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 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 CV values can be attributed to high variation among
individuals and not to factors pertaining to the kits. We found no
correlations between 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 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 TLR-4expression. 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 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.