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. 2016 Sep 6;9(1):36.
doi: 10.1186/s13072-016-0085-1. eCollection 2016.

The cnidarian Hydractinia echinata employs canonical and highly adapted histones to pack its DNA

Anna Török  1 , Philipp H Schiffer  2 , Christine E Schnitzler  3 , Kris Ford  4 , James C Mullikin  5 , Andreas D Baxevanis  6 , Antony Bacic  7 , Uri Frank  1 , Sebastian G Gornik  1
Affiliations

The cnidarian Hydractinia echinata employs canonical and highly adapted histones to pack its DNA

Anna Török et al. Epigenetics Chromatin. .

Abstract

Background: Cnidarians are a group of early branching animals including corals, jellyfish and hydroids that are renowned for their high regenerative ability, growth plasticity and longevity. Because cnidarian genomes are conventional in terms of protein-coding genes, their remarkable features are likely a consequence of epigenetic regulation. To facilitate epigenetics research in cnidarians, we analysed the histone complement of the cnidarian model organism Hydractinia echinata using phylogenomics, proteomics, transcriptomics and mRNA in situ hybridisations.

Results: We find that the Hydractinia genome encodes 19 histones and analyse their spatial expression patterns, genomic loci and replication-dependency. Alongside core and other replication-independent histone variants, we find several histone replication-dependent variants, including a rare replication-dependent H3.3, a female germ cell-specific H2A.X and an unusual set of five H2B variants, four of which are male germ cell-specific. We further confirm the absence of protamines in Hydractinia.

Conclusions: Since no protamines are found in hydroids, we suggest that the novel H2B variants are pivotal for sperm DNA packaging in this class of Cnidaria. This study adds to the limited number of full histone gene complements available in animals and sets a comprehensive framework for future studies on the role of histones and their post-translational modifications in cnidarian epigenetics. Finally, it provides insight into the evolution of spermatogenesis.

Keywords: Chromatin; Cnidaria; Histone; Histone variants; Sperm-specific histones.

