Chromosomal Position Research Articles

Rapid and Reproducible Karyotyping with Nanopore Sequencing in AML Patients

Introduction: We evaluated low coverage whole genome sequencing (WGS) of acute myeloid leukemia (AML) patients using long read Oxford Nanopore Technology (ONTseq) for karyotyping and compared the results and the frequency of patients with myelodysplasia-related cytogenetic abnormalities according to previous and current AML classifications with conventional cytogenetics (CG). Methods: Diagnostic samples from peripheral blood or bone marrow of 100 AML patients were sequenced in 3 medical centers on a GridION sequencer using SQK-LSK109 kits according to the manufacturer's instructions, and analyzed by CG. Ten samples were sequenced independently in two different laboratories. Fifty samples were sequenced prospectively from newly diagnosed de novo AML patients. Sequencing reads were analyzed by an in-house bioinformatics pipeline based on publicly available tools such as nglmr aligner, samtools, igv_tools, R package ACE, the CyDAS webserver, and custom scripts for transforming ACE copy number models to karyotype formulas. CNVs from ONTseq data were mapped to 290 chromosome bands to allow comparison with CG. We compared the following cytogenetic categories: Complex karyotype (cKT) ≥ 3 chromosomal abnormalities; monosomal karyotype (mKT) as defined by Breems et al.; karyotype with myelodysplasia-related cytogenetic abnormalities that defines MRC-AML according WHO 2016 classification (MRC-KT), and ICC 2022 classification (MRCA-KT). Results: ONTseq of 100 AML patients with a 4.4-fold genome coverage (range: 2.1-6.2) allowed us to resolve CNVs at a high resolution of ≥0.1 mega-basepairs (Mbp). Analysis of the ten duplicate samples established a high reproducibility of ONTseq. 110 CNVs were detected by both laboratories at identical chromosome positions and similar length (R2=0.999). Only 8 CNVs (7%) differed considerably in length between the replicates (median length difference 2 Mbp, range 0.4-16 Mbp). Of 100 patients with a median age of 65 years (range 18-97), ≥1 chromosome band was gained or lost in 69 patients according to ONTseq analysis, while CNVs or balanced translocations were found by cytogenetics in 72 patients. Five patients had a normal karyotype by ONTseq, but not by CG, while two patients had a normal karyotype by CG but not by ONTseq. Two of these 7 discrepant patients had translocations as their sole cytogenetic abnormality [t(8;21) and t(9;11)], which cannot be detected by low coverage genome ONTseq. Patients with chromosomal abnormalities had a median of 29 (range 1-140) and 30 (range 3-136) chromosomal bands with CNVs according to ONTseq and CG, respectively. Considering all patients, gains and losses at the level of chromosomal bands correlated very well (gains: R2=0.922; losses: R2=0.923) (Figure 1). All patients were then classified according the definitions for cKT, mKT, MRC-KT, and MRCA-KT (Table 1). Overall, patients were similarly classified by ONTseq and CG results (P<0.001). MRC and MRCA-KTs were consistently identified by ONTseq and CG in 57 patients. In 2 and 2 patients, MRC and MRCA-KTs, respectively, were identified by CG only. In 1 and 1 patient, MRC and MRCA-KT, respectively, was identified by ONTseq only. The largest discrepancies were found for mKTs due to the higher resolution of ONTseq, which more often identified subchromosomal instead of whole chromosome CNVs compared to CG, which were not considered for mKT. Fifty of the 100 patients were sequenced prospectively at time of diagnosis. The median age of these patients was 70 years (range 61-97) and all patients had de novo AML. At least one CNV was detected by ONTseq in 24 (48%) and by CG in 26 (52%) patients. cKT, mKT, MRC-KT, and MRCA-KT were detected by ONTseq in 12, 7, 17, and 17 patients and by CG in 14, 11, 17, and 17 patients, respectively. The median time for library preparation for prospectively analyzed patients was 3:45 hours, for ONTseq 24 hours and for bioinformatic analysis 6:30 hours. In this setting, clinical reports can be delivered on the fourth day of sample receipt. Conclusion: Low coverage WGS with ONT provides a highly reproducible method for rapid karyotyping with high resolution. The identification of cKT and myelodysplasia-related cytogenetic abnormalities correlated very well with conventional cytogenetics, even though balanced translocations cannot be detected by our ONTseq protocol. Low coverage WGS is a promising approach for diagnostic karyotyping in AML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genome-wide identification of copy neutral loss of heterozygosity reveals its possible association with spatial positioning of chromosomes.

