Transcriptional control of immune-metabolic functional switching during the innate immune response
Lead Research Organisation:
Imperial College London
Department Name: Life Sciences
Abstract
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.
References
1. 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).
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.
References
1. 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).
Organisations
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M011178/1 | 01/10/2015 | 25/02/2025 | |||
| 1948951 | Studentship | BB/M011178/1 | 30/09/2017 | 23/12/2021 |
| Description | Immunity and metabolism are highly interconnected processes. The aim of this project was to better understand how metabolism is altered during infection of Drosophila melanogaster, and the mechanisms by which this is regulated transcriptionally. Targeted DamID was used to generate a transcriptional profile of the fat body, an organ which orchestrates both immune and metabolic functions, during infection. Two metabolic genes with potentially linked roles in amino acid and purine metabolism, Nmdmc and astray, were found to be upregulated. We discovered that this upregulation is dependent on Dif, an immune-activated Nf-kB transcription factor, and Foxo, a major metabolic regulator. We have data that suggest that Dif and Foxo may act in a co-ordinated manner to drive Nmdmc and astray upregulation, and we are in the process of using confocal microscopy to investigate this further. We have also identified that MEF2, a transcription factor which is known to regulate both immune and metabolic genes, likely contributes to Nmdmc and astray repression in healthy flies. The second aspect of this project has been investigating the functional role of Nmdmc and astray during infection. We found that fat body knock down of astray led to a survival defect in response to both Staphylococcus aureus and Enterococcus faecalis infection, whilst bacterial numbers remained unchanged. To investigate Nmdmc function, we made a CRISPR knock out of the mitochondrial isoform, which we had previously identified as the only isoform upregulated during infection. These mitochondrial Nmdmc mutants showed improved survival during S. aureus infection, and a lower bacterial load. However the mutants did show transcriptional compensation from the cytosolic isoforms of Nmdmc, so we have generated full CRISPR mutants lacking all Nmdmc isoforms. Immune phenotyping of these mutants will begin shortly. Together, this data has furthers our understanding of the metabolic changes that are induced by infection, and demonstrates the complex impact of these changes on infection outcome. |
| Exploitation Route | Metabolic disruption can be a major cause of pathology during infection. For example, infection with Mycobacterium tuberculosis results in dramatic lethal wasting of fat tissue. It is therefore crucial to understand the means by which the immune and metabolic systems communicate, and how metabolism is remodelled as a result. Having a bigger picture of this immune-metabolic interplay could pave the way for investigation into ways to alleviate some of the pathologies associated with dysregulated metabolism during infection. For example, the mammalian homologue of one of the key genes investigated in this project, Nmdmc, is an important target of some cancer therapeutics. The role of Nmdmc in supporting the immune system is not yet fully understood, but if it proved to be important, some of the inhibitor drugs already available for cancer treatment could hypothetically be re-purposed to aid in treatment of infection or inflammatory disease. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |