Mitotic Gene Research Articles

Mitotic checkpoint gene expression is tuned by codon usage bias

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half‐lives and long protein half‐lives supports stable SAC protein levels. For the SAC genes mad2+ and mad3+, their short mRNA half‐lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1+ mRNA has a short half‐life despite a higher frequency of optimal codons, and despite the lack of known RNA‐destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co‐translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine‐tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.

Genomic instability genes in lung and colon adenocarcinoma indicate organ specificity of transcriptomic impact on Copy Number Alterations

Genomic instability (GI) in cancer facilitates cancer evolution and is an exploitable target for therapy purposes. However, specific genes involved in cancer GI remain elusive. Causal genes for GI via expressions have not been comprehensively identified in colorectal cancers (CRCs). To fill the gap in knowledge, we developed a data mining strategy (Gene Expression to Copy Number Alterations; “GE-CNA”). Here we applied the GE-CNA approach to 592 TCGA CRC datasets, and identified 500 genes whose expression levels associate with CNA. Among these, 18 were survival-critical (i.e., expression levels correlate with significant differences in patients’ survival). Comparison with previous results indicated striking differences between lung adenocarcinoma and CRC: (a) less involvement of overexpression of mitotic genes in generating genomic instability in the colon and (b) the presence of CNA-suppressing pathways, including immune-surveillance, was only partly similar to those in the lung. Following 13 genes (TIGD6, TMED6, APOBEC3D, EP400NL, B3GNT4, ZNF683, FOXD4, FOXD4L1, PKIB, DDB2, MT1G, CLCN3, CAPS) were evaluated as potential drug development targets (hazard ratio [> 1.3 or < 0.5]). Identification of specific CRC genomic instability genes enables researchers to develop GI targeting approach. The new results suggest that the “targeting genomic instability and/or aneuploidy” approach must be tailored for specific organs.

The metastatic spread of breast cancer accelerates during sleep.

The metastatic spread of cancer is achieved by the haematogenous dissemination of circulating tumour cells (CTCs). Generally, however, the temporal dynamics that dictate the generation of metastasis-competent CTCs are largely uncharacterized, and it is often assumed that CTCs are constantly shed from growing tumours or areshed as a consequence of mechanical insults1. Here we observe a striking and unexpected pattern of CTC generation dynamics in both patients with breast cancer and mouse models, highlighting that most spontaneous CTC intravasation events occur duringsleep. Further, we demonstrate that rest-phase CTCs are highly prone to metastasize, whereas CTCs generated during the active phase are devoid of metastatic ability. Mechanistically, single-cell RNA sequencing analysis of CTCs reveals a marked upregulation of mitotic genes exclusively during the rest phase in both patients and mouse models, enabling metastasis proficiency. Systemically, we find that key circadian rhythm hormones such as melatonin, testosterone and glucocorticoids dictate CTC generation dynamics, and as a consequence, that insulin directly promotes tumour cell proliferation in vivo, yet in a time-dependent manner. Thus, the spontaneous generation of CTCs with a high proclivity to metastasize does not occur continuously, but it is concentrated within the rest phase of the affected individual, providing a new rationale for time-controlled interrogation and treatment of metastasis-prone cancers.

Abstract 1616: Dissecting the biology and therapeutic vulnerabilities of RB1-mutant osteosarcoma using hereditary retinoblastoma iPSCs