Loss of heterozygosity (LOH) is a genetic alteration that results from the loss of one allele at a heterozygous locus. In particular, copy neutral LOH (CN-LOH) events are generated, for example, by mitotic homologous recombination after monoallelic defection or gene conversion, resulting in novel homozygous locus having two copies of the normal counterpart allele. This phenomenon can serve as a source of genome diversity and is associated with various diseases. To clarify the nature of the CN-LOH such as the frequency, genomic distribution and inheritance pattern, we made use of whole-genome sequencing data of the three-generation CEPH/Utah family cohort, with the pedigree consisting of grandparents, parents and offspring. We identified an average of 40.7 CN-LOH events per individual taking advantage of 285 healthy individuals from 33 families in the cohort. On average 65% of them were classified as gonosomal-mosaicism-associated CN-LOH, which exists in both germline and somatic cells. We also confirmed that the incidence of the CN-LOH has little to do with the parents' age and sex. Furthermore, through the analysis of the genomic region including the CN-LOH, we found that the chance of the occurrence of the CN-LOH tends to increase at the GC-rich locus and/or on the chromosome having a relatively close inter-homolog distance. We expect that these results provide significant insights into the association between genetic alteration and spatial position of chromosomes as well as the intrinsic genetic property of the CN-LOH.

Revealing the Genetic Architecture of Yield-Related and Quality Traits in Indian Mustard [Brassica juncea (L.) Czern. and Coss.] Using Meta-QTL Analysis

A meta-QTL analysis was conducted in Indian mustard to identify robust and stable meta-QTLs (MQTLs) by utilizing 1504 available QTLs, which included 891 QTLs for yield-related traits and 613 QTLs for quality traits. For yield-related traits, a total of 57 MQTLs (YRTs_MQTLs) were uncovered from the clustering of 560 projected QTLs, which had a 4.18-fold smaller confidence interval (CI) than that of the initial QTLs, whereas, for quality traits, as many as 51 MQTLs (Quality_MQTLs) were derived from 324 projected QTLs, which had a 2.65-fold smaller CI than that of the initial QTLs. Sixteen YRTs_MQTLs were observed to share chromosomal positions with 16 Quality_MQTLs. Moreover, four most promising YRTs_MQTLs and eight Quality-MQTLs were also selected and recommended for use in breeding programs. Four of these selected MQTLs were also validated with significant SNPs that were identified in previously published genome-wide association studies. Further, in silico functional analysis of some promising MQTLs allowed the detection of as many as 1435 genes, which also involved 15 high-confidence candidate genes (CGs) for yield-related traits and 46 high-confidence CGs for quality traits. After validation, the identified CGs can also be exploited to model the plant architecture and to improve quality traits through marker-assisted breeding, genetic engineering, and genome editing approaches.

Sequencing and Genomic Analysis of Sorghum DNA Introgression Variant Line R21 and Recipient Rice Jin Hui 1 Revealed Repetitive Element Variation.

