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Zhongguo Dang Dai Er Ke Za Zhi =... Apr 2024The first patient, a 10-year-old girl, presented with pancytopenia and recurrent epistaxis, along with a history of repeated upper respiratory infections, café-au-lait...
The first patient, a 10-year-old girl, presented with pancytopenia and recurrent epistaxis, along with a history of repeated upper respiratory infections, café-au-lait spots, and microcephaly. Genetic testing revealed compound heterozygous mutations in the DNA ligase IV () gene, leading to a diagnosis of LIG4 syndrome. The second patient, a 6-year-old girl, was seen for persistent thrombocytopenia lasting over two years and was noted to have short stature, hyperpigmented skin, and hand malformations. She had a positive result from chromosome breakage test. She was diagnosed with Fanconi anemia complementation group A. Despite similar clinical presentations, the two children were diagnosed with different disorders, suggesting that children with hemocytopenia and malformations should not only be evaluated for hematological diseases but also be screened for other potential underlying conditions such as immune system disorders.
Topics: Humans; Female; Child; Abnormalities, Multiple; Pancytopenia; DNA Ligase ATP; Thrombocytopenia; Cytopenia
PubMed: 38660906
DOI: 10.7499/j.issn.1008-8830.2311058 -
Proceedings of the National Academy of... Apr 2024The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1...
The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1 activation. However, it is unclear how the activity of PARP1 is regulated. Here, we found that UFMylation is required for the efficient activation of CHK1 by UFMylating PARP1 at K548 during replication stress. Inactivation of UFL1, the E3 enzyme essential for UFMylation, delayed CHK1 activation and inhibits nascent DNA degradation during replication blockage as seen in PARP1-deficient cells. An in vitro study indicated that PARP1 is UFMylated at K548, which enhances its catalytic activity. Correspondingly, a PARP1 UFMylation-deficient mutant (K548R) and pathogenic mutant (F553L) compromised CHK1 activation, the restart of stalled replication forks following replication blockage, and chromosome stability. Defective PARP1 UFMylation also resulted in excessive nascent DNA degradation at stalled replication forks. Finally, we observed that PARP1 UFMylation-deficient knock-in mice exhibited increased sensitivity to replication stress caused by anticancer treatments. Thus, we demonstrate that PARP1 UFMylation promotes CHK1 activation and replication fork stability during replication stress, thus safeguarding genome integrity.
Topics: DNA Replication; Animals; Poly (ADP-Ribose) Polymerase-1; Checkpoint Kinase 1; Mice; Humans; DNA Damage; Ubiquitin-Protein Ligases
PubMed: 38657044
DOI: 10.1073/pnas.2322520121 -
Genome Biology Apr 2024The proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years, but investigations on its biological functions have lagged....
BACKGROUND
The proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years, but investigations on its biological functions have lagged. Recently, Ki-67 has been shown to regulate both the composition of the chromosome periphery and chromosome behaviour in mitosis as well as to play a role in heterochromatin organisation and gene transcription. However, how the different roles for Ki-67 across the cell cycle are regulated and coordinated remain poorly understood. The progress towards understanding Ki-67 function have been limited by the tools available to deplete the protein, coupled to its abundance and fluctuation during the cell cycle.
RESULTS
Here, we use a doxycycline-inducible E3 ligase together with an auxin-inducible degron tag to achieve a rapid, acute and homogeneous degradation of Ki-67 in HCT116 cells. This system, coupled with APEX2 proteomics and phospho-proteomics approaches, allows us to show that Ki-67 plays a role during DNA replication. In its absence, DNA replication is severely delayed, the replication machinery is unloaded, causing DNA damage that is not sensed by the canonical pathways and dependent on HUWE1 ligase. This leads to defects in replication and sister chromatids cohesion, but it also triggers an interferon response mediated by the cGAS/STING pathway in all the cell lines tested.
CONCLUSIONS
We unveil a new function of Ki-67 in DNA replication and genome maintenance that is independent of its previously known role in mitosis and gene regulation.
Topics: Humans; DNA Damage; DNA Replication; Genomic Instability; HCT116 Cells; Ki-67 Antigen; Ubiquitin-Protein Ligases
PubMed: 38649976
DOI: 10.1186/s13059-024-03243-5 -
The EMBO Journal Jun 2024Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in...
Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in a compartmentalized manner, however, the mechanism underlying metabolic regulation of mtDNA function remains unknown. Here, we report that expression of tricarboxylic acid cycle enzyme succinate-CoA ligase SUCLG1 strongly correlates with ETC genes across various TCGA cancer transcriptomes. Mechanistically, SUCLG1 restricts succinyl-CoA levels to suppress the succinylation of mitochondrial RNA polymerase (POLRMT). Lysine 622 succinylation disrupts the interaction of POLRMT with mtDNA and mitochondrial transcription factors. SUCLG1-mediated POLRMT hyposuccinylation maintains mtDNA transcription, mitochondrial biogenesis, and leukemia cell proliferation. Specifically, leukemia-promoting FMS-like tyrosine kinase 3 (FLT3) mutations modulate nuclear transcription and upregulate SUCLG1 expression to reduce succinyl-CoA and POLRMT succinylation, resulting in enhanced mitobiogenesis. In line, genetic depletion of POLRMT or SUCLG1 significantly delays disease progression in mouse and humanized leukemia models. Importantly, succinyl-CoA level and POLRMT succinylation are downregulated in FLT3-mutated clinical leukemia samples, linking enhanced mitobiogenesis to cancer progression. Together, SUCLG1 connects succinyl-CoA with POLRMT succinylation to modulate mitochondrial function and cancer development.
Topics: Humans; Animals; Mice; Succinate-CoA Ligases; Organelle Biogenesis; Mitochondria; Leukemia; DNA-Directed RNA Polymerases; Disease Progression; Acyl Coenzyme A; DNA, Mitochondrial; Mitochondrial Proteins; Cell Line, Tumor; Cell Proliferation
PubMed: 38649537
DOI: 10.1038/s44318-024-00101-9 -
Nature Cell Biology May 2024DNA-protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based...
DNA-protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4 and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription.
Topics: DNA Repair; DNA Repair Enzymes; Humans; Transcription, Genetic; Poly-ADP-Ribose Binding Proteins; DNA Helicases; RNA Polymerase II; Proteolysis; Ubiquitin-Protein Ligases; DNA-Binding Proteins; DNA; HEK293 Cells; Transcription Factors; DNA Damage; Proteasome Endopeptidase Complex; Carrier Proteins; Receptors, Interleukin-17
PubMed: 38600236
DOI: 10.1038/s41556-024-01394-y -
BioRxiv : the Preprint Server For... Mar 2024DNA ligases repair the strand breaks are made continually and naturally throughout the genome, if left unrepaired and allowed to persist, they can lead to genome...
DNA ligases repair the strand breaks are made continually and naturally throughout the genome, if left unrepaired and allowed to persist, they can lead to genome instability in the forms of lethal double-strand (ds) breaks, deletions, and duplications. DNA ligase 1 (LIG1) joins Okazaki fragments during the replication machinery and seals nicks at the end of most DNA repair pathways. Yet, how LIG1 recognizes its target substrate is entirely missing. Here, we uncover the dynamics of nick DNA binding by LIG1 at the single-molecule level. Our findings reveal that LIG1 binds to dsDNA both specifically and non-specifically and exhibits diffusive behavior to form a stable complex at the nick. Furthermore, by comparing with the LIG1 C-terminal protein, we demonstrate that the N-terminal non-catalytic region promotes binding enriched at nick sites and facilitates an efficient nick search process by promoting 1D diffusion along the DNA. Our findings provide a novel single-molecule insight into the nick binding by LIG1, which is critical to repair broken phosphodiester bonds in the DNA backbone to maintain genome integrity.
PubMed: 38586032
DOI: 10.1101/2024.03.28.587287 -
Nature Communications Apr 2024DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely...
DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.
Topics: Humans; CCAAT-Enhancer-Binding Proteins; Chromatin; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Neoplasms; Ubiquitin-Protein Ligases
PubMed: 38580649
DOI: 10.1038/s41467-024-47314-4 -
Cellular and Molecular Life Sciences :... Apr 2024The DNA methylation is gradually acquired during oogenesis, a process sustained by successful follicle development. However, the functional roles of methyl-CpG-binding...
