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\begin{document}

\title{Screening H3 histone acetylation in a wild bird, the house sparrow
(Passer domesticus)}



\author[1]{Daniella Ray}%
\author[2]{Elizabeth Sheldon}%
\author[3]{Cedric  Zimmer }%
\author[4]{Lynn B. Martin}%
\author[5]{Aaron Schrey}%
\affil[1]{Georgia Southern University}%
\affil[2]{Macquarie University Faculty of Science}%
\affil[3]{Université Sorbonne Paris Nord}%
\affil[4]{University of South Florida}%
\affil[5]{Georgia Southern University - Armstrong Campus}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\selectlanguage{english}
\begin{abstract}
Epigenetic mechanisms are increasingly understood to have major impacts
across ecology. However, one molecular epigenetic mechanism, DNA
methylation, currently dominates the literature. A second mechanism,
histone modification, is likely important to ecologically relevant
phenotypes and thus warrants investigation, especially because molecular
interplay between methylation and histone acetylation can strongly
affect gene expression. There are a limited number of histone
acetylation studies on non-model organisms, yet those that exist show
that it can impact gene expression and phenotypic plasticity. Wild birds
provide an excellent system to investigate histone acetylation, as
free-living individuals must rapidly adjust to environmental change.
Here, we screen histone acetylation in the house sparrow (Passer
domesticus); we studied this species because DNA methylation was
important in the spread of this bird globally. This species has one of
the broadest geographic distributions in the world, and part of this
success is related to the way that it uses methylation to regulate its
gene expression. Here, we verify that a commercially available assay
that was developed for mammals can be used in house sparrows. We
detected high variance in histone acetylation among individuals in both
liver and spleen tissue. Further, house sparrows with higher epigenetic
potential in the Toll Like Receptor-4 (TLR-4) promoter (i.e., CpG
content) had higher histone acetylation in liver. Also, there was a
negative correlation between histone acetylation in spleen and TLR-4
expression. In addition to validating a method for measuring histone
acetylation in wild songbirds, this study also shows that histone
acetylation varies in an ecologically relevant way, adding a new study
option for ecological epigenetics.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Epigenetic mechanisms can alter gene expression without variation in
underlying DNA sequence (Bossdorf et al. 2008). Of the three molecular
epigenetic mechanisms: DNA methylation, histone modification, and
chromatin structure, only DNA methylation has received much attention in
non-model organisms, especially vertebrates (Schrey et al. 2014; Sheldon
et al. 2018).

Histones are highly conserved proteins that package DNA into the
nucleosome (reviewed by Bartova et al. 2008). The ammino acid residues
comprising histone tails can be covalently modified by up to 15
different subunits (i.e., methylation, acetylation, phosphorylation,
ubiquitylation). Modifications to histone residues alter the reactivity
of that histone-DNA complex and hence the conformation of DNA within the
nucleus. Histone acetylation is generally an activating modification,
promoting transcription by making the DNA more euchromatin like. By
decreasing a histone's affinity for DNA, transcription factor binding
sites become more accessible, thus bolstering transcription (Bartova et
al. 2008).

There are a limited number of histone acetylation studies on non-model
organisms, and those that exist indicate that histone acetylation can
have a major impact on gene expression, phenotypic plasticity, and
plasticity of life history traits (Choppin et al. 2021, Reynolds et al.
2016). For instance, altered histone acetylation facilitated the shift
in physiology of worker ants (\emph{Temnothorax rugatulus} ) following
the loss of a queen, increasing their fecundity, expression of immune
genes, and longevity of the hive (Choppin et al. 2021). Changes in
histone acetylation were also associated with pupal diapause (i.e., a
period of dormancy that is induced by harsh environmental conditions) in
the flesh fly (\emph{Sarcophaga bullata} ; Reynolds et al. 2016).

Introduced species provide an excellent system to investigate the role
of histone acetylation in variation in gene expression. Introduced
individuals must rapidly adjust to new environments, a particularly
fraught challenge because introduced populations tend to have low
genetic variation and differentiation compared to native populations
(Schrey et al. 2011; Liebl et al. 2015; Hanson et al. 2022). Ample
evidence exists to support that DNA methylation, is important in this
process (Liebl et al. 2013; Mounger et al. 2021). Indeed, the house
sparrow (\emph{Passer domesticus} ) has been successfully introduced
throughout the world (Liebl et al. 2015), and DNA methylation is
important at the individual level to variation in gene expression
(Kilvitis et al. 2019) and at the population level in terms of success
in colonizing new areas (Liebl et al. 2013; Sheldon et al. 2018;
Swaegers et al. 2023). Partly, these population-level effects arise
because individuals possess genomes with different dispositions to be
epigenetically modified, a trait termed epigenetic potential (Kilvitis
et al. 2017; Sheldon et al. 2023). One form of epigenetic potential has
been estimated as the number of CpG sites in promoters. In past work, we
have found that epigenetic potential varies among individuals (Hanson et
al. 2020), is related to gene expression levels (Hanson et al. 2021),
and is greater in house sparrows collected in more recently introduced
areas than native ones (Hanson et al. 2022). We propose that histone
modification is also important to introduced house sparrows, given that
it has an impact on gene expression and phenotypic plasticity (Choppin
et al. 2021, Reynolds et al. 2016).

Here, we screened histone modification in wild-collected house sparrows.
Our goal was to facilitate investigations of histone acetylation and
encourage the use of additional epigenetic markers beyond DNA
methylation in ecological epigenetics. To do that we determined whether
1) histone acetylation can be measured in wild collected house sparrows
using a commercially available assay developed for mammals, and 2)
histone acetylation in house sparrows varies in an
ecologically-intelligible manner. We measured histone acetylation in
house sparrows that had been previously collected for a study on the
response of individuals to \emph{Salmonella} infection (Sheldon et
al.\emph{in press} ). These experimental samples provide us with the
ecologically-relevant framework, in a controlled setting, to investigate
relationships among epigenetic potential, acetylation and gene
expression.

\textbf{Methods}

\emph{Samples Screened}

We screened H3 histone acetylation in paired liver and spleen tissues
from house sparrows (n = 12) previously collected from Tampa, Florida
(Table 1). These samples were used in a separate study of house sparrow
response to \emph{Salmonella enterica} infection (as described in
Sheldon et al. 2023). We screened only males with similar body masses
(Table 1). For each individual, we compared H3 acetylation to the level
of the \emph{Toll Like Receptor-4 (TLR-4} ) gene expression in the cecum
estimated by qRT-PCR from RNA extracts using primers targeting
the\emph{TLR-4} transcript (Sheldon et al. 2023). Also, we compared H3
acetylation to EP of the putative \emph{TLR-4} promoter, estimated by
sequencing 500 bp upstream from the transcription start site, and
categorizing individuals as low EP if they had 7 or fewer CpG sites, or
high EP if they had 8 or more CpG sites (Sheldon et al. 2023). We
used\emph{TLR-4} expression and EP because they are ecologically
relevant, variable in the individuals screened, and differ in response
to infection (Sheldon et al. 2023).

We used \emph{TLR-4} expression as an indicator of change in gene
expression among individuals in response to infection, in an attempt to
link variation in histone acetylation to individual phenotype. Given
that our H3 acetylation estimates are genome-wide, and not localized to
particular genes, we do not expect there to be a direct causal
relationship between H3 acetylation and \emph{TLR-4} expression. Nor do
we expect there to be a direct causal relationship between H3
acetylation and EP, as EP was estimated based on the number of CpG
sites, which are directly relevant to DNA methylation. However, as
histone modification and DNA methylation are epigenetic mechanisms, it
may be reasonable to expect that individuals with higher EP for DNA
methylation might also have higher EP for histone modification. Our
intention in comparing histone acetylation to \emph{TLR-4} expression
and EP was to ground the global estimates of H3 acetylation to
ecologically relevant factors at the individual level.

\emph{Measuring Histone Acetylation}

We extracted proteins from tissues using the EpiQuick Total Histone
Extraction Kit (EpiGentek, Farmingdale, NY). We then stored the extracts
at -80\selectlanguage{ngerman}°C overnight, and measured H3 histone acetylation in triplicate
the next day using the Total Histone H3 Acetylation Detection Fast Kit
(EpiGentek, Farmingdale, NY). Briefly, the Total Histone H3 Acetylation
Detection Fast Kit detects H3 acetylation by ELISA. Strip wells are
coated with an anti-acetyl histone H3 antibody that captures acetyl
histone H3 from the protein extract and allows the experimenter to
colorimetrically detect if histone H3 is acetylated, and
colorimetrically quantify the amount of the acetylated histone H3. The
H3 histone presence is detected and measured using a labeled detection
antibody and a color development reagent. We measured the resultant
color change with a plate reader (Bio Tek, Synergy HTX Multimode Reader,
Agilent, Santa Clara CA. USA).

