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The Journal of Clinical Investigation Mar 2024The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been...
The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.
Topics: Animals; Humans; Mice; Ataxia Telangiectasia Mutated Proteins; B-Lymphocytes; Carcinogenesis; Checkpoint Kinase 1; DNA Replication; Genomic Instability; Mice, Knockout; Nuclear Proteins; Proto-Oncogene Proteins c-myc; Signal Transduction; Sumoylation; Ubiquitin-Protein Ligases
PubMed: 38530355
DOI: 10.1172/JCI167419 -
Biotechnology Journal Mar 2024DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5' phosphates and 3' hydroxyl groups on...
DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5' phosphates and 3' hydroxyl groups on one strand of the duplex. While DNA ligases preferentially ligate single breaks in double-stranded DNA (dsDNA), they are capable of ligating a multitude of other nucleic acid substrates like blunt-ended dsDNA, TA overhangs, short overhangs and various DNA-RNA hybrids. Here we report a novel DNA ligase from Cronobacter phage CR 9 (R2D Ligase) with an unexpected DNA-to-RNA ligation activity. The R2D ligase shows excellent efficiency when ligating DNA to either end of RNA molecules using a DNA template. Furthermore, we show that DNA can be ligated simultaneously to both the 5' and 3' ends of microRNA-like molecules in a single reaction mixture. Abortive adenylated side product formation is suppressed at lower ATP concentrations and the ligase reaction reaches near completion when ligating RNA-to-DNA or DNA-to-RNA. The ligation of a DNA strand to the 5'-PO end of RNA is unique among the commercially available ligases and may facilitate novel workflows in microRNA analysis, RNA sequencing and the preparation of chimeric guide DNA-RNA for gene editing applications.
Topics: DNA Ligases; Ligases; DNA; Base Sequence; MicroRNAs
PubMed: 38528369
DOI: 10.1002/biot.202300711 -
Scientific Reports Mar 2024Maintenance of genome integrity is instrumental in preventing cancer. In addition to DNA repair pathways that prevent damage to DNA, damage tolerance pathways allow for...
Maintenance of genome integrity is instrumental in preventing cancer. In addition to DNA repair pathways that prevent damage to DNA, damage tolerance pathways allow for the survival of cells that encounter DNA damage during replication. The Rad6/18 pathway is instrumental in this process, mediating damage bypass by ubiquitination of proliferating cell nuclear antigen. Previous studies have shown different roles of Rad18 in vivo and in tumorigenesis. Here, we show that B cells induce Rad18 expression upon proliferation induction. We have therefore analysed the role of Rad18 in B cell activation as well as in B cell lymphomagenesis mediated by an Eµ-Myc transgene. We find no activation defects or survival differences between Rad18 WT mice and two different models of Rad18 deficient tumour mice. Also, tumour subtypes do not differ between the mouse models. Accordingly, functions of Rad18 in B cell activation and tumorigenesis may be compensated for by other pathways in B cells.
Topics: Animals; Mice; Carcinogenesis; DNA Damage; DNA Repair; DNA Replication; Neoplasms; Proliferating Cell Nuclear Antigen; Ubiquitin-Protein Ligases; Ubiquitination; DNA-Binding Proteins; B-Lymphocytes; Lymphocyte Activation
PubMed: 38528023
DOI: 10.1038/s41598-024-57018-w -
Advanced Science (Weinheim,... Jun 2024Bladder cancer (BC) is one of the most common tumors characterized by a high rate of relapse and a lack of targeted therapy. Here, YEATS domain-containing protein 4...
Bladder cancer (BC) is one of the most common tumors characterized by a high rate of relapse and a lack of targeted therapy. Here, YEATS domain-containing protein 4 (YEATS4) is an essential gene for BC cell viability using CRISPR-Cas9 library screening is reported, and that HUWE1 is an E3 ligase responsible for YEATS4 ubiquitination and proteasomal degradation by the Protein Stability Regulators Screening Assay. KAT8-mediated acetylation of YEATS4 impaired its interaction with HUWE1 and consequently prevented its ubiquitination and degradation. The protein levels of YEATS4 and KAT8 are positively correlated and high levels of these two proteins are associated with poor overall survival in BC patients. Importantly, suppression of YEATS4 acetylation with the KAT8 inhibitor MG149 decreased YEATS4 acetylation, reduced cell viability, and sensitized BC cells to cisplatin treatment. The findings reveal a critical role of the KAT8/YEATS4 axis in both tumor growth and cisplatin sensitivity in BC cells, potentially generating a novel therapeutic strategy for BC patients.
Topics: Urinary Bladder Neoplasms; Humans; Cisplatin; Cell Line, Tumor; Mice; Histone Acetyltransferases; Animals; Ubiquitin-Protein Ligases; Antineoplastic Agents; Tumor Suppressor Proteins; Acetylation; Disease Models, Animal; Drug Resistance, Neoplasm
PubMed: 38526153
DOI: 10.1002/advs.202310146 -
The Journal of Biological Chemistry May 2024Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of...
Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of the broken strand breaks in the three steps of the ligation reaction. LIG1 can tolerate the RNA strand upstream of the nick, yet an atomic insight into the sugar discrimination mechanism by LIG1 against a ribonucleotide at the 3'-terminus of nick DNA is unknown. Here, we determined X-ray structures of LIG1/3'-RNA-DNA hybrids and captured the ligase during pre- and post-step 3 the ligation reaction. Furthermore, the overlays of 3'-rA:T and 3'-rG:C step 3 structures with step 2 structures of canonical 3'-dA:T and 3'-dG:C uncover a network of LIG1/DNA interactions through Asp570 and Arg871 side chains with 2'-OH of the ribose at nick showing a final phosphodiester bond formation and the other ligase active site residues surrounding the AMP site. Finally, we demonstrated that LIG1 can ligate the nick DNA substrates with pre-inserted 3'-ribonucleotides as efficiently as Watson-Crick base-paired ends in vitro. Together, our findings uncover a novel atomic insight into a lack of sugar discrimination by LIG1 and the impact of improper sugar on the nick sealing of ribonucleotides at the last step of DNA replication and repair.
Topics: Humans; DNA Ligase ATP; DNA; Ribonucleotides; Crystallography, X-Ray; DNA Repair
PubMed: 38522520
DOI: 10.1016/j.jbc.2024.107216 -
Analytica Chimica Acta Apr 20245-hydroxymethylcytosine (5hmC) as an epigenetic modification can regulate gene expression, and its abnormal level is related with various tumor invasiveness and poor...
Construction of a glycosylation-mediated fluorescent biosensor for label-free measurement of site-specific 5-hydroxymethylcytosine in cancer cells with zero background signal.
BACKGROUND
5-hydroxymethylcytosine (5hmC) as an epigenetic modification can regulate gene expression, and its abnormal level is related with various tumor invasiveness and poor prognosis. Nevertheless, the current methods for 5hmC assay usually involve expensive instruments/antibodies, radioactive risk, high background, laborious bisulfite treatment procedures, and non-specific/long amplification time.
RESULTS
We develop a glycosylation-mediated fluorescent biosensor based on helicase-dependent amplification (HDA) for label-free detection of site-specific 5hmC in cancer cells with zero background signal. The glycosylated 5hmC-DNA (5ghmC) catalyzed by β-glucosyltransferase (β-GT) can be cleaved by AbaSI restriction endonuclease to generate two dsDNA fragments with sticky ends. The resultant dsDNA fragments are complementary to the biotinylated probes and ligated by DNA ligases, followed by being captured by magnetic beads. After magnetic separation, the eluted ligation products act as the templates to initiate HDA reaction, generating abundant double-stranded DNA (dsDNA) products within 20 min. The dsDNA products are measured in a label-free manner with SYBR Green I as an indicator. This biosensor can measure 5hmC with a detection limit of 2.75 fM and a wide linear range from 1 × 10 to 1 × 10 M, and it can discriminate as low as 0.001% 5hmC level in complex mixture. Moreover, this biosensor can measure site-specific 5hmC in cancer cells, and distinguish tumor cells from normal cells.
SIGNIFICANCE
This biosensor can achieve a zero-background signal without the need of either 5hmC specific antibody or bisulfite treatment, and it holds potential applications in biological research and disease diagnosis.
Topics: Glycosylation; DNA; 5-Methylcytosine; Biosensing Techniques; Neoplasms; Sulfites
PubMed: 38521572
DOI: 10.1016/j.aca.2024.342463 -
Biochimica Et Biophysica Acta.... Apr 2024Inflammatory environments can trigger endoplasmic reticulum (ER) stress and lead to pyroptosis in various tissues and cells, including liver, brain, and immune cells. As...
Inflammatory environments can trigger endoplasmic reticulum (ER) stress and lead to pyroptosis in various tissues and cells, including liver, brain, and immune cells. As a key factor of ER stress, DNA damage-inducible transcript 3 (DDIT3)/CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is upregulated in osteoblasts during inflammatory stimulation. DDIT3/CHOP may therefore regulate osteoblast pyroptosis in inflammatory conditions. During this investigation, we found that lipopolysaccharides (LPS)/adenosine 5'-triphosphate (ATP) stimulation in vitro induced osteoblasts to undergo pyroptosis, and the expression of DDIT3/CHOP was increased during this process. The overexpression of DDIT3/CHOP further promoted osteoblast pyroptosis as evidenced by the increased expression of the inflammasome NLR family pyrin domain containing 3 (NLRP3) and ratios of caspase-1 p20/caspase-1 and cleaved gasdermin D (GSDMD)/GSDMD. To explore the specific mechanism of this effect, we found through fluorescence imaging and Western blot analysis that LPS/ATP stimulation promoted PTEN-induced kinase 1 (PINK1)/E3 ubiquitin-protein ligase parkin (Parkin)-mediated mitophagy in osteoblasts, and this alteration was suppressed by the DDIT3/CHOP overexpression, resulting in increased ratio of pyroptosis compared with the control groups. The impact of DDIT3/CHOP on pyroptosis in osteoblasts was reversed by the application of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a specific mitophagy agonist. Therefore, our data demonstrated that DDIT3/CHOP promotes osteoblast pyroptosis by inhibiting PINK1/Parkin-mediated mitophagy in an inflammatory environment.
