Transcriptional plasticity buffers genetic variation in zinc homeostasis

AbstractIn roots of Arabidopsis thaliana, Zn can be either loaded into the xylem for translocation to the shoot or stored in vacuoles. Vacuolar storage is achieved through the action of the Zn/Cd transporter HMA3 (Heavy Metal Atpase 3). The Col-0 accession has an HMA3 loss-of-function allele resulting in high shoot Cd, when compared to accession CSHL-5 which has a functional allele and low shoot Cd. Interestingly, both Col-0 and CSHL-5 have similar shoot Zn concentrations. We hypothesize that plants sense changes in cytosolic Zn that are due to variation in HMA3 function, and respond by altering expression of genes related to Zn uptake, transport and compartmentalisation, in order to maintain Zn homeostasis. The expression level of genes known to be involved in Zn homeostasis were quantified in both wild-type Col-0 and Col-0::HMA3CSHL-5 plants transformed with the functional CSHL-5 allele of HMA3. We observed significant positive correlations between expression of HMA3 and of genes known to be involved in Zn homeostasis, including ZIP3, ZIP4, MTP1, and bZIP19. The results support our hypothesis that alteration in the level of function of HMA3 is counterbalanced by the fine regulation of the Zn homeostasis gene network in roots of A. thaliana.

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Brain transcriptome analysis reveals subtle effects on mitochondrial function and iron homeostasis of mutations in the SORL1 gene implicated in early onset familial Alzheimer’s disease

Molecular Brain ◽

10.1186/s13041-020-00681-7 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Karissa Barthelson ◽

Stephen Martin Pederson ◽

Morgan Newman ◽

Michael Lardelli

Keyword(s):

Gene Expression ◽

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Early Onset ◽

Iron Homeostasis ◽

Enrichment Analysis ◽

Loss Of Function ◽

Wild Type ◽

Expression Of Genes ◽

Insight Into

Abstract To prevent or delay the onset of Alzheimer’s disease (AD), we must understand its molecular basis. The great majority of AD cases arise sporadically with a late onset after 65 years of age (LOAD). However, rare familial cases of AD can occur due to dominant mutations in a small number of genes that cause an early onset prior to 65 years of age (EOfAD). As EOfAD and LOAD share similar pathologies and disease progression, analysis of EOfAD genetic models may give insight into both subtypes of AD. Sortilin-related receptor 1 (SORL1) is genetically associated with both EOfAD and LOAD and provides a unique opportunity to investigate the relationships between both forms of AD. Currently, the role of SORL1 mutations in AD pathogenesis is unclear. To understand the molecular consequences of SORL1 mutation, we performed targeted mutagenesis of the orthologous gene in zebrafish. We generated an EOfAD-like mutation, V1482Afs, and a putatively null mutation, to investigate whether EOfAD-like mutations in sorl1 display haploinsufficiency by acting through loss-of-function mechanisms. We performed mRNA-sequencing on whole brains, comparing wild type fish with their siblings heterozygous for EOfAD-like or putatively loss-of-function mutations in sorl1, or transheterozygous for these mutations. Differential gene expression analysis identified a small number of differentially expressed genes due to the sorl1 genotypes. We also performed enrichment analysis on all detectable genes to obtain a more complete view on changes to gene expression by performing three methods of gene set enrichment analysis, then calculated an overall significance value using the harmonic mean p-value. This identified subtle effects on expression of genes involved in energy production, mRNA translation and mTORC1 signalling in both the EOfAD-like and null mutant brains, implying that these effects are due to sorl1 haploinsufficiency. Surprisingly, we also observed changes to expression of genes occurring only in the EOfAD-mutation carrier brains, suggesting gain-of-function effects. Transheterozygosity for the EOfAD-like and null mutations (i.e. lacking wild type sorl1), caused apparent effects on iron homeostasis and other transcriptome changes distinct from the single-mutation heterozygous fish. Our results provide insight into the possible early brain molecular effects of an EOfAD mutation in human SORL1. Differential effects of heterozygosity and complete loss of normal SORL1 expression are revealed.