Transferring the genome of distant species to crops is an efficient way to create new germplasms. However, the molecular mechanisms involved are unclear. In this study, a new rice restorer line R21 with heat tolerance was created by introgressing the genomic DNA of sorghum into the recipient restorer line Jin Hui 1. Assembly of rice R21 and Jin Hui 1 genomes was performed using PacBio sequencing technology. Comparative genome analysis and coverage statistics showed that the repetitive sequence atr0026 was a candidate introgression fragment of sorghum DNA. Sequence similarity analysis revealed that atr0026 was distributed at different copy numbers on the telomeric position of chromosomes 9 or 10 in R21, Jin Hui 1, and several rice varieties, indicating that the repetitive sequence from sorghum was highly conserved in rice. The repeat annotation in Gramineae indicated that ribosomal DNA loci that existed in atr0026 may be cause a rearrangement of chromosomes 9 and 10 of the R21 genome, resulting in a copy number variation at the 5′ end of it. Our study lays the foundation for further elucidation of the molecular mechanisms underlying the heat tolerance of sorghum DNA introgression variant line R21, which is of great significance for guiding crop genetic breeding.

Separate optogenetic manipulation of Nerve/glial antigen 2 (NG2) glia and mural cells using the NG2 promoter.

Nerve/glial antigen 2 (NG2) is a protein marker of NG2 glia and mural cells, and NG2 promoter activity is utilized to target these cells. However, the NG2 promoter cannot target NG2 glia and mural cells separately. This has been an obstacle for NG2 glia-specific manipulation. Here, we developed transgenic mice in which either cell type can be targeted using the NG2 promoter. We selected a tetracycline-controllable gene induction system for cell type-specific transgene expression, and generated NG2-tetracycline transactivator (tTA) transgenic lines. We crossed tTA lines with the tetO-ChR2 (channelrhodopsin-2)-EYFP line to characterize tTA-dependent transgene induction. We isolated two unique NG2-tTA mouse lines: one that induced ChR2-EYFP only in mural cells, likely due to the chromosomal position effect of NG2-tTA insertion, and the other that induced it in both cell types. We then applied a Cre-mediated set-subtraction strategy to the latter case and eliminated ChR2-EYFP from mural cells, resulting in NG2 glia-specific transgene induction. We further demonstrated that tTA-dependent ChR2 expression could manipulate cell function. Optogenetic mural cell activation decreased cerebral blood flow, as previously reported, indicating that tTA-mediated ChR2 expression was sufficient to impact cellular function. ChR2-mediated depolarization was observed in NG2 glia in acute hippocampal slices. In addition, ChR2-mediated depolarization of NG2 glia inhibited their proliferation but promoted their differentiation in juvenile mice. Since the tTA-tetO combination is expandable, the mural cell-specific NG2-tTA line and the NG2 glia-specific NG2-tTA line will permit us to conduct observational and manipulation studies to examine in vivo function of these cells separately.

Genome-Wide Identification of Membrane-Bound Fatty Acid Desaturase Genes in Three Peanut Species and Their Expression in Arachis hypogaea during Drought Stress.

As a crop irrigated primarily by rain, the quality and yield of peanuts are significantly limited by drought. To date, many studies have indicated that fatty acid desaturase (FAD) genes enhance plant tolerance to drought stresses. In this study, 16, 15, and 31 FADs were identified in Arachis duranensis, Arachis ipaensis, and Arachis hypogaea, respectively. All the FADs were divided into four subfamilies, which had relatively conserved gene structures, motifs, and domains. The synteny relationships and chromosomal position analysis showed that the FADs in subgenome pairs, A. duranensis-A. hypogaea (AA) and A. ipaensis-A. hypogaea (BB), were homologous, and their physical locations were consistent. The Ka/Ks results indicated that nine FAD genes underwent a purifying selection, and Ah|FAD3.2 experienced positive selection during tetraploid peanut speciation. Various cis-acting elements related to hormone signaling and stress responsiveness in promoters and the predicted miRNA targeting Ah|FADs suggested that these genes play crucial roles in drought tolerance. The expression profiles of Ah|FADs in 22 tissues and drought-tolerant and -sensitive cultivars under drought stress suggested that 4 and 6 FADs were putative genes related to oil accumulation and drought, respectively. These findings will help provide insight into the potential functional roles of the FAD genes, which may aid in dealing with plant drought stress.

The Chromosome Number and rDNA Loci Evolution in Onobrychis (Fabaceae).

