ABSTRACT
Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4CSA complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.
Subject(s)
DNA Repair , DNA-Binding Proteins , Transcription, Genetic , Ubiquitination , Humans , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Cullin Proteins/metabolism , Cullin Proteins/genetics , DNA Repair Enzymes/metabolism , DNA Repair Enzymes/genetics , Cryoelectron Microscopy , HEK293 Cells , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , DNA Damage , RNA Polymerase II/metabolism , Protein Binding , Excision Repair , Carrier Proteins , DNA Helicases , Transcription Factors , Receptors, Interleukin-17ABSTRACT
Autosomal recessive congenital ichthyoses (ARCI) is a genetically heterogeneous condition that can be caused by pathogenic variants in at least 12 genes, including ABCA12. ARCI mainly consists of congenital ichthyosiform erythroderma (CIE), lamellar ichthyosis (LI) and harlequin ichthyosis (HI). The objective was to determine previously unreported pathogenic variants in ABCA12 and to update genotype-phenotype correlations for patients with pathogenic ABCA12 variants. Pathogenic variants in ABCA12 were detected using Sanger sequencing or a combination of Sanger sequencing and whole-exome sequencing. To verify the pathogenicity of a previously unreported large deletion and intron variant, cDNA analysis was performed using total RNA extracted from hair roots. Genetic analyses were performed on the patients with CIE, LI, HI and non-congenital ichthyosis with unusual phenotypes (NIUP), and 11 previously unreported ABCA12 variants were identified. Sequencing of cDNA confirmed the aberrant splicing of the variant ABCA12 in the patients with the previously unreported large deletion and intron variant. Our findings expand the phenotype spectrum of ichthyosis patients with ABCA12 pathogenic variants. The present missense variants in ABCA12 are considered to be heterogenous in pathogenicity, and they lead to varying disease severities in patients with ARCI and non-congenital ichthyosis with unusual phenotypes (NIUP).
Subject(s)
Ichthyosiform Erythroderma, Congenital , Ichthyosis, Lamellar , Ichthyosis , Humans , Ichthyosis, Lamellar/genetics , Ichthyosis, Lamellar/pathology , DNA, Complementary , Genes, Recessive , Mutation , Ichthyosis/genetics , Ichthyosiform Erythroderma, Congenital/genetics , Genetic Association Studies , ATP-Binding Cassette Transporters/geneticsABSTRACT
CONTEXT: Telomerase reverse transcriptase promoter (TERT-p) mutations, which upregulate TERT expression, are strongly associated with tumor aggressiveness and worse prognosis in papillary thyroid carcinomas (PTCs). TERT expression is also observed in a proportion of PTCs without TERT-p mutations, but such tumors show less aggressiveness and better prognosis compared with TERT-p mutation-positive tumors. OBJECTIVE: TERT has multiple splicing variants whose relationships with the TERT-p status and clinicopathological characteristics remain poorly understood. We examined the relationship between the TERT-p mutational status, the TERT splicing pattern, and clinicopathological features. METHODS: We investigated the expression of two major variants, α deletion (dA) and ß deletion (dB), in a series of 207 PTCs operated between November 2001 and March 2020 in Nagasaki University Hospital and Kuma Hospital. RESULTS: The TERT-p mutations were found in 33 cases, and among 174 mutation-negative cases, 24 showed TERT expression. All cases were classified into three groups: the TERT-p mutation-negative/expression-negative group (mut-/exp-), the TERT-p mutation-negative/expression-positive group (mut-/exp+), and the TERT-p mutation-positive group (mut+/exp+). The +A + B/dB ratio in mut+/exp + was significantly higher than that in mut-/exp + PTCs. Analysis with clinicopathological data revealed that +A + B expression was associated with higher PTC aggressiveness, whereas dB expression counteracted this effect. Functional in vitro study demonstrated that dB strongly inhibited cell growth, migration, and clonogenicity, suggesting its tumor suppressive role. CONCLUSION: These results provide evidence that the TERT-p mutations alter the expression of different TERT splice variants, which, in turn, associates with different tumor aggressiveness.
