Developing a Tool for the Investigation of

Hydrogen Sulfide Response in C. elegans

Silvia-Antonia Rus, Christopher Braden, Dana L. Miller
University of Washington, Department of Biochemistry
Objectives Auxin Induced Degradation (AID) Creating Transgenic Nematodes
To obtain temporal control over our experiment, we decided to use We are creating our own nematode lines that contain the tag in their
We are interested in observing the functions of the epigenetic
Auxin-Induced Degradation (AID) to modulate the activity of the genome, attached to set-2 and spr-5 by using CRISPR/Cas9
factors involved in H2S response, therefore we are developing a
bookmark genes due to its relatively short effect time and reversible technology. Then, we will inject the animals so that their progeny
tool that will allow temporal and spatial control in our studies. Our
capability. express the genetic edits.
research would facilitate other studies on H2S response and
expand on the knowledge of this specific epigenetic pathway and
its effects in various situations such as ischemic reperfusion
injuries.

The H2S Bookmark Auxin not present Auxin present

Fig.3- Schematic of auxin induced degradation

The detrimental, highly toxic role of H2S is more widely known as it
competes with O2; however, prior research placed H2S as a factor in
increasing lifespan in C. elegans.1,3
Previous studies in our laboratory indicate that H2S modulates
response to high H2S levels through manipulating the condensation of
chromatin, inducing epigenetic alterations that form or maintain
memories to environmental stressors (Fig. 1).3
Auxin is a plant hormone that, among other functions, is involved in
bringing proteins up for degradation by binding both the protein of
interest and another protein known as TIR-1. TIR-1 is an important Fig. 5- Injection Schematic
enzyme in the activation process of ubiquitin, a molecule involved in
marking proteins for degradation.5
Future Directions
Testing the AID Tag Once we obtain successful gene editing events, we intend to place
To test the efficiency of the AID tag, we collected the protein samples from the transgenic animals on auxin and observe the effects of the AID-
synchronized animals at the L4 developmental stage, then separated them in tag on the functions of SET-2 and SPR-5, which will facilitate the
three treatment groups: AID::GFP TIR1::mRuby (treated, untreated), and study of H2S bookmarking.
untreated N2 , after which we washed them off the plates and proceeded into
creating protein stocks. Are set-2 and spr-5 required to
A) maintain the H2S bookmark?

Fig.1- Animal survival upon low H2S exposure

prior to high H2S exposure
Fig. 2-Survival of animals carrying the
bookmark
https://www.k-
Are set-2 and spr-5 required to
state.edu/hermanlab/ form the H2S bookmark?
We focus on two proteins
that act on histones: SET-2 Fig. 6- Experimental Setting
AID::GFP TIR1::mRuby
(H3K4 methyltransferase),
and SPR-5 (H3K4me2
demethylase).3 The low B) Acknowledgements
survival in mutants at high
Special thanks to Frazer Heinis, Nelson Ruth, Ellie Garcia, Nicole
H2S after the 48 h retention
Iranon, Bailey Jochim, for all their help and support, as well as
time from low H2S exposure
Emily Fawcett who provided the base for our project. This project is
suggests that both SET-2
funded by NIH grant R01ES02495803.
and SPR-5 are involved in
maintaining the “bookmark”
of H2S exposure (Fig. 2) 3. References
Fig. 4- Degradation of AID::GFP after 2 h on 2 mM auxin plate A) 1Miller,

2018.
D. L., and M. B. Roth. "Hydrogen Sulfide Increases Thermotolerance And Lifespan In Caenorhabditis Elegans". PNAS,

Question: WHEN are set-2 and spr-5 each required for a Fluorescent microscopy of L4 larvae B) Western blot of protein 2Zhang, L. et al. "The Auxin-Inducible Degradation (AID) System Enables Versatile Conditional Protein Depletion In C. Elegans."

2018.
functional H2S bookmark? samples as labeled 3Fawcett, E. (2015). A beneficial toxin: The response to hydrogen sulfide improves survival in a changing environment. Ph.D.

University of Washington, Seattle.

4Weinhold, B. Epigenetics: The Science of Change. Mar. 2006, Environmental Health Perspectives.
5Dharmasiri, Nihal, et al. “The F-Box Protein TIR1 Is an Auxin Receptor.” Nature News, Nature Publishing Group, 26 May 2005.
6Ding, Y., H. Li, L.-L. Chen, and K. Xie. Recent Advances in Genome Editing Using CRISPR/Cas9. 24 May 2016.
7Schwartz, Matthew L., and Erik M. Jorgensen. Advances in Pediatrics., U.S. National Library of Medicine, Apr. 2016.