The DNA methylation is gradually acquired during oogenesis, a process sustained by successful follicle development. However, the functional roles of methyl-CpG-binding protein 2 (MeCP2), an epigenetic regulator displaying specifical binding with methylated DNA, remains unknown in oogenesis. In this study, we found MeCP2 protein was highly expressed in primordial and primary follicle, but was almost undetectable in secondary follicles. However, in aged ovary, MeCP2 protein is significantly increased in both oocyte and granulosa cells. Overexpression of MeCP2 in growing oocyte caused transcription dysregulation, DNA hypermethylation, and genome instability, ultimately leading to follicle growth arrest and apoptosis. MeCP2 is targeted by DCAF13, a substrate recognition adaptor of the Cullin 4-RING (CRL4) E3 ligase, and polyubiquitinated for degradation in both cells and oocytes. Dcaf13-null oocyte exhibited an accumulation of MeCP2 protein, and the partial rescue of follicle growth arrest induced by Dcaf13 deletion was observed following MeCP2 knockdown. The RNA-seq results revealed that large amounts of genes were regulated by the DCAF13-MeCP2 axis in growing oocytes. Our study demonstrated that CRL4 E3 ubiquitin ligase targets MeCP2 for degradation to ensure normal DNA methylome and transcription in growing oocytes. Moreover, in aged ovarian follicles, deceased DCAF13 and DDB1 protein were observed, indicating a potential novel mechanism that regulates ovary aging.
Topics: Female; Humans; Cullin Proteins; DNA; DNA Methylation; Methyl-CpG-Binding Protein 2; Oocytes; Ubiquitin-Protein Ligases
PubMed: 38578457
DOI: 10.1007/s00018-024-05185-4 -
Arthritis Research & Therapy Apr 2024Transitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such...
BACKGROUND
Transitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such as reporter assays and genome editing. In this report, we undertook such studies on the osteoarthritis (OA) risk that is marked by single nucleotide polymorphism (SNP) rs34195470 (A > G). The OA risk-conferring G allele of this SNP associates with increased DNA methylation (DNAm) at two CpG dinucleotides within WWP2. This gene encodes a ubiquitin ligase and is the host gene of microRNA-140 (miR-140). WWP2 and miR-140 are both regulators of TGFβ signaling.
METHODS
Nucleic acids were extracted from adult OA (arthroplasty) and foetal cartilage. Samples were genotyped and DNAm quantified by pyrosequencing at the two CpGs plus 14 flanking CpGs. CpGs were tested for transcriptional regulatory effects using a chondrocyte cell line and reporter gene assay. DNAm was altered using epigenetic editing, with the impact on gene expression determined using RT-qPCR. In silico analysis complemented laboratory experiments.
RESULTS
rs34195470 genotype associates with differential methylation at 14 of the 16 CpGs in OA cartilage, forming a methylation quantitative trait locus (mQTL). The mQTL is less pronounced in foetal cartilage (5/16 CpGs). The reporter assay revealed that the CpGs reside within a transcriptional regulator. Epigenetic editing to increase their DNAm resulted in altered expression of the full-length and N-terminal transcript isoforms of WWP2. No changes in expression were observed for the C-terminal isoform of WWP2 or for miR-140.
CONCLUSIONS
As far as we are aware, this is the first experimental demonstration of an OA association signal targeting specific transcript isoforms of a gene. The WWP2 isoforms encode proteins with varying substrate specificities for the components of the TGFβ signaling pathway. Future analysis should focus on the substrates regulated by the two WWP2 isoforms that are the targets of this genetic risk.
Topics: Adult; Humans; Base Sequence; Ubiquitin; Protein Isoforms; Ubiquitin-Protein Ligases; DNA Methylation; MicroRNAs; Osteoarthritis; Transforming Growth Factor beta
PubMed: 38570801
DOI: 10.1186/s13075-024-03315-8 -
Zhongguo Dang Dai Er Ke Za Zhi =... Mar 2024Central precocious puberty (CPP) is a developmental disorder caused by early activation of the hypothalamic-pituitary-gonadal axis. The incidence of CPP is rapidly... (Review)
Review
Central precocious puberty (CPP) is a developmental disorder caused by early activation of the hypothalamic-pituitary-gonadal axis. The incidence of CPP is rapidly increasing, but the underlying mechanisms are not fully understood. Previous studies have shown that gain-of-function mutations in the and genes and loss-of-function mutations in the , , and genes may lead to early initiation of pubertal development. Recent research has also revealed the significant role of epigenetic factors such as DNA methylation and microRNAs in the regulation of gonadotropin-releasing hormone neurons, as well as the modulating effect of gene networks involving multiple variant genes on pubertal initiation. This review summarizes the genetic etiology and pathogenic mechanisms underlying CPP.
Topics: Humans; Puberty, Precocious; Gonadotropin-Releasing Hormone; Mutation; MicroRNAs; Puberty; Ubiquitin-Protein Ligases
PubMed: 38557384
DOI: 10.7499/j.issn.1008-8830.2309098