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Proposed Research Question:
How are the genes associated with resistance to extreme conditions, specifically heat shock
proteins in the hsp70 gene region (taHSP70), regulated during late embryonic development in
tardigrades, Milnesium Tardigradum during anhydrobiotic and hydrated conditions? More
specifically, are the histone modifications H3K9me3, H3K9ac and H3K4me2 associated with the
H3 core histones of taHSP70 in embryonic stage 4 (96 hours after egg lay) tardigrades, M.
Tardigradum before and after dehydration?
Using Drosophila melanogaster as a model to base predictions, we will analyze the histone
modifications of taHSP70 in hydrated and dehydrated embryonic tardigrades using chromatin
immunoprecipitation (ChIP).
Background:
Tardigrades – Anhydrobiosis and the “tun” State
Tardigrades, also known as water bears, are microscopic metazoans approximately 0.11.2mm long that are closely related to the model organisms Caenorhabditis elegans and
Drosophila melanogaster (Goldstein & Blaxter. 2002, Møbjerg et al. 2011). They are a highly
diverse phylum with over 1000 described species that can be found all over the world in both
marine and terrestrial environments (Møbjerg et al. 2011, Degma et al. 2010). These organisms
are famous for their ability to survive in the most diverse and extreme conditions and have
emerged as a prime model organism for studying extreme stress resistance (Goldstein & King.
2016, Møbjerg et al. 2011).
Tardigrade survival in extreme conditions is achieved by anhydrobiosis, where adult
tardigrades enter a so-called “tun” state (Welnicz, et al. 2011). In the tun state tardigrades are
almost completely dehydrated (about 2% water) and metabolically dormant (Welnicz, et al.
2011, Møbjerg et al. 2011, Westh & Ramlov. 1991). Over the past 15 plus years many studies
have been published looking at the biochemical, physiological and genetic/epigenetic changes
that occur during dehydration to the tun state and the rehydration process that follows (Welnicz,
et al. 2011, Møbjerg et al. 2011). Sufficient information is also available on morphological
characteristics of adult tardigrades however, such detailed information does not exist for their
developmental stages or how the embryonic state can tolerate desiccation by anhydrobiosis
(Gross & Mayer. 2015, Schill & Fritz. 2008). Rebecchi et al. (2006) reported successful hatches
after embryonic desiccation of the species M. tardigradum as did Schill & Fritz (2008).
Information on gene regulation during development for these organisms is also lacking.
Heat Shock Proteins
Heat shock proteins (hsps) are a subgroup of molecular chaperones that protect proteins
from denaturation during stressful conditions and guide the proper folding conformation of
already denatured proteins (Wang et al. 2014, Reuner et al. 2008, Schokraie et al. 2011). In
addition to their role in stress response hsps also play a role in many other biological processes
such as cellular differentiation and development (Sakurai & Enoki. 2010, Gonsalves et al. 2011).
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Hsp70 – Tardigrades and Drosophila
The fly, Drosophila melanogaster and the tardigrade, Milnesium tardigradum belong to
the same superclade Ecdysozoa (Møbjerg et al. 2011). Because hsp70 is highly conserved among
the metazoan kingdom, we will be using Drosophila as a model to make predictions of how the
hsp70 gene region (taHSP70) is regulated in the embryonic tardigrade both in the hydrated and
dehydrated state (Tenlen et al. 2012, Daugaard et al. 2007). Our current knowledge of the
regulation of HSP genes in tardigrades is limited but is likely controlled in-part, at the level of
chromatin structure (Wang et al. 2014). Hsp70 isoform 2 has been shown to be upregulated in
the tardigrade species M. tardigradum in transition from the hydrated to tun state ( Förster et al
2012, Møbjerg et al. 2011, Welnicz, et al. 2011). Hsp70 gene regulation is very well studied in
polytene chromosomes in Drosophila, both in response to heat stress and under control
conditions making it an ideal model to base predictions (Petesch & Lis. 2008, Reyes-Carmona et
al. 2011, Tillib et al. 2004).
Hsp70 Gene Regulation by Histone Modifications in Drosophila Embryos
In the developing Drosophila embryo (late gastrulation stage) the inactive taHSP70
contains the histone modification H3K9me3 which is associated with heterochromatic regions
(Reyes-Carmona et al. 2011). RNA Polymerase II is found already bound to the hsp70 gene in a
paused state, ready to begin transcription (Mazina. 2016). When activated by heat stress, heat
shock factor 1 (HSF1) binds to heat shock elements (HSE) upstream of the transcription start site
and the histone modification H3K9me3 becomes disassociated from the chromatin (ReyesCarmona et al. 2011, Tillib et al. 2004). During this process the H3 core histones become both
methylated (H3K4me2) and acetylated (H3K9ac) (Tillib et al. 2004, Chen et al. 2002). This
disruption in the nucleosomes is followed by rapid loss of nucleosomes across the whole gene
region and beyond, stopping at the scs and scs’ insulating elements (Petesch & Lis. 2008). This
is then followed up by cycles of nucleosome assembly and disassembly where nucleosomes
remain associated with the active hsp70 gene (Teves & Henikoff. 2013).