\emph{Data Analysis}

We calculated mean H3 acetylation by averaging the replicates and
controlling for total protein concentration. We evaluated the
repeatability of the raw results by calculating the coefficient of
variation (\emph{CV} ) among replicates for each individual within
tissues. We used the \emph{CV} estimates to define outliers and
performed all subsequent tests on a dataset that excluded the most
divergent replicate from any replicate sets with \emph{CV} of 25\% or
higher. We also calculated \emph{CV} among individuals for each tissue,
before and after removing outliers. We tested for differences in H3
acetylation mean and variance between liver and spleen by
\emph{t-tests}and \emph{f-tests} . We also tested for differences in H3
acetylation levels in both tissues between individuals with high and low
epigenetic potential. Additionally, we compared H3 acetylation to
\emph{TLR-4}expression using Pearson's correlation for both liver and
spleen tissues.

\textbf{Results}

We found that the EpiQuick Total Histone Extraction Kit (EpiGentek) and
the Total Histone H3 Acetylation Detection Fast Kit (EpiGentek) were
effective in house sparrows. Estimates of H3 acetylation ranged from
0.01 to 2.08 OD/mg/ml in liver and from 0.08 to 2.18 OD/mg/ml in
spleen.\emph{CV} among replicates ranged from 5.0 to 48.2\% in liver and
3.0 to 46.6\% in spleen. Our criteria of \emph{CV} 25\%, or higher,
identified 3 outlier estimates in liver and 7 outlier estimates in
spleen. The\emph{CV} among replicates was much lower than the \emph{CV}
among individuals before and after removing outliers. Among replicates
before removing outliers, \emph{CV} was 16.9\% for liver and 26.5\% for
spleen, and \emph{CV} among individuals within tissue was 49.9\% for
liver and 65.5\% for spleen. Among replicates after removing outliers,
\emph{CV}was 12.2\% for liver and 10.7\% for spleen, and among
individuals was 51.1\% for liver and 66.1\% for spleen. There were no
correlations between \emph{CV} (among replicates and after removing
outliers) and tissue mass (Liver: \emph{r} = -0.09, \emph{P} = 0.38,
Spleen: \emph{r}= 0.17, \emph{P} = 0.29), histone concentration (Liver:
\emph{r} = 0.30,\emph{P} = 0.16, Spleen: \emph{r} = 0.26, \emph{P} =
0.20), or mean H3 (Liver: \emph{r} = -0.45, \emph{P} = 0.06, Spleen:
\emph{r} = -0.03,\emph{P} = 0.46).

We detected high level of variation among individuals (Table 1). Mean H3
histone acetylation in liver was 3.82 OD/mg/ml (\selectlanguage{greek}σ \selectlanguage{english}= 2.58); in spleen,
mean H3 histone acetylation was 3.51 OD/mg/ml (\selectlanguage{greek}σ \selectlanguage{english}= 2.45). We failed to
detect significant differences between tissues (\emph{t-test P} =
0.38,\emph{f-test} P = 0.87). We caution that based on the high
variation observed, these results should not be interpreted as
indicating no differences exist in H3 acetylation among tissue, rather
that they indicate high variation among individuals.

Interestingly, house sparrows with higher epigenetic potential had
higher H3 acetylation in liver tissue (High EP \emph{Mean} \emph{H3AC} =
4.13 OD/mg/ml; Low EP \emph{Mean} \emph{H3AC} = 1.56
OD/mg/ml;\emph{t-test} = 0.046), and a similar trend occurred in spleen
(High EP\emph{Mean H3AC} = 4.04 OD/mg/ml; Low EP \emph{Mean H3AC} = 1.78
OD/mg/ml; \emph{t-test} = 0.108; Figure 1). Also, we found a negative
correlation between H3 acetylation in spleen and \emph{TLR-4} expression
in cecum among house sparrows (\emph{r} = -0.595, \emph{P} = 0.02).

\textbf{Discussion}

The present study shows that histone H3 acetylation can be screened
among wild bird samples using commercially available kits from
EpiGentek. We found that H3 acetylation in house sparrows is highly
variable. We detected a high \emph{CV} and several outliers (CV above
25\%); however, the variation among replicates is much lower than
variation among individuals within both tissues, suggesting that the
high \emph{CV} values can be attributed to high variation among
individuals and not to factors pertaining to the kits. We found no
correlations between \emph{CV} and potential sources of error (either
technically or biologically). Liver values were more consistent than
spleen values, suggesting a difference between tissues. To our
knowledge, this is the first study of its kind.

