An inverse relationship to germline transcription defines centromeric chromatin in C. elegans

Centromeres are chromosomal loci that direct segregation of the genome during cell division. The histone H3 variant CENP-A (also known as CenH3) defines centromeres in monocentric organisms, which confine centromere activity to a discrete chromosomal region, and holocentric organisms, which distribute centromere activity along the chromosome length1–3. Because the highly repetitive DNA found at most centromeres is neither necessary nor sufficient for centromere function, stable inheritance of CENP-A nucleosomal chromatin is postulated to epigenetically propagate centromere identity4. Here, we show that in the holocentric nematode Caenorhabditis elegans pre-existing CENP-A nucleosomes are not necessary to guide recruitment of new CENP-A nucleosomes. This is indicated by lack of CENP-A transmission by sperm during fertilization and by removal and subsequent reloading of CENP-A during oogenic meiotic prophase. Genome-wide mapping of CENP-A location in embryos and quantification of CENP-A molecules in nuclei revealed that CENP-A is incorporated at low density in domains that cumulatively encompass half the genome. Embryonic CENP-A domains are established in a pattern inverse to regions that are transcribed in the germline and early embryo, and ectopic transcription of genes in a mutant germline altered the pattern of CENP-A incorporation in embryos. Furthermore, regions transcribed in the germline but not embryos fail to incorporate CENP-A throughout embryogenesis. We propose that germline transcription defines genomic regions that exclude CENP-A incorporation in progeny, and that zygotic transcription during early embryogenesis remodels and reinforces this basal pattern. These findings link centromere identity to transcription and shed light on the evolutionary plasticity of centromeres

Separase Cleaves the N-Tail of the CENP-A Related Protein CPAR-1 at the Meiosis I Metaphase-Anaphase Transition in <i>C</i>. <i>elegans</i>

<div><p>Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP-A. Most species have a single gene encoding a centromeric histone variant whereas <i>C</i>. <i>elegans</i> has two: HCP-3 (also known as CeCENP-A) and CPAR-1. Prior RNAi replacement experiments showed that HCP-3 is the functionally dominant isoform, consistent with CPAR-1 not being detectable in embryos. GFP::CPAR-1 is loaded onto meiotic chromosomes in diakinesis and is enriched on bivalents until meiosis I. Here we show that GFP::CPAR-1 signal loss from chromosomes precisely coincides with homolog segregation during anaphase I. This loss of GFP::CPAR-1 signal reflects proteolytic cleavage between GFP and the histone fold of CPAR-1, as CPAR-1::GFP, in which GFP is fused to the C-terminus of CPAR-1, does not exhibit any loss of GFP signal. A focused candidate screen implicated separase, the protease that initiates anaphase by cleaving the kleisin subunit of cohesin, in this cleavage reaction. Examination of the N-terminal tail sequence of CPAR-1 revealed a putative separase cleavage site and mutation of the signature residues in this site eliminated the cleavage reaction, as visualized by retention of GFP::CPAR-1 signal on separating homologous chromosomes at the metaphase-anaphase transition of meiosis I. Neither cleaved nor uncleavable CPAR-1 were centromere-localized in mitosis and instead localized throughout chromatin, indicating that centromere activity has not been retained in CPAR-1. Although the functions of CPAR-1 and of its separase-dependent cleavage remain to be elucidated, this effort reveals a new substrate of separase and provides an <i>in vivo</i> biosensor to monitor separase activity at the onset of meiosis I anaphase.</p></div

The GFP signal of CPAR-1::GFP is retained on meiotic chromosomes but is not centromeric in mitosis.