Abstract Patients with hereditary retinoblastoma (RB), an inherited autosomal dominant cancer disordercaused by germline mutations/deletions in the RB1 gene, have a &amp;gt;400 fold increased incidenceof osteosarcoma (OS), suggesting a strong mechanistic link between RB1 loss andosteosarcomagenesis. Although mice offer many advantages when conducting cancer research,unlike humans, Rb1 knockout mice do not develop OS, suggesting the urgent requirement foralternative disease models to understand how RB1 mutation leads to osteosarcomagenesis.Here, we generated patient-derived induced pluripotent stem cells (iPSCs) by reprogramming RBand healthy control fibroblasts and then created isogenic controls by correcting RB1 mutationusing CRISPR/Cas9. Performing in vitro soft-agar assay and in vivo xenografts, we foundpotential tumorigenic ability in RB osteoblasts (OBs) but not control OBs. In order to gain insightsinto RB1 loss associated osteosarcomagenesis, we compared global transcripts among RB andcontrol OBs. GSEA analysis indicated that OS associated genes are specifically enriched in RBOBs in comparison with control OBs, demonstrating that RB OBs acquire OS characteristics inthe absence of additional gene alterations. GO analysis revealed that genes involved in the mitoticcell cycle are enriched in RB OBs. Although RB1 is known to controlling G1/S related geneexpression by inhibiting E2F transcription factors, it is still nebulous if RB1 plays a role inregulating mitotic genes.To comprehensively identify preferential binding of RB1 to specific promoters in OBs, weconducted RB1 ChIP-seq to map genome-wide RB1-binding sites in iPSC-derived OBs. SinceE2F3a, a RB1 repressed transcription factor, is enriched expressed in OBs, we also performedE2F3a ChIP-seq to define whether these RB1-repressed targets are E2F3a-activated targets.Using the nonbiased de novo motif search algorithm DME, we further identified the most highlyenriched RB1-binding motif in all conditions and cross-compare with the E2F3a motifs. Weconfirmed that RB1 and E2F3a co-occupy the mitotic regulator promoters by ChIP-PCR. IHCstudies of clinical OS specimens supported the clinical correlation between RB1/E2F3a andmitotic regulators. High mitotic regulator genes expression is correlated with poor prognosis. Ourresults demonstrated that the mitotic regulators are co-regulated by both RB1 and E2F3a andthey play roles in osteosarcomagenesis.Taken together, our findings demonstrated multiple OS-related phenotypes in human RB iPSC-derived OBs and RB1/E2F3a-regulating mitotic regulators can be a therapeutic vulnerability inRB1-mutant OS. Citation Format: Dandan Zhu, Jian Tu, Zijun Huo, An Xu, Mo-Fan Huang, Ying Liu, Yi-Hung Chen, Ruiying Zhao, Dung-Fang Lee. Dissecting the biology and therapeutic vulnerabilities of RB1-mutant osteosarcoma using hereditary retinoblastoma iPSCs [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 1616.

Abstract 3949: Identification of a novel HASPIN inhibitor and the synthetic lethal partner by computational analysis

Abstract HASPIN, a mitotic kinase to phosphorylate Histone H3 at centromere, has been studied as a promising target for anti-cancer therapy. However, as HASPIN belongs to atypical kinase, lacking the Asp-Phe-Gly (DFG) motif, development from the chemical library of kinase inhibitors with conventional pharmacophore, would be technically challenging. In particular, one of adenosine analogues (LJ4827), a potent inhibitor of multi-kinases, showing clear anti-cancer activity in vitro and in vivo, was predicted as a HASPIN inhibitor by computation analysis of its transcriptome profile in cancer cells to drug-omic data set in the connectivity MAP (cMAP) as it shared similar transcriptome profile of 5-iodotubercidin (5ITU). The specificity and potency as HASPIN inhibitor of LJ4827 (IC50 = 0.45 nM) validated by in vitro kinase screening and consequent 3D structure modeling. As expected, treatment of LJ4827 in cancer cell lines efficiently delayed mitotic progression without double strand break (DSB) unlike 5ITU and significantly attenuated Aurora B localization at centromere. Along with clinical significance of HASPIN expression in lung cancer patients, mitotic gene signature closely associated to high expression of HASPIN, revealed the poor prognosis. Additional computational analysis of kinase perturbation data to predict the dependency of mitotic kinase in the absence of HASPIN activity, revealed the synthetic lethal effect of cotreatment of the chemical inhibitor of BUB1, PLK1 or AURKA with LJ4827. These data suggest that combined inhibition of HASPIN with the novel inhibitor and key mitotic kinases for centromere/kinetochore regulation would be effectivity therapeutic approach for cancer therapy. Citation Format: Eun-Ji Kwon, Karishma Mashelkar, Hyuk Woo Lee, Yoon-Ze Shin, Lak Shin Jeong, Hyuk-Jin Cha. Identification of a novel HASPIN inhibitor and the synthetic lethal partner by computational analysis [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 3949.

