Transcriptional control of immune-metabolic functional switching during the innate immune response

免疫和新陈代谢是紧密相连的过程。感染或持续的免疫激活可能会对新陈代谢造成重大干扰，反之，新陈代谢障碍可能会对免疫产生深远影响。因此，更好地了解新陈代谢和免疫之间的相互作用对传染病和代谢性疾病的治疗具有重要意义。模式生物黑腹果蝇已被广泛用于研究免疫代谢关系。在像人类这样的高等生物中，新陈代谢和免疫功能是由独立的器官执行的。然而，昆虫有一个独特的脂肪身体，协调免疫和新陈代谢功能。在健康的果蝇中，脂肪身体是糖原和甘油三酯的储存场所。在感染期间，主要的转录变化被诱导，导致许多代谢基因的下调和免疫基因的诱导，如抗菌肽。在确定参与这种转录免疫-代谢转换的特定转录因子方面已经取得了进展，但尚未获得基因组规模的了解。该项目的目的是描述感染期间脂肪体的转录状态，包括基因表达和基因组结构的全球变化。为了实现这一点，将执行目标大坝ID(TADA)。这项技术依赖于细菌酶DNA腺嘌呤甲基酶(DAM)。当在体内表达时，Dam将其附近的任何GATC序列甲基化。由于黑腹果蝇没有内源性DNA甲基化，对这些甲基化标记的鉴定揭示了Dam与基因组2的相互作用。由于DNA必须能够被Dam访问才能被甲基化，甲基化特征将给出染色质可访问性的指示。此外，DAM可以与RNA聚合酶II的催化亚单位(POL II)融合。当表达Dam-Pol II结构时，Dam将被特异性地招募到Pol II结合部位。因此，这些甲基化图谱将给出哪些基因可能被转录的指示。通过驱动Dam结构在脂肪体中的特异性表达，并比较感染和未感染果蝇的图谱，将有可能确定感染如何改变基因表达和基因组结构。塔达实验的结果将决定该项目的进展情况。在感染期间，免疫或代谢基因的启动子之间可能会观察到染色质可及性的差异。Dam-Pol II的数据将揭示这是否与任何染色质修饰物表达的变化有关，然后可以进一步研究。或者，Dam-Pol II数据可能揭示了几个相关基因表达的变化。计算技术可以用来预测负责驱动观察到的变化的关键转录因子。然后，可以进行存活分析、细菌数量测定和代谢组学，以确定所涉及的染色质修饰物和转录因子在感染过程中的真实生物学功能。Clark，R.I.等人。MEF2是体内的一种免疫代谢开关。电话155,435-447(2013)。2.马歇尔，O.J.等人。使用具有下一代测序的靶向DAMID，无需细胞分离即可对蛋白质-DNA相互作用进行特定细胞类型的分析。纳特。普罗托克。11,1586-98(2016)。

Immunity and metabolism are tightly interlinked processes. Infection or persistent immune activation can cause major disruptions to metabolism, and conversely, disturbances to metabolism can have profound effects on immunity. Better understanding the interplay between metabolism and immunity therefore has implications for treatment of both infectious and metabolic diseases. The model organism Drosophila melanogaster has been widely used to study the immune-metabolic relationship. In higher organisms like humans, metabolic and immune functions are carried out by independent organs. However, insects have a unique fat body which orchestrates both immune and metabolic functions. In healthy flies, the fat body acts as a storage site for glycogen and triglycerides. During infection, major transcriptional changes are induced which result in downregulation of numerous metabolic genes and the induction of immune genes like antimicrobial peptides. Progress has been made in identifying specific transcription factors that contribute to this transcriptional immune-metabolic switch1, but a genome-scale understanding is yet to be obtained. The aim of this project is to profile the transcriptional state of the fat body during infection, including global changes in gene expression and genome structure. To achieve this, Targeted Dam ID (TaDa) will be performed. This technique relies on the bacterial enzyme DNA adenine methylase (Dam). When expressed in vivo, Dam methylates any GATC sequences in its vicinity. Since D. melanogaster do not have endogenous DNA methylation, identification of these methylation markings reveals where Dam has interacted with the genome2. Since DNA must be accessible to Dam to be methylated, methylation profiles will give an indication of chromatin accessibility. Further, Dam can be fused to the catalytic subunit of RNA polymerase II (Pol II). When the Dam-Pol II construct is expressed, Dam will be specifically recruited to Pol II binding sites. These methylation profiles will therefore give an indication of which genes are likely being transcribed. By driving expression of Dam constructs specifically in the fat body and comparing the profiles of infected and uninfected flies, it will be possible to determine how gene expression and genome structure are altered by infection. The results of the TaDa experiment will determine how the project progresses. Differences in chromatin accessibility may be observed between the promoters of immune or metabolic genes during infection. The Dam-Pol II data will reveal whether this correlates with a change in the expression of any chromatin modifiers, which could then be further investigated. Alternatively, the Dam-Pol II data may reveal changes in expression of several related genes. Computational techniques could be used to predict key transcription factors responsible for driving the observed changes. Survival assays, bacterial quantification and metabolomics could then be performed to determine the true biological functions of the implicated chromatin modifiers and transcription factors during infection.References1. Clark, R. I. et al. MEF2 is an in vivo immune-metabolic switch. Cell 155, 435-447 (2013). 2. Marshall, O. J., et al. Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing. Nat. Protoc. 11, 1586-98 (2016).