Topics: Pyroptosis; Lipopolysaccharides; Mitophagy; Caspase 1; Adenosine Triphosphate; Osteoblasts; Protein Kinases; Ubiquitin-Protein Ligases
PubMed: 38521466
DOI: 10.1016/j.bbamcr.2024.119712 -
Nature Communications Mar 2024Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the...
Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation. To obtain structural insights of H2BK120 ubiquitination and its regulation, we report the cryo-electron microscopy structure of the human Bre1 complex bound to the nucleosome. The two RING domains of Bre1A and Bre1B recognize the acidic patch and the nucleosomal DNA phosphates around SHL 6.0-6.5, which are ideally located to recruit the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. Mutational experiments suggest that the two RING domains bind in two orientations and that ubiquitination occurs when Bre1A binds to the acidic patch. Our results provide insights into the H2BK120-specific ubiquitination by the Bre1 proteins and suggest that H2B monoubiquitination can be regulated by nuclesomal DNA flexibility.
Topics: Humans; Cryoelectron Microscopy; DNA; Histones; Neoplasms; Nucleosomes; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38519511
DOI: 10.1038/s41467-024-46910-8 -
Cell Reports Apr 2024Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying...
Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying enzymes leading to changes in histone acetylation, citrullination, and methylation at target genes. Here, we demonstrate that hypoxia-inducible gene expression in estrogen receptor (ER)-positive MCF7 and ER-negative SUM159 human breast cancer cells requires the histone H2A/H2B chaperone facilitates chromatin transcription (FACT) and the H2B ubiquitin ligase RING finger protein 20/40 (RNF20/40). Knockdown of FACT or RNF20/40 expression leads to decreased transcription initiation and elongation at HIF-1 target genes. Mechanistically, FACT and RNF20/40 are recruited to hypoxia response elements (HREs) by HIF-1 and stabilize binding of HIF-1 (and each other) at HREs. Hypoxia induces the monoubiquitination of histone H2B at lysine 120 at HIF-1 target genes in an HIF-1-dependent manner. Together, these findings delineate a cooperative molecular mechanism by which FACT and RNF20/40 stabilize multiprotein complex formation at HREs and mediate histone ubiquitination to facilitate HIF-1 transcriptional activity.
Topics: Humans; Cell Hypoxia; Cell Line, Tumor; DNA-Binding Proteins; Histones; Hypoxia-Inducible Factor 1; MCF-7 Cells; Protein Binding; Response Elements; Transcription Factors; Transcriptional Activation; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38517892
DOI: 10.1016/j.celrep.2024.113972 -
Journal of Cellular Physiology Jun 2024The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is attribute to the aggressive local invasion, distant metastasis and drug resistance of PDAC patients,...
The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is attribute to the aggressive local invasion, distant metastasis and drug resistance of PDAC patients, which was strongly accelerated by epithelial-mesenchymal transition (EMT). In current study, we systematically investigate the role of ZNF263/RNF126 axis in the initiation of EMT in PDAC in vitro and vivo. ZNF263 is firstly identified as a novel transactivation factor of RNF126. Both ZNF263 and RNF126 were overexpressed in PDAC tissues, which were associated with multiple advanced clinical stages and poor prognosis of PDAC patients. ZNF263 overexpression promoted cell proliferation, drug resistance and EMT in vitro via activating RNF126 following by the upregulation of Cyclin D1, N-cad, and MMP9, and the downregulation of E-cad, p21, and p27. ZNF263 silencing contributed to the opposite phenotype. Mechanistically, ZNF263 transactivated RNF126 via binding to its promoter. Further investigations revealed that ZNF263 interacted with ZNF31 to coregulate the transcription of RNF126, which in turn promoted ubiquitination-mediated degradation of PTEN. The downregulation of PTEN activated AKT/Cyclin D1 and AKT/GSK-3β/β-catenin signaling, thereby promoting the malignant phenotype of PDAC. Finally, the coordination of ZNF263 and RNF126 promotes subcutaneous tumor size and distant liver metastasis in vivo. ZNF263, as an oncogene, promotes proliferation, drug resistance and EMT of PDAC through transactivating RNF126.
Topics: Animals; Female; Humans; Male; Mice; Middle Aged; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Mice, Inbred BALB C; Mice, Nude; Pancreatic Neoplasms; PTEN Phosphohydrolase; Signal Transduction; Transcription Factors; Transcriptional Activation; Ubiquitin-Protein Ligases; RING Finger Domains; Zinc Fingers; Prognosis; Liver Neoplasms
PubMed: 38515383
DOI: 10.1002/jcp.31259