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1. Deciphering the Mechanism of Glyphosate Resistance in Amaranthus palmeri by Cytogenomics

   https://doi.org/10.1159/000521409  

   Koo, Dal-Hoe ; Sathishraj, Rajendran ; Friebe, Bernd ; Gill, Bikram S. ( February 2022 , Cytogenetic and Genome Research)

   In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an important herbicide invariably used in the cultivation of glyphosate-resistant crops to control weeds. Overuse of glyphosate results in the evolution of glyphosate-resistant weeds. Evolution of glyphosate resistance (GR) in Amaranthus palmeri (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5- to >160-fold increase in copies of the EPSPS gene than in a glyphosate-susceptible (GS) population. This increased copy number of the EPSPS gene increased the genome size ranging from 3.5 to 11.8%, depending on the copy number compared to the genome size of GS AP. FISH analysis using a 399-kb EPSPS cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified EPSPS copies in GR AP exist in extrachromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The EPSPS gene-containing eccDNA having a size of ∼400 kb is termed EPSPS-eccDNA and showed somatic mosacism in size and copy number. EPSPS-eccDNA has a genetic mechanism to tether randomlymore »to mitotic or meiotic chromosomes during cell division or gamete formation and is inherited to daughter cells or progeny generating copy number variation. These eccDNAs are stable genetic elements that can replicate and exist independently. The genomic characterization of the EPSPS locus, along with the flanking regions, identified the presence of a complex array of repeats and mobile genetic elements. The cytogenomics approach in understanding the biology of EPSPS-eccDNA sheds light on various characteristics of EPSPS-eccDNA that favor GR in AP.« less
2. HolistIC: leveraging Hi–C and whole genome shotgun sequencing for double minute chromosome discovery

   https://doi.org/10.1093/bioinformatics/btab816  

   Hayes, Matthew ; Nguyen, Angela ; Islam, Rahib ; Butler, Caryn ; Tran, Ethan ; Mullins, Derrick ; Hicks, Chindo ; Birol, ed., Inanc ( December 2021 , Bioinformatics)

   Double minute (DM) chromosomes are acentric extrachromosomal DNA artifacts that are frequently observed in the cells of numerous cancers. They are highly amplified and contain oncogenes and drug-resistance genes, making their presence a challenge for effective cancer treatment. Algorithmic discovery of DM can potentially improve bench-derived therapies for cancer treatment. A hindrance to this task is that DMs evolve, yielding circular chromatin that shares segments from progenitor DMs. This creates DMs with overlapping amplicon coordinates. Existing DM discovery algorithms use whole genome shotgun sequencing (WGS) in isolation, which can potentially incorrectly classify DMs that share overlapping coordinates.

   In this study, we describe an algorithm called ‘HolistIC’ that can predict DMs in tumor genomes by integrating WGS and Hi–C sequencing data. The consolidation of these sources of information resolves ambiguity in DM amplicon prediction that exists in DM prediction with WGS data used in isolation. We implemented and tested our algorithm on the tandem Hi–C and WGS datasets of three cancer datasets and a simulated dataset. Results on the cancer datasets demonstrated HolistIC’s ability to predict DMs from Hi–C and WGS data in tandem. The results on the simulated data showed the HolistIC can accurately distinguish DMs that have overlappingmore »amplicon coordinates, an advance over methods that predict extrachromosomal amplification using WGS data in isolation.

   Our software, named ‘HolistIC’, is available at http://www.github.com/mhayes20/HolistIC.

   Supplementary data are available at Bioinformatics online.

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3. Live-Cell Imaging Shows Uneven Segregation of Extrachromosomal DNA Elements and Transcriptionally Active Extrachromosomal DNA Hubs in Cancer

   https://doi.org/10.1158/2159-8290.CD-21-1376  

   Yi, Eunhee ; Gujar, Amit D. ; Guthrie, Molly ; Kim, Hoon ; Zhao, Dacheng ; Johnson, Kevin C. ; Amin, Samirkumar B. ; Costa, Megan L. ; Yu, Qianru ; Das, Sunit ; et al ( February 2022 , Cancer Discovery)

   Abstract Oncogenic extrachromosomal DNA elements (ecDNA) play an important role in tumor evolution, but our understanding of ecDNA biology is limited. We determined the distribution of single-cell ecDNA copy number across patient tissues and cell line models and observed how cell-to-cell ecDNA frequency varies greatly. The exceptional intratumoral heterogeneity of ecDNA suggested ecDNA-specific replication and propagation mechanisms. To evaluate the transfer of ecDNA genetic material from parental to offspring cells during mitosis, we established the CRISPR-based ecTag method. ecTag leverages ecDNA-specific breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying ecTag during mitosis revealed disjointed ecDNA inheritance patterns, enabling rapid ecDNA accumulation in individual cells. After mitosis, ecDNAs clustered into ecDNA hubs, and ecDNA hubs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and shed new light on mechanisms through which ecDNAs contribute to oncogenesis. Significance: ecDNAs are vehicles for oncogene amplification. The circular nature of ecDNA affords unique properties, such as mobility and ecDNA-specific replication and segregation behavior. We uncovered fundamental ecDNA properties by tracking ecDNAs in live cells, highlighting uneven and random segregation and ecDNA hubs that drive cargo gene transcription. See relatedmore »commentary by Henssen, p. 293. This article is highlighted in the In This Issue feature, p. 275« less
4. Natural Heteroplasmy and Mitochondrial Inheritance in Bivalve Molluscs