A transcriptomic analysis of retroperitoneal well differentiated liposarcoma (WDLPS) and well differentiated (WD) and dedifferentiated (DD) components of dedifferentiated liposarcoma (DDLPS).

e23520 Background: Retroperitoneal DDLPS is an aggressive tumor characterized by a WD component, morphologically similar to a WDLPS, and a DD component that drives patient prognosis. We aimed at unraveling whether WDLPS and DDLPS are distinct entities or entail a progressive evolution from WDLPS to DDLPS through a transcriptomic analysis. Methods: A transcriptomic analysis was performed in a retrospective series of 107 patients with primary retroperitoneal WDLPS (N = 68, 63.5%) or DDLPS (N = 39, 36.5%) who underwent surgery (2011-15). Paired DD, WD, and normal fat (NF) components were sampled in DDLPS, while paired WD and NF components were sampled in WDLPS. RNA-Seq data were normalized according to the trimmed mean of M-value (TMM) algorithm and differential expression was evaluated with the voom method implemented into the edgeR package. Enrichment in hallmark gene sets from Molecular Signatures Database (MSigDB) was evaluated with gene sets enrichment analysis (GSEA), by using t-statistic as measure of ranking. A false discovery rate (FDR) adjusted p-value &lt; 0.05 was considered for statistical significance. Results: Differential expression analysis revealed marked transcriptional changes within paired components of DDLPS (DD, WD, NF) and WDLPS (WD, NF). Changes of WD and NF components between WDLPS and DDLPS were not statistically significant. Gene sets were analyzed to evaluate the ensemble and overcome the FDR correction applied to single genes. Hallmarks deregulated in WD component of DDLPS compared to WDLPS were detected also in their paired DD component. Among them, G2M checkpoint and mitotic spindle were up-regulated, while adipogenesis, fatty acid metabolism, cholesterol homeostasis, oxidative phosphorylation, and peroxisome were down-regulated. These differences persisted also when NF components were compared with their paired WD component of DDLPS and WDLPS. G2M checkpoint and mitotic spindle gene sets did not differ between NF of DDLPS and NF of WDLPS, suggesting these two hallmarks as tumor-specific. Conversely, expression of adipogenesis, fatty acid metabolism, and oxidative phosphorylation was up-regulated together with other gene sets related to metabolism in NF of DDLPS. Tumor inflammation and interferon response were up-regulated in WD components compared to their paired NF components. Also, interferon response was down-regulated in WD component of DDLPS compared to WDLPS, and inflammation was down-regulated in DD component compared to both WD components. Conclusions: Transcriptomic changes that distinguished WDLPS and WD component of DDLPS increased progressively also in the paired DD component of DDLPS, supporting the hypothesis of a progression from WDLPS to DDLPS in some liposarcomas. Changes observed in NF may suggest a paracrine effect sustaining tumor dedifferentiation.

Structure and function of MuvB complexes.

Proper progression through the cell-division cycle is critical to normal development and homeostasis and is necessarily misregulated in cancer. The key to cell-cycle regulation is the control of two waves of transcription that occur at the onset of DNA replication (S phase) and mitosis (M phase). MuvB complexes play a central role in the regulation of these genes. When cells are not actively dividing, the MuvB complex DREAM represses G1/S and G2/M genes. Remarkably, MuvB also forms activator complexes together with the oncogenic transcription factors B-MYB and FOXM1 that are required for the expression of the mitotic genes in G2/M. Despite this essential role in the control of cell division and the relationship to cancer, it has been unclear how MuvB complexes inhibit and stimulate gene expression. Here we review recent discoveries of MuvB structure and molecular interactions, including with nucleosomes and other chromatin-binding proteins, which have led to the first mechanistic models for the biochemical function of MuvB complexes.