ABSTRACT
DNA-protein crosslinks (DPCs) induced by aldehydes interfere with replication and transcription. Hereditary deficiencies in DPC repair and aldehyde clearance processes cause progeria, including Ruijs-Aalfs syndrome (RJALS) and AMeD syndrome (AMeDS) in humans. Although the elimination of DPC during replication has been well established, how cells overcome DPC lesions in transcription remains elusive. Here we show that endogenous aldehyde-induced DPC roadblocks are efficiently resolved by transcription-coupled repair (TCR). We develop a high-throughput sequencing technique to measure the genome-wide distribution of DPCs (DPC-seq). Using proteomics and DPC-seq, we demonstrate that the conventional TCR complex as well as VCP/p97 and the proteasome are required for the removal of formaldehyde-induced DPCs. TFIIS-dependent cleavage of RNAPII transcripts protects against transcription obstacles. Finally, a mouse model lacking both aldehyde clearance and TCR confirms endogenous DPC accumulation in actively transcribed regions. Collectively, our data provide evidence that transcription-coupled DPC repair (TC-DPCR) as well as aldehyde clearance are crucial for protecting against metabolic genotoxin, thus explaining the molecular pathogenesis of AMeDS and other disorders associated with defects in TCR, such as Cockayne syndrome.
Subject(s)
Aldehydes , DNA Repair , Transcription, Genetic , Animals , Humans , Aldehydes/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/genetics , Mice , DNA/metabolism , DNA/genetics , DNA Damage , Mice, Knockout , Valosin Containing Protein/metabolism , Valosin Containing Protein/genetics , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Mice, Inbred C57BL , Formaldehyde/toxicity , Formaldehyde/pharmacology , Excision RepairABSTRACT
Recent single-cell analyses have revealed the complexity of microglial heterogeneity in brain development, aging, and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Disease-associated microglia (DAMs) have been identified in ALS mice model, but their role in ALS pathology remains unclear. The effect of genetic background variations on microglial heterogeneity and functions remains unknown. Herein, we established and analyzed two mice models of ALS with distinct genetic backgrounds of C57BL/6 and BALB/c. We observed that the change in genetic background from C57BL/6 to BALB/c affected microglial heterogeneity and ALS pathology and its progression, likely due to the defective induction of neurotrophic factor-secreting DAMs and impaired microglial survival. Single-cell analyses of ALS mice revealed new markers for each microglial subtype and a possible association between microglial heterogeneity and systemic immune environments. Thus, we highlighted the role of microglia in ALS pathology and importance of genetic background variations in modulating microglial functions.
ABSTRACT
Cardiospondylocarpofacial syndrome (CSCFS) is a congenital malformation characterized by growth retardation, facial features, short toes with carpal and tarsal fusion, extensive posterior neck vertebral fusion, congenital heart disease, and deafness. Here, we report a severe case of CSCFS with a novel variant, p.Thr187Ile, in MAP3K7. Thr187 is the main phosphorylation site for TGF-beta-activated kinase 1 encoded by MAP3K7, and this variant may cause significant abnormalities in downstream signaling.
Subject(s)
Epidermolysis Bullosa Simplex , Keratin-14 , Humans , Epidermolysis Bullosa Simplex/genetics , Epidermolysis Bullosa Simplex/pathology , Epidermolysis Bullosa Simplex/diagnosis , Keratin-14/genetics , Male , Skin/pathology , Female , Protein Domains , DNA Mutational Analysis , Mutation, MissenseABSTRACT
Dyssegmental dysplasia (DD) is a severe skeletal dysplasia comprised of two subtypes: lethal Silverman-Handmaker type (DDSH) and nonlethal Rolland-Desbuquois type (DDRD). DDSH is caused by biallelic pathogenic variants in HSPG2 encoding perlecan, whereas the genetic cause of DDRD remains undetermined. Schwartz-Jampel syndrome (SJS) is also caused by biallelic pathogenic variants in HSPG2 and is an allelic disorder of DDSH. In SJS and DDSH, 44 and 8 pathogenic variants have been reported in HSPG2, respectively. Here, we report that five patients with DDRD carried four pathogenic variants in HSPG2: c.9970 G > A (p.G3324R), c.559 C > T (p.R187X), c7006 + 1 G > A, and c.11562 + 2 T > G. Two patients were homozygous for p.G3324R, and three patients were heterozygous for p.G3324R. Haplotype analysis revealed a founder haplotype spanning 85,973 bp shared in the five patients. SJS, DDRD, and DDSH are allelic disorders with pathogenic variants in HSPG2.
