Chromosome Analysis and Sorting Using Conventional Flow Cytometers.

The fluorescent dyes Hoechst (HO) and Chromomycin A3 (CA3) are commonly used for bivariate flow karyotyping to distinguish individual chromosomes from one another based on differences in base composition and DNA content. However, analysis of chromosomes using this fluorescent dye combination requires a flow cytometer equipped with lasers of specific wavelengths and higher power than is typical of conventional flow cytometers. This unit presents a chromosome staining technique with a dye combination of DAPI and propidium iodide (PI). Chromosomes stained using this dye combination can be analyzed on conventional flow cytometers equipped with a typical configuration of lasers and optics. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cell culture and metaphase harvest of suspension cell line Alternate Protocol 1: Cell culture and metaphase harvest of adherent cell line Basic Protocol 2: Preparation of chromosome suspension using polyamine isolation buffer Basic Protocol 3: Staining chromosomes with DAPI and propidium iodide Alternate Protocol 2: Staining chromosomes with Hoechst and Chromomycin A3 Basic Protocol 4: Bivariate flow karyotyping on a cell analyzer Basic Protocol 5: Bivariate flow karyotyping on a cell sorter Basic Protocol 6: Purification of flow-sorted chromosomes.

Hi-C scaffolded short- and long-read genome assemblies of the California sea lion are broadly consistent for syntenic inference across 45 million years of evolution.

With the advent of chromatin-interaction maps, chromosome-level genome assemblies have become a reality for a wide range of organisms. Scaffolding quality is, however, difficult to judge. To explore this gap, we generated multiple chromosome-scale genome assemblies of an emerging wild animal model for carcinogenesis, the California sea lion (Zalophus californianus). Short-read assemblies were scaffolded with two independent chromatin interaction mapping data sets (Hi-C and Chicago), and long-read assemblies with three data types (Hi-C, optical maps and 10X linked reads) following the "Vertebrate Genomes Project (VGP)" pipeline. In both approaches, 18 major scaffolds recovered the karyotype (2n = 36), with scaffold N50s of 138 and 147 Mb, respectively. Synteny relationships at the chromosome level with other pinniped genomes (2n = 32-36), ferret (2n = 34), red panda (2n = 36) and domestic dog (2n = 78) were consistent across approaches and recovered known fissions and fusions. Comparative chromosome painting and multicolour chromosome tiling with a panel of 264 genome-integrated single-locus canine bacterial artificial chromosome probes provided independent evaluation of genome organization. Broad-scale discrepancies between the approaches were observed within chromosomes, most commonly in translocations centred around centromeres and telomeres, which were better resolved in the VGP assembly. Genomic and cytological approaches agreed on near-perfect synteny of the X chromosome, and in combination allowed detailed investigation of autosomal rearrangements between dog and sea lion. This study presents high-quality genomes of an emerging cancer model and highlights that even highly fragmented short-read assemblies scaffolded with Hi-C can yield reliable chromosome-level scaffolds suitable for comparative genomic analyses.

Simvastatin enhances the efficacy of nilotinib in chronic myeloid leukaemia by post-translational modification and drug transporter modulation.

The resistance of chronic myeloid leukaemia (CML) to tyrosine kinase inhibitors (TKIs) remains a significant clinical problem. Targeting alternative pathways, such as protein prenylation, is known to be effective in overcoming resistance. Simvastatin inhibits 3-hydroxy-3-methylglutaryl-CoA reductase (a key enzyme in isoprenoid-regulation), thereby inhibiting prenylation. We demonstrate that simvastatin alone effectively inhibits proliferation in a panel of TKI-resistant CML cell lines, regardless of mechanism of resistance. We further show that the combination of nilotinib and simvastatin synergistically kills CML cells via an increase in apoptosis and decrease in prosurvival proteins and cellular proliferation. Mechanistically, simvastatin inhibits protein prenylation as shown by increased levels of unprenylated Ras and rescue experiments with mevalonate resulted in abrogation of synergism. The combination also leads to an increase in the intracellular uptake and retention of radio-labelled nilotinib, which further enhances the inhibition of Bcr-Abl kinase activity. In primary CML samples, this combination inhibits clonogenicity in both imatinib-naive and resistant cells. Such combinatorial effects provide the basis for utilising these Food and Drug Administration-approved drugs as a potential clinical approach in overcoming resistance and improving CML treatment.

Single-cell atlas of the first intra-mammalian developmental stage of the human parasite Schistosoma mansoni.

