式知之甚少，还有许多悬而未决的问题。越来越多的证据表明，植物TPL/TPR蛋白作为包含组蛋白脱乙酰酶(HDAC)蛋白的更大复合体的一部分，能够抑制基因表达。HDAC会改变目标基因的染色质结构，使其处于非活性状态。在动物体内，HDAC还与抑制复合体有关，其中最丰富的是核小体重塑和脱乙酰酶复合体(NuRD)。目前，还没有在植物中发现这样的复合体。动物NuRD的一些核心成分存在于植物基因组中，我们最近发现动物NuRD成分的同源物是TPL/TPR相互作用伙伴。这提出了一种有趣的可能性，即对正常植物功能如此重要的TPL/TPR，作为目前未知的植物NuRD类复合体的一部分，抑制转录。这种复合体必然不同于在动物中发现的那些复合体，因为植物缺乏一些核心的动物调节复合体成分的同源物。因此，这项建议的一个目标是分离和鉴定第一个植物TPL/TPR-HDAC复合体，既要了解它是如何控制基因表达的，又要将它与不同但相似的动物复合体进行比较。利用新发现的植物TPL/TPR-HDAC复合体的成分，或先前确定的候选成分，我们将继续利用分子遗传学来研究它们在基因表达中的作用。动物NuRD-复合体与抑制基因和活性基因的关联支持了转录调控的平衡模型。这个模型也为植物中一些令人困惑的结果提供了一个潜在的解释。因此，我们的最终目标是研究这些复合体是否同时参与植物的激活和抑制。通过对DNA结合和基因表达的全局观察，我们将能够识别与TPL/TPR辅阻遏子和/或HDAC相关的基因组区域，并将其与全基因组转录活性图谱进行比较。这一强大的方法将使我们能够量化基因组与TPL/TPR单独、HDAC单独以及两者一起的关联。然后我们可以将DNA关联分配到转录状态(激活或抑制)，在某些情况下，将其与基因的组蛋白乙酰化状态进行比较。这个项目将提供一个机制框架，在植物中建立抑制和激活之间的平衡，可以操纵这种平衡来改变植物的发育和反应。它还将揭示新的植物调控复合体是否同时参与基因表达的抑制和激活。

All life critically depends on the ability of organisms to set the expression levels of each of their genes. Textbook models claim that gene regulation is controlled by transcription factors (TFs); proteins that bind specific regions of the genome to 'turn on' or 'turn off' expression of target genes. The TFs provide specificity, identifying the correct genes to regulate, but they recruit other proteins to actually set the levels of gene expression. Many TFs turn off gene expression by recruiting proteins known as corepressors. Corepressors cannot bind DNA, but, once brought to a target by a specific TF, they can shut down expression of that target gene. More recent studies reject this simplistic model, in favour of a more nuanced model involving a regulated balance between activation and repression, to deliver the appropriate expression levels. Paradoxically then, in animals, repressive factors have been found to be part of complexes required to both repress and activate. An important class of animal and plant corepressors is the GRO/TUP1 family. The major group of these corepressors in plants is called TPL/TPR. TPL/TPRs are involved in multiple diverse plant processes and are absolutely fundamental to normal growth and development. Despite the fact that these regulators are so critical, their mode of action is poorly understood, with many open questions.There is accumulating evidence that plant TPL/TPR proteins inactivate gene expression as part of a larger complex containing histone deacetylase (HDAC) proteins. HDACs modify the chromatin structure of the target gene, rendering it inactive. In animals, HDACs are also associated with repression complexes, the most abundant of which is the nucleosome remodelling and deacetylase complex (NuRD). Currently, no such complex is known in plants. Some, but not all, of the core components of animal NuRDs are present in plant genomes and we recently identified homologues of animal NuRD components as TPL/TPR interaction partners. This raises the intriguing possibility that TPL/TPRs, which are so essential for normal plant function, repress transcription as part of a currently unknown plant NuRD-like complex. Such a complex would necessarily differ from those found in animals, because plants lack homologues of some of the core animal regulatory complex components. One aim of this proposal is therefore to isolate and characterise the first plant TPL/TPR-HDAC complex, both to understand how it controls gene expression and to compare it with the distinct, but analogous animal complexes. Using either newly identified components of the plant TPL/TPR-HDAC complex, or previously identified candidate components, we will then go on to use molecular genetics to investigate their role in gene expression.The balance model of transcriptional regulation is supported by the association of the animal NuRD-complex with both repressed and active genes. This model also provides a potential explanation for some perplexing results in plants. Our final aim is therefore to investigate whether these complexes are involved in both activation and repression in plants. By taking a global view of DNA-binding and gene expression, we will be able to identify the genomic regions that are associated with TPL/TPR corepressors and/or HDAC and to compare this to a genome-wide map of transcriptional activity. This powerful approach will allow us to quantify the genome association with TPL/TPR alone, HDAC alone and both together. We can then assign DNA association to a transcriptional status (activated or repressed) and, in some cases, compare this to the histone acetylation status of the gene.This project will provide a mechanistic framework for establishing a balance between repression and activation in plants that could be manipulated to alter plant development and responses. It will also reveal whether the novel plant regulatory complexes are involved in both repression and activation of gene expression.