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Brain transcriptome analysis reveals subtle effects on mitochondrial function and iron homeostasis of mutations in the SORL1 gene implicated in early onset familial Alzheimer’s disease

10.1101/2020.07.17.207787 ◽

2020 ◽

Cited By ~ 1

Author(s):

Karissa Barthelson ◽

Stephen Martin Pederson ◽

Morgan Newman ◽

Michael Lardelli

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Early Onset ◽

Iron Homeostasis ◽

Mrna Translation ◽

Complete Loss ◽

Loss Of Function ◽

Wild Type ◽

Expression Of Genes ◽

Insight Into

AbstractBackgroundTo prevent or delay the onset of Alzheimer’s disease (AD), we must understand its molecular basis. The great majority of AD cases arise sporadically with a late onset after 65 years of age (LOAD). However, rare familial cases of AD can occur due to dominant mutations in a small number of genes that cause an early onset prior to 65 years of age (EOfAD). As EOfAD and LOAD share similar pathologies and disease progression, analysis of EOfAD genetic models may give insight into both subtypes of AD. Sortilin-related receptor 1 (SORL1) is genetically associated with both EOfAD and LOAD and provides a unique opportunity to investigate the relationships between both forms of AD. Currently, the role of SORL1 mutations in AD pathogenesis is unclear.MethodsTo understand the molecular consequences of SORL1 mutation, we performed targeted mutagenesis of the orthologous gene in zebrafish. We generated an EOfAD-like mutation, V1482Afs, and a putatively null mutation, to investigate whether EOfAD-like mutations in sorl1 display haploinsufficiency by acting through loss-of-function mechanisms. We performed mRNA-sequencing on whole brains comparing normal (wild type) fish with their siblings heterozygous for EOfAD-like or complete loss-of-function mutations in sorl1 or transheterozygous for these mutations. Differential gene expression and gene set enrichment analyses identified, respectively, changes in young adult zebrafish brain transcriptomes, and putative effects on neural subcellular functions.ResultsWe identified subtle effects on expression of genes involved in energy production, mRNA translation and mTORC1 signalling in both the EOfAD-like and null mutant brains, implying that these effects are due to sorl1 haploinsufficiency. Surprisingly, we also observed changes to expression of genes occurring only in the EOfAD-mutation carrier brains, suggesting gain-of-function effects. Transheterozygosity for the EOfAD-like and null mutations (i.e. lacking wild type sorl1), caused apparent effects on iron homeostasis and other transcriptome changes distinct from the single-mutation heterozygous fish.ConclusionsOur results provide insight into the possible early brain molecular effects of an EOfAD mutation in human SORL1. Differential effects of heterozygosity and complete loss of normal SORL1 expression are revealed.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Alterations in ZnT1 expression and function lead to impaired intracellular zinc homeostasis in cancer

Cell Death Discovery ◽

10.1038/s41420-019-0224-0 ◽

2019 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Adrian Israel Lehvy ◽

Guy Horev ◽

Yarden Golan ◽

Fabian Glaser ◽

Yael Shammai ◽

...

Keyword(s):