Over 250 million people suffer from schistosomiasis, a tropical disease caused by parasitic flatworms known as schistosomes. Humans become infected by free-swimming, water-borne larvae, which penetrate the skin. The earliest intra-mammalian stage, called the schistosomulum, undergoes a series of developmental transitions. These changes are critical for the parasite to adapt to its new environment as it navigates through host tissues to reach its niche, where it will grow to reproductive maturity. Unravelling the mechanisms that drive intra-mammalian development requires knowledge of the spatial organisation and transcriptional dynamics of different cell types that comprise the schistomulum body. To fill these important knowledge gaps, we perform single-cell RNA sequencing on two-day old schistosomula of Schistosoma mansoni. We identify likely gene expression profiles for muscle, nervous system, tegument, oesophageal gland, parenchymal/primordial gut cells, and stem cells. In addition, we validate cell markers for all these clusters by in situ hybridisation in schistosomula and adult parasites. Taken together, this study provides a comprehensive cell-type atlas for the early intra-mammalian stage of this devastating metazoan parasite.

Chromosome-level genome assembly for giant panda provides novel insights into Carnivora chromosome evolution.

BACKGROUND: Chromosome evolution is an important driver of speciation and species evolution. Previous studies have detected chromosome rearrangement events among different Carnivora species using chromosome painting strategies. However, few of these studies have focused on chromosome evolution at a nucleotide resolution due to the limited availability of chromosome-level Carnivora genomes. Although the de novo genome assembly of the giant panda is available, current short read-based assemblies are limited to moderately sized scaffolds, making the study of chromosome evolution difficult. RESULTS: Here, we present a chromosome-level giant panda draft genome with a total size of 2.29 Gb. Based on the giant panda genome and published chromosome-level dog and cat genomes, we conduct six large-scale pairwise synteny alignments and identify evolutionary breakpoint regions. Interestingly, gene functional enrichment analysis shows that for all of the three Carnivora genomes, some genes located in evolutionary breakpoint regions are significantly enriched in pathways or terms related to sensory perception of smell. In addition, we find that the sweet receptor gene TAS1R2, which has been proven to be a pseudogene in the cat genome, is located in an evolutionary breakpoint region of the giant panda, suggesting that interchromosomal rearrangement may play a role in the cat TAS1R2 pseudogenization. CONCLUSIONS: We show that the combined strategies employed in this study can be used to generate efficient chromosome-level genome assemblies. Moreover, our comparative genomics analyses provide novel insights into Carnivora chromosome evolution, linking chromosome evolution to functional gene evolution.

Disruption of chromatin organisation causes MEF2C gene overexpression in intellectual disability: a case report.

BACKGROUND: Balanced structural variants are mostly described in disease with gene disruption or subtle rearrangement at breakpoints. CASE PRESENTATION: Here we report a patient with mild intellectual deficiency who carries a de novo balanced translocation t(3;5). Breakpoints were fully explored by microarray, Array Painting and Sanger sequencing. No gene disruption was found but the chromosome 5 breakpoint was localized 228-kb upstream of the MEF2C gene. The predicted Topologically Associated Domains analysis shows that it contains only the MEF2C gene and a long non-coding RNA LINC01226. RNA studies looking for MEF2C gene expression revealed an overexpression of MEF2C in the lymphoblastoid cell line of the patient. CONCLUSIONS: Pathogenicity of MEF2C overexpression is still unclear as only four patients with mild intellectual deficiency carrying 5q14.3 microduplications containing MEF2C are described in the literature. The microduplications in these individuals also contain other genes expressed in the brain. The patient presented the same phenotype as 5q14.3 microduplication patients. We report the first case of a balanced translocation leading to an overexpression of MEF2C similar to a functional duplication.

Derivation and maintenance of mouse haploid embryonic stem cells.

Ploidy represents the number of chromosome sets in a cell. Although gametes have a haploid genome (n), most mammalian cells have diploid genomes (2n). The diploid status of most cells correlates with the number of probable alleles for each autosomal gene and makes it difficult to target these genes via mutagenesis techniques. Here, we describe a 7-week protocol for the derivation of mouse haploid embryonic stem cells (hESCs) from female gametes that also outlines how to maintain the cells once derived. We detail additional procedures that can be used with cell lines obtained from the mouse Haplobank, a biobank of >100,000 individual mouse hESC lines with targeted mutations in 16,970 genes. hESCs can spontaneously diploidize and can be maintained in both haploid and diploid states. Mouse hESCs are genomically and karyotypically stable, are innately immortal and isogenic, and can be derived in an array of differentiated cell types; they are thus highly amenable to genetic screens and to defining molecular connectivity pathways.

