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)