Chromosome-scale genome assemblies of aphids reveal extensively rearranged autosomes and long-term conservation of the X chromosome

Chromosome rearrangements are arguably the most dramatic type of mutations, often leading to rapid evolution and speciation. However, chromosome dynamics have only been studied at the sequence level in a small number of model systems. In insects, Diptera and Lepidoptera have conserved genome structure at the scale of whole chromosomes or chromosome arms. Whether this reflects the diversity of insect genome evolution is questionable given that many species exhibit rapid karyotype evolution. Here, we investigate chromosome evolution in aphids-an important group of hemipteran plant pests-using newly generated chromosome-scale genome assemblies of the green peach aphid (Myzus persicae) and the pea aphid (Acyrthosiphon pisum), and a previously published assembly of the corn-leaf aphid (Rhopalosiphum maidis). We find that aphid autosomes have undergone dramatic reorganization over the last 30 My, to the extent that chromosome homology cannot be determined between aphids from the tribes Macrosiphini (Myzus persicae and Acyrthosiphon pisum) and Aphidini (Rhopalosiphum maidis). In contrast, gene content of the aphid sex (X) chromosome remained unchanged despite rapid sequence evolution, low gene expression, and high transposable element load. To test whether rapid evolution of genome structure is a hallmark of Hemiptera, we compared our aphid assemblies with chromosome-scale assemblies of two blood-feeding Hemiptera (Rhodnius prolixus and Triatoma rubrofasciata). Despite being more diverged, the blood-feeding hemipterans have conserved synteny. The exceptional rate of structural evolution of aphid autosomes renders them an important emerging model system for studying the role of large-scale genome rearrangements in evolution

Hybridisation has shaped a recent radiation of grass-feeding aphids

Background: Aphids are common crop pests. These insects reproduce by facultative parthenogenesis involving several rounds of clonal reproduction interspersed with an occasional sexual cycle. Furthermore, clonal aphids give birth to live young that are already pregnant. These qualities enable rapid population growth and have facilitated the colonisation of crops globally. In several cases, so-called “super clones” have come to dominate agricultural systems. However, the extent to which the sexual stage of the aphid life cycle has shaped global pest populations has remained unclear, as have the origins of successful lineages. Here, we used chromosome-scale genome assemblies to disentangle the evolution of two global pests of cereals—the English (Sitobion avenae) and Indian (Sitobion miscanthi) grain aphids.   Results: Genome-wide divergence between S. avenae and S. miscanthi is low. Moreover, comparison of haplotype-resolved assemblies revealed that the S. miscanthi isolate used for genome sequencing is likely a hybrid, with one of its diploid genome copies closely related to S. avenae (~ 0.5% divergence) and the other substantially more divergent (> 1%). Population genomics analyses of UK and China grain aphids showed that S. avenae and S. miscanthi are part of a cryptic species complex with many highly differentiated lineages that predate the origins of agriculture. The complex consists of hybrid lineages that display a tangled history of hybridisation and genetic introgression.   Conclusions: Our analyses reveal that hybridisation has substantially contributed to grain aphid diversity, and hence, to the evolutionary potential of this important pest species. Furthermore, we propose that aphids are particularly well placed to exploit hybridisation events via the rapid propagation of live-born “frozen hybrids” via asexual reproduction, increasing the likelihood of hybrid lineage formation

A transcriptionally distinct CXCL13+CD103+CD8+ T-cell population is associated with B-cell recruitment and neoantigen load in human cancer

The chemokine CXCL13 mediates recruitment of B cells to tumors and is essential for the formation of tertiary lymphoid structures (TLSs). TLSs are thought to support antitumor immunity and are associated with improved prognosis. However, it remains unknown whether TLSs are formed in response to the general inflammatory character of the tumor microenvironment, or rather, are induced by (neo)antigen-specific adaptive immunity. We here report on the finding that the transforming growth factor beta (TGFβ)-dependent CD103+CD8+ tumor-infiltrating T-cell (TIL) subpopulation expressed and produced CXCL13. Accordingly, CD8+ T cells from peripheral blood activated in the presence of TGFβ upregulated CD103 and secreted CXCL13. Conversely, inhibition of TGFβ receptor signaling abrogated CXCL13 production. CXCL13+CD103+CD8+ TILs correlated with B-cell recruitment, TLSs, and neoantigen burden in six cohorts of human tumors. Altogether, our findings indicated that TGFβ plays a non-canonical role in coordinating immune responses against human tumors and suggest a potential role for CXCL13+CD103+CD8+ TILs in mediating B-cell recruitment and TLS formation in human tumors