Automating the Shared Resource Laboratory Using Computer Scripts: A Case Report.

Shared resource laboratories (SRLs) offer instrumentation, training, and support to investigators and play an important role in the progress and development of science. To facilitate daily tasks and to provide an effective service, we have made use of computer scripts; a list of computer commands that are processed sequentially, to automate tasks in our flow cytometry facility. Using Python and an application programming interface (API), we automate user communication and produce a daily schedule display screen. We exploit the accessible nature of open standards to use R and Python to analyze and backup data from the BD Influx cell sorter. Finally, we show that through simple scripting, we can add value to an existing service by producing sort statistics from the Beckman Coulter XDP cell sorter. With these five examples, we demonstrate and wish to inspire other SRLs that the use of scripts helps to improve work efficiency, can solve problems, and can enhance the service provided by the SRL. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Characterizing Mutational Signatures in Human Cancer Cell Lines Reveals Episodic APOBEC Mutagenesis.

Multiple signatures of somatic mutations have been identified in cancer genomes. Exome sequences of 1,001 human cancer cell lines and 577 xenografts revealed most common mutational signatures, indicating past activity of the underlying processes, usually in appropriate cancer types. To investigate ongoing patterns of mutational-signature generation, cell lines were cultured for extended periods and subsequently DNA sequenced. Signatures of discontinued exposures, including tobacco smoke and ultraviolet light, were not generated in vitro. Signatures of normal and defective DNA repair and replication continued to be generated at roughly stable mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing exhibited substantial fluctuations in mutation rate over time with episodic bursts of mutations. The initiating factors for the bursts are unclear, although retrotransposon mobilization may contribute. The examined cell lines constitute a resource of live experimental models of mutational processes, which potentially retain patterns of activity and regulation operative in primary human cancers.

Relationship Between Sequence Homology, Genome Architecture, and Meiotic Behavior of the Sex Chromosomes in North American Voles.

In most mammals, the X and Y chromosomes synapse and recombine along a conserved region of homology known as the pseudoautosomal region (PAR). These homology-driven interactions are required for meiotic progression and are essential for male fertility. Although the PAR fulfills key meiotic functions in most mammals, several exceptional species lack PAR-mediated sex chromosome associations at meiosis. Here, we leveraged the natural variation in meiotic sex chromosome programs present in North American voles (Microtus) to investigate the relationship between meiotic sex chromosome dynamics and X/Y sequence homology. To this end, we developed a novel, reference-blind computational method to analyze sparse sequencing data from flow-sorted X and Y chromosomes isolated from vole species with sex chromosomes that always (Microtus montanus), never (Microtus mogollonensis), and occasionally synapse (Microtus ochrogaster) at meiosis. Unexpectedly, we find more shared X/Y homology in the two vole species with no and sporadic X/Y synapsis compared to the species with obligate synapsis. Sex chromosome homology in the asynaptic and occasionally synaptic species is interspersed along chromosomes and largely restricted to low-complexity sequences, including a striking enrichment for the telomeric repeat sequence, TTAGGG. In contrast, homology is concentrated in high complexity, and presumably euchromatic, sequence on the X and Y chromosomes of the synaptic vole species, M. montanus Taken together, our findings suggest key conditions required to sustain the standard program of X/Y synapsis at meiosis and reveal an intriguing connection between heterochromatic repeat architecture and noncanonical, asynaptic mechanisms of sex chromosome segregation in voles.

Chromosomal breaks at FRA18C: association with reduced DOK6 expression, altered oncogenic signaling and increased gastric cancer survival.

Chromosomal rearrangements are common in cancer. More than 50% occur in common fragile sites and disrupt tumor suppressors. However, such rearrangements are not known in gastric cancer. Here we report recurrent 18q2 breakpoints in 6 of 17 gastric cancer cell lines. The rearranged chromosome 18, t(9;18), in MKN7 cells was flow sorted and identified by reverse chromosome painting. High-resolution tiling array hybridization mapped breakpoints to DOK6 (docking protein 6) intron 4 in FRA18C (18q22.2) and an intergenic region in 9q22.2. The same rearrangement was detected by FISH in 22% of 99 primary gastric cancers. Intron 4 truncation was associated with reduced DOK6 transcription. Analysis of The Cancer Genome Atlas stomach adenocarcinoma cohort showed significant correlation of DOK6 expression with histological and molecular phenotypes. Multiple oncogenic signaling pathways (gastrin-CREB, NGF-neurotrophin, PDGF, EGFR, ERK, ERBB4, FGFR1, RAS, VEGFR2 and RAF/MAP kinase) known to be active in aggressive gastric cancers were strikingly diminished in gastric cancers with low DOK6 expression. Median survival of patients with low DOK6-expressing tumors was 2100 days compared with 533 days in patients with high DOK6-expressing tumors (log-rank P = 0.0027). The level of DOK6 expression in tumors predicted patient survival independent of TNM stage. These findings point to new functions of human DOK6 as an adaptor that interacts with diverse molecular components of signaling pathways. Our data suggest that DOK6 expression is an integrated biomarker of multiple oncogenic signals in gastric cancer and identify FRA18C as a new cancer-associated fragile site.