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Figures

Fig. 1
Fig. 1
Unrooted maximum likelihood phylogenies of Hydractinia echinata histones. Black circles indicate nodes with bootstrap support above 50. H2A.Z and macroH2A and H3.3 variants from clear clades, whereas H2A.X, all H2B variants and CENP-A (H3-derived) are not clearly separated from their canonical counterparts. The H1 phylogeny is unresolved
Fig. 2
Fig. 2
Annotated genomic locus and expression profiles of the H1.1 and core histones H2A.1, H2B.1, H3.1 and H4.1 of Hydractinia echinata. a Expression of H1.1, H2B.1, H3.1 and H4.1 in feeding polyps. H2B.1 was analysed using a red fluorescent probe. The white wedge highlights an individual cell expressing H4.1. b Organisation of the genomic locus depicting coding sequences, mapped RNA reads (showing the number of reads mapped), predicted TATA-boxes and 3′-UTR stem-loops. Extended RNA read mapping can be found in Additional file 6: S6. c Co-localisation of gene expression (green) and S-phase (EdU, red). A white wedge highlights histone-expressing cells in S-phase. d Predicted structure of the histone cluster arginine tRNA
Fig. 3
Fig. 3
Analysis of the 3′-UTR stem-loop of Hydractinia echinata histone mRNAs. a Stem-loop sequence alignment, consensus sequence and sequence logo. Nucleotide sequences start after the termination codon (not shown), and 6–17 non-conserved base pairs are omitted before the stem-loop sequences begin. The alignment continues past the histone downstream element (HDE). Sequence differences are highlighted. b Predicted structure of the Hydractinia echinata histone 3′-UTR stem-loop, c Comparison of the human and Hydractinia echinata histone 3′-UTR stem-loop consensus sequences
Fig. 4
Fig. 4
Annotated genomic loci and expression profiles of Hydractinia echinata H1.2, H2A.Z, macroH2A and CENP-A. a H1.2 expression in feeding (Ai) and male sexual polyps (Aii). RNA-Seq mapping shows that H1.2 transcripts are found in larva, female, feeding polyps and highly abundant in male polyps (green wedge; RNA-Seq track). The annotated genomic locus of H1.2 shows its coding sequence, mapped RNA reads (showing the number of reads mapped), a predicted TATA-boxes and a polyA signal. The gene contains two exons. H1.2 expression is replication-independent, and its transcripts do not exclusively co-localise with EdU-positive S-phase cells (red wedge; Aiii). b H2A.Z expression in feeding polyps. RNA-Seq shows that the gene contains five exons, of which 2 are non-coding. The H2A.Z transcript is abundant in all life stages. The annotated genomic locus of H2A.Z shows its coding sequence, mapped RNA reads (showing the number of reads mapped), a predicted TATA-box and a polyA signal. c The annotated genomic locus of macroH2A showing its coding sequence, mapped RNA reads (showing the number of reads mapped), a predicted TATA-box and polyA signal. The gene contains eight exons. d The annotated genomic locus of CENP-A shows its coding sequence, mapped RNA reads (showing the number of reads mapped), a predicted TATA-box and a polyA signal. The gene contains two exons
Fig. 5
Fig. 5
Annotated genomic loci and expression profiles of Hydractinia echinata H2A.X.1 and H2A.X.2. a H2A.X.1 expression in feeding, male and female sexual polyps (red wedges; RNA-Seq mapping track). The annotated genomic locus of H2A.X.1 shows its coding sequence, mapped RNA reads (showing the number of reads mapped), a predicted TATA-boxes and a 3′-UTR stem-loop. The gene contains two exons, and one of them is non-coding. b H2A.X.2 expression is restricted to female sexual polyps (red wedges; RNA-Seq mapping track) and absent in feeding and male sexual polyps. The annotated genomic locus of H2A.X.2 shows its coding sequence, mapped RNA reads (showing the number of reads mapped), a predicted TATA-box and a polyA signal. The gene contains five exons, two of which are non-coding
Fig. 6
Fig. 6
Annotated genomic loci and expression profiles of Hydractinia echinata H3.3.1 and H3.3.2. a H3.3.1 expression in feeding polyps and male and female sexual polyps. H3.3.1 is highly abundant in female germ cells (red wedge). The annotated genomic locus of H3.3.1 shows its coding sequence, mapped RNA reads (showing the number of reads mapped), a predicted TATA-box and a polyA signal. The gene contains two exons. b H3.3.1 expression is replication-independent and its transcripts do not co-localise with EdU-positive S-phase cells in both feeding polyps (red wedge in Bi) and male sexual polyps (white asterisk in Bii). Replicating cells do not contain H3.3.1 transcripts (yellow asterisk in Bii). c H3.3.2 expression in feeding polyps. The annotated genomic locus of H3.3.2 shows its coding sequence, mapped RNA reads (showing the number of reads mapped), a predicted TATA-box and a 3′-UTR stem-loop. The gene contains one exon. H3.3.2 expression is replication-dependent, and its transcripts co-localise with EdU-positive S-phase cells (red wedge)
Fig. 7
Fig. 7
Annotated genomic loci and expression profiles of Hydractinia echinata H2B.2-6. a H2B.3/4 expression in embryo and male sexual polyps. The annotated genomic loci of H2B.3 and H2B.4 show their coding sequence, mapped RNA reads (showing the number of reads mapped), predicted TATA-boxes and 3′-UTR stem-loops. Both genes contain one exon. RNA-Seq mapping shows that H2B.3 transcripts are only found in male polyps (green wedge) and that H2B.4 transcripts are expressed in male sexual polyps, feeding polyps and larva (red wedge). Two expression patterns exist, but due to sequence similarities it cannot be determined which pattern is derived from which gene; thus, both expression patterns are shown (black wedges in Ai and Aii) using a shared H2B.3/4 annotation. b Co-localisations of H2B.1 or S-phase cells with H2B.3/4. Expression patterns of H2B.1 and H2B.3/4 do not overlap (Bi), indicating that H2B.3/4 genes are expressed independent of H2B.1—the Hydractinia canonical core H2B. Histone H2B.3/4 expression is replication-dependent, and transcripts co-localise with EdU-positive S-phase cells in male gonads (yellow asterisk in Bii). c H2B.3/4 expression in male polyps using fluorescent probes. The white wedges pinpoint an individual cell expressing H2B.3/4 at different magnification (Ci and Cii). See above for an explanation of the expression patterns in (Ai) and (Aii). d H2B.5/6 expression in male polyps. Endogenous H2B.2 expression could not be determined. Genes for H2B.5 and H2B.6 group with H2B.2 and form a genomic cluster. The annotated genomic locus shows their coding sequence, mapped RNA reads (showing the number of reads mapped), predicted TATA-boxes and 3′-UTR stem-loops. All three genes contain one exon. RNA-Seq mapping shows that their transcripts are only found in male polyps (green wedges). e Micrococcal nuclease (MNase) digestion of Hydractinia sperm cells. Lane 1 shows sperm genomic DNA extracted in the absence of MNase. Lanes 2–4 shows sperm genomic DNA extracted after nuclei were subjected to increased concentration of MNase. Nucleosomal DNA bands representing one to five nucleosomal arrays (labelled 1n to 5n) are clearly visible in lanes 2 and 3, while in lane 3 the majority of DNA is present as a mono-nucelosomal (1n) band. No DNA smear or other bands are visible, indicating that the majority of sperm DNA packed by nucleosomes. f Coomassie-stained SDS-PAGE of Hydractinia sperm acid extracts and recombinant human histones (H2A, H2B, H3 and H4). Hydractinia sperm protein bands (labelled with numbers 1–8) were subjected to trypsin digest and consecutive mass spectrometry. Both the major and minor components of each band as determined by mass spectrometry are given. Note, no major band containing H2Bs is apparent; instead, H2B.3-6 proteins are dispersed across the gel (red bracket, red highlight)
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