hMMS2 serves a redundant role in human PCNA polyubiquitination

<p>Abstract</p> <p>Background</p> <p>In yeast, DNA damage leads to the mono and polyubiquitination of the sliding clamp PCNA. Monoubiquitination of PCNA is controlled by RAD18 (E3 ligase) and RAD6 (E2 conjugating enzyme), while the extension of the monoubiquitinated PCNA into a polyubiquitinated substrate is governed by RAD5, and the heterodimer of UBC13/MMS2. Each modification directs a different branch of the DNA damage tolerance pathway (DDT). While PCNA monoubiquitination leads to error-prone bypass via TLS, biochemical studies have identified MMS2 along with its heteromeric partner UBC13 to govern the error-free repair of DNA lesions by catalyzing the formation of lysine 63-linked polyubiquitin chains (K63-polyUb). Recently, it was shown that PCNA polyubiquitination is conserved in human cells and that this modification is dependent on RAD18, UBC13 and SHPRH. However, the role of hMMS2 in this process was not specifically addressed.</p> <p>Results</p> <p>In this report we show that mammalian cells in which MMS2 was reduced by siRNA-mediated knockdown maintains PCNA polyubiquitination while a knockdown of RAD18 or UBC13 abrogates PCNA ubiquitination. Moreover, the additional knockdown of a UEV1A (MMS2 homolog) does not deplete PCNA polyubiquitination. Finally, mouse embryonic stem cells null for MMS2 with or without the additional depletion of mUEV1A continue to polyubiquitinated PCNA with normal kinetics.</p> <p>Conclusion</p> <p>Our results point to a high level of redundancy in the DDT pathway and suggest the existence of another hMMS2 variant (hMMSv) or complex that can compensate for its loss.</p

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

K63RUb expression induces S-phase specific repair defects.

<p>(A) WT and K63R Ub cells were fixed and immuno-stained for γH2AX and 53BP1 foci at the indicated time points following 2 Gy IR or untreated (Ctrl). Mean values ± sd. of 2 independent exp's, >150 cells were analysed per time-point (B) 30 min EdU (10 µM) incorporation before 2 Gy IR, cells were fixed 30 min or 24 hrs post IR, EdU and 53BP1 foci were visualized by fluorescent staining and quantified. Mean values ± s.e.m. of 2 independent exp's (n>100 per treatment). (C) 30 min EdU (10 µM) incorporation alone (untreated) or before 2.5 h 100 nM CPT treatment. Cells were fixed and immuno-stained for EdU and γH2AX 3 h post treatment. (D) Quantification of 53BP1 foci. Cells were fixed and immuno-stained for 53BP1 directly or 24 h after 1 h 100 nM CPT treatment, >80 cells were analysed per sample. (E) Quantification of γH2AX immuno-staining of untreated (Ctrl) or 1 h 100 nM CPT treated cells. Mean values ± s.e.m. Data is representative exp (n>130) of 3 independent exp's, *P<0.05.</p

Dual inhibition of PCNA and RNF8 signalling pathways does not mimic the K63RUb phenotype.

<p>(A) Depletion of HLTF or SHPRH in combination with knock-down of RNF8 was achieved using lentiviral shRNA's and was confirmed using real-time PCR. (B) Spontaneous or CPT (20 nM) induced mutations were determined at the <i>HPRT</i> locus, mean ± s.d. (n = 5) per exp). (C) Clonogenic survival of A549 cells after double knock-down of HLTF and RNF8 or SHPRH and RNF8 was determined after treatment with 100 nM CPT (24 h). Data are mean ± s.d. (n = 3), *P<0.05, **P<0.01, ***P<0.001.</p

RNF8 depletion does not reproduce K63RUb phenotype.

<p>(A-C) WT and K63R Ub cells were transfected with siRNA against RNF8 or RAP80 on day 1 and 3. (A) Knockdown of RNF8 and RAP80 was assessed by mRNA expression levels determined by real-time PCR. Clonogenic survival was assessed after (B) 4 Gy IR and (C) PARPi (continuous) (B-C) Data are mean ± s.d. of 2 independent exp's (n = 3 per exp). (D) Spontaneous and IR-induced (4 Gy) mutations were determined at the <i>HPRT</i> locus, mean ± s.e.m. of 3 independent exp's (n = 10 per exp), *P<0.05, **P<0.01, ***P<0.001.</p

K63RUb phenotype is not due to NHEJ.

<p>(A-C) Clonogenic survival of WT and K63R Ub cells after treatment with (A) 1 µM DNA-PKi alone for indicated times, (B) 1 µM DNA-PKi for 24 or 48 hrs, started 1 h before combined 24 hrs treatment with 50 nM CPT or (C) 1 µM DNA-PKi for 24 hrs, started 1 h before 2 Gy IR (A-C) mean ± s.d. (n = 3). (D) Mutations at the <i>HPRT</i> locus were determined after continuous treatment with 1 µM DNA-PKi started 1 h before combined with additional treatments either spontaneous or 20 nM CPT for 6 days total, mean ± s.d. (n = 5).</p

K63RUb expression sensitizes cells to replication-associated DNA damage.

<p>(A-C) Clonogenic survival of A549 cells expressing empty vector (EV), WTUb or K63RUb was determined after (A) CPT (24h) treatment started following cell attachment. Data are mean ± sd. of 2 independent exp's (n = 3 per exp). (B) Continuous PARPi treatment, data are mean ± sd. of 3 independent exp's (n = 3 per exp). (C) IR, data are mean ± sd. of 3 independent exp's (n = 3 per exp) *P<0.05, **P<0.01, ***P<0.001.</p