The evolution of chromosome number and ribosomal DNA (rDNA) loci number and localisation were studied in Onobrychis Mill. Diploid and tetraploid species, as well as two basic chromosome numbers, x = 7 and x = 8, were observed among analysed taxa. The chromosomal distribution of rDNA loci was presented here for the first time using fluorescence in situ hybridisation (FISH) with 5S and 35S rDNA probes. Onobrychis species showed a high polymorphism in the number and localisation of rDNA loci among diploids, whereas the rDNA loci pattern was very similar in polyploids. Phylogenetic relationships among the species, inferred from nrITS sequences, were used as a framework to reconstruct the patterns of basic chromosome number and rDNA loci evolution. Analysis of the evolution of the basic chromosome numbers allowed the inference of x = 8 as the ancestral number and the descending dysploidy and polyploidisation as the major mechanisms of the chromosome number evolution. Analyses of chromosomal patterns of rRNA gene loci in a phylogenetic context resulted in the reconstruction of one locus of 5S rDNA and one locus of 35S rDNA in the interstitial chromosomal position as the ancestral state in this genus.

Fine mapping of the reduced height gene Rht22 in tetraploid wheat landrace Jianyangailanmai (Triticum turgidum L.)

Rht22 was fine mapped in the interval of 0.53-1.48Mb on 7AS, which reduces cell number of internode to cause semi-dwarfism in Jianyangailanmai. As a valuable germplasm resource for wheat genetic improvement, tetraploid wheat has several reduced height (Rht) and enhanced harvest index genes. Rht22, discovered in Jianyangailanmai (JAM, Triticum turgidum L., 2n = 4x = 28, AABB), significantly increases the spikelet number per spike, but its accurate chromosomal position is still unknown. In this study, a high-density genetic map was constructed using specific-length amplified fragment sequencing in an F7 RIL_DJ population, which was derived from a cross between dwarf Polish wheat (T. polonicum L., 2n = 4x = 28, AABB) and JAM. Two plant height loci, Qph.sicau-4B and Qph.sicau-7A, were mapped on chromosomes 4BS and 7AS, respectively. Qph.sicau-7A was mapped to the 0.33-4.46Mb interval on 7AS and likely represents the candidate region of Rht22. Fine mapping confirmed and narrowed Rht22 on chromosome arm 7AS between Xbag295.s53 and Xb295.191 in three different populations. The physical region ranged from 0.53 to 1.48Mb and included 18 candidate genes. Transcriptome analysis of two pairs of near-isogenic lines revealed that 135 differentially expressed genes (DEGs) were associated with semi-dwarfism. Of these, the expression of 83 annotated DEGs involved in hormones synthesis and signal transduction, cell wall composition, DNA replication, microtubule and phragmoplast arrays was significantly down-regulated in the semi-dwarf line. Therefore, Rht22 causes semi-dwarfism in JAM by disrupting these cellular processes, which impairs cell proliferation and reduces internode cell number.

Genome-Wide Identification of Polyamine Oxidase (PAO) Family Genes: Roles of CaPAO2 and CaPAO4 in the Cold Tolerance of Pepper (Capsicum annuum L.).

Polyamine oxidases (PAOs), which are flavin adenine dinucleotide-dependent enzymes, catalyze polyamine (PA) catabolism, producing hydrogen peroxide (H2O2). Several PAO family members have been identified in plants, but their expression in pepper plants remains unclear. Here, six PAO genes were identified in the ‘Zunla-1’ pepper genome (named CaPAO1–CaPAO6 according to their chromosomal positions). The PAO proteins were divided into four subfamilies according to phylogenetics: CaPAO1 belongs to subfamily I; CaPAO3 and CaPAO5 belong to subfamily III; and CaPAO2, CaPAO4, and CaPAO6 belong to subfamily IV (none belong to subfamily II). CaPAO2, CaPAO4, and CaPAO6 were ubiquitously and highly expressed in all tissues, CaPAO1 was mainly expressed in flowers, whereas CaPAO3 and CaPAO5 were expressed at very low levels in all tissues. RNA-seq analysis revealed that CaPAO2 and CaPAO4 were notably upregulated by cold stress. CaPAO2 and CaPAO4 were localized in the peroxisome, and spermine was the preferred substrate for PA catabolism. CaPAO2 and CaPAO4 overexpression in Arabidopsis thaliana significantly enhanced freezing-stress tolerance by increasing antioxidant enzyme activity and decreasing malondialdehyde, H2O2, and superoxide accumulation, accompanied by the upregulation of cold-responsive genes (AtCOR15A, AtRD29A, AtCOR47, and AtKIN1). Thus, we identified candidate PAO genes for breeding cold-stress-tolerant transgenic pepper cultivars.

