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. 2012 Apr 8;484(7395):534-7.
doi: 10.1038/nature10973.

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

Reto Gassmann  1 , Andreas Rechtsteiner, Karen W Yuen, Andrew Muroyama, Thea Egelhofer, Laura Gaydos, Francie Barron, Paul Maddox, Anthony Essex, Joost Monen, Sevinc Ercan, Jason D Lieb, Karen Oegema, Susan Strome, Arshad Desai
Affiliations

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

Reto Gassmann et al. Nature. .

Abstract

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 length. 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 propagate centromere identity epigenetically. 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.

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Figures

Figure 1
Figure 1. CeCENP-A dynamics in meiotic prophase, at fertilization and across embryonic divisions
a, Gonad region of an adult hermaphrodite co-expressing GFP–CeCENP-A and mCherry–histone H2b in a CeCENP-AΔ (hcp-3(ok1892; see also Supplementary Figs 1 and 2) background (see also Supplementary Figs 1 and 2). Scale bar, 20 μm. b, Quantitative immunoblot showing that sperm lack a significant pool of CeCENP-A (see also Supplementary Fig. 2c, d). c, Fertilized one-cell control or CeCENP-A-depleted embryos at different stages of the first mitotic division were immunostained for CeCENP-A and α-tubulin (MT). Wild-type (N2) males were mated to fem-1 mutant worms to ensure all embryos were cross-progeny. Scale bar, 5 μm. d, Schematic of photobleaching experiment to assay CeCENP-A inheritance across early embryonic divisions. par-6 RNA interference (RNAi) abolishes developmental asynchrony in the two-cell embryo. Unbleached (U) and bleached (B) chromatid sets are indicated. Scale bar, 2 μm. e, Representative images and quantification of the photobleaching experiment. Higher magnification views highlight bleached and unbleached chromatid sets. Error bars are 95% confidence intervals for the means. Scale bars, 5 μm.
Figure 2
Figure 2. Genome-wide mapping of CeCENP-A-enriched chromatin
a, Regions enriched for CeCENP-A and its loading factor KNL-2 in a representative portion of chromosome I. For each track, the average z-score probe signal of two independent biological replicates is plotted. b, Genome-wide correlation plot of CeCENP-A and KNL-2 occupancy. The correlation coefficient (r) is in the upper left corner. c, Regions enriched for CeCENP-A with the positions of annotated genes. CeCENP-A domains were defined by a sliding window algorithm. d, Features of CeCENP-A domains for individual chromosomes. Boxplots: boxes indicate 25th to 75th percentile, whiskers 2.5th to 97.5th percentile. Wedges around the medians indicate 95% confidence intervals for the medians (see also Supplementary Fig. 5d–f). e, Two independent nuclei preparations (Prep) from early embryos (<100 nuclei) were blotted alongside a purified CeCENP-A standard (see also Supplementary Figs 2c, d and 6a–c). f, Hypothetical model for CeCENP-A permissive domain.
Figure 3
Figure 3. Relationship between CeCENP-A and gene expression
a, Chromosomal region containing the cle-1 gene, which is expressed in neurons, and flanking genes that are expressed during embryogenesis. b, Genome browser view showing inverse correlation between CeCENP-A and RNA Pol II occupancy. c, Genome-wide correlation plot of CeCENP-A and RNA Pol II occupancy. The correlation coefficient (r) is in the upper right corner. d, CeCENP-A, KNL-2, RNA Pol II and H3K36me3 occupancy for various gene sets defined on the basis of expression data. The number of genes in each set is shown in parentheses. Boxplots (as in Fig. 2d) show the range of z-scores averaged over gene bodies. e, CeCENP-A, KNL-2, RNA Pol II and H3K36me3 occupancy for the germline-only gene set. f, Genome browser views of CeCENP-A, RNA Pol II, and H3K36me3 occupancy on germline-only genes, flanked by genes expressed in embryos.
Figure 4
Figure 4. Germline expression controls CeCENP-A occupancy in the progeny embryos
a, Portion of chromosome I featuring specific regions with ectopic H3K36me3 signal in the met-1 mutant (see also Supplementary Figs 11 and 12). b, Screen shots of the regions boxed in (a). Real-time quantitative reverse transcription PCR was performed on hand-dissected wild-type and met-1 mutant gonads. Mean met-1:wild-type expression ratio (four independent biological replicates each) is listed above genes (see table in Supplementary Fig. 12b for all genes analysed).
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