H3 acetylation in house sparrows varies in a manner suggesting
ecological relevance. House sparrows with higher epigenetic potential
(i.e., more CpG sites in \emph{TLR-4} promoter region) had significantly
more H3 acetylation in liver compared to house sparrows with low
epigenetic potential. A non-significant, but similar trend also occurred
in the spleen. Previous studies on epigenetic potential in house
sparrows, based on DNA methylation, found that higher epigenetic
potential is important for house sparrows to rapidly adapt to novel
environments (Hanson et al. 2021, 2022). It is yet to be determined if
estimating epigenetic potential as the number of CpG sites is directly
relevant to histone modification, yet our data suggest the two may be
related in some manner. At the very least, we expect differences in
histone acetylation among individuals with differing epigenetic
potential, measured as the number of CpG sites.

We also found a relationship between H3 acetylation and
\emph{TLR-4}expression. \emph{TLR-4} is a receptor that detects
Gram-negative bacteria and ultimately leads to an inflammatory response
(Kilvitis et al. 2019). Histone acetylation being related to
\emph{TLR-4} expression suggests that histone acetylation has an effect
on gene expression in house sparrows. Because we measured total
acetylation levels, at this time, we cannot be certain exactly how H3
acetylation is associated with gene expression.

For those interested in using EpiGentek's Total Histone H3 Acetylation
Detection Fast Kit for ecologically-based studies of birds, we note
that, as this kit does not identify specific genomic locations of
acetylated H3, the use of experimental designs asking questions relevant
to total H3 acetylation levels is critical. For example, asking
questions about how different experimental treatments affect total H3
and H4 histone acetylation (Liu et al. 2016). Or, determining if
relationships exist between global histone acetylation levels and
individual phenotypes (Mosashvilli et al. 2010). Also, we recommend
using three replicates and a large sample size, as values were highly
variable among individuals.

In conclusion, we describe a fast and inexpensive method for measuring
H3 acetylation in birds, and suggest that histone acetylation varies in
an ecologically relevant way. Screening histone acetylation will likely
add an important new perspective to ecological epigenetics, which has,
to date, been dominated by studies of DNA methylation. Given the role
that histone modification plays in gene expression and phenotypic
plasticity, and its apparent level of individual-level variation, it is
very important that we factor this epigenetic mechanism in to future
ecological epigenetics studies.

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\textbf{Tables and Figures}

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/636049/articles/653147-screening-h3-histone-acetylation-in-a-wild-bird-the-house-sparrow-passer-domesticus}

Figure 1. In liver of house sparrows, H3 acetylation differed between
individuals with high (Mean H3AC = 4.13) and low (Mean H3AC = 1.56)
epigenetic potential (\emph{t-test} \emph{P} = 0.046). A similar trend
occurred in spleen of house sparrows, high (Mean H3AC = 4.04) versus low
(Mean H3AC = 1.78) epigenetic potential, yet was not statistically
significant (\emph{t-test} \emph{P} = 0.108).

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/636049/articles/653147-screening-h3-histone-acetylation-in-a-wild-bird-the-house-sparrow-passer-domesticus}

Figure 2. Correlation between H3 acetylation in spleen and
\emph{TLR-4}gene expression in cecum of infected house sparrows
(\emph{r} = -0.60;\emph{P} = 0.02).