Screening of Amorpha fruticosa and Ailanthus altissima extracts for genotoxicity/antigenotoxicity, mutagenicity/antimutagenicity and carcinogenicity/anticarcinogenicity

The aim of the present study was to evaluate the potential genotoxic/antigenotoxic, mutagenic/antimutagenic, and carcinogenic/anticarcinogenic effect of Amorpha fruticosa (AF) fruit, Ailanthus altissima bark hexane (AAEH) and methanol (AAEM) extracts on a model system Saccharomyces cerevisiae. Plants were identified and extracted by Ekaterina Kozuharova. Three concentrations of each extract were tested – 10, 100 and 1000 µg/ml. In vitro pro-oxidant/antioxidant activities were evaluated by DPPH and DNA topology assay. The potential genotoxic/antigenotoxic, mutagenic/antimutagenic and carcinogenic/anticarcinogenic effects were revealed in vivo by: Zimmermman’s test on Saccharomyces cerevisiae diploid strain D7ts1, and Ty1 retrotransposition test on S. cerevisiae haploid strain 551. Zeocin was used as a positive control. Based on the in vitro antioxidant activity the extracts could be arranged as follows: AF&amp;gt;AAEM&amp;gt;AAEH. AAEH possessed moderate oxidative potential. No genotoxic and mutagenic capacity was obtained in vivo for extracts tested. The levels of total aberrants, convertants and revertants were comparable with the control ones. No Ty1 retrotransposition was induced by extracts treatment. Further, the extracts possessed well-expressed antigenotoxic, antimutagenic and anticarcinogenic activity. Significant reduction of the total aberrants, reverse point mutations and Ty1 retrotransposition was obtained. Only the AF extract was found to reduce the levels of zeocin-induced mitotic gene conversion. The three extracts did not possess any genotoxic, mutagenic and carcinogenic effect on Saccharomyces cerevisiae. Based on their protective activity, they can be arranged as follows: AF&amp;gt;AAEM&amp;gt;AAEH which corresponds well with their phytochemical composition. Further experiments could provide more detailed information concerning the mode of action of extracts, as well as their main constituents.

Austropuccinia psidii uses tetrapolar mating and produces meiotic spores in older infections on Eucalyptus grandis

Austropuccinia psidii is the causal agent of myrtle rust, a fungal disease that infects over 480 species in the Myrtaceae. A. psidii is a biotrophic pathogen that reproduces sexually and asexually. Sexual reproduction has been previously shown on Syzygium jambos and little is known about its reproductive biology on other hosts or whether populations that were formerly structured by host range can outcross on universally susceptible hosts. We investigated if mating genes in three genomes of A. psidii were under selection as a proxy for whether different strains can reproduce sexually on a shared host. We examined three homologs of the STE3.2 gene, sequences of which were near-identical in the three genomes, and the homeodomain locus, which contained two alleles of two homeodomain genes in each genome. A. psidii likely uses tetrapolar mating. Pheromone/receptor loci were distal to homeodomain loci, and based on haplotypes of a phased assembly, mate compatibility is regulated by multiallelic HD genes and biallelic STE3.2 genes; the third homolog of STE3.2 (STE3.2-1) was present in both haplotypes, and our study supports hypotheses this gene does not regulate mate recognition. Populations of A. psidii formerly structured by host range could potentially outcross on universal hosts based on their related mating genes, however this hypothesis should remain theoretical given the implications for biosecurity. Additionally, we searched for core meiotic genes in genomes of A. psidii, four species of Puccinia, and Sphaerophragmium acaciae through comparative genomics based on 136 meiosis-related orthologous genes modeled from Mycosarcoma maydis. Meiotic genes are conserved in rust fungi at family rank. We analyzed the expression of two meiotic and four mitotic genes of A. psidii on E. grandis over a 28-day time course to validate that identified meiotic genes were upregulated in teliospores. Three mitotic genes were significantly downregulated in samples collected 28 days after inoculation (DAI) compared to 14 DAI. Expression of meiotic genes was significantly up-regulated in samples collected 28 DAI compared to 14 DAI, indicating a temporal switch from production of uredinia (mitotic stage) to telia in the life cycle, which we hypothesize may be in response to leaf ageing.