   https://doi.org/10.1093/icb/icz061  

   Ghiselli, Fabrizio ; Maurizii, Maria Gabriella ; Reunov, Arkadiy ; Ariño-Bassols, Helena ; Cifaldi, Carmine ; Pecci, Andrea ; Alexandrova, Yana ; Bettini, Simone ; Passamonti, Marco ; Franceschini, Valeria ; et al ( May 2019 , Integrative and Comparative Biology)

   Heteroplasmy is the presence of more than one type of mitochondrial genome within an individual, a condition commonly reported as unfavorable and affecting mitonuclear interactions. So far, no study has investigated heteroplasmy at protein level, and whether it occurs within tissues, cells, or even organelles. The only known evolutionarily stable and natural heteroplasmic system in Metazoa is the Doubly Uniparental Inheritance (DUI)—reported so far in ∼100 bivalve species—in which two mitochondrial lineages are present: one transmitted through eggs (F-type) and the other through sperm (M-type). Because of such segregation, mitochondrial oxidative phosphorylation proteins reach a high amino acid sequence divergence (up to 52%) between the two lineages in the same species. Natural heteroplasmy coupled with high sequence divergence between F- and M-type proteins provides a unique opportunity to study their expression and assess the level and extent of heteroplasmy. Here, for the first time, we immunolocalized F- and M-type variants of three mitochondrially-encoded proteins in the DUI species Ruditapes philippinarum, in germline and somatic tissues at different developmental stages. We found heteroplasmy at organelle level in undifferentiated germ cells of both sexes, and in male soma, whereas gametes were homoplasmic: eggs for the F-type and sperm for the M-type.more »Thus, during gametogenesis, only the sex-specific mitochondrial variant is maintained, likely due to a process of meiotic drive. We examine the implications of our results for DUI proposing a revised model, and we discuss interactions of mitochondria with germ plasm and their role in germline development. Molecular and phylogenetic evidence suggests that DUI evolved from the common Strictly Maternal Inheritance, so the two systems likely share the same underlying molecular mechanism, making DUI a useful system for studying mitochondrial biology.

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5. Abstract 2634: The role of Eph A receptor 3 tyrosine kinase signaling in prostate cancer progression

   https://doi.org/10.1158/1538-7445.AM2019-2634  

   Al-Mathkour, Marwah M. ; Cinar, Bekir ( July 2019 , AACR Annual Meeting 2019)

   Dysregulation of the receptor tyrosine kinases (RTKs) by means of mutation, amplification or overexpression plays a crucial role in cell growth, cell survival, cell motility during cancer progression and metastasis. EPHA3 (erythropoietin-producing hepatocellular carcinoma cell surface type A receptor 3) is a member of the RTKs. Evidence indicates that the upregulation of the EPHA3 activity is implicated in the pathobiology of various cancers, including prostate cancer, and thus, it is a prime therapeutic target in cancer. However, the role of EPHA3 signaling in prostate cancer progression remains obscure. Currently, the development of castration-resistant prostate cancer (CRPC) poses a clinical challenge because it is lethal. The molecular mechanisms that contribute to lethal prostate cancer are largely unknown. The objective of this study is to investigate whether EPHA3 signaling plays a critical role in prostate cancer progression and therapeutic relapse. Our analysis of the prostate cancer public datasets revealed that the EPHA3 gene was amplified up to 19% of metastatic CRPC cases with the neuroendocrine phenotype. Our immunological assay confirmed the positive staining of EPHA3 protein in human prostate cancer specimens. Our semi-quantitative and quantitative PCR assays demonstrated that the levels of EPHA3 vary among established prostate cancer cell lines. Nevertheless, wemore »consistently found that the levels of EPHA3 mRNA in CRPC cell line, C4-2, were 3-fold higher than its castration-sensitive parental LNCaP cells. Furthermore, we demonstrated that an increase in expression of EPHA3 mRNA in C4-2 compared with LNCaP cells coincided with the upregulation of the EPHA3 protein, as independently confirmed by western blotting and immunofluorescence imaging. These findings indicate that EPHA3 may confer an aggressive prostate cancer cell phenotype. Because androgen receptor (AR) signaling is a potent mediator of CRPC cell growth and survival, the targeting of EPHA3 signaling alone or together with AR may improve the efficacy of current therapies for patients with advanced prostate cancer.« less