Spatiotemporal coordination of gene expression, replication, repair, and developmental processes in eukaryotes are coordinated by the interplay of the genome and epigenetic signatures at various hierarchical levels, such as CpG signaling (that is, DNA cytosine methylation/hydroxymethylation)[1, 2] and post-translational modification (PTM) contributing to chromatin structure regulation[3, 4]. Evidence is accumulating that selective deposition of variant histones into nucleosomes and eventually chromatin via interactions with histone chaperones represents a further crucial level of chromatin structure regulation. It is believed that the selective incorporation of histone variants into nucleosomal arrays leads to the establishment of cell type-specific ‘barcodes’, which can be transmitted to daughter nuclei in proliferating cells, thus contributing to the maintenance of cell type-specific gene expression patterns. According to earlier studies, the histone variant H3.3 associates preferentially with euchromatin. However, H3.3 is also suspected to fulfill more versatile functions during mammalian embryogenesis, in which murine paternal and maternal pronuclei adopt asymmetric H3.3 signatures. In detail, H3.3 associates preferentially with the paternal pronucleus, but is largely devoid of H3K4me3. Instead, H3.3 seems to be involved in the establishment of pericentric heterochromatin, which is required for proper chromosome segregation during the first mitosis, which follows pronuclei formation[9–11].
It has been argued that constitutively expressed variants may initially have evolved solely as replacement variants in non-cycling cells or between S-phases, when replication-dependent variants are absent. However, observations that diverse H3 variants evolved frequently but independently within related species in almost all eukaryotic supergroups oppose this view. Instead, it is likely that that multiple H3 variants evolved to fulfill diverse functions in the cell cycle and development of various eukaryotic lineages, despite their extremely high degree of protein sequence conservation.
In addition to being found in metazoa, histone H3 variants are commonly found in single-celled ciliated protozoa, such as Tetrahymena or Euplotes. Even within the ciliophora phylum, Stylonychia occupies an exceptional position. Recently, we characterized full-length macronuclear genomic sequences encoding eight histone H3 variants, which had been fragmentarily identified more than a decade ago. To date, this is the highest number of H3 variants found in a single species, except for humans. Thus, this ciliate species could be an attractive model for the study of the spatiotemporally coordinated expression of histone variants, their assembly into chromatin, and their biological relevance.
Ciliates are characterized by nuclear dualisms, whereby each cell contains two different nuclear types: somatic macronuclei and germline micronuclei (see Additional file1: Figure S1A, step 1). Transcripts required for vegetative growth are synthesized in the macronucleus, whereas the transcriptionally inert micronuclei consist of condensed chromatin. The macronuclear DNA of the stichotrichous ciliate species Stylonychia lemnae is organized in short molecules, known as nanochromosomes, ranging in size from 0.4 to 75 kb. Each of these nanochromosomes usually contains one open reading frame and all the sequences required for expression and replication. Sexual reproduction (conjugation) leads to the differentiation of a new macronucleus from a micronuclear derivative, while the parental macronucleus becomes degraded (see Additional file1: Figure S1A, steps 2 to 6). The latter starts at the onset of conjugation and at the same time, micronucleus meiosis takes place (see Additional file1: Figure S1A, step 2). Subsequently, haploid migratory micronuclei become exchanged between conjugation partners (see Additional file1: Figure S1A, step 3, Ainset). By fusion, these migratory nuclei build a synkaryon with their stationary counterparts, which is followed by mitosis. One of the resulting products of this mitosis will build a new micronucleus, whereas the other product (anlage) will develop into a new macronucleus (see Additional file1: Figure S1A, step 4). In Stylonychia, a first phase of sequential DNA replication events leads to polytene chromosomes formation in the macronuclear anlagen followed by a programmed loss of micronucleus-specific DNA sequences (Additional file1: Figure S1A, steps 5–6). Thus the DNA content in the developing macronucleus changes dramatically over time (Additional file1: Figure S1B). Micronucleus-specific DNA largely consists of ‘bulk’ repetitive and transposon-like elements, and of internal eliminated sequences (IES), which interrupt macronucleus-destined sequences (MDSs) in a large portion of scrambled genes, whose modules have to be reordered during macronuclear development. During these processes, dramatic DNA reorganization and elimination processes take place. Over 90% of micronuclear sequences become organized into condensed chromatin domains, which are eventually excised from the genome[18, 19]. Macronucleus maturation is accompanied by a second phase of sequential DNA replication events, leading to the final copy numbers of nanochromosomes. Conjugation is associated with a short-term boost of differential gene expression, and many of these expressed genes are suspected to be involved in the regulation of programmed genome reorganization. Among these genes are histone variants and a Piwi family protein[20, 21]. Furthermore, small non-coding RNAs (ncRNAs) accumulate, which may result from short-term transcription of the micronuclear genome, as reported for Tetrahymena. By contrast, recent studies suggest a parental macronuclear origin of ncRNA in Oxytricha, a species closely related to Stylonychia[23, 24]. For Stylonychia, the nuclear localization of ncRNA synthesis remains unsolved, and some earlier observations support a possible micronuclear origin[16, 25]. However, it is believed that these ncRNAs eventually interact with Piwi, and undergo a selection process via comparison with the parental macronuclear genome, resulting in a subfraction of ncRNAs homologous to specific sequences. Finally, Piwi-bound ncRNAs target homologous sequences in the developing macronucleus, which are then converted into discrete chromatin structures.
Here we provide detailed insight into differential H3 gene expression patterns and the accumulation of three H3 variant proteins during macronuclear differentiation in Stylonychia. We show that some H3 variants are spatiotemporally regulated, and possess specific PTM signatures. In polytene anlagen, acetylated H3.7 is associated with specific sequence classes. Perturbation of the Piwi-ncRNA pathway leads to impaired HIS33 gene expression, and entails decreased deposition of H3.3 protein levels in anlagen chromatin, suggesting a link between the mechanisms responsible for RNA-directed chromatin-reorganization and the expression of some H3 variants.