Inhibition of DYRK1a Enhances Cardiomyocyte Cycling After Myocardial Infarction.

DYRK1a (dual-specificity tyrosine phosphorylation-regulated kinase 1a) contributes to the control of cycling cells, including cardiomyocytes. However, the effects of inhibition of DYRK1a on cardiac function and cycling cardiomyocytes after myocardial infarction (MI) remain unknown. We investigated the impacts of pharmacological inhibition and conditional genetic ablation of DYRK1a on endogenous cardiomyocyte cycling and left ventricular systolic function in ischemia-reperfusion (I/R) MI using αMHC-MerDreMer-Ki67p-RoxedCre::Rox-Lox-tdTomato-eGFP (RLTG) (denoted αDKRC::RLTG) and αMHC-Cre::Fucci2aR::DYRK1aflox/flox mice. We observed that harmine, an inhibitor of DYRK1a, improved left ventricular ejection fraction (39.5±1.6% and 29.1±1.6%, harmine versus placebo, respectively), 2 weeks after I/R MI. Harmine also increased cardiomyocyte cycling after I/R MI in αDKRC::RLTG mice, 10.8±1.5 versus 24.3±2.6 enhanced Green Fluorescent Protein (eGFP)+ cardiomyocytes, placebo versus harmine, respectively, P=1.0×10-3. The effects of harmine on left ventricular ejection fraction were attenuated in αDKRC::DTA mice that expressed an inducible diphtheria toxin in adult cycling cardiomyocytes. The conditional cardiomyocyte-specific genetic ablation of DYRK1a in αMHC-Cre::Fucci2aR::DYRK1aflox/flox (denoted DYRK1a k/o) mice caused cardiomyocyte hyperplasia at baseline (210±28 versus 126±5 cardiomyocytes per 40× field, DYRK1a k/o versus controls, respectively, P=1.7×10-2) without changes in cardiac function compared with controls, or compensatory changes in the expression of other DYRK isoforms. After I/R MI, DYRK1a k/o mice had improved left ventricular function (left ventricular ejection fraction 41.8±2.2% and 26.4±0.8%, DYRK1a k/o versus control, respectively, P=3.7×10-2). RNAseq of cardiomyocytes isolated from αMHC-Cre::Fucci2aR::DYRK1aflox/flox and αMHC-Cre::Fucci2aR mice after I/R MI or Sham surgeries identified enrichment in mitotic cell cycle genes in αMHC-Cre::Fucci2aR::DYRK1aflox/flox compared with αMHC-Cre::Fucci2aR. The pharmacological inhibition or cardiomyocyte-specific ablation of DYRK1a caused baseline hyperplasia and improved cardiac function after I/R MI, with an increase in cell cycle gene expression, suggesting the inhibition of DYRK1a may serve as a therapeutic target to treat MI.

Functional dissection of human mitotic genes using CRISPR-Cas9 tiling screens.