Malignant Tumors ◽

Zinc Homeostasis ◽

Healthy Population ◽

Missense Mutations ◽

Protein Alignment ◽

Loss Of Function ◽

Intracellular Zinc ◽

Dismal Prognosis ◽

Zinc Levels ◽

And Function

Abstract Zinc is vital for the structure and function of ~3000 human proteins and hence plays key physiological roles. Consequently, impaired zinc homeostasis is associated with various human diseases including cancer. Intracellular zinc levels are tightly regulated by two families of zinc transporters: ZIPs and ZnTs; ZIPs import zinc into the cytosol from the extracellular milieu, or from the lumen of organelles into the cytoplasm. In contrast, the vast majority of ZnTs compartmentalize zinc within organelles, whereas the ubiquitously expressed ZnT1 is the sole zinc exporter. Herein, we explored the hypothesis that qualitative and quantitative alterations in ZnT1 activity impair cellular zinc homeostasis in cancer. Towards this end, we first used bioinformatics to analyze inactivating mutations in ZIPs and ZNTs, catalogued in the COSMIC and gnomAD databases, representing tumor specimens and healthy population controls, respectively. ZnT1, ZnT10, ZIP8, and ZIP10 showed extremely high rates of loss of function mutations in cancer as compared to healthy controls. Analysis of the putative functional impact of missense mutations in ZnT1-ZnT10 and ZIP1-ZIP14, using homologous protein alignment and structural predictions, revealed that ZnT1 displays a markedly increased frequency of predicted functionally deleterious mutations in malignant tumors, as compared to a healthy population. Furthermore, examination of ZnT1 expression in 30 cancer types in the TCGA database revealed five tumor types with significant ZnT1 overexpression, which predicted dismal prognosis for cancer patient survival. Novel functional zinc transport assays, which allowed for the indirect measurement of cytosolic zinc levels, established that wild type ZnT1 overexpression results in low intracellular zinc levels. In contrast, overexpression of predicted deleterious ZnT1 missense mutations did not reduce intracellular zinc levels, validating eight missense mutations as loss of function (LoF) mutations. Thus, alterations in ZnT1 expression and LoF mutations in ZnT1 provide a molecular mechanism for impaired zinc homeostasis in cancer formation and/or progression.

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The NALCN Channel Regulator UNC-80 Functions in a Subset of Interneurons To Regulate Caenorhabditis elegans Reversal Behavior

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400692 ◽

2019 ◽

Vol 10 (1) ◽

pp. 199-210 ◽

Cited By ~ 1

Author(s):

Chuanman Zhou ◽

Jintao Luo ◽

Xiaohui He ◽

Qian Zhou ◽

Yunxia He ◽

...

Keyword(s):

Plant Hormone ◽

Neuronal Excitability ◽

Resting Membrane Potential ◽

Loss Of Function ◽

Wild Type ◽

C Elegans ◽

Link Type ◽

Complex Functions ◽

And Function ◽

Multiple Loss

NALCN (Na+leak channel, non-selective) is a conserved, voltage-insensitive cation channel that regulates resting membrane potential and neuronal excitability. UNC79 and UNC80 are key regulators of the channel function. However, the behavioral effects of the channel complex are not entirely clear and the neurons in which the channel functions remain to be identified. In a forward genetic screen for C. elegans mutants with defective avoidance response to the plant hormone methyl salicylate (MeSa), we isolated multiple loss-of-function mutations in unc-80 and unc-79. C. elegans NALCN mutants exhibited similarly defective MeSa avoidance. Interestingly, NALCN, unc-80 and unc-79 mutants all showed wild type-like responses to other attractive or repelling odorants, suggesting that NALCN does not broadly affect odor detection or related forward and reversal behaviors. To understand in which neurons the channel functions, we determined the identities of a subset of unc-80-expressing neurons. We found that unc-79 and unc-80 are expressed and function in overlapping neurons, which verified previous assumptions. Neuron-specific transgene rescue and knockdown experiments suggest that the command interneurons AVA and AVE and the anterior guidepost neuron AVG can play a sufficient role in mediating unc-80 regulation of the MeSa avoidance. Though primarily based on genetic analyses, our results further imply that MeSa might activate NALCN by direct or indirect actions. Altogether, we provide an initial look into the key neurons in which the NALCN channel complex functions and identify a novel function of the channel in regulating C. elegans reversal behavior through command interneurons.

