Identification of ESRE Regulatory Molecules Anna Justis Dr. David Fay, Mentor EPSCoR Research Fellowship Project Identification of the ESRE stress-response pathway regulatory molecules Involved : 1. RNAi screen for ESRE-pathway participants 2. Constructing new strains with ESRE-gene expression reporters

The ESRE Pathway in C. elegans Stress SLR-2 ? JMJC-1 Activated by heat shock, osmotic stress, redox stress, ethanol, & bacterial pathogen exposure Slr-2 and jmjc-1 are upstream participants Acts through a promoter motif termed ESRE (for ethanol and stress response element) -ESRE regulatory pathway is a novel genetic pathway that responds to a variety of environmental stresses, including heat shock, osmotic stress, oxidation stress, ethanol exposure, and bacterial pathogen exposure. -Previous work in Dr. Fay’s lab has showed that slr-2 and jmjc-1 genes are involved in this pathway, but do not bind to the DNA motif that is the pathway’s namesake. -The ESRE promoter motif, termed Ethanol and Stress Response Element is found in the promoters of a broad array of genes needed to respond to stress.

Research Questions How does the ESRE pathway work? Stress SLR-2 ? JMJC-1 How does the ESRE pathway work? Who are the players? Identify the EBP (ESRE-binding protein) Identify other participants in the ESRE pathway -To better understand how the ESRE motif allows animals to respond to a variety of stresses, we want to know what other genes are involved in the pathway and what they do. -We especially would like to identify the ESRE-Binding Protein (EBP). This is an as yet unidentified transcription factor that binds directly to the ESRE motif and turns on ESRE-gene expression. -We expect to find other regulator elements in the pathway that make act downstream of or in parallel with slr-2 and jmjc-1.

The ESRE motif sequence, slr- 2, and jmjc-1 are highly conserved among worms, flies, and mammals -The ESRE motif is highly conserved in worms, flies, and mammals (including humans). -Understanding this network in worms could have applications to human health areas like cancer and aging.

ESRE pathway participants are likely also conserved 59 (24.6%) 158 (65.8%) 23 (9.6%) -Because the ESRE motif is conserved, we guessed that the ESRE-binding protein is also conserved. -943 transcription factors in C. elegans. -We tested 217 transcription factors, which have orthologs in Drosophila and in mammals because the ESRE sequence, SLR-2, and JMJC-1 are conserved.

RNAi Screen Double-stranded RNA used to knock down one gene at a time 3xESRE::GFP, rol-6 reporter to visualize ESRE driven gene expression promoter rol-6 3xESRE GFP When Where Double-stranded RNA is fed to the worms, and triggers the RNA-Interference pathway, that knocks down the targeted gene in their progeny. Gene promoter controls what tissue the reporter is expressed in: “where” -what tissue type ESRE controls when the reporter is expressed: “when” – in response to stress Roller Non-Roller

RNAi Screen Tested 217 clones of evolutionarily conserved transcription factors in worms Tested 173 clones of known signaling molecules ? The lab tested 217 conserved transcription factors. I tested an additional 173 signaling molecules. Signaling molecules are candidates for the ESRE pathway.

RNAi Screen Results C08B11.3 RNAi knocks down 3xESRE::GFP expression by 2-fold SWI/SNF nucleosome remodeling complex component No other genes identified from screen ESRE pathway components are essential Functional redundancy Complex that binds ESRE ? From the transcription factors tested, one was shown to affect the expression of ESRE reporters. Knocking down C08B11.3 results in a 2-fold decrease in 3xESRE::GFP expression. This gene encodes a member of a nucleosome remodeling complex. This modifies super-structure of DNA and therefore gene expression. No other transcription factors or signaling molecules tested resulted a change in ESRE reporter expression. This could be because: 1. Incomplete knockdown by RNAi 2. The relevant genes have not yet been annotated as transcription factors, so we haven’t tested them yet. 3. ESRE pathway components are essential. In this screen, I didn’t have a way to differentiate between non-green due to knockdown of ESRE pathway components and non-green due to lethality of RNAi for the targeted gene. 4. Functional redundancy. Knocking down one pathway may be masked by a second, redundant pathway. 5. Complex. Knocking down one member of a complex may not result in a strong phenotype. **Our working hypothesis is that there may be two EBPs or redundant pathways.

Strain Construction: Microinjection

Strains 3xESRE::GFP, myo-2 3xESRE::GFP, myo-3 Muscles of the pharynx Body wall muscles promoter myo-2 3xESRE GFP When Where Unc-119 used as co-injection marker

Acknowledgements Dr. David Fay Aleksandra Kuzmanov Questions?