Homeostatic control mechanisms are vital components of biological maintenance within the cell. Reactive oxygen species (ROS) are kept under homeostatic control, in part, by ?Cap?n?Collar?(CnC) transcription factors, like SKN-1 in C. elegans. A mutagenesis screen uncovered SKN-1 gain-of-function(gf) alleles that display increased resistance to oxidative stress, but surprisingly, deregulated metabolism and shortened lifespans. These early findings suggest a link between uncontrolled regulation of cytoprotective transcription factors and disease pathology. Regulation of SKN-1 has been documented primarily through genetic studies. This has left many mechanistic details of SKN-1 regulation unstudied. This proposal leverages the opportunity to examine a constitutively active transcription factor to characterize aberrant regulation normally applied in the wild type condition. This proposal will CRISPR/cas9- generated GFP tagged variants of SKN-1 and SKN-1gf at the endogenous locus, which avoids problems with multi- copy transgenic arrays used in previous studies.
In Aim 1, I will utilize ChIP-seq to assess target selection as well as a Biacore surface plasmon resonance (SPR) studies to define binding affinity and dissociation constants, which based on our RNAseq studies are predicted to be different across target genes. Preliminarily ChIP-qPCR experiments have been performed for AIM1 and have revealed that SKN-1gf occupies target gene promoters more than wildtype. We will expand this approach to a genome-wide assessment and begin with these established targets for our binding studies.
In Aim 2, I will examine the interplay of phosphorylation and OGlcNAcylation post-translational modifications on SKN-1 activity and target selection. Phosphorylation on SKN-1 is critical for its regulation as well as OGlcNAcylation. A preliminary assessment of phosphorylation stoichiometry has been performed via PhosTag SDS PAGE, where an increase in phosphorylation on SKN-1gf compared to wildtype was observed. It is unclear what kinases are causing this shift, but my combined genetic and biochemical approach will define these important regulators. Taken together these biochemical assessments will fill a critical knowledge gap for SKN-1 activity and target gene selection, which is a topic of great interest to the study of transcription factor regulation.
Homeostatic control mechanisms are a crucial components of the cellular infrastructure and ensure that normal biological functions are maintained. This proposal is focused on a SKN-1 gain-of-function mutant in which the reactive oxygen species homeostat has lapsed into an ?always on? state. By examining SKN-1 gain-of-function, and its interactions within the genome and on regulatory pathways, we can elucidate new control mechanisms for SKN-1 and apply this knowledge to mammalian NRF2 of which constitutive activation is prevalent in oncogenesis.
