Molecular Characterization of Core Histones in the Silkworm, Bombyx mori

In eukaryotic cells, the DNA is packaged into chromatin. The basic module of chromatin, the nucleosome, consists of 146 base pairs of DNA wrapped around a histone octamer (Luger et al., 1997). The octamer is composed of two each of the core histones H2A, H2B, H3 and H4. Each of the four core histones has a central “histone fold” domain that consists of three alpha helixes connected by two loops (Arents & Moudrianakis, 1995; Luger et al., 1997). An array of nucleosomes generally contains the additional histone, H1, which is also known as a linker histone. The amino acid sequence of canonical core histones is highly conserved in eukaryotes. The N-terminal tails of the core histones protrude from the nucleosomal surface and are subject to multiple covalent modifications including methylation, phosphorylation and acetylation. These modifications have the potential to regulate the chromatin architecture and can thereby affect all aspects of DNA processing. According to the so-called “histone code” hypothesis, these modifications could be read by proteins that bind to specific modifications and can then regulate downstream events (Turner, 1993; Strahl & Allis, 2000). With the exception of H4, all canonical core histone proteins are known to have variant counterparts, which often differ in surprisingly few amino acids (Pusarla & Bhargava, 2005). The incorporation of histone variants into nucleosomes as a mode of marking chromatin regions has been proven to have a high impact on gene regulation, DNA repair and meiotic events. These histone variants have been implicated in the epigenetic inheritance mechanisms of chromatin markings (Ahmad & Henikoff, 2002a; Henikoff et al., 2004) and shown to play significant roles in gene expression, antisilencing, heterochromatinization and the formation of specialized regions of chromatin (Ausio & Abbott, 2002; Smith, 2002; Kamakaka & Biggins, 2005; Sarma & Reinberg, 2005). H2A has the largest macro-heterogeneous family of variants, and all of them were found to have a crucial role in gene expression and nuclear dynamics (Ausio & Abbott, 2002). Homologues of H2AX are found across all phyla, including fungi, animals, plants and the most primitive eukaryotes such as Giardia (Malik & Henikoff, 2003). H2AX is randomly incorporated into nucleosomes and represents 10-15% of total cellular H2A. Phosphorylation of H2AX provides an epigenetic mark for broken DNA to recruit repair machineries. The carboxy terminus of H2AX differs from that of bulk H2A by being longer and having a four-amino-acid sequence element SQEL at the extreme C-terminal end of the protein. H2AZ, which is sometimes referred to as H2A.Z/F, is the most conserved variant during eukaryotic evolution. H2AZ has different functions in various organisms that include maintenance of pericentric and telomeric heterochromatin, transcriptional activation and viability. H3.3, one of the histone H3 variants, is almost identical to H3 and differs at only four positions: one in the N-terminal tail (Ala31) and three in the histone In eukaryotes, genomic DNA is wrapped around an octamer of the core histone proteins H2A, H2B, H3 and H4. Silkworms have been known to bear holocentric chromosomes and their regulatory mechanisms of chromatin remodeling remain unclear. We have cloned the silkworm canonical core histones and their variants. The H2A variants, H2AX and H2AZ, were highly conserved in the silkworm, whereas the fly has only one H2A variant, H2Av, which is a chimera of H2AX and H2AZ with the function of both molecules. In the silkworm, all histones except for H2AX were ubiquitously expressed in all tested tissues, and H2AX was expressed only in the genital organs. A subcellular localization analysis of the cloned histones using an EGFP fusion construction demonstrated that the behaviors of the histones are the same as those of genomic DNA throughout the cell cycle. The fluorescence intensities of H2AX and H3.3 were weaker than those of the other histones. The incorporation of these histones into chromatin might be restricted due to their specific function in DNA repair and transcription activity. There were two H3 variants, H3.2 and H3.3, in the silkworm; H3.1 was not present. Lysine 9 on H3 was methylated and acetylated in silkworms bearing holocentric chromosomes.