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.