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Identification of a novel homozygous loss-of-function mutation in FUCA1 gene causing severe fucosidosis: A case report

Journal of International Medical Research ◽

10.1177/03000605211005975 ◽

2021 ◽

Vol 49 (4) ◽

pp. 030006052110059

Author(s):

Xinwen Zhang ◽

Shaozhi Zhao ◽

Hongwei Liu ◽

Xiaoyan Wang ◽

Xiaolei Wang ◽

...

Keyword(s):

Amino Acids ◽

Lysosomal Storage Disorder ◽

Autosomal Recessive ◽

Signs And Symptoms ◽

Lysosomal Storage ◽

Loss Of Function ◽

Wild Type ◽

Single Nucleotide ◽

Whole Exome ◽

Exon 1

Fucosidosis is a rare lysosomal storage disorder characterized by deficiency of α-L-fucosidase with an autosomal recessive mode of inheritance. Here, we describe a 4-year-old Chinese boy with signs and symptoms of fucosidosis but his parents were phenotypically normal. Whole exome sequencing (WES) identified a novel homozygous single nucleotide deletion (c.82delG) in the exon 1 of the FUCA1 gene. This mutation will lead to a frameshift which will result in the formation of a truncated FUCA1 protein (p.Val28Cysfs*105) of 132 amino acids approximately one-third the size of the wild type FUCA1 protein (466 amino acids). Both parents were carrying the mutation in a heterozygous state. This study expands the mutational spectrum of the FUCA1 gene associated with fucosidosis and emphasises the benefits of WES for accurate and timely clinical diagnosis of this rare disease.

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Frequent gain- and loss-of-function mutations of the BjMYB113 gene accounted for leaf color variation in Brassica juncea

BMC Plant Biology ◽

10.1186/s12870-021-03084-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Guanghui An ◽

Jiongjiong Chen

Keyword(s):

Brassica Juncea ◽

Anthocyanin Biosynthesis ◽

Color Variation ◽

Nucleotide Polymorphisms ◽

Loss Of Function ◽

High Accumulation ◽

Single Nucleotide ◽

Breeding Programs ◽

Promoter Sequences ◽

Expression Of Genes

Abstract Background Mustard (Brassica juncea) is an important economic vegetable, and some cultivars have purple leaves and accumulate more anthocyanins than the green. The genetic and evolution of purple trait in mustard has not been well studied. Result In this study, free-hand sections and metabolomics showed that the purple leaves of mustard accumulated more anthocyanins than green ones. The gene controlling purple leaves in mustard, Mustard Purple Leaves (MPL), was genetically mapped and a MYB113-like homolog was identified as the candidate gene. We identified three alleles of the MYB113-like gene, BjMYB113a from a purple cultivar, BjMYB113b and BjMYB113c from green cultivars. A total of 45 single nucleotide polymorphisms (SNPs) and 8 InDels were found between the promoter sequences of the purple allele BjMYB113a and the green allele BjMYB113b. On the other hand, the only sequence variation between the purple allele BjMYB113a and the green allele BjMYB113c is an insertion of 1,033-bp fragment in the 3’region of BjMYB113c. Transgenic assay and promoter activity studies showed that the polymorphism in the promoter region was responsible for the up-regulation of the purple allele BjMYB113a and high accumulation of anthocyanin in the purple cultivar. The up-regulation of BjMYB113a increased the expression of genes in the anthocyanin biosynthesis pathway including BjCHS, BjF3H, BjF3’H, BjDFR, BjANS and BjUGFT, and consequently led to high accumulation of anthocyanin. However, the up-regulation of BjMYB113 was compromised by the insertion of 1,033-bp in 3’region of the allele BjMYB113c. Conclusions Our results contribute to a better understanding of the genetics and evolution of the BjMYB113 gene controlling purple leaves and provide useful information for further breeding programs of mustard.

