Naturallyoccurring differences in cenh3 affect chromosome segregation in zygotic mitosis of hybrids

The point of attachment of spindle microtubules to metaphase chromosomes is known as the centromere. Plant and animal centromeres are epigenetically specified by a centromere-specific variant of Histone H3, CENH3 (a.k.a. CENP-A). Unlike canonical histones that are invariant, CENH3 proteins are accumulating substitutions at an accelerated rate. This diversification of CENH3 is a conundrum since its role as the key determinant of centromere identity remains a constant across species. Here, we ask whether naturally occurring divergence in CENH3 has functional consequences. We performed functional complementation assays on cenh3-1, a null mutation in Arabidopsis thaliana, using untagged CENH3s from increasingly distant relatives. Contrary to previous results using GFP-tagged CENH3, we find that the essential functions of CENH3 are conserved across a broad evolutionary landscape. CENH3 from a species as distant as the monocot Zea mays can functionally replace A. thaliana CENH3. Plants expressing variant CENH3s that are fertile when selfed show dramatic segregation errors when crossed to a wild-type individual. The progeny of this cross include hybrid diploids, aneuploids with novel genetic rearrangements and haploids that inherit only the genome of the wild-type parent. Importantly, it is always chromosomes from the plant expressing the divergent CENH3 that missegregate. Using chimeras, we show that it is divergence in the fast-evolving N-terminal tail of CENH3 that is causing segregation errors and genome elimination. Furthermore, we analyzed N-terminal tail sequences from plant CENH3s and discovered a modular pattern of sequence conservation. From this we hypothesize that while the essential functions of CENH3 are largely conserved, the N-terminal tail is evolving to adapt to lineage-specific centromeric constraints. Our results demonstrate that this lineage-specific evolution of CENH3 causes inviability and sterility of progeny in crosses, at the same time producing karyotypic variation. Thus, CENH3 evolution can contribute to postzygotic reproductive barriers

Single-cell sequencing of genomic DNA resolves sub-clonal heterogeneity in a melanoma cell line

Abstract: We performed shallow single-cell sequencing of genomic DNA across 1475 cells from a cell-line, COLO829, to resolve overall complexity and clonality. This melanoma tumor-line has been previously characterized by multiple technologies and is a benchmark for evaluating somatic alterations. In some of these studies, COLO829 has shown conflicting and/or indeterminate copy number and, thus, single-cell sequencing provides a tool for gaining insight. Following shallow single-cell sequencing, we first identified at least four major sub-clones by discriminant analysis of principal components of single-cell copy number data. Based on clustering, break-point and loss of heterozygosity analysis of aggregated data from sub-clones, we identified distinct hallmark events that were validated within bulk sequencing and spectral karyotyping. In summary, COLO829 exhibits a classical Dutrillaux’s monosomic/trisomic pattern of karyotype evolution with endoreduplication, where consistent sub-clones emerge from the loss/gain of abnormal chromosomes. Overall, our results demonstrate how shallow copy number profiling can uncover hidden biological insights

Cis-by-Trans Regulatory Divergence Causes the Asymmetric Lethal Effects of an Ancestral Hybrid Incompatibility Gene

The Dobzhansky and Muller (D-M) model explains the evolution of hybrid incompatibility (HI) through the interaction between lineage-specific derived alleles at two or more loci. In agreement with the expectation that HI results from functional divergence, many protein-coding genes that contribute to incompatibilities between species show signatures of adaptive evolution, including Lhr, which encodes a heterochromatin protein whose amino acid sequence has diverged extensively between Drosophila melanogaster and D. simulans by natural selection. The lethality of D. melanogaster/D. simulans F1 hybrid sons is rescued by removing D. simulans Lhr, but not D. melanogaster Lhr, suggesting that the lethal effect results from adaptive evolution in the D. simulans lineage. It has been proposed that adaptive protein divergence in Lhr reflects antagonistic coevolution with species-specific heterochromatin sequences and that defects in LHR protein localization cause hybrid lethality. Here we present surprising results that are inconsistent with this coding-sequence-based model. Using Lhr transgenes expressed under native conditions, we find no evidence that LHR localization differs between D. melanogaster and D. simulans, nor do we find evidence that it mislocalizes in their interspecific hybrids. Rather, we demonstrate that Lhr orthologs are differentially expressed in the hybrid background, with the levels of D. simulans Lhr double that of D. melanogaster Lhr. We further show that this asymmetric expression is caused by cis-by-trans regulatory divergence of Lhr. Therefore, the non-equivalent hybrid lethal effects of Lhr orthologs can be explained by asymmetric expression of a molecular function that is shared by both orthologs and thus was presumably inherited from the ancestral allele of Lhr. We present a model whereby hybrid lethality occurs by the interaction between evolutionarily ancestral and derived alleles

LHR orthologs have conserved localization properties despite species-specific divergence of heterochromatin.