Relevance and Importance:
According to Daugaard et al. (2007), hsp70 is the most conserved protein in evolution
and some form of it can be found in all organisms on the planet. Its conservation through
evolution is constant enough that Drosophila hsp70 is sufficient to rescue mammalian cells from
heat stress (Daugaard et al. 2007) When expressed in high amounts, hsp70 can cause cells to
survive otherwise lethal conditions (Kumar et al. 2016). This can be problematic when the
surviving cell populations are the targets of chemotherapy or other cancer treatments.
Overexpression of hsp70 could be responsible for tumorigenesis, negating the apoptosis
mechanism in cells and tumor progression by providing resistance to chemotherapy (Kumar et
al. 2016). Like some tumor cells tardigrades are very resilient to environmental stressors
(Møbjerg et al. 2011). Understanding the mechanism by which tardigrades regulate their hsp70
genes could provide insights into future cancer treatment methods but hsp70 research
applications are not just limited to cancer. Reduced levels of hsp70 are found in Huntington’s
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models in mice demonstrating the versatility of this protein and the importance of studying it in a
variety of model systems. (Labbadia et al. 2011).
Although not directly relevant to hsp70, tardigrade research is already starting to show
promising applications to human cells. A unique tardigrade protein has been found to suppress
X-ray DNA damage in human cell cultures (Hashimoto et al. 2016). This shows that tardigrade
research has direct applications to human cells and suggests that future research into tardigrade
protein expression will yield more promising applications.
Hypotheses:
Regulation of the Hsp70 gene region (taHSP70) in the stage 4 tardigrade (M.
Tardigradum) embryo is controlled, in part, at the level of chromatin structure. The histone
modification H3K9me3 is associated with taHSP70 chromatin, while H3K9ac and H3K4me2 are
not associated with taHSP70 chromatin in hydrated stage 4 tardigrade embryos. The histone
modifications H3K4me2 and H3K9ac are associated with taHSP70 chromatin, while H3K9me3
is not associated with the taHSP70 chromatin of dehydrated stage 4 tardigrade embryos.
Methods and Predicted Results:
We will be using the embryonic stage of the tardigrade species Milnesium tardigradum
Doyère, 1840 (Eutardigrada, Apochela). Rearing protocols for adults and embryos outlined by
Suzuki (2003). After egg lay, tardigrade embryos will be reared to stage 4, 96 hours after egg lay
outlined by Schill & Fritz (2008). Tardigrades that do not meet the developmental characteristics
for stage 4 after 96 hours will be discarded. Embryos to be dehydrated will be transferred to a
paper filter to remove excess water and dehydrated in an oversaturated salt solution of potassium
acetate at 20 °C for 24 hours (Schill & Fritz. 2008). Stage 4 embryos will be used to ensure
sufficient survivability after drying and to provide information on late stage embryonic
development (Suzuki 2003). Drying in an oversaturated potassium acetate solution is done to
ensure even drying of all specimens at a relative 20% humidity (Schill & Fritz. 2008). We chose
to treat embryos at 20% relative humidity for 24 hours because we expect that relative humidity
level and time to be sufficient for desiccation stress response (novel).
Three chromatin immunoprecipitation assays will be performed on the chromatin of the
hydrated and dehydrated embryos. Hydrated embryos at the 96-hour mark and dehydrated
embryos at the 120-hour mark (96-hour incubation, 24-hour treatment) will be treated with
formaldehyde at 2% concentration (Tillib et al. 2004). This is done to cross-link DNA and
associated proteins (complete sample preparation outlined in Tillib et al. 2004) Cells are then
lysed and DNA-protein complexes are sheared to obtain fragments between 100-500bp long
(Tillib et al. 2004). For all ChIP assays performed a positive control using a non-specific binding
antibody will be used to ensure negative results are meaningful. In addition, a negative control
using an antibody specific to a protein not found in tardigrade chromatin will also be performed
to ensure positive results are meaningful. We choose to use ChIP because it is a relatively
inexpensive and simple technique that will clearly show the presence, or absence of specific
histone modifications on taHSP70 chromatin.
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For hydrated and dehydrated embryos ChIP assays, we will first use anti-acetyl-histone
H3K4 (antibody) to test for the presence or absence of the histone modification H3K9ac (Tillib
et al. 2004). Next assays, anti-dimethyl-histone H3 (antibody) will be used to test for the
presence or absence of the histone modification H3K4me2 (Tillib et al. 2004). For the final
assays, anti-methyl-histone H3K9 (antibody) will be used to test for the presence/absence of the
histone modification H3K9me3 (Tillib et al. 2004). Results are in the form of bands on gels with
chromatin associated to treatment antibodies being higher on the gel because of the added
molecular weight.
We predict, based on the drosophila model that hydrated tardigrade embryos will show
H3K4me3 associated to the chromatin of taHSP70 and neither H3K4me2 or H3K9ac associated
to taHSP70 chromatin. Dehydrated embryos should show the opposite, with H3K4me2 and
H3K9ac associated to taHSP70 chromatin and H3K9me3 absent from the ChIP assay bands.