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Somatic production of reactive oxygen species does not predict its production in sperm cells across Drosophila melanogaster lines

BMC Research Notes ◽

10.1186/s13104-021-05550-7 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Biz R. Turnell ◽

Luisa Kumpitsch ◽

Anne-Cécile Ribou ◽

Klaus Reinhardt

Keyword(s):

Reactive Oxygen Species ◽

Drosophila Melanogaster ◽

Reactive Oxygen ◽

Future Research ◽

Ros Production ◽

Oxygen Species ◽

Loss Of Function ◽

Wild Type ◽

Ros Scavenging ◽

Transport Chain

Abstract Objective Sperm ageing has major evolutionary implications but has received comparatively little attention. Ageing in sperm and other cells is driven largely by oxidative damage from reactive oxygen species (ROS) generated by the mitochondria. Rates of organismal ageing differ across species and are theorized to be linked to somatic ROS levels. However, it is unknown whether sperm ageing rates are correlated with organismal ageing rates. Here, we investigate this question by comparing sperm ROS production in four lines of Drosophila melanogaster that have previously been shown to differ in somatic mitochondrial ROS production, including two commonly used wild-type lines and two lines with genetic modifications standardly used in ageing research. Results Somatic ROS production was previously shown to be lower in wild-type Oregon-R than in wild-type Dahomey flies; decreased by the expression of alternative oxidase (AOX), a protein that shortens the electron transport chain; and increased by a loss-of-function mutation in dj-1β, a gene involved in ROS scavenging. Contrary to predictions, we found no differences among these four lines in the rate of sperm ROS production. We discuss the implications of our results, the limitations of our study, and possible directions for future research.

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Alopecia in Harlequin mutant mice is associated with reduced AIF protein levels and expression of retroviral elements

Mammalian Genome ◽

10.1007/s00335-020-09854-0 ◽

2020 ◽

Author(s):

Maik Hintze ◽

Sebastian Griesing ◽

Marion Michels ◽

Birgit Blanck ◽

Lena Wischhof ◽

...

Keyword(s):

Hair Growth ◽

Transcriptional Regulators ◽

Mutant Mice ◽

Wild Type ◽

Protein Levels ◽

Expression Of Genes ◽

Genes Encoding ◽

Keratinocyte Cell ◽

Apoptosis Inducing Factor ◽

Hair Cortex

AbstractWe investigated the contribution of apoptosis-inducing factor (AIF), a key regulator of mitochondrial biogenesis, in supporting hair growth. We report that pelage abnormalities developed during hair follicle (HF) morphogenesis in Harlequin (Hq) mutant mice. Fragility of the hair cortex was associated with decreased expression of genes encoding structural hair proteins, though key transcriptional regulators of HF development were expressed at normal levels. Notably, Aifm1 (R200 del) knockin males and Aifm1(R200 del)/Hq females showed minor hair defects, despite substantially reduced AIF levels. Furthermore, we cloned the integrated ecotropic provirus of the Aifm1Hq allele. We found that its overexpression in wild-type keratinocyte cell lines led to down-regulation of HF-specific Krt84 and Krtap3-3 genes without altering Aifm1 or epidermal Krt5 expression. Together, our findings imply that pelage paucity in Hq mutant mice is mechanistically linked to severe AIF deficiency and is associated with the expression of retroviral elements that might potentially influence the transcriptional regulation of structural hair proteins.

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First genome edited poinsettias: targeted mutagenesis of flavonoid 3′-hydroxylase using CRISPR/Cas9 results in a colour shift

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-021-02103-5 ◽

2021 ◽

Author(s):

Daria Nitarska ◽

Robert Boehm ◽

Thomas Debener ◽

Rares Calin Lucaciu ◽

Heidi Halbwirth

Keyword(s):

Transgenic Plants ◽

Genome Editing ◽

Ornamental Plants ◽

Targeted Mutagenesis ◽

Cloning And Expression ◽

Euphorbia Pulcherrima ◽

Loss Of Function ◽

Wild Type ◽

Promising Tool ◽

Reddish Orange

AbstractThe CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3′-hydroxylase (F3′H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar ‘Christmas Eve’, in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3′H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3′H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3′H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3′H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia.

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