MEF2 targets and their functions in Drosophila immunity

Infections cause many changes in their hosts. Some of these changes involve mostly the immune system and are oriented purely at preventing infection - for example, when people get infections, some kinds of white blood cells will multiply to make for a more effective immune response. Some of these changes are not immune-system specific, but can clearly help cure infection - for example, fevers can help clear invading viruses or bacteria. Finally, some of these changes are not purely immune-system specific, and don't serve any obvious function in the response to infection - for example, the wasting syndrome often seen in people with chronic illnesses. All of these changes are ultimately linked to the activation of the immune response, but in general it is hard to understand the mechanisms that link them together and it is hard to pinpoint many of the genes that are actually involved in these changes. Nonetheless, it is tremendously useful for us to understand the genes that regulate these processes and how they work together, partly because they may provide useful new molecular targets for drugs to fight infections or reduce pathology and partly because knowing how these genes work together to drive the effects they do can begin to tell us which aspects of the immune response are actually important in fighting which infections.

We have found an important part of this puzzle by studying infections in a fruit-fly, Drosophila melanogaster. Like people, flies waste away when they have chronic infections. We have found that a protein called MEF2 has two different roles in fruit-fly fat cells. In flies that do not have an infection, MEF2 is important for normal energy storage - flies that have low MEF2 activity in their fat cells cannot store fat and are abnormally lean. When flies are infected with bacteria, MEF2 is required for a normal immune response - flies that have low MEF2 activity in their fat cells die rapidly, from many kinds of infections, because they cannot raise an immune response. Importantly, these two different functions of MEF2 are mutually exclusive: flies that are using MEF2 to generate an immune response cannot use MEF2 to store fat. This is part of the reason that some infections cause wasting.

There are still many parts of this switch between metabolism and immunity that we do not understand. In particular, we know some of the genes that MEF2 controls in each process, but we do not know all of them. The goal of this proposal is to identify all the genes MEF2 controls in fat cells from healthy and sick flies. We will then test the functions of these genes - can we identify the genes that are particularly important for immune function? This will also help us figure out how to identify MEF2 immune targets in other animals, like people, so that we can begin to try to tell whether MEF2 works the same way in people as in flies.

This proposal follows from our recent work, in which we have explored the function of the transcription factor MEF2 in immune-regulated gene expression in the Drosophila fat body. We have shown that MEF2 regulates two distinct suites of genes. In flies not experiencing an acute immune challenge, MEF2 is phosphorylated at a conserved site to promote the expression of enzymes of triglyceride and glycogen synthesis. Upon infection, MEF2 is dephosphorylated and interacts with the TATA binding protein (TBP) to bind a distinct sequence at the TATA box of many antimicrobial peptides; it is required for the expression of these genes as well. Both functions are critical: flies with MEF2 knocked down in the fat body have essentially no fat or glycogen and are severely immunocompromised against a wide variety of pathogens. The loss of phospho-MEF2 causes the loss of metabolic transcripts seen after infection with Gram-negative bacteria.

Here, we propose to use ChIP-chip to identify MEF2 binding sites in fat body from infected and uninfected flies, and RNAseq to determine which transcriptional changes depend on MEF2 activity. These datasets will allow us to identify binding sites for the MEF2-TBP complex as well as direct and indirect MEF2 targets. We will then explore the biological roles of transcription factors that interact with MEF2 using in vivo functional screens. Finally, we will integrate all of these data to derive a transcriptional circuit describing the functions of MEF2 and its interactors in Drosophila immune regulation and metabolism. The project will thus identify new transcriptional circuitry downstream of, and in parallel to, this novel and exciting immune-metabolic transcriptional switch, and characterize at least some of these elements at a functional level.

Academic impact has been covered under academic beneficiaries.

Beneficiaries/Societal
In the long term, if successful, our work will have significant societal impact.
Direct medical and veterinary implications include, most conspicuously, the identification of new therapeutic targets for intervention in bacterial infection. These targets open up new avenues of therapy, focused on host rather than bacterial processes and thus possibly less susceptible to the evolution of resistance on the part of the pathogen. Drugs ultimately targeted to these systems may be useful in humans or in other animals.
Indirect scientific impacts may also produce significant feed-through into societal impact. By improving our understanding of Drosophila inflammation/immunity and host-pathogen interactions, we will expand scientific knowledge of how this powerful experimental system can be used to address issues of human and animal health and disease. This in turn will facilitate the use of this system by others to address other, different issues of health and disease.
Impacts on the 3R's stand to be particularly important from this work. By demonstrating the use of Drosophila to examine important issues in infection biology, we hope to popularize this system as a "first-line" screening system for interventions in infectious disease, alongside existing tissue-culture and whole-animal models. With support, we may be able to provide significant information permitting refinement of genetic hypotheses in whole-animal infection experiments.
Therefore, these studies may have long-term global health and economic impacts. Again, dissemination to academic and industry researchers will be important for societal impact.

Industrial interactions/IP
This research may generate commercially exploitable intellectual property and research tools. One of our ams is to identify host targets of pathogenic effectors in intracellular infection. As mentioned above, these targets may be accessible as targets of therapeutic intervention in these infections. We will make use of the existing structures at KCL for industrial outreach to help identify these opportunities and we will attempt to forge interactions that may result, for example, in CASE awards for researchers working at the academic/industrial interface.

Training potential
This project will provide very useful training for the postdoctoral research associate as well as for the PhD students in the laboratory. This project lies at the interdisciplinary intersection of genetics, systems biology, and immunology; the researchers will learn techniques lying in all of these disparate areas. This training is very important for the scientists of the future, who will gain diverse skill sets and be better able to address complex research problems.
The postdoctoral fellow will also receive training in additional career skills, such as organization of projects, supervision of students, grantsmanship, manuscript writing, interpersonal skills and presentation skills. This training will be undertaken within the lab, on a day-to-day basis, as well as formally. For example, postdocs will be encouraged to attend media training. Postdoctoral researchers and PhD students are expected to present their work at internal seminars (at least once per year) and at at least one external conference per year. Informal presentations within the group will take place on a weekly basis.
The training potential in the lab is good: our previous BBSRC-funded postdoc entered the lab with no experience in Drosophila or in disease studies; she is now a researcher at the UCLA School of Medicine, working on using Drosophila to explore issues related to age-related disease.