<p>(A,B) The dodeca satellite has diverged in its chromosomal location and interphase organization between <i>D. melanogaster</i> and <i>D. simulans</i>. FISH to mitotic (A) and interphase (B) nuclei from 3^rd instar larval brain cells with probes to dodeca (green) and 2L3L (red). Right panels in part B show quantification of the nuclear distribution of the interphase dodeca FISH signals. The mean values are indicated by the green lines (n = 10 for each sample).Boxes span the interquartile range and whiskers extend to the maximum and minimum values. Significance was tested by Wilcoxon rank-sum test. mel = <i>D. melanogaster</i>; sim = <i>D. simulans</i>. (C, D) Conserved heterochromatic localization properties of LHR orthologs. (C) <i>sim-Lhr-HA</i> transgene in <i>D. simulans</i> embryos. Top panel, Anti-HA (green) detects sim-LHR-HA colocalizing with HP1 (red) in the apical heterochromatin. Bottom panel, sim-LHR-HA (green) partially colocalizes with dodeca satellite (blue). (D) sim-LHR-HA (green) expressed in <i>D. melanogaster</i> embryos colocalizes with H3K9me2 (red) and with mel-LHR-YFP, detected with anti-GFP (red). sim-LHR-HA also partially overlaps with the <i>D. melanogaster</i> dodeca satellite (blue).</p

<i>D. melanogaster</i> and <i>D. simulans Lhr</i> orthologs suppress hybrid rescue by <i>D. simulans Lhr</i>.<i>¹</i>

<p>Crosses were between <i>D. melanogaster</i> females heterozygous for the different transgenes tested (<i>w; φ{transgene, w⁺}</i>) and <i>D. simulans Lhr¹</i> males. Transgenes are denoted as <i>φ{}</i> in the table. The transgenes carry a copy of the <i>w⁺</i> gene so the hybrid sons inheriting the transgene (genotype 2) were distinguished from their control siblings (genotype 1) by their eye-colour, except for cross 8 where <i>D. melanogaster</i> females homozygous for the integration site without an inserted transgene were mated to <i>D. simulans Lhr¹</i> males. NA = not applicable. RT = room temperature.</p

<i>D. simulans Lhr</i> interacts more strongly with <i>Hmr</i> than <i>D. melanogaster Lhr</i>.

<p>Transgenic <i>Lhr</i> orthologs were tested for interaction with two <i>Hmr</i> alleles: a null and a hypomorph, specified in the table as <i>Df(1)Hmr⁻</i> and <i>Hmr¹</i> respectively. <i>D. melanogaster</i> female parent genotypes were: null mutation, <i>y w Df(1)Hmr⁻ v/FM6</i>; <i>φ{transgene, w⁺}/+</i> and hypomorph, <i>w Hmr¹ v</i>; <i>φ{transgene, w⁺}/+</i>. Transgenes are denoted as <i>φ{}</i> in the table. Each genotype was mated separately to males from <i>D. simulans v</i> or <i>D. mauritiana Iso105</i>. Hybrid male progeny that inherit the transgene are orange eyed, while the sibling brothers are white eyed. The FET is comparing the relative viability of hybrid sons inheriting the <i>D. melanogaster Lhr</i> transgene <i>vs</i> the relative viability of sons inheriting the <i>D. simulans Lhr</i> transgene in parallel crosses. (n.d. = not determined;</p><p>*, <i>p</i>≤0.05;</p><p>**, <i>p</i>≤0.01;</p><p>***, <i>p</i>≤0.001). RT = room temperature.</p

Asymmetric expression of <i>Lhr</i> orthologs in hybrids.

<p>Pyrosequencing across a SNP fixed between <i>Lhr</i> orthologs was used to measure the ratio of allelic transcription in pure-species and hybrid larvae that were 3–5 days old. <i>Lhr</i> transcript from pure species <i>D. melanogaster</i> is 100% for the <i>D. melanogaster</i>-specific variant of the SNP. For <i>D. simulans</i> 2 out of 3 of the technical replicates from one of the cDNA preparations showed 100% of transcripts with the <i>D. simulans</i>-specific variant of the SNP; a small amount of the <i>D. melanogaster</i>-specific SNP detected in the 3rd replicate most likely reflects error or contamination. The <i>D. simulans</i>-specific SNP is detected at levels significantly greater than the expected 50% in both male and female hybrids (<i>p</i><.0001 and <i>p</i> = .005 for hybrid males and females, respectively, by two-tailed <i>t</i>-test).</p