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导致代谢的重大破坏，相反，代谢的干扰可对免疫产生深远影响。因此，更好地理解代谢和免疫之间的相互作用对感染性疾病和代谢性疾病的治疗都有意义。 模式生物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，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体，协调免疫和代谢功能。在健康的果蝇中，脂肪体充当糖原和甘油三酯的储存场所。在感染过程中，诱导主要的转录变化，这导致许多代谢基因的下调和免疫基因如抗微生物肽的诱导。在确定有助于这种转录免疫代谢开关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理解。该项目的目的是分析感染过程中脂肪体的转录状态，包括基因表达和基因组结构的整体变化。为了实现这一点，将进行目标母兽ID（TaDa）。该技术依赖于细菌酶DNA腺嘌呤甲基化酶（Dam）。当在体内表达时，Dam将其附近的任何GATC序列甲基化。自D.黑腹果蝇没有内源性DNA甲基化，这些甲基化标记的鉴定揭示了Dam与基因组相互作用的位置2。由于DNA必须能够被Dam甲基化，因此甲基化图谱将指示染色质可及性。此外，Dam可以与RNA聚合酶II（Pol II）的催化亚基融合。当表达Dam-Pol II构建体时，Dam将被特异性募集到Pol II结合位点。因此，这些甲基化图谱将指示哪些基因可能被转录。通过驱动Dam构建体在脂肪体中的特异性表达并比较感染和未感染果蝇的概况，将有可能确定基因表达和基因组结构如何被感染改变。TaDa实验的结果将决定该项目的进展。在感染过程中，可以在免疫或代谢基因的启动子之间观察到染色质可及性的差异。Dam-Pol II数据将揭示这是否与任何染色质修饰剂的表达变化相关，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。计算技术可用于预测负责驱动观察到的变化的关键转录因子。然后可以进行存活测定、细菌定量和代谢组学以确定感染期间所涉及的染色质修饰剂和转录因子的真实生物学功能。克拉克河I. MEF2是体内免疫代谢开关。Cell 155，435 - 447（2013）. 2.马歇尔，O. J.，等人，Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing.国家方案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).