CTCF and cohesin promote focal detachment of DNA from the nuclear lamina

BackgroundLamina-associated domains (LADs) are large genomic regions that are positioned at the nuclear lamina. It has remained largely unclear what drives the positioning and demarcation of LADs. Because the insulator protein CTCF is enriched at LAD borders, it was postulated that CTCF binding could position some LAD boundaries, possibly through its function in stalling cohesin and hence preventing cohesin invading into the LAD. To test this, we mapped genome–nuclear lamina interactions in mouse embryonic stem cells after rapid depletion of CTCF and other perturbations of cohesin dynamics.ResultsCTCF and cohesin contribute to a sharp transition in lamina interactions at LAD borders, while LADs are maintained after depletion of these proteins, also at borders marked by CTCF. CTCF and cohesin may thus reinforce LAD borders, but do not position these. CTCF binding sites within LADs are locally detached from the lamina and enriched for accessible DNA and active histone modifications. Remarkably, despite lamina positioning being strongly correlated with genome inactivity, this DNA remains accessible after the local detachment is lost following CTCF depletion. At a chromosomal scale, cohesin depletion and cohesin stabilization by depletion of the unloading factor WAPL quantitatively affect lamina interactions, indicative of perturbed chromosomal positioning in the nucleus. Finally, while H3K27me3 is locally enriched at CTCF-marked LAD borders, we find no evidence for an interplay between CTCF and H3K27me3 on lamina interactions.ConclusionsThese findings illustrate that CTCF and cohesin are not primary determinants of LAD patterns. Rather, these proteins locally modulate NL interactions.

Characterization of Host-Specific Genes from Pine- and Grass-Associated Species of the Fusarium fujikuroi Species Complex.

The Fusarium fujikuroi species complex (FFSC) includes socioeconomically important pathogens that cause disease for numerous crops and synthesize a variety of secondary metabolites that can contaminate feedstocks and food. Here, we used comparative genomics to elucidate processes underlying the ability of pine-associated and grass-associated FFSC species to colonize tissues of their respective plant hosts. We characterized the identity, possible functions, evolutionary origins, and chromosomal positions of the host-range-associated genes encoded by the two groups of fungi. The 72 and 47 genes identified as unique to the respective genome groups were potentially involved in diverse processes, ranging from transcription, regulation, and substrate transport through to virulence/pathogenicity. Most genes arose early during the evolution of Fusarium/FFSC and were only subsequently retained in some lineages, while some had origins outside Fusarium. Although differences in the densities of these genes were especially noticeable on the conditionally dispensable chromosome of F. temperatum (representing the grass-associates) and F. circinatum (representing the pine-associates), the host-range-associated genes tended to be located towards the subtelomeric regions of chromosomes. Taken together, these results demonstrate that multiple mechanisms drive the emergence of genes in the grass- and pine-associated FFSC taxa examined. It also highlighted the diversity of the molecular processes potentially underlying niche-specificity in these and other Fusarium species.

Identification and characterization of auxin response factor (ARF) family members involved in fig (Ficus carica L.) fruit development.