The pig X and Y Chromosomes: structure, sequence, and evolution.

We have generated an improved assembly and gene annotation of the pig X Chromosome, and a first draft assembly of the pig Y Chromosome, by sequencing BAC and fosmid clones from Duroc animals and incorporating information from optical mapping and fiber-FISH. The X Chromosome carries 1033 annotated genes, 690 of which are protein coding. Gene order closely matches that found in primates (including humans) and carnivores (including cats and dogs), which is inferred to be ancestral. Nevertheless, several protein-coding genes present on the human X Chromosome were absent from the pig, and 38 pig-specific X-chromosomal genes were annotated, 22 of which were olfactory receptors. The pig Y-specific Chromosome sequence generated here comprises 30 megabases (Mb). A 15-Mb subset of this sequence was assembled, revealing two clusters of male-specific low copy number genes, separated by an ampliconic region including the HSFY gene family, which together make up most of the short arm. Both clusters contain palindromes with high sequence identity, presumably maintained by gene conversion. Many of the ancestral X-related genes previously reported in at least one mammalian Y Chromosome are represented either as active genes or partial sequences. This sequencing project has allowed us to identify genes--both single copy and amplified--on the pig Y Chromosome, to compare the pig X and Y Chromosomes for homologous sequences, and thereby to reveal mechanisms underlying pig X and Y Chromosome evolution.

Inhibition of isoprenylcysteine carboxylmethyltransferase augments BCR-ABL1 tyrosine kinase inhibition-induced apoptosis in chronic myeloid leukemia.

Despite the success of BCR-ABL1 tyrosine kinase inhibitors in patients with chronic myeloid leukemia (CML), resistance to tyrosine kinase inhibitors remains a therapeutic challenge. One strategy used to overcome resistance is combination of existing BCR-ABL1 tyrosine kinase inhibitors with agents that target alternative pathways. We report that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), a key enzyme in the protein prenylation pathway, with the selective inhibitor cysmethynil enhances the effect of BCR-ABL1 tyrosine kinase inhibitors in killing CML cells. Cysmethynil augments tyrosine kinase inhibitor-induced apoptosis in both BCR-ABL1 wild type and BCR-ABL1 kinase domain mutant-expressing cell lines. Importantly, the enhanced apoptosis observed with the combination of cysmethynil and imatinib is significant only in primary CML CD34+ progenitor cells, not normal cord blood progenitor cells. The combination was also selective in inhibiting colony formation in CML CD34+ cells. The enhanced apoptosis appears to be due to combination of immediate and persistent inhibition of MAPK signaling. Consistent with in vitro studies, cysmethynil and imatinib, in combination, enhance the in vivo effects of either drug used alone. We found that simultaneous inhibition of BCR-ABL1 and Icmt may represent a potential therapeutic strategy for CML.

Identification of disrupted AUTS2 and EPHA6 genes by array painting in a patient carrying a de novo balanced translocation t(3;7) with intellectual disability and neurodevelopment disorder.

Intellectual disability (ID) is a frequent feature but is highly clinically and genetically heterogeneous. The establishment of the precise diagnosis in patients with ID is challenging due to this heterogeneity but crucial for genetic counseling and appropriate care for the patients. Among the etiologies of patients with ID, apparently balanced de novo rearrangements represent 0.6%. Several mechanisms explain the ID in patients with apparently balanced de novo rearrangement. Among them, disruption of a disease gene at the breakpoint, is frequently evoked. In this context, technologies recently developed are used to characterize precisely such chromosomal rearrangements. Here, we report the case of a boy with ID, facial features and autistic behavior who is carrying a de novo balanced reciprocal translocation t(3;7)(q11.2;q11.22)dn. Using microarray analysis, array painting (AP) technology combined with molecular study, we have identified the interruption of the autism susceptibility candidate 2 gene (AUTS2) and EPH receptor A6 gene (EPHA6). We consider that the disruption of AUTS2 explains the phenotype of the patient; the exact role of EPHA6 in human pathology is not well defined. Based on the observation of recurrent germinal and somatic translocations involving AUTS2 and the molecular environment content, we put forward the hypothesis that the likely chromosomal mechanism responsible for the translocation could be due either to replicative stress or to recombination-based mechanisms.