Discussion:
Because of the novelty of this proposed study there are many possible results that this
experiment could show that cannot all be explained here. If the hypothesis is correct we can
directly conclude that H3K9me3 and not H3K4me2 or H3K9ac is associated with the chromatin
of hydrated tardigrade embryos. We can also conclude that H3K4me2, H3K9ac and not
H3K4me3 is associated with the chromatin of dehydrated tardigrade embryos. We cannot
directly conclude anything about the transcriptional state of taHSP70, only that the histone
modifications mentioned are associated to taHSP70 chromatin.
We can infer from these results that taHSP70 is heterochromatic and likely
transcriptionally inactive in hydrated embryos because of the presents of the H3K9me3 histone
modification (Filion et al. 2010, Hediger & Gasser. 2006). We can also infer that the taHSP70 is
euchromatic and possibly transcriptionally active in dehydrated embryos because of the presents
of the H3K4me2 and H3K9ac histone modifications (Riu et al. 2007). Future experiments could
further test aspects of the described drosophila model on tardigrades, such as the presence of
paused RNA Polymerase II upstream of the promoter and the loss of nucleosomes following
nucleosome disruption (Petesch & Lis. 2008, Mazina. 2016). Evidence for these aspects of the
model could help to explain tardigrades rapid response to environmental stressors (Møbjerg et al.
2011).
Other possible results may show that the chromatin of taHSP70 in hydrated embryos is
associated with H3K4me2 and H3K9ac. This would infer that hsp70 gene chromatin is
euchromatic and the gene is transcriptionally active (Riu et al. 2007). We could further infer
from this possible result that the hsp70 gene is expressed without desiccation stress in stage 4
tardigrade embryos (Schill & Fritz. 2008). Another possible result is that H3K9me3 is associated
to hsp70 chromatin in dehydrated tardigrade embryos. This infers that the chromatin is
heterochromatic and transcriptionally inactive, inferring that hsp70 is not expressed in response
to desiccation stress in stage 4 tardigrade embryos (Schill & Fritz. 2008, Filion et al. 2010). It is
very possible that other histone modifications are involved in this process in tardigrades such as
H4K27me3 for example. Further experiments will need to be done to determine if other
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modifications are involved in the regulation of the hsp70 gene region (taHSP70) in tardigrades,
M. tardigradum.
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What is THE toughest living entity on our planet?
Tardigrade “Tun” State
Chances are you have never heard of a “tardigrade”
before but you may have heard them called “water bear”
or “moss piglet”. Not much to look at, tardigrades cannot
be seen with the naked eye. Don’t let their tiny size fool
you, these wee guys are far tougher than you, me or even
Chuck Norris. Tardigrades can survive some of the most
extreme conditions far better than any other animal on
the planet by removing all the water from their body and
transitioning to a dormant state called a “tun”. This
process is known as cryptobiosis or anhydrobiosis to
biologists.
1
In their dormant state, they can survive conditions that
would kill a human instantly. They can endure temperatures as cold as -273°C and as hot as 151°C.
Radiation hundreds of times above a lethal dose, pressure that would crush a person and even the
vacuum of space is no match for the robust tardigrade.
They have been around on this planet for a very long
time and have survived all 5 mass extinctions that killed
most life on earth 5 times over.
2
But what makes these petite creatures so tough? The
answer is found in the proteins tardigrades produce in
their bodies. Certain proteins called heat shock proteins
(hsps), work like little molecule sized machines to help
the tardigrade fix and prevent damage done by extreme
conditions. Hsps are not just found in tardigrades, all
living things on earth have some form of these proteins
in their body.
Heat shock proteins are not always a good thing. In
humans, heat shock proteins have been linked to the
resistance of cancer cells to chemotherapy, working in
the same way to fix and prevent damage done by the
cancer killing chemicals in chemotherapy.
What can tardigrades do for us? For one, they can give
us a model to observe how these heat shock proteins
are expressed so we can one day apply what we
learned. In this research study, we will look at how the gene (DNA) of a specific heat shock protein is
controlled in the embryonic stage of tardigrades before and after drying them out. Our goal is to provide
new information on how heat shock proteins are controlled in developing tardigrades when they
experience extremely low water conditions.
3
Research into tardigrades has revealed interesting applications that have already been applied to human
tissue. Using a protein unique to tardigrades, researchers have been able to enhance human tissue to
withstand a lethal amount of X-ray radiation. It is Our hope is that further investigation into these
astonishing creatures will uncover invaluable applications that one day may benefit humans and other
species to improve survival and fight disease.
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Images
1. Wełnicz, W., Grohme, M. A., Kaczmarek, Ł., Schill, R. O., & Frohme, M. (2011). Anhydrobiosis in
tardigrades—The last decade. Journal of Insect Physiology, 57(5), 577-583.
2/3. Sketchy Science. (2014) http://www.sketchyscience.com/2014/10/the-trouble-with-tardigradestrials-and.html Accessed: 12/1/2016