The identity of human protein-coding genes is well known, yet our in-depth knowledge of their molecular functions and domain architecture remains limited by shortcomings in homology-based predictions and experimental approaches focused on whole-gene depletion. To bridge this knowledge gap, we developed a method that leverages CRISPR-Cas9-induced mutations across protein-coding genes for the a priori identification of functional regions at the sequence level. As a test case, we applied this method to 48 human mitotic genes, revealing hundreds of regions required for cell proliferation, including domains that were experimentally characterized, ones that were predicted based on homology, and novel ones. We validated screen outcomes for 15 regions, including amino acids 387-402 of Mad1, which were previously uncharacterized but contribute to Mad1 kinetochore localization and chromosome segregation fidelity. Altogether, we demonstrate that CRISPR-Cas9-based tiling mutagenesis identifies key functional domains in protein-coding genes de novo, which elucidates separation of function mutants and allows functional annotation across the human proteome.

Mitotic spindle formation in the absence of Polo kinase

SignificanceMitosis is an essential process in all eukaryotes, but paradoxically, genes required for mitosis vary among species. The essentiality of many mitotic genes was bypassed by activating alternative mechanisms during evolution. However, bypass events have rarely been recapitulated experimentally. Here, using the fission yeast Schizosaccharomyces pombe, the essentiality of a kinase (Plo1) required for bipolar spindle formation was bypassed by other mutations, many of which are associated with glucose metabolism. The Plo1 bypass by the reduction in glucose uptake was dependent on another kinase (casein kinase I), which potentiated spindle microtubule formation. This study illustrates a rare experimental bypass of essentiality for mitotic genes and provides insights into the molecular diversity of mitosis.

Atorvastatin Inhibits Viability and Migration of MCF7 Breast Cancer Cells.

Objective:Atorvastatin is commonly used as a lipid lowering drug. The emerging interest in statins as anticancer agents is based on their pleiotropic effects on cancer cells. Among the statins, atorvastatin, and in cancers, breast malignancies have received less attention in preclinical investigations. In order to enhance the efficacy of cancer treatment, adjuvant, less expensive therapeutic strategies have been recently noticed. In this case, we investigated the in-vitro effect of atorvastatin on viability and migration of MCF7 breast cancer cell line. Methods:We tested the cytotoxicity of atorvastatin on breast cancer cells survival by MTT assay. Annexin-V / PI staining and then flow cytometry of cancer cells in addition to quantitative real-time PCR tests quantified the apoptosis and necrosis of cancer cells. We figured out the impact of atorvastatin on cancer cell migration capability through scratch-wound healing assay and transwell migration examination. Inverted light microscope and fluorescent imaging displayed the morphological changes following treatment of MCF7 cells with atorvastatin.Result:We resulted that atorvastatin can trigger MCF7 cancer cells to undergo necrosis and caspase-dependent apoptosis based on the viable/dead cell number, mitotic cell cycle, gene expression, and morphological assays. The results were dose- and time-dependent and the half- maximal inhibitory concentration of atorvastatin for cancer cells’ viability inhibition was 9.1 μM/L(nM/mL). Moreover, the migration of MCF7 cells were inhibited in the treated group as we figured out in two- and three-dimensional migration methods. Conclusion:In-vitro inspection of drug-cancer cell interactions paves the way for future in-vivo research studies. These in-vitro results revealed that atorvastatin has anti-viability and anti-migration effects on breast cancer cells.

Chlamydomonas CHT7 is involved in repressing DNA replication and mitotic genes during synchronous growth.

In the green alga Chlamydomonas reinhardtii, regulation of the cell cycle in response to external cues is critical for survival in a changing environment. The loss of the nuclear COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS 7 (CHT7) protein affects the expression of many genes especially in response to nitrogen availability. Cells lacking CHT7 exhibit abnormal cell morphology following nitrogen deprivation and fail to resume normal cell division after N resupply. To investigate the function of CHT7 in the regulation of cell cycle-related pathways, cells were synchronized, and RNA-seq analysis was performed during various stages of the cell cycle. In the cht7 mutant following nitrogen deprivation, the cells were not dividing, but a subset of cell cycle genes involved in DNA replication and mitosis were found to be derepressed, suggesting that the CHT7 protein plays a role in cell cycle regulation that is opposite to that of the mitotic cyclin-dependent kinases. Furthermore, genes for cell wall synthesis and remodeling were found to be abnormally induced in nondividing cht7 cells; this misregulation may deplete cellular resources and thus contribute to cell death following nitrogen deprivation. Lastly, 43 minimally characterized kinases were found to be highly misregulated in cht7. Further analysis suggested that some of these CHT7-regulated kinases may be related to the MAP3K and Aurora-like kinases, while others are unique. Together, these results suggest a role of CHT7 in transcriptional regulation of the cell cycle and reveal several pathways and genes whose expression appears to be subject to a CHT7-mediated regulatory network.