<p><b>(A)</b> Comparison of GFP::CPAR-1 (OD82) to CPAR-1::GFP (OD145) in meiosis I anaphase. HFD refers to the histone fold domain of CPAR-1. The GFP signal is abruptly lost at meiosis I anaphase onset for GFP::CPAR-1 but is unchanged for CPAR-1::GFP. Similar results were observed for n = 12 time-lapse sequences. Scale bars, 2 μm. <b>(B)</b> Immunofluorescence analysis of one-cell mitotic embryos expressing CPAR-1::GFP (OD145). Embryos were fixed and stained for DNA, HCP-3, and GFP, to detect the cleaved CPAR-1::GFP. A representative image of a metaphase embryo is shown. Scale bar, 1 μm. Linescan analysis on 6 embryos was performed using a 47-pixel wide line drawn as depicted. Linescans were aligned using peak Hoechst intensity as the midline, normalized and the averaged profiles plotted. Error bars are the standard deviations. <b>(C)</b> Comparison of GFP::HCP-3 (OD421) to CPAR-1::GFP (OD145) in multi-cellular mitotic embryos. GFP::HCP-3 exhibits robust nuclear localization in all cells while CPAR-1::GFP is very weakly detected in embryos, with prominent signal in polar bodies. The images were collected with equal exposure and illumination intensity and processed identically after acquisition.</p

A small candidate screen implicates separase in the loss of chromosomal GFP::CPAR-1 signal observed at meiosis I anaphase onset.

<p><b>(A)</b> Schematic of the experimental strategy employed to screen candidate genes for their role in the loss of chromosomal GFP::CPAR-1 signal. <b>(B)</b> Summary of the results for the tested candidate genes. Only depletion of separase/SEP-1 resulted in presence of GFP signal on chromosomes of mitotic embryos, indicating that the normal cleavage-associated loss of GFP::CPAR-1 from chromosomes at meiosis I anaphase did not occur. <b>(C)</b> Example images of GFP::CPAR-1 (OD82) on oocyte and embryo chromosomes in control and <i>sep-1(RNAi)</i> worms. GFP::CPAR-1 intensities in oocytes and embryos were quantified by taking average projections of 10 μm thick sections (6 slices total) in a 5 μm by 5 μm region of interest encompassing the chromosomes. Background signal intensity was subtracted out of each projected stack and integrated GFP signal intensities were normalized relative to the average value in oocytes. GFP fluorescence was reduced to 0.070 +/- 0.035 (n = 6) in embryos compared to oocytes in controls; in contrast, for <i>sep-1(RNAi)</i> GFP fluorescence was 0.528 +/- 0.185 (n = 5) in embryos compared to oocytes (p < 0.0001 relative to control). Arrow points to retention of GFP signal on embryo chromosomes in <i>sep-1(RNAi)</i>. Scale bar, 5 μm.</p

GFP::CPAR-1 signal is lost from chromosomes at the metaphase-anaphase transition of oocyte meiosis I.

<p>Image frames from time-lapse sequences of GFP::CPAR-1 (OD416) and GFP::HCP-3 (OD421) meiosis I embryos (single copy insertions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.g001" target="_blank">Fig 1</a>). Chromosomes were labeled with mCherry::H2b. In the GFP::HCP-3 strain, the GFP fusion is the sole source of HCP-3. The images shown are from prometaphase through late anaphase. Metaphase is defined as the first frame in which chromosomes are aligned and spindle is rotated to be perpendicular to cortex. Schematics on the left indicate the stages shown. Similar results were obtained for n = 10 time-lapse sequences. Scale bar, 5 μm.</p

Duplicated CENP-A related genes in Caenorhabditis species.