Subject(s)
Haplotypes , Heparan Sulfate Proteoglycans , Osteochondrodysplasias , Female , Humans , Male , Alleles , Bone Diseases, Developmental/genetics , Bone Diseases, Developmental/pathology , Founder Effect , Heparan Sulfate Proteoglycans/genetics , Mutation , Osteochondrodysplasias/genetics , Osteochondrodysplasias/pathology , Fetal DiseasesABSTRACT
Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases including congenital myasthenic syndromes (CMS). Germline mutations in CHRNE encoding the acetylcholine receptor (AChR) ε subunit are the most common cause of CMS. An active form of vitamin D, calcitriol, binds to vitamin D receptor (VDR) and regulates gene expressions. We found that calcitriol enhanced MuSK phosphorylation, AChR clustering, and myotube twitching in co-cultured C2C12 myotubes and NSC34 motor neurons. RNA-seq analysis of co-cultured cells showed that calcitriol increased the expressions of Rspo2, Rapsn, and Dusp6. ChIP-seq of VDR revealed that VDR binds to a region approximately 15 âkbp upstream to Rspo2. Biallelic deletion of the VDR-binding site of Rspo2 by CRISPR/Cas9 in C2C12 myoblasts/myotubes nullified the calcitriol-mediated induction of Rspo2 expression and MuSK phosphorylation. We generated Chrne knockout (Chrne KO) mouse by CRISPR/Cas9. Intraperitoneal administration of calcitriol markedly increased the number of AChR clusters, as well as the area, the intensity, and the number of synaptophysin-positive synaptic vesicles, in Chrne KO mice. In addition, calcitriol ameliorated motor deficits and prolonged survival of Chrne KO mice. In the skeletal muscle, calcitriol increased the gene expressions of Rspo2, Rapsn, and Dusp6. We propose that calcitriol is a potential therapeutic agent for CMS and other diseases with defective neuromuscular signal transmission.
Subject(s)
Myasthenic Syndromes, Congenital , Animals , Mice , Myasthenic Syndromes, Congenital/drug therapy , Myasthenic Syndromes, Congenital/genetics , Myasthenic Syndromes, Congenital/metabolism , Calcitriol/metabolism , Neuromuscular Junction/metabolism , Receptors, Cholinergic/genetics , Receptors, Cholinergic/metabolism , Motor Neurons/metabolismABSTRACT
Pathogenic variants in ABCA12 are important causative genetic defects for autosomal recessive congenital ichthyoses (ARCI), which include congenital ichthyosiform erythroderma (CIE), harlequin ichthyosis, and lamellar ichthyosis. In addition, pathogenic variants in ABCA12 are known to cause a localized nevoid form of CIE due to recessive mosaicism. We previously reported siblings who carried an ABCA12 variant but did not show a "congenital" phenotype. They were considered to have pityriasis rubra pilaris (PRP). Here, we present a further patient with ABCA12 variants whose phenotype was not congenital ichthyosis, in an independent family. Notably, these three patients had geographic unaffected areas. Such areas are not usually found in patients with ARCI who have ABCA12 variants, suggesting mild phenotypes for these patients. Interestingly, the histological features of the ichthyotic lesions in these patients resembled those of PRP. All three patients had homozygous pathogenic missense variants in ABCA12. Our findings expand the phenotypic spectrum of patients with ABCA12 variants.