Replication-related genes are upregulated in XP-A cells after UV-C irradiation

BackgroundXeroderma pigmentosum (XP) is hereditary disorder characterized by photosensitivity, predisposition to skin cancers of sun-exposed body sites and progressive neurologic symptoms in some cases. Cells from XP patients show higher sensitivity to ultraviolet radiation (UV) than normal cells. ObjectiveWe aimed to ascertain the genes differentially regulated in XP complementation group A (XP-A) cells after UV irradiation. MethodsXP-A cells were harvested at 4 or 12 h after a single exposure to low-dose UV-C radiation and subjected to transcriptome analysis by microarray. ResultsThe number of genes with significantly altered expression (≥2-fold difference) at 12 h was markedly higher in XP-A cells than that in normal cells, suggesting that the number of altered genes could be correlated to the amount of DNA damage. ConclusionWe recently reported that mitotic genes are induced in normal human fibroblasts after UV-C exposure, and similar results were observed in XP-A cells as normal cells. In addition, a majority of replication-related genes were significantly upregulated in XP-A cells, whereas no such expression pattern was observed in the normal control cells. Collectively, these results indicate that the XPA protein can transcriptionally inhibit the series of replication-related genes, and could possibly regulate replication and/or re-replication after UV irradiation.

Revertant Mosaicism in Epidermolysis Bullosa.

Epidermolysis bullosa (EB) is a group of genetic blistering diseases characterized by mechanically fragile skin and mucocutaneous involvement. Historically, disease management has focused on supportive care. The development of new genetic, cellular, and recombinant protein therapies has shown promise, and this review summarizes a unique gene and cell therapy phenomenon termed revertant mosaicism (RM). RM is the spontaneous correction of a disease-causing mutation. It has been reported in most EB subtypes, some with relatively high frequency, and has been observed in both keratinocytes and fibroblasts. RM manifests as identifiable patches of unaffected, blister-resistant skin and can occur through a variety of molecular mechanisms, including true back mutation, intragenic crossover, mitotic gene conversion, and second-site mutation. RM cells represent a powerful autologous platform for therapy, and leveraging RM cells as a therapeutic substrate may avoid the inherent mutational risks of gene therapy/editing. However, further examination of the genomic integrity and long-term functionality of RM-derived cells, as well in vivo testing of systemic therapies with RM cells, is required to realize the full therapeutic promise of naturally occurring RM in EB.

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates.1-3 The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle,4-7 together with variations in substrate docking to sites on associated cyclin and Cks subunits.3,6,8-10 Many substrates are modified at multiple sites to provide more complex regulation.10-14 Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of budding yeast genes required for mitotic progression.11,12 As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback.13-16 Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that when high Cdk1 activity accumulates in a mitotic arrest, CLB2 expression is inhibited. Inhibition is accompanied by Ndd1 degradation, and we present evidence that degradation is triggered by multisite Ndd1 phosphorylation by high mitotic Cdk1-Clb2 activity. Complete Ndd1 phosphorylation by Clb2-Cdk1-Cks1 requires the phosphothreonine-binding site of Cks1, as well as a recently identified phosphate-binding pocket on the cyclin Clb2.17 We therefore propose that initial phosphorylation by Cdk1 primes Ndd1 for delayed secondary phosphorylation at suboptimal sites that promote degradation. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.