<p><b>(A)</b> Tree generated by primary sequence alignments of CENP-A related proteins in the indicated <i>Caenorhabditis</i> species. The sequences were obtained from Wormbase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.ref040" target="_blank">40</a>]. Alignments were performed using Muscle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.ref044" target="_blank">44</a>] implemented in Jalview 2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.ref045" target="_blank">45</a>]. The tree was constructed in the Clustal W Phylogeny tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.ref046" target="_blank">46</a>], employing the neighbor-joining method and default parameters. The alignment was imported into FigTree v1.3.1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.ref047" target="_blank">47</a>] for formatting and export. <b>(B)</b> Primary sequence features of the two CENP-A related proteins in <i>C</i>. <i>elegans</i> and <i>C</i>. <i>remanei</i>. In all 4 proteins an N-terminal tail (N-tail), significantly longer than the N-tail of canonical histone H3 or human Cenp A, is followed by a histone fold domain (HFD). The tail & histone fold alignments were done using Blast and percent identity and similarity (in brackets) is reported; gaps are not reported. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.g004" target="_blank">Fig 4A</a>. HCP-3 and CPAR-1 comparison adapted from <i>Monen et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.ref016" target="_blank">16</a>]. <b>(C)</b> Images of adult <i>C</i>. <i>elegans</i> worms expressing single copy GFP transgene insertions of HCP-3 (OD421) and CPAR-1 (OD416) under their endogenous 5’ and 3’ UTR. GFP was fused to the N-terminus of each CENP-A related protein [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.ref007" target="_blank">7</a>]; the GFP::HCP-3-expressing transgene was crossed into an <i>hcp-3Δ</i> mutant, which it fully rescues. The region of the germline where oocytes are fertilized, pass through the spermatheca and begin early embryogenesis is shown. The boxed regions magnified on the right are of oocyte chromosomes (box 1, 1’) and of prometaphase one-cell embryo chromosomes (box 2, 2’). Scale bars are 20 μm; blowups are magnified an additional 2-fold.</p

Mutation of a predicted separase cleavage site in the CPAR-1 N-terminal tail prevents loss of GFP::CPAR-1 from chromosome at the meiosis I metaphase-anaphase transition.

<p><b>(A)</b> Summary of known separase cleavage motifs in the kleisin subunits of the cohesin complex, in the fungal spindle protein Slk19, in vertebrate separase, and in the pericentriolar material protein kendrin. Blue shading indicates conservation of amino acid residues. Sequence alignment performed using Muscle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.ref044" target="_blank">44</a>] in Jalview 2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.ref045" target="_blank">45</a>]. A sequence logo summarizing the distribution of amino acids in the cleavage sites is shown below. Color indicates amino acid charge. Sequence logo was generated with Weblogo (<a href="http://weblogo.threeplusone.com/create.cgi" target="_blank">http://weblogo.threeplusone.com/create.cgi</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.ref048" target="_blank">48</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125382#pone.0125382.ref049" target="_blank">49</a>]. <b>(B)</b> Alignment of the N-terminal tails of HCP-3 and CPAR-1. Both N-tails harbor an EQAR motif that resembles a separase cleavage site. Note the 33 amino acid sequence immediately upstream of this motif in HCP-3 that is absent in CPAR-1. <b>(C)</b> Image frames from time-lapse sequences of meiosis I embryos expressing either GFP::CPAR-1^WT (OD416) or GFP::CPAR-1^E68L;R71Q (OD180). GFP::CPAR-1 intensities were quantified by taking average projections of 8 μm thick sections (5 slices total) in a 5 μm by 5 μm region of interest encompassing the chromosomes. Background signal intensity was subtracted out of each projected stack and integrated GFP signal was normalized relative to average integrated GFP signal on chromosomes in prometaphase I. For WT GFP::CPAR-1, mCherry::H2b was crossed in and imaged at the same time to highlight the abrupt loss of GFP fluorescence. No loss of GFP fluorescence was observed at meiosis I anaphase onset for the E68L;R71Q mutant form of GFP::CPAR-1. GFP signal loss from chromosomes was quantified on chromosomes 60 seconds post anaphase I onset relative to chromosomes at prometaphase I within the same cell. For GFP::CPAR-1, chromosomal GFP signal dropped 60 seconds after the metaphase-to-anaphase transition to 0.04 +/- 0.05 (n = 12), relative to prometaphase I. In contrast, for GFP::CPAR-1^E68L;R71Q, chromosomal GFP signal persisted at 0.85 +/- 0.09 (n = 10), relative to prometaphase I. Scale bars, 2 μm. * p-value < 0.0001. <b>(D)</b> Frames from a time-lapse sequence of a one-cell mitotic embryo expressing GFP::CPAR-1^E68L;R71Q (OD180). Similar results were observed in n = 3 embryos. Scale bar, 5 μm. Lower panel shows a field of multi-cellular embryos expressing GFP::CPAR-1^E68L;R71Q.</p