Post-operative survival following metastasectomy for patients receiving BRAF inhibitor therapy is associated with duration of pre-operative treatment and elective indication.

INTRODUCTION: Metastasectomy can provide durable disease control for selected patients with metastatic melanoma. Vemurafenib is a BRAF kinase inhibitor which has demonstrated significant improvement in disease-specific survival in patients with metastatic melanoma with a BRAF gene mutation. This study examined the efficacy and safety of metastasectomy during treatment with vemurafenib. METHODS: A retrospective review was performed of all patients receiving vemurafenib at Peter MacCallum Cancer Centre. Patient records were reviewed to identify patients undergoing surgery within 30 days of vemurafenib therapy. Descriptive statistics and survival analysis were performed. RESULTS: Nineteen patients underwent 21 metastasectomies including craniotomy (57%), spinal decompression (14%), small bowel resection (14%), lung resection (9.5%) and neck dissection (4.5%). Indications for surgery were: an isolated residual focus of disease (n = 2); isolated progressive disease in the setting of stability elsewhere (n = 9); and symptomatic disease (n = 8). Grade 2 or higher surgical complications occurred in 19% of cases and there was one peri-operative death. Median post-operative survival was seven months. There was a trend toward improved post-operative survival for patients with longer duration of vemurafenib therapy (P = 0.04) and for those undergoing elective surgery (P = 0.07). CONCLUSION: Resection of oligometastatic disease during BRAF-targeted therapy is safe. Selected patients have durable post-operative disease control.

A high-throughput in vivo micronucleus assay for genome instability screening in mice.

We describe a sensitive, robust, high-throughput method for quantifying the formation of micronuclei, markers of genome instability, in mouse erythrocytes. Micronuclei are whole chromosomes or chromosome segments that have been separated from the nucleus. Other methods of detection rely on labor-intensive, microscopy-based techniques. Here we describe a 2-d, 96-well plate-based flow cytometric method of micronucleus scoring that is simple enough for a research technician experienced in flow cytometry to perform. The assay detects low levels of genome instability that cannot be readily identified by classic phenotyping, using 25 µl of blood. By using this assay, we have screened >10,000 blood samples and discovered novel genes that contribute to vertebrate genome maintenance, as well as novel disease models and mechanisms of genome instability disorders. We discuss experimental design considerations, including statistical power calculation, we provide troubleshooting tips and we discuss factors that contribute to a false-positive increase in the number of micronucleated red blood cells and to experimental variability.

Massively parallel sequencing reveals the complex structure of an irradiated human chromosome on a mouse background in the Tc1 model of Down syndrome.

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype--phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.

Genetic basis of Y-linked hearing impairment.

A single Mendelian trait has been mapped to the human Y chromosome: Y-linked hearing impairment. The molecular basis of this disorder is unknown. Here, we report the detailed characterization of the DFNY1 Y chromosome and its comparison with a closely related Y chromosome from an unaffected branch of the family. The DFNY1 chromosome carries a complex rearrangement, including duplication of several noncontiguous segments of the Y chromosome and insertion of ∼160 kb of DNA from chromosome 1, in the pericentric region of Yp. This segment of chromosome 1 is derived entirely from within a known hearing impairment locus, DFNA49. We suggest that a third copy of one or more genes from the shared segment of chromosome 1 might be responsible for the hearing-loss phenotype.

Genome sequencing and analysis of the Tasmanian devil and its transmissible cancer.

The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.

Reprogramming of T cells to natural killer-like cells upon Bcl11b deletion.

T cells develop in the thymus and are critical for adaptive immunity. Natural killer (NK) lymphocytes constitute an essential component of the innate immune system in tumor surveillance, reproduction, and defense against microbes and viruses. Here, we show that the transcription factor Bcl11b was expressed in all T cell compartments and was indispensable for T lineage development. When Bcl11b was deleted, T cells from all developmental stages acquired NK cell properties and concomitantly lost or decreased T cell-associated gene expression. These induced T-to-natural killer (ITNK) cells, which were morphologically and genetically similar to conventional NK cells, killed tumor cells in vitro, and effectively prevented tumor metastasis in vivo. Therefore, ITNKs may represent a new cell source for cell-based therapies.