|Abstract or Summary
- Fungi are capable of growth on a wide variety of carbon sources, both living and dead. They can produce an arsenal of enzymes and transporters for harvesting sugars, polysaccharides, amino acids, lipids and micronutrients from their environments . Within the nucleus of a cell, transcription factors (TF) control whether genes will be transcribed, after which the transcripts can be translated into functional protein. TF contact with DNA can be influenced by nucleosomal occupancy, DNA binding affinity, and competition with other DNA binding proteins , . Sequence specific DNA binding transcription factors associate with promoter sequences in order to tune core metabolic pathways in response to nutrient availability, for example N. crassa’s major nitrogen regulator NIT-2, , or in response to oxidative stress, the alternative oxidase regulator, AOD-2 . Change in the relative abundance of proteins within the cell or proteome can have broad effects from redirection of metabolic flux via carbon and nitrogen use, production of enzymes for detoxification, and altered growth and development from the examples above. A survey of transcription factor binding influenced by light is underway in Neurospora crassa as part of the Neurospora Functional Genomics and Systems Biology (NcFGSB) program project funded by the NIH (P01GM). This work began by testing the link of FAR-1, or Fatty Acid Regulator -1 (NCU08000) to light regulation, but has continued toward investigation of how both FAR-1 and a second TF, Fatty Acid Regulator-2, or FAR-2 (NCU03643) influence central metabolism, oxidative stress response, and development.
Transcription factor networks consist of describing TFs that bind to multiple genes and individual genes controlled by multiple regulators. Further, the regulation of regulators describes how the transcription of transcription factor genes is controlled. Chromatin immunoprecipitation, or 'ChIP' experiments show that a variety of factors are enriched at the same promoter region as can be seen by comparing multiple datasets , . Transcriptional regulators may promote or inhibit one another from binding DNA, leading to a total regulation as the sum of TF activity. This complexity cannot be explained by single genetic experiments and study of a limited number of loci. Even in the event of TF association with specific promoter DNA as analyzed by ChIP, proximal binding of a transcriptional activator does not directly mean that transcript levels will increase, as we have seen with the identification of WC-2 binding sites .
I used a reverse genetics approach, assaying deletion mutants of selected loci, to determine the cellular effects of FAR-1 and FAR-2 transcriptional activity in different carbon sources. The tools of high throughput sequencing, and bioinformatics data analyses were combined with experiments to characterize phenotypes observed. Here I report (1) a collection of binding sites found in the Neurospora genome for FAR-1 and FAR-2 in sucrose, butyrate, and oleate, (2) changes in transcription as a result of the loss of FAR-1 and/or FAR-2 function, and (3) how the combination of binding sites and transcriptional activities are reflected in phenotypes. This work has developed and utilized methods for combining genome-scale ChIP- and RNA-sequencing data to describe direct and indirect transcriptional regulation, and has added to the definition of transcription factor networks in N. crassa.