Investigating the molecular basis of evolved stress resilience in a subterrestrial nematode

A critical and well-studied cellular stress response pathway, the Unfolded Protein Response (UPR), protects
organisms against several stressors including heat, hypoxia, starvation, and toxins. Helping to repair cellular damage, the
UPR can also trigger apoptosis if the stress is ongoing, severe, and unrecoverable. Therefore, proper regulation of this
pathway is essential, particularly since its malfunction contributes to human pathologies including autoimmune disorders,
cancer, and neurodegenerative diseases.
The Bracht lab recently published the genome of a nematode, Halicephalobus mephisto, isolated from the deep
terrestrial subsurface of South Africa, over a kilometer underground. This organism has adapted to a stressful
environment: hot, hypoxic, and rich in methane. Therefore the organism displays a naturally evolved resilience to stresses
that would normally cause lethality; we also found that its UPR pathway is a site where adaptation has occurred. We have
confirmed that RNA Interference (RNAi) by feeding can be used to modulate gene expression in this organism, setting the
stage for a molecular investigation of stress resilience.
Aim 1. Test the hypothesis that ARMET/MANF is not just an inhibitor of UPR signaling in H. mephisto.
A UPR signaling gene discovered in 2003, Arginine-Rich, Mutated in Early-stage Tumors / Mesencephalic
Astrocyte derived Neurotrophic Factor (ARMET / MANF), remains mysterious. While its precise molecular function has
proven elusive, we identified it as the second most highly upregulated gene under heat stress in H. mephisto. In this aim,
we will perform analysis of the transcriptomic changes when ARMET / MANF is knocked down by RNAi.
Aim 2. Test the hypothesis that HSF1 has acquired an expanded role in heat resilience in H. mephisto.
Heat-shock factor 1 (HSF1) is a well-characterized, conserved transcriptional regulator of the heat response across
metazoa. However, we identified the potential for this protein to regulate 75% of the genes through a helitron-driven
expansion of its recognition site. This aim is structured to test this apparent re-wiring of the HSF1 regulatory network.
Aim 3. Test the role of HeaT-Upregulated-Protein-1 (HTUP-1) in heat and tunicamycin resilience.
HTUP-1 is the most upregulated gene on heat in H. mephisto and it is unlike any other known protein--no blast
matches and no recognizable domains. We hypothesize that HTUP-1 is a novel modulator of the evolved UPR response in
H. mephisto. To study HTUP-1 function, we will inactivate it by RNAi, measure growth phenotypes under heat or
tunicamycin stress, verify knockdown by qRT-PCR, and then perform RNA-seq to examine the pathways affected.
Aim 4. Construct multi-copy arrays of H. mephisto genes in C. elegans as a mechanism of heat resilience.
Hsp70 genes are extremely well characterized. However, in H. mephisto we uncovered a new family of Hsp70
genes: Hspa15; here we propose to evaluate whether these genes can confer heat tolerance de novo by heterologous
expression in C. elegans. Because C. elegans is not thermotolerant, any acquired heat tolerance will be easily detected in
this genetic background.

Grant Number: 1R15GM146207-01
NIH Institute/Center: NIH
Principal Investigator: John Bracht