The auxin response factor (ARF) combines with AuxREs cis-acting elements in response to auxin to regulate plant development. To date, no comprehensive analysis of ARF genes expressed during fruit development has been conducted for common fig (Ficus carica L.). In this study, members of the FcARF gene family were screened, identified in the fig genome database and their features characterized using bioinformatics. Twenty FcARF genes were clustered into three classes, with almost similar highly conserved DBD (B3-like DNA binding domain), AUX/IAA (auxin/indole-3-acetic acid gene family) and MR domain structure among class members. Analysis of amino acid species in MR domain revealed 10 potential transcription activators and 10 transcription inhibitors, and 17 FcARF members were predicted to be located in the nucleus. DNA sequence analysis showed that the ARF gene family consisted of 4–25 exons, and the promoter region contained 16 cis-acting elements involved in stress response, hormone response and flavonoid biosynthesis. ARF genes were expressed in most tissues of fig, especially flower and peel. Transcriptomics analysis results showed that FcARF2, FcARF11 and FcARF12, belonging to class-Ia, were stably and highly expressed in the early development stage of flower and peel of ‘Purple peel’ fig. However, their expression levels decreased after maturity. Expression of class-Ic member FcARF3 conformed to the regularity of fig fruit development. These four potential transcription inhibitors may regulate fruit growth and development of ‘Purple Peel’ fig. This study provides comprehensive information on the fig ARF gene family, including gene structure, chromosome position, phylogenetic relationship and expression pattern. Our work provides a foundation for further research on auxin-mediated fig fruit development.

Nuclear chromosome locations dictate segregation error frequencies

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis1–5. Selective pressures then shape distinct aneuploidy and rearrangement patterns—for example, in cancer6,7—but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing8 after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes9 and aneuploidies in early development10 occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development.

Integrative genome modeling platform reveals essentiality of rare contact events in 3D genome organizations

A multitude of sequencing-based and microscopy technologies provide the means to unravel the relationship between the three-dimensional organization of genomes and key regulatory processes of genome function. Here, we develop a multimodal data integration approach to produce populations of single-cell genome structures that are highly predictive for nuclear locations of genes and nuclear bodies, local chromatin compaction and spatial segregation of functionally related chromatin. We demonstrate that multimodal data integration can compensate for systematic errors in some of the data and can greatly increase accuracy and coverage of genome structure models. We also show that alternative combinations of different orthogonal data sources can converge to models with similar predictive power. Moreover, our study reveals the key contributions of low-frequency (‘rare’) interchromosomal contacts to accurately predicting the global nuclear architecture, including the positioning of genes and chromosomes. Overall, our results highlight the benefits of multimodal data integration for genome structure analysis, available through the Integrative Genome Modeling software package.

Genome-wide analysis and characterization of heat shock transcription factors (Hsfs) in common bean (Phaseolus vulgaris L.).

Heat shock transcription factors (Hsfs) play an essential role as transcriptional regulatory proteins against heat stress by controlling the expression of heat-responsive genes. Common bean is a highly thermosensitive crop, and, therefore, its genome sequence information is segregated, characterized here in terms of heat shock transcription factors and its evolutionary significance. In this study, a complete comprehensive set of 29 non-redundant full-length Hsf genes were identified and characterized from Phaseolus vulgaris L. (PvHsf) genome sequence. Detailed gene information such as chromosomal localization, domain position, motif organization, and exon-intron identification were analyzed. All the 29 PvHsf genes were mapped on 8 out of 11 chromosomes, indicating the gene duplication occurred in the common bean genome. Motif analysis and exon-intron structure were conserved in each group, which showed that the cytoplasmic proteins highly influence the conserved structure of PvHsfs and heat-induced response. The HSF genes were grouped into three classes, i.e., A to C and 14 groups, based on structural features and phylogenetic relationships. Only one pair of paralog sequences suggests that it may be derived from the duplication event during evolution. A comparative genomics study indicated the influence of whole-genome duplication and purifying selection on the common bean genome during development. In silico expression analysis showed the active role of class A and B family during abiotic stress conditions and higher expression in floral organs. The qRT-PCR analysis revealed PvHSFA8 as the master regulator and PvHSFB1A and PvHSFB2A induction during heat exposure in French beans.