Table 1. House sparrow samples with sex, mass, gene expression level
of\emph{TLR-4} , epigenetic potential (EP), weight of starting tissue,
histone extraction concentration (Conc.), results of three replicates
(Rep 1, 2, 3), mean histone acetylation (Mean), standard deviation, and
coefficient of variation (CV\%).\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllllll@{}}
\toprule
Bird & Sex & Mass & \emph{TLR-4} & EP & Tissue (mg) & Conc. (mg/ml) &
Rep 1 (OD/mg/ml) & Rep 2 (OD/mg/ml) & Rep3 (OD/mg/ml) & Mean (OD/mg/ml)
& Std Dev & CV\%\tabularnewline
\midrule
\endhead
3139-L & M & 27.1 & 2.62 & high & 9.8 & 0.44 & 1.50 & 1.48 & 1.28 & 1.42
& 0.12 & 8.4\tabularnewline
3155-L & M & 25.3 & 1.01 & high & 9.6 & 0.49 & 1.59 & 1.79 & 1.51 & 1.63
& 0.14 & 8.7\tabularnewline
3157-L & M & 20.5 & 1.02 & high & 9.9 & 0.33 & 2.00 & 2.54 & 1.72 & 2.08
& 0.42 & 20.3\tabularnewline
3158-L & M & 21.7 & 9.22 & high & 12 & 0.29 & 1.64 & 2.50 & 1.59 & 1.91
& 0.51 & 26.7\tabularnewline
3159-L & M & 22.5 & 1.27 & high & 9.6 & 0.38 & 1.81 & 1.83 & 1.56 & 1.73
& 0.15 & 8.7\tabularnewline
3174-L & M & 24.1 & 0.77 & high & 25.4 & 0.71 & 2.16 & 1.95 & 2.08 &
2.06 & 0.10 & 5.0\tabularnewline
3179-L & M & 21.5 & 1.22 & high & 8 & 0.38 & 1.77 & 2.27 & 2.09 & 2.05 &
0.25 & 12.3\tabularnewline
3190-L & M & 24 & 2.38 & high & 6.9 & 0.14 & 1.24 & 1.29 & 1.43 & 1.32 &
0.10 & 7.4\tabularnewline
3191-L & M & 22.1 & 2.37 & high & 4.8 & 0.56 & 0.07 & 0.10 & 0.09 & 0.08
& 0.01 & 10.1\tabularnewline
3162-L & M & 20.4 & 14.93 & low & 18.6 & 0.58 & 1.28 & 1.65 & 1.09 &
1.34 & 0.28 & 21.1\tabularnewline
3176-L & M & NA & 4.53 & low & 22.2 & 0.32 & 0.96 & 1.40 & 0.87 & 1.08 &
0.28 & 26.2\tabularnewline
3187-L & M & 24.8 & 1.95 & low & 5.1 & 0.79 & 0.04 & 0.11 & 0.06 & 0.07
& 0.03 & 48.2\tabularnewline
3139-S & M & 27.1 & 2.62 & high & 21.7 & 0.55 & 1.88 & 2.34 & 0.95 &
1.72 & 0.71 & 41.2\tabularnewline
3155-S & M & 25.3 & 1.01 & high & 3.1 & 0.57 & 1.94 & 2.32 & 0.93 & 1.73
& 0.72 & 41.5\tabularnewline
3157-S & M & 20.5 & 1.02 & high & 8 & 0.30 & 2.70 & 2.49 & 1.34 & 2.18 &
0.73 & 33.5\tabularnewline
3158-S & M & 21.7 & 9.22 & high & 0.4 & 0.80 & 0.10 & 0.12 & 0.04 & 0.08
& 0.04 & 46.6\tabularnewline
3159-S & M & 22.5 & 1.27 & high & 16.5 & 1.13 & 0.52 & 0.50 & 0.30 &
0.44 & 0.12 & 28.2\tabularnewline
3174-S & M & 24.1 & 0.77 & high & 3.5 & 0.40 & 2.21 & 2.23 & 1.86 & 2.10
& 0.21 & 10.0\tabularnewline
3179-S & M & 21.5 & 1.22 & high & 0.6 & 0.19 & 1.10 & 1.06 & 0.63 & 0.93
& 0.27 & 28.5\tabularnewline
3190-S & M & 24 & 2.38 & high & 1.9 & 0.40 & 2.16 & 2.03 & 1.62 & 1.94 &
0.28 & 14.6\tabularnewline
3191-S & M & 22.1 & 2.37 & high & 9.1 & 0.19 & 1.00 & 0.71 & 0.37 & 0.69
& 0.32 & 45.9\tabularnewline
3162-S & M & 20.4 & 14.93 & low & 10.1 & 0.32 & 0.20 & 0.21 & 0.21 &
0.20 & 0.01 & 3.0\tabularnewline
3176-S & M & NA & 4.53 & low & 3.5 & 0.30 & 1.05 & 0.89 & 0.98 & 0.97 &
0.08 & 8.2\tabularnewline
3187-S & M & 24.8 & 1.95 & low & 6.1 & 0.42 & 0.90 & 0.83 & 0.63 & 0.79
& 0.14 & 17.8\tabularnewline
\bottomrule
\end{longtable}

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