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Preventing dedifferentiation of neurons: a role for H3K9me- and HP1 associated heterochromatin?

防止神经元去分化：H3K9me 和 HP1 相关异染色质的作用？

基本信息

批准号：
BB/X00256X/1
负责人：
Tony Southall
金额：
$ 116.23万
依托单位：
Imperial College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2023
资助国家：
英国
起止时间：
2023 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FX00256X%2F1
关键词：
Preventing dedifferentiation neurons role H3K9me

项目摘要

As an organism develops, and when it repairs its tissues, stem cells divide to produce daughter cells that differentiate into specific cell types. These cells are the building blocks of the body and can have very diverse functions, such as hair follicle cells in the skin, or nerve cells (neurons) in the brain. As a cell differentiates, it shuts down expression of stem cell genes and turns on genes required for its specific function. It will also make sure that other genes (related to other functions or properties) are kept switched off. These 'epigenetic' mechanisms that prevent these genes from being expressed are very important. If silent genes are re-expressed, it can prevent the cell from functioning properly, cause cells to proliferate uncontrollably, or lead to cell death. It is becoming increasingly evident that erosion of epigenetic silencing, and the expression of unwanted genes, is a key driver of aging. Furthermore, metabolism, which is influenced by the genetic background, the environment and diet, can affect the epigenetic control of gene expression.Here, we will investigate the mechanisms that keeps neural stem cell (NSC) genes off in neurons. We have previously shown that the mutation of a specific transcription factor (a protein that binds to DNA to regulate gene expression) can cause developing neurons to have an identity crisis and revert back to a NSC-like state. This only happens in immature neurons, suggesting that there are epigenetic alterations that 'lock' NSC genes into a highly repressed state. A strong candidate is HP1 heterochromatin, which is characterised by the presence of the HP1 protein and a modification on histones (the proteins that DNA is wrapped around) called 'H3K9me'. Evidence from studies in Drosophila (fruit fly) and mouse cells show that HP1 heterochromatin is present at NSC genes in neurons. Our key hypothesis is that HP1 heterochromatin prevents neurons from 'dedifferentiating' back into NSCs. We will test this hypothesis and investigate how one-carbon metabolism, a central metabolic pathway involved in aging and histone modification, affects HP1 heterochromatin in neurons.We will first comprehensively profile HP1 heterochromatin in developing neurons, young adult neurons and old neurons. This will be done in Drosophila and mice using a technique called Targeted DamID that we developed. These data will determine whether mechanisms required for maintaining cell fate identity deteriorate with age. Then, to test our main hypothesis, we will use a cutting-edge technique called 'epigenome editing'. This utilises CRISPR-Cas9 technology (more widely known for its use in altering the sequence of DNA in the genome) that has been adapted to recruit effector proteins to specific sites in the genome. A histone demethylase will be recruited to NSC genes in neurons to remove the H3K9 methylation and prevent the formation of HP1 heterochromatin. We can then check whether NSC genes are de-repressed and whether it can cause or enhance dedifferentiation of neurons into NSCs. Finally, we will characterise and genetically manipulate one-carbon metabolism enzymes to modulate levels of SAM and SAH. These are the metabolites involved in H3K9me modification of histones and are known to change during ageing. We will assess the impact on H3K9me abundance and cell fate maintenance when the levels of these metabolites change in developing and ageing neurons. Together, these studies will provide important insights into neuronal cell fate plasticity and mechanisms that prevent ageing in the brain.

随着生物体的发育，当它修复其组织时，干细胞分裂产生分化成特定细胞类型的子细胞。这些细胞是身体的基石，可以有非常不同的功能，如皮肤中的毛囊细胞，或大脑中的神经细胞（神经元）。当细胞分化时，它会关闭干细胞基因的表达，并打开其特定功能所需的基因。它还将确保其他基因（与其他功能或属性相关）保持关闭。这些阻止这些基因表达的“表观遗传”机制非常重要。如果沉默基因重新表达，它可以阻止细胞正常运作，导致细胞不受控制地增殖，或导致细胞死亡。越来越明显的是，表观遗传沉默的侵蚀和不需要的基因的表达是衰老的关键驱动力。此外，受遗传背景、环境和饮食影响的代谢可以影响基因表达的表观遗传控制。在这里，我们将研究使神经干细胞（NSC）基因在神经元中关闭的机制。我们之前已经证明，特定转录因子（一种与DNA结合以调节基因表达的蛋白质）的突变可以导致发育中的神经元发生身份危机并恢复到神经干细胞样状态。这只发生在未成熟的神经元中，表明存在表观遗传改变，将NSC基因“锁定”在高度受抑制的状态。一个强有力的候选者是HP1异染色质，其特征是存在HP1蛋白和组蛋白（DNA包裹的蛋白质）上的修饰，称为“H3K9me”。来自果蝇和小鼠细胞研究的证据表明，HP1异染色质存在于神经元的NSC基因中。我们的关键假设是，HP1异染色质阻止神经元“去分化”回神经干细胞。我们将检验这一假设，并研究一碳代谢，一个参与衰老和组蛋白修饰的中心代谢途径，如何影响神经元中的HP1异染色质。我们将首先全面分析发育中的神经元，年轻的成年神经元和老年神经元中的HP1异染色质。这将在果蝇和小鼠中进行，使用我们开发的一种称为靶向DamID的技术。这些数据将确定维持细胞命运同一性所需的机制是否随年龄而恶化。然后，为了验证我们的主要假设，我们将使用一种称为“表观基因组编辑”的尖端技术。这利用了CRISPR-Cas9技术（因其用于改变基因组中DNA序列而广为人知），该技术已被改造为将效应蛋白募集到基因组中的特定位点。组蛋白去甲基化酶将被募集到神经元中的NSC基因以去除H3K9甲基化并防止HP 1异染色质的形成。然后，我们可以检查NSC基因是否被去抑制，以及它是否可以引起或增强神经元向NSC的去分化。最后，我们将通过基因工程和基因操作一碳代谢酶来调节SAM和SAH的水平。这些是参与组蛋白的H3K9me修饰的代谢物，并且已知在衰老过程中发生变化。我们将评估当这些代谢物的水平在发育和老化的神经元中发生变化时，对H3K9me丰度和细胞命运维持的影响。总之，这些研究将为神经元细胞命运可塑性和防止大脑衰老的机制提供重要见解。