5. Epigenomics

5.4. Chromatin organization and histone modifications

Among a variety topics of epigenomics, transcriptional activities occurring at tissue and cellular levels are regulated by transcription factors (TFs) (Li-Byarlay et al., 2013; Qiu et al., 2013; Spitz & Furlong, 2012). TFs work with cis-acting regulatory elements (CRE) for cell regulation od promoters, epigenetic patterns, and genome structure (Stadhouders et al., 2019).

A histone is an octomeric (8-subunit) protein complex which is a crucial component of chromatin. These complexes serve as a spool that genomic DNA is wrapped around. Post-translational modifications determine how tightly the DNA is associated with histones. Methylation of lysine 9 on the third subunit of the histone proteins (denoted H3K9me3) results in tighter association of the DNA to the histones, and limits the ability of transcription factors to access the DNA for transcription. Acetylation of several amino acids causes the DNA and histones to be associated more loosely, making the DNA more accessible to transcription factors and therefore more likely to be transcribed. Modification of the histones proteins provides an additional mechanism of gene regulation, beyond the binding of transcription factors to the promoter region of a gene.

5.4.1. Chromatin immunoprecipitation sequencing and transcription factor binding motifs

Chromatin immunoprecipitation sequencing (ChIP-seq) is one way that sites of histone modifications can be mapped to the genome. Like many modern molecular techniques, existing protocols and guidelines can be, more or less, directly adapted for use in honey bees with little modification (e.g. see Nakato & Sakata (2021)).

5.4.2. Hi-C & chromatin conformation

The Hi-C technology that captures genome-wide chromatin interactions and structure method has been already discussed in Section 3.3.1.1. Beyond genome assembly improvement, HI-C has identified that the variation of the genomic structure of A. mellifera is associated with the variation of phenotype (Jin et al., 2023), and is highly regulated with gene activities or adaptations in different environment (Kirkpatrick & Barton, 2006; Wallberg et al., 2017). In Drosophila fruit flies, or Heliconius butterflies, chromosomal inversions is related to adaptation to environmental adaptation (Joron et al., 2011; Krimbas & Powell, 1992). Future research using Hi-C technology is needed to obtain more information on the chromatin structure and how they interact with gene regulations.

5.4.3. Chromatin accessibility and transcriptional factor motifs

The chromatin accessibility is the percentage of time any given fragment of the genome is occupied by a nucleosome. Although not necessarily heritable, it plays an important role in gene regulation and can change in response to environmental cues (Turner, 2008). Three assays used to study chromatin accessibility are: 1) assay for transposase accessible chromatin by sequencing (ATAC-seq), 2) DNase I hypersensitive site -seq (DNase-seq), and 3) micrococcal nuclear sequencing (MNase-seq). High read depth in these assays can reveal regions that where TFs bind (Tsompana & Buck, 2014).

Recent research using ATAC-seq, RNA-seq and ChIP-seq has identified many regulatory regions, including accessible chromatin regions, nucleosome occupancy, and specific patterns of TF gene networks in the genome of queen, worker, and drone adult bees (Lowe et al., 2022; Zhang et al., 2023). Another ATAC-seq and RNA-seq study comparing the TFs among the brains of foragers, newborn workers, and nurses revealed different regulatory landscape and new interactions within the transcriptional regulatory network underlying the division of labor (Fang et al., 2022). Previous meta-analyses and cis-metanalysis tools also revealed that transcriptional regulatory mechanisms that are underlying the behavioral maturation of honey bee workers (Ament et al., 2012). The genome-wide scanning and gene regulatory network activity also identified TF motifs in the honey bee brains at the colony and individual levels, and how they are associated with the regulation of social behavior and its evolution (Jones et al., 2020; Rittschof et al., 2014; Shpigler et al., 2019).

Additional ChIP-seq experiments have informed on transcriptional factor binding sites of Vitellogenin gene, which affect the immunity and behavior of honey bees (Salmela et al., 2022). Other transcription factors such as usp, ubx, Mblk-1/E93 are also critical for phenotypic plasticity and development of honey bees and other insects (Ament, Wang, et al., 2012; Matsumura et al., 2022; Prasad et al., 2016; H. Yan et al., 2014). While these cutting-edge methods are still rare in Apis, the few studies that exist provide very detailed protocols for performing ChIP-seq experiments of histone methylation in honey bee larvae, including the extensive optimization such as crosslinking time and buffer compositions (Wojciechowski et al., 2018).

5.4.4. Detecting histone modifications by mass spectrometry

Histone modifications, a key epigenetic mechanism, significantly contribute significantly to phenotypic dimorphism and caste difference in A. mellifera honey bees (Dickman et al., 2013; Jin et al., 2023; Zhang et al., 2023). Several studies have reported that histone modifications are associated with social behavior, evolution, development, and ecology of A. mellifera (Alghamdi & Alattal, 2024; Dickman et al., 2013; Wojciechowski et al., 2018; Zhang et al., 2023). Histone deacetylase inhibitors also play a role in caste determination in honey bees (Spannhoff et al., 2011).

Mass spectrometry-based proteomics can be used to detect and quantify modified histones. Since a typical proteomics workflow will dissociate histones from the DNA with which they interact, this approach is not suitable for identifying DNA binding sites. However, the power of mass spectrometry is its ability to detect numerous types of covalent modifications, with dozens of potential post-translational modifications of histones (Huang et al., 2015). While a generic shotgun proteomics dataset will contain ions corresponding to modified peptides, signal intensity and data quality are improved if immunoprecipitation is conducted to enrich the peptide or protein sample for sequences carrying the desired modification. The resulting mass spectrometry data processing will differ as well, as the mass shift and affected amino acid residue associated with the modification will need to be specified. Sample preparation procedures are essentially as described in Section 8, but further details can be found in see Huang et al. (2015).