La Tierra, un planeta con vida

The DNA is packaged by an octamer of core histones, consisting of two H2A-H2B dimers and an H3-H4 tetramer (Figure 1.2) to form a 10-nm nucleosome as the primary component of chromatin. Histones are proteins comprising 100-140 amino acid long and structurally consist of 3 motifs: i) the histone fold, ii) the extra fold structured elements unique to the different histones, and iii) the labile termini, which vary from 13 to 42 amino acids in length (Arents & Moudrianakis 1995; Luger et al. 1997). The 10 nm polynucleosome arrays are folded and stabilized by linker H1 histones. As discussed above, histones are constantly modified, shifted, evicted and re-deposited as chromatin continues to remodel, in order to maintain proper organization and gene regulation (Marzluff et al. 2008). Major chromatin disruption requiring new histone synthesis also occurs during DNA/chromatin replication at S-phase. These continuous disassembly-reassembly of chromatin requires rapid transcription and translation of histone genes.

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The core histone proteins are encoded by a family of replication-dependent genes. In eukaryotes, the mRNA of these genes is the only known cellular non-polyadenylated mRNA that has a unique 3' stem-loop (26 bp), crucial in modulating mRNA stability, transport, and translation. These mRNAs are rapidly expressed at the beginning of S phase and are maintained at high levels throughout the S phase to coincide with DNA replication (Marzluff et al. 2008).

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Figure 1-2: Nucleosome core particle schematic diagram: half ribbon traces (73 bp) of the 146-bp DNA phosphodiester backbones (brown and turquoise) and eight histone protein main chains (blue: H3; green: H4; yellow: H2A; red: H2B. Adapted from Luger et al. (1997).

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Histone variants, unlike the core histones, are expressed and incorporated into chromatin throughout the cell cycle. The replication-independent gene and mRNA structure of histone variants also differ from core histones. The histone variant genes often contain introns, and the transcripts are often polyadenylated; features that are thought to be important in the post-transcriptional regulation (Kamakaka & Biggins 2005). The most prominent sequence divergence is found in H2A and H2B, with H2A having the most variants. In contrast, H4 has no reported variants, and H3 variants have only minimal sequence variations (Mattiroli et al. 2015).

Histone variants have evolved to contribute to the complexity of chromatin and have specialized functions in regulating chromatin dynamics. Their sequence divergence can alter chromatin states and the fate of cell decisions (Weber & Henikoff 2014). The role of histone variants in regulating transcription has been widely reported. It was initially understood that in order for transcription to take place, a nucleosome- depleted region (NDR) must form at the TSS, to allow binding of transcription machinery. Based on experimental evidence, the accessibility of micrococcal nuclease supported this notion, however, this has been disputed upon finding that nucleosomes containing labile H2A variants flanked the NDR. Jin et al. (2009) used chromatin immunoprecipitation (ChIP) and re-ChIP, followed by high throughput Solexa sequencing on HeLa cells expressing Flag-tagged H3.3 histone to reveal that hybrids of H3.3-H2A.Z nucleosome core particle (NCP) localized to TSS of active genes. Subsequently, this laboratory has shown that heterotypic H2A.Z-H2A nucleosomes occupy the TSS in stem cells thus raising the possibility that H3.3-H2A.Z nucleosomes are in fact heterotypic with respect to H2A.Z (Nekrasov et al. 2012; Soboleva et al. 2014). As mentioned above, we also found that H2A.Lap1-containing nucleosomes are found at the TSS of active genes in the testis (Soboleva et al. 2012; Soboleva et al. 2014). These unstable nucleosomes may serve as a ‘place holder’ to prevent the region from being covered by adjacent stable NCP and/or nonspecific factors, when the region is nucleosome-free. Moreover, these fragile nucleosomes could be more easily displaced by transcription factors because of its instability (Jin et al. 2009).

Histone variants have also been reported to participate in their own epigenetic inheritance, maintaining correct localization on newly synthesized daughter strands following DNA replication. For instance, during the cell cycle of mouse trophoblast stem cells, a decrease in the level of H2A.Z, at promoters, during S phase occurred. This coincided with the H2A.Z-H2A.Z nucleosome, flanking the TSS, becoming

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heterotypic. This nucleosome persisted as heterotypic until M phase (Nekrasov et al. 2012). The depleted expression of H2A.Z at promoter, after DNA replication, is possibly due to its redistribution to constitutive heterochromatin and/or at the centromere. This cell cycle-dependent possible dynamic movement of H2A.Z suggests its functional importance in providing transcriptional memory, that is, active marks established at the G1 phase and remaining at M phase may facilitate the reestablishment of gene transcription after cell division (Nekrasov et al. 2012).

Histone variants have also been linked to the response to DNA damage, following exposure to gamma-radiation (Volle & Dalal 2014). H2A variants, γH2A.X. H2A.Z and macroH2A, specifically, have been implicated in this role. The various functions of histone variants in eukaryotes are briefly presented in Table 1.

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Table 1-1: List of reported functions of histone variants in eukaryotic organisms (Yuan & Zhu 2012)

Histone variants Function

CENP-A or cenH3 Epigenetic marker of the centromere

H3.3 Involved in transcription

Enriched at transcriptionally active regions, telomerase, and pericentric regions

H3.Z Regulation of cellular response to outside stimuli H3.Y Regulation of cellular response to outside stimuli H2A.Z Transcriptional control rheostat

Heterochromatin formation and maintenance

H2A.X Double strand break repair

Meiotic remodelling of sex chromosome

macroH2A Gene silencing

X-chromosome inactivation H2A.Bbd/H2A.B or

H2A.Lap1

Epigenetic mark of active chromatin. Found at the TSS and intron-exon boundaries.

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