Functional genomics identifies a Myb domain–containing protein family required for assembly of CENP-A chromatin

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2–associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain–containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading

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

Meiotic chromosome segregation in C. elegans : discovering a new look for CENP-A

In this dissertation, I use the nematode, Caenorhabditis elegans, to understand mechanisms involved in chromosome segregation during meiosis, by exploring the role that known mitotic kinetochore proteins play during meiotic segregation. During mitosis, the histone-H3 variant CENP-A is known to be the nucleosomal subunit at the centromere that is responsible for directing kinetochore assembly. In cells lacking CENP-A, chromosomes are unable to segregate leading to aneuploidy and cell-death. Interestingly, I demonstrate that during meiosis CeCENP-A no longer directs outer kinetochore assembly. Furthermore, in embryos depleted of CeCENP-A, meiotic chromosome segregation appears completely normal, whereas subsequent mitotic divisions completely fail. Outer kinetochore components localize to a cup-like structure, which likely is involved in aligning chromosomes at the metaphase plate. I speculate that this new mechanism for chromosome segregation in meiosis may be a requirement to facilitate proper segregation of recombined chromosome pairs. During these meiotic studies, I discovered a striking CeCENP-A cleavage event, which I spend the remainder of this dissertation describing, and postulate that this cleavage event may be involved in maintenance of the centromere during mitotic divisions. Centromere specification is thought to be propagated by an epigenetic mark produced by CENP-A. The mechanism for how CENP-A achieves this mark is unknown. In a variety of studies presented here, I show that CeCENP-A is a substrate for Separase-mediated cleavage and discuss its possible implications on maintaining the epigenetic mark. This cleavage event is best demonstrated during meiosis in embryos expressing N- terminally tagged GFP::CeCENP-A. During mitosis, I show that Separase is unable to cleave centromeric CeCENP-A under wild-type conditions. However, in the absence of the kinetochore, centromeric CeCENP-A is cleaved, indicating that the kinetochore protects CeCENP-A while non- centromeric CeCENP-A is susceptible to cleavage. Worms expressing an uncleavable mutant form of CeCENP-A show an increase in embryonic lethality, and worms solely expressing a pre-cleaved form of CeCENP-A completely lose CeCENP-A localization and function, resulting in complete embryonic lethality. These data suggest that CeCENP-A cleavage may be a way to inactivate CeCENP-A loading. I propose that cleavage of improperly loaded CeCENP-A onto chromosome arms may be a mechanism used by the cell to ensure that the epigenetic mark for CeCENP-A loading remains strictly at the centromer

Meiotic chromosome segregation in C. elegans : discovering a new look for CENP-A

In this dissertation, I use the nematode, Caenorhabditis elegans, to understand mechanisms involved in chromosome segregation during meiosis, by exploring the role that known mitotic kinetochore proteins play during meiotic segregation. During mitosis, the histone-H3 variant CENP-A is known to be the nucleosomal subunit at the centromere that is responsible for directing kinetochore assembly. In cells lacking CENP-A, chromosomes are unable to segregate leading to aneuploidy and cell-death. Interestingly, I demonstrate that during meiosis CeCENP-A no longer directs outer kinetochore assembly. Furthermore, in embryos depleted of CeCENP-A, meiotic chromosome segregation appears completely normal, whereas subsequent mitotic divisions completely fail. Outer kinetochore components localize to a cup-like structure, which likely is involved in aligning chromosomes at the metaphase plate. I speculate that this new mechanism for chromosome segregation in meiosis may be a requirement to facilitate proper segregation of recombined chromosome pairs. During these meiotic studies, I discovered a striking CeCENP-A cleavage event, which I spend the remainder of this dissertation describing, and postulate that this cleavage event may be involved in maintenance of the centromere during mitotic divisions. Centromere specification is thought to be propagated by an epigenetic mark produced by CENP-A. The mechanism for how CENP-A achieves this mark is unknown. In a variety of studies presented here, I show that CeCENP-A is a substrate for Separase-mediated cleavage and discuss its possible implications on maintaining the epigenetic mark. This cleavage event is best demonstrated during meiosis in embryos expressing N- terminally tagged GFP::CeCENP-A. During mitosis, I show that Separase is unable to cleave centromeric CeCENP-A under wild-type conditions. However, in the absence of the kinetochore, centromeric CeCENP-A is cleaved, indicating that the kinetochore protects CeCENP-A while non- centromeric CeCENP-A is susceptible to cleavage. Worms expressing an uncleavable mutant form of CeCENP-A show an increase in embryonic lethality, and worms solely expressing a pre-cleaved form of CeCENP-A completely lose CeCENP-A localization and function, resulting in complete embryonic lethality. These data suggest that CeCENP-A cleavage may be a way to inactivate CeCENP-A loading. I propose that cleavage of improperly loaded CeCENP-A onto chromosome arms may be a mechanism used by the cell to ensure that the epigenetic mark for CeCENP-A loading remains strictly at the centromer

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

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

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