Subject(s)
Ichthyosiform Erythroderma, Congenital , Ichthyosis, Lamellar , Ichthyosis , Pityriasis Rubra Pilaris , Humans , Pityriasis Rubra Pilaris/genetics , Ichthyosis, Lamellar/genetics , Ichthyosiform Erythroderma, Congenital/genetics , Ichthyosiform Erythroderma, Congenital/pathology , Phenotype , Mutation , ATP-Binding Cassette Transporters/geneticsSubject(s)
Psoriasis , Humans , Acute Disease , Causality , Chronic Disease , Psoriasis/epidemiology , Psoriasis/genetics , Pyrin/geneticsSubject(s)
Epidermolysis Bullosa Simplex , Humans , Male , Epidermolysis Bullosa Simplex/complications , Epidermolysis Bullosa Simplex/genetics , Epidermolysis Bullosa Simplex/diagnosis , Epidermolysis Bullosa Simplex/pathology , Growth Disorders/etiology , Growth Disorders/diagnosis , Growth Disorders/complications , Toes/abnormalities , InfantSubject(s)
Calcium-Transporting ATPases , Pemphigus, Benign Familial , Humans , Pemphigus, Benign Familial/genetics , Pemphigus, Benign Familial/pathology , Pemphigus, Benign Familial/diagnosis , Calcium-Transporting ATPases/genetics , Female , Male , Genetic Association Studies , Middle Aged , Adult , MutationABSTRACT
The organelle contact site of the endoplasmic reticulum and mitochondria, known as the mitochondria-associated membrane (MAM), is a multifunctional microdomain in cellular homeostasis. We previously reported that MAM disruption is a common pathological feature in amyotrophic lateral sclerosis (ALS); however, the precise role of MAM in ALS was uncovered. Here, we show that the MAM is essential for TANK-binding kinase 1 (TBK1) activation under proteostatic stress conditions. A MAM-specific E3 ubiquitin ligase, autocrine motility factor receptor, ubiquitinated nascent proteins to activate TBK1 at the MAM, which results in ribosomal protein degradation. MAM or TBK1 deficiency under proteostatic stress conditions resulted in increased cellular vulnerability in vitro and motor impairment in vivo. Thus, MAM disruption exacerbates proteostatic stress via TBK1 inactivation in ALS. Our study has revealed a proteostatic mechanism mediated by the MAM-TBK1 axis, highlighting the physiological importance of the organelle contact sites.
Subject(s)
Amyotrophic Lateral Sclerosis , Humans , Amyotrophic Lateral Sclerosis/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Endoplasmic Reticulum/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we applied a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis showed that DDA1 is an integral component of the CRL4CSA complex. Functional analysis revealed that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.
ABSTRACT
Transcription-blocking lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which prevents DNA damage-induced cellular toxicity and maintains proper transcriptional processes. TC-NER is initiated by the stalling of RNA polymerase II (RNAPII), which triggers the assembly of TC-NER-specific proteins, namely CSB, CSA and UVSSA, which collectively control and drive TC-NER progression. Previous research has revealed molecular functions for these proteins, however, exact mechanisms governing the initiation and regulation of TC-NER, particularly at low UV doses have remained elusive, partly due to technical constraints. In this study, we employ knock-in cell lines designed to target the endogenous CSB gene locus with mClover, a GFP variant. Through live cell imaging, we uncover the intricate molecular dynamics of CSB in response to physiologically relevant UV doses. We showed that the DNA damage-induced association of CSB with chromatin is tightly regulated by the CSA-containing ubiquitin-ligase CRL complex (CRL4CSA). Combining the CSB-mClover knock-in cell line with SILAC-based GFP-mediated complex isolation and mass-spectrometry-based proteomics, revealed novel putative CSB interactors as well as discernible variations in complex composition during distinct stages of TC-NER progression. Our work not only provides molecular insight into TC-NER, but also illustrates the versatility of endogenously tagging fluorescent and affinity tags.
Subject(s)
DNA Damage , DNA Repair , Cell Line , Chromatin , Mass SpectrometryABSTRACT
Accumulation of lipotoxic lipids, such as free cholesterol, induces hepatocyte death and subsequent inflammation and fibrosis in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the underlying mechanisms remain unclear. We have previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated that lysosomal cholesterol overload triggers lysosomal dysfunction and profibrotic activation of macrophages during the development of NASH. ß-cyclodextrin polyrotaxane (ßCD-PRX), a unique supramolecule, is designed to elicit free cholesterol from lysosomes. Treatment with ßCD-PRX ameliorated cholesterol accumulation and profibrotic activation of macrophages surrounding dead hepatocytes with cholesterol crystals, thereby suppressing liver fibrosis in a NASH model, without affecting the hepatic cholesterol levels. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in macrophages predisposed to the steatotic microenvironment. This study provides evidence that dysregulated cholesterol metabolism in macrophages would be a novel mechanism of NASH.