Abstract 204: Notch2NL is a novel regulator of radiation sensitivity in non-small cell lung cancer and a positive regulator of MYC activity

Abstract Background: A gain-of-function genetic screen identified NOTCH2 N-terminal like (NOTCH2NL) as a candidate novel gene involved in radiation resistance in non-small cell lung cancer (NSCLC). NOTCH2NL is located on chromosome position 1q21.1 which was incorrectly aligned in previous builds of the reference human genome sequence. NOTCH2NL was derived from a segmental duplication event in NOTCH2 during the evolutionary divergence between humans and non-human primates, forming three Notch2NL proteins: Notch2NL-A, Notch2NL-B and Notch2NL-C. Consequently, the only species whose genomes contain NOTCH2NL are modern and ancient humans. Notch2NL-B is a secreted protein and has been shown to promote Notch signalling in cortical stem cell progenitors via inhibition of inhibitory cis-DLL1/NOTCH signalling. Methods: Stable overexpression of NOTCH2NLB and secreted Notch2NL-B protein were validated by RT-qPCR and Western Blotting. The effects of NOTCH2NLB overexpression; transfer of Notch2NL-B protein from conditioned media onto wild-type cell lines; and siRNA knockdown of NOTCH1-4 transmembrane receptors on Notch signalling were quantified by a Notch luciferase reporter assay, RT-qPCR of classical NOTCH target genes and cancer stem cell marker (CSC) and RNA-Seq. Clonogenic cell survival assays determined the impact of NOTCH2NLB overexpression on radiation response. Cell proliferation was measured using the IncuCyte Zoom system. shRNA stable cell lines to knockdown NOTCH2NL were generated to mitigate the NOTCH2NLB overexpression phenotype we observed. RNA-Seq data from the first 100 patients from the TRACERx lung cancer study was used to quantify hallmark gene expression signatures and NOTCH2NLB expression. Results: NOTCH2NLB overexpression upregulates CSL-dependent Notch reporter luciferase activity; confers a radiation-resistant phenotype and impacts proliferation rate in NSCLC cell lines. Furthermore, we demonstrate that NOTCH2NLB overexpression and secreted Notch2NL-B protein transfer increase expression of classical NOTCH target genes (c-MYC, HES1 and HEY1) and CSC marker, ALDH1A1. Analysis of RNA-Seq data from the Lung TRACERx cohort study shows that NOTCH2NL is commonly expressed in NSCLC. NOTCH2NL expression positively correlates with notch hallmark gene expression signature in squamous (R=0.59, p=5.6e-9) and non-squamous carcinoma (R=0.51, p=4.7e-10). Interestingly, NOTCH2NL expression also positively correlated with the myc hallmark gene expression signature in squamous cell carcinoma (R=0.45, p=2.4e-5). Conclusions: Notch2NL is a novel determinant of radiation therapy response and impacts canonical Notch signalling in lung cancer. It is commonly expressed in NSCLC and can act in a paracrine manner as a secreted protein. Notch2NL, through canonical notch signalling, can regulate MYC expression and increase activity in the MYC pathway. Citation Format: Simranpreet Kaur Summan, Barbara Chow, Rhona Millar, Su Kit Chew, Philip East, Robert Hynds, Carlos Martinez Ruiz, Eva Gronroos, Mariam Jamal-Hanjani, Kevin Litchfield, Nicholas McGranahan, Nnennaya Kanu, Charles Swanton, Crispin Thomas Hiley. Notch2NL is a novel regulator of radiation sensitivity in non-small cell lung cancer and a positive regulator of MYC activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 204.