HaplotypeTools: a toolkit for accurately identifying recombination and recombinant genotypes

BackgroundIdentifying haplotypes is central to sequence analysis in diploid or polyploid genomes. Despite this, there remains a lack of research and tools designed for physical phasing and its downstream analysis.ResultsHaplotypeTools is a new toolset to phase variant sites using VCF and BAM files and to analyse phased VCFs. Phasing is achieved via the identification of reads overlapping ≥ 2 heterozygous positions and then extended by additional reads, a process that can be parallelized across a computer cluster. HaplotypeTools includes various utility scripts for downstream analysis including crossover detection and phylogenetic placement of haplotypes to other lineages or species. HaplotypeTools was assessed for accuracy against WhatsHap using simulated short and long reads, demonstrating higher accuracy, albeit with reduced haplotype length. HaplotypeTools was also tested on real Illumina data to determine the ancestry of hybrid fungal isolate Batrachochytrium dendrobatidis (Bd) SA-EC3, finding 80% of haplotypes across the genome phylogenetically cluster with parental lineages BdGPL (39%) and BdCAPE (41%), indicating those are the parental lineages. Finally, ~ 99% of phasing was conserved between overlapping phase groups between SA-EC3 and either parental lineage, indicating mitotic gene conversion/parasexuality as the mechanism of recombination for this hybrid isolate. HaplotypeTools is open source and freely available from https://github.com/rhysf/HaplotypeTools under the MIT License.ConclusionsHaplotypeTools is a powerful resource for analyzing hybrid or recombinant diploid or polyploid genomes and identifying parental ancestry for sub-genomic regions.

On the Origin and Evolution of Drosophila New Genes during Spermatogenesis.

The origin of functional new genes is a basic biological process that has significant contribution to organismal diversity. Previous studies in both Drosophila and mammals showed that new genes tend to be expressed in testes and avoid the X chromosome, presumably because of meiotic sex chromosome inactivation (MSCI). Here, we analyze the published single-cell transcriptome data of Drosophila adult testis and find an enrichment of male germline mitotic genes, but an underrepresentation of meiotic genes on the X chromosome. This can be attributed to an excess of autosomal meiotic genes that were derived from their X-linked mitotic progenitors, which provides direct cell-level evidence for MSCI in Drosophila. We reveal that new genes, particularly those produced by retrotransposition, tend to exhibit an expression shift toward late spermatogenesis compared with their parental copies, probably due to the more intensive sperm competition or sexual conflict. Our results dissect the complex factors including age, the origination mechanisms and the chromosomal locations that influence the new gene origination and evolution in testes, and identify new gene cases that show divergent cell-level expression patterns from their progenitors for future functional studies.

Bioinformatics Analysis Identifies Precision Treatment with Paclitaxel for Hepatocellular Carcinoma Patients Harboring Mutant TP53 or Wild-Type CTNNB1 Gene.

Hepatocellular carcinoma (HCC) is an aggressive and chemoresistant cancer type. The development of novel therapeutic strategies is still urgently needed. Personalized or precision medicine is a new trend in cancer therapy, which treats cancer patients with specific genetic alterations. In this study, a gene signature was identified from the transcriptome of HCC patients, which was correlated with the patients’ poorer prognoses. This gene signature is functionally related to mitotic cell cycle regulation, and its higher or lower expression is linked to the mutation in tumor protein p53 (TP53) or catenin beta 1 (CTNNB1), respectively. Gene–drug association analysis indicated that the taxanes, such as the clinically approved anticancer drug paclitaxel, are potential drugs targeting this mitotic gene signature. Accordingly, HCC cell lines harboring mutant TP53 or wild-type CTNNB1 genes are more sensitive to paclitaxel treatment. Therefore, our results imply that HCC patients with mutant TP53 or wild-type CTNNB1 genes may benefit from the paclitaxel therapy.