Development of a nuclear alpha-ketoglutarate biosensor system to study metabolic control of the epigenome

About This Grant

PROJECT SUMMARY Alpha-ketoglutarate (aKG, also known as 2-oxoglutarate) is a key metabolic regulator of chromatin structure and, by extension, cell fate and function. aKG is imbued with this regulatory capacity based on its role as a requisite substrate for a broad class of epigenetic ‘eraser’ enzymes that catalyze the removal of methyl marks from DNA and histones. The importance of aKG in establishing and maintaining cellular identity is underscored by a series of recent discoveries that cut across normal and diseased biological states. For example, elevated aKG levels in naïve embryonic stem cells are critical for maintaining a genomic architecture that facilitates pluripotency. Conversely, many cells display aKG antagonism phenotypes that promote malignancy or alter immune cell function, which is achieved through elevated aKG catabolism or by production of metabolites that competitively inhibit aKG binding to proteins. Despite the fundamental role for aKG in establishing cellular identity, we have limited understanding of the molecular mechanisms that control the pool of aKG that is available in the nucleus to support DNA and histone demethylation. This limitation is tied to one central roadblock: a paucity of biomedical research tools that can be used to quantify changes in nuclear aKG pool size with high sensitivity and specificity. To address this issue, we have designed and collected feasibility data supporting a new biomedical research tool to measure nuclear aKG. This tool, which we term the aKG-ON biosensor system, is similar to the classical TET-ON system. Rather than responding to exogenous tetracyclines, however, the aKG-ON biosensor system responds to changes in nuclear aKG. The aKG-ON biosensor system has two components: 1) a chimeric transcription factor based on a cyanobacterial protein, NtcA, whose DNA binding activity is allosterically regulated by aKG, and 2) a synthetic reporter gene comprising a promoter with NtcA DNA binding sites and a GFP cDNA. The output of this system, GFP expression, can be used to assess levels of aKG in nuclei of live mammalian cells. Our central hypothesis is that the transcription factor NtcA presents an attractive starting point for biosensor engineering and synthetic biology given its evolutionarily conserved role in aKG sensing in cyanobacteria. We seek to develop and validate an optimized prototype of the aKG-ON biosensor system via three Aims. In Specific Aim #1, we seek to improve the dynamic range of this system through signal amplification and directed evolution approaches to produce a prototype of the aKG-ON biosensor system. In Specific Aim #2, we seek to validate utility of the aKG-ON biosensor system prototype in unbiased forward genetic studies. In Specific Aim #3, we seek to engineer the aKG-ON biosensor system into mice and validate its utility for measuring nuclear aKG levels in vivo. If successful, our work will create a new biomedical research tool that permits direct assessment of the aKG pool that is available to chromatin-modifying enzymes, thereby enabling studies of mechanisms of metabolic-epigenetic crosstalk that drive cell state transitions in development and disease.