Mendel's First Law: partisan interests and the parliament of genes

Mendel’s First Law requires explanation because of the possibility of ‘meiotic drivers’, genes that distort fair segregation for selfish gain. The suppression of drive, and the restoration of fair segregation, is often attributed to genes at loci unlinked to the drive locus—such genes cannot benefit from drive but do suffer its associated fitness costs. However, selection can also favour suppressors at loci linked to the drive locus, raising the question of whether suppression of drive usually comes from linked or unlinked loci. Here, I study linked and unlinked suppression in a two-locus model with initial stable polymorphism at the drive locus. I find that the invasion rate of suppressors is a decreasing function of the recombination fraction between the drive and suppressor loci. Surprisingly, the relative likelihood of unlinked vs. linked suppression increases with the strength of drive and is insensitive to the fitness costs of the driver allele. I find that the chromosomal position of the driver influences how rapidly it is suppressed, with a driver in the middle of a chromosome suppressed more rapidly than a driver near the tip. When drive is strong, only a small number of chromosomes are required for suppression usually to derive from unlinked loci. In contrast, when drive is weak, and especially when suppressor alleles are associated with fitness costs, suppression will usually come from linked loci unless the genome comprises many chromosomes.

Cohesin-dependent chromosome loop extrusion is limited by transcription and stalled replication forks.

Genome function depends on regulated chromosome folding, and loop extrusion by the protein complex cohesin is essential for this multilayered organization. The chromosomal positioning of cohesin is controlled by transcription, and the complex also localizes to stalled replication forks. However, the role of transcription and replication in chromosome looping remains unclear. Here, we show that reduction of chromosome-bound RNA polymerase weakens normal cohesin loop extrusion boundaries, allowing cohesin to form new long-range chromosome cis interactions. Stress response genes induced by transcription inhibition are also shown to act as new loop extrusion boundaries. Furthermore, cohesin loop extrusion during early S phase is jointly controlled by transcription and replication units. Together, the results reveal that replication and transcription machineries are chromosome-folding regulators that block the progression of loop-extruding cohesin, opening for new perspectives on cohesin’s roles in genome function and stability.

Genome wide analysis of the heavy-metal-associated (HMA) gene family in tomato and expression profiles under different stresses

The heavy-metal-associated (HMA) family plays a major role in the transportation of metals. Despite having the genome sequence of the tomato (Solanum lycopersicum), the HMA gene family has not been studied yet. In this study, we identified 48 HMA genes and categorized them into Cu/Ag P1B-ATPase and Zn/Co/Cd/Pb P1BATPase sub-families according to their phylogenic relationship with Arabidopsis and rice. The SlHMA genes were distributed throughout the 12 chromosomes. Analysis of gene structure, chromosomal position, and synteny, revealed that segmental duplications bestowed their evolution. The high numbers of stress-related cis-elements were found to be present in the putative promoter regions indicate the involvement of SlHMAs in stress modulation pathways. RNA-seq data revealed that SlHMAs had divergent expression in different tissues and developmental stages, where members of Cu/Ag P1B-ATPase subfamily were strongly expressed in the roots. RT-qPCR analysis of nine selected SlHMAs showed that most of the genes were up-regulated in response to heavy metals and moderately regulated in response to different abiotic stresses such as salt, drought, and cold.

Partners in crime: Tbf1 and Vid22 promote expansions of long human telomeric repeats at an interstitial chromosome position in yeast.

In humans, telomeric repeats (TTAGGG)n are known to be present at internal chromosomal sites. These interstitial telomeric sequences (ITSs) are an important source of genomic instability, including repeat length polymorphism, but the molecular mechanisms responsible for this instability remain to be understood. Here, we studied the mechanisms responsible for expansions of human telomeric (Htel) repeats that were artificially inserted inside a yeast chromosome. We found that Htel repeats in an interstitial chromosome position are prone to expansions. The propensity of Htel repeats to expand depends on the presence of a complex of two yeast proteins: Tbf1 and Vid22. These two proteins are physically bound to an interstitial Htel repeat, and together they slow replication fork progression through it. We propose that slow progression of the replication fork through the protein complex formed by the Tbf1 and Vid22 partners at the Htel repeat cause DNA strand slippage, ultimately resulting in repeat expansions.