Subject(s)
Non-alcoholic Fatty Liver Disease , Animals , Mice , Disease Models, Animal , Liver Cirrhosis , Macrophages , Cholesterol , LysosomesABSTRACT
The pervasive weak electromagnetic fields (EMF) inundate the industrialized society, but the biological effects of EMF as weak as 10 µT have been scarcely analyzed. Heat shock proteins (HSPs) are molecular chaperones that mediate a sequential stress response. HSP70 and HSP90 provide cells under undesirable situations with either assisting covalent folding of proteins or degrading improperly folded proteins in an ATP-dependent manner. Here we examined the effect of extremely low-frequency (ELF)-EMF on AML12 and HEK293 cells. Although the protein expression levels of HSP70 and HSP90 were reduced after an exposure to ELF-EMF for 3 h, acetylations of HSP70 and HSP90 were increased, which was followed by an enhanced binding affinities of HSP70 and HSP90 for HSP70/HSP90-organizing protein (HOP/STIP1). After 3 h exposure to ELF-EMF, the amount of mitochondria was reduced but the ATP level and the maximal mitochondrial oxygen consumption were increased, which was followed by the reduced protein aggregates and the increased cell viability. Thus, ELF-EMF exposure for 3 h activated acetylation of HSPs to enhance protein folding, which was returned to the basal level at 12 h. The proteostatic effects of ELF-EMF will be able to be applied to treat pathological states in humans.
Subject(s)
Electromagnetic Fields , Heat-Shock Proteins , Humans , Acetylation , Electromagnetic Fields/adverse effects , HEK293 Cells , Protein Folding , HSP70 Heat-Shock Proteins , HSP90 Heat-Shock Proteins , Adenosine TriphosphateABSTRACT
CRISPR/Cas9-mediated gene editing has great potential utility for treating genetic diseases. However, its therapeutic applications are limited by unintended genomic alterations arising from DNA double-strand breaks and random integration of exogenous DNA. In this study, we propose NICER, a method for correcting heterozygous mutations that employs multiple nicks (MNs) induced by Cas9 nickase and a homologous chromosome as an endogenous repair template. Although a single nick near the mutation site rarely leads to successful gene correction, additional nicks on homologous chromosomes strongly enhance gene correction efficiency via interhomolog homologous recombination (IH-HR). This process partially depends on BRCA1 and BRCA2, suggesting the existence of several distinct pathways for MN-induced IH-HR. According to a genomic analysis, NICER rarely induces unintended genomic alterations. Furthermore, NICER restores the expression of disease-causing genes in cells derived from genetic diseases with compound heterozygous mutations. Overall, NICER provides a precise strategy for gene correction.
Subject(s)
Anti-Bacterial Agents , Homologous Recombination , Mutation , DNA Breaks, Double-Stranded , Deoxyribonuclease IABSTRACT
Xeroderma pigmentosum (XP) is a genodermatosis defined by cutaneous photosensitivity with an increased risk of skin tumors because of DNA repair deficiency. The worldwide prevalence of XP is ~1 to 4 in million, with higher incidence in some countries and regions including Japan (1 in 22,000) and North Africa due to founder mutations and a high degree of consanguinity. Among XP, the complementation group F (XP-F), is a rare form (1% of worldwide XP); however, this is underdiagnosed, because the ERCC4/XPF gene is essential for fetal development and most of previously reported ERCC4/XPF pathogenic variants are hypomorphs causing relatively mild phenotypes. From the largest Japanese XP cohort study, we report 17 XP-F cases bearing two pathogenic variants, both identified in deep intronic regions of the ERCC4/XPF gene. The first variant, located in intron 1, is a Japanese founder mutation, which additionally accounts for ~10% of the entire Japanese XP cases (MAF = 0.00196), causing an aberrant pre-mRNA splicing due to a miss-binding of U1snRNA. The second mutation located in intron eight induces an alternative polyadenylation. Both mutations cause a reduction of the ERCC4/XPF gene expression, resulting in XP clinical manifestations. Most cases developed early-onset skin cancers, indicating that these variants need critical attention. We further demonstrate that antisense oligonucleotides designed for the mutations can restore the XPF protein expression and DNA repair capacity in the patients' cells. Collectively, these pathogenic variants can be potential therapeutic targets for XP.