The RNA content is crucial for the formation of nuclear compartments, such as nuclear speckles and nucleoli. Phosphatidylinositol 4,5-bisphosphate (PIP2) is found in nuclear speckles, nucleoli, and nuclear lipid islets and is involved in RNA polymerase I/II transcription. Intriguingly, the nuclear localization of PIP2 was also shown to be RNA-dependent. We therefore investigated whether PIP2 and RNA cooperate in the establishment of nuclear architecture. In this study, we unveiled the RNA-dependent PIP2-associated (RDPA) nuclear proteome in human cells by mass spectrometry. We found that intrinsically disordered regions (IDRs) with polybasic PIP2-binding K/R motifs are prevalent features of RDPA proteins. Moreover, these IDRs of RDPA proteins exhibit enrichment for phosphorylation, acetylation, and ubiquitination sites. Our results show for the first time that the RDPA protein Bromodomain-containing protein 4 (BRD4) associates with PIP2 in the RNA-dependent manner via electrostatic interactions, and that altered PIP2 levels affect the number of nuclear foci of BRD4 protein. Thus, we propose that PIP2 spatiotemporally orchestrates nuclear processes through association with RNA and RDPA proteins and affects their ability to form foci presumably via phase separation. This suggests the pivotal role of PIP2 in the establishment of a functional nuclear architecture competent for gene expression.
- MeSH
- buněčné jádro * metabolismus genetika MeSH
- fosfatidylinositol-4,5-difosfát * metabolismus MeSH
- fosforylace MeSH
- jaderné proteiny * metabolismus genetika MeSH
- lidé MeSH
- proteiny buněčného cyklu metabolismus genetika MeSH
- proteiny obsahující bromodoménu MeSH
- RNA metabolismus genetika MeSH
- transkripční faktory * metabolismus genetika MeSH
- vazba proteinů MeSH
- vnitřně neuspořádané proteiny * metabolismus genetika chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Acute lymphoblastic leukemia expressing the gamma delta T-cell receptor (γδ T-ALL) is a poorly understood disease. We studied 200 children with γδ T-ALL from 13 clinical study groups to understand the clinical and genetic features of this disease. We found age and genetic drivers were significantly associated with outcome. γδ T-ALL diagnosed in children under 3 years of age was extremely high-risk and enriched for genetic alterations that result in both LMO2 activation and STAG2 inactivation. Mechanistically, using patient samples and isogenic cell lines, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping, resulting in deregulation of gene expression associated with T-cell differentiation. High-throughput drug screening identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which can be targeted by poly(ADP-ribose) polymerase inhibition. These data provide a diagnostic framework for classification and risk stratification of pediatric γδ T-ALL. Significance: Patients with acute lymphoblastic leukemia expressing the gamma delta T-cell receptor under 3 years old or measurable residual disease ≥1% at end of induction showed dismal outcomes and should be classified as having high-risk disease. The STAG2/LMO2 subtype was enriched in this very young age group. STAG2 inactivation may perturb chromatin conformation and cell differentiation and confer vulnerability to poly(ADP-ribose) polymerase inhibition.
- MeSH
- adaptorové proteiny signální transdukční * genetika metabolismus MeSH
- dítě MeSH
- genová přestavba MeSH
- kojenec MeSH
- lidé MeSH
- lymfoblastická leukemie-lymfom z prekurzorových T-buněk genetika patologie MeSH
- předškolní dítě MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- proteiny s doménou LIM * genetika MeSH
- protoonkogenní proteiny MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
RAD18 is an E3 ubiquitin ligase that prevents replication fork collapse by promoting DNA translesion synthesis and template switching. Besides this classical role, RAD18 has been implicated in homologous recombination; however, this function is incompletely understood. Here, we show that RAD18 is recruited to DNA lesions by monoubiquitination of histone H2A at K15 and counteracts accumulation of 53BP1. Super-resolution microscopy revealed that RAD18 localizes to the proximity of DNA double strand breaks and limits the distribution of 53BP1 to the peripheral chromatin nanodomains. Whereas auto-ubiquitination of RAD18 mediated by RAD6 inhibits its recruitment to DNA breaks, interaction with SLF1 promotes RAD18 accumulation at DNA breaks in the post-replicative chromatin by recognition of histone H4K20me0. Surprisingly, suppression of 53BP1 function by RAD18 is not involved in homologous recombination and rather leads to reduction of non-homologous end joining. Instead, we provide evidence that RAD18 promotes HR repair by recruiting the SMC5/6 complex to DNA breaks. Finally, we identified several new loss-of-function mutations in RAD18 in cancer patients suggesting that RAD18 could be involved in cancer development.
- MeSH
- 53BP1 * metabolismus genetika MeSH
- chromatin * metabolismus genetika MeSH
- DNA vazebné proteiny * metabolismus genetika MeSH
- dvouřetězcové zlomy DNA * MeSH
- histony * metabolismus MeSH
- homologní rekombinace genetika MeSH
- lidé MeSH
- oprava DNA spojením konců MeSH
- oprava DNA MeSH
- proteiny buněčného cyklu metabolismus genetika MeSH
- rekombinační oprava DNA MeSH
- replikace DNA MeSH
- ubikvitinace * MeSH
- ubikvitinligasy * metabolismus genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Psoriasis is a chronic non-contagious autoimmune disease. Gallic acid is a natural compound with potential health benefits, including antioxidant, anticancer, antiviral and antibacterial properties. Nevertheless, the influence of gallic acid on psoriasis has not been fully determined. This investigation aimed to discover the effect of gallic acid on psoriasis. Thirty-one pairs of psoriatic skin tissues and healthy adult human skin tissues were collected. Human keratinocytes (HaCaT cells) were transfected with interleukin 17A (IL-17A) to create the psoriatic keratinocyte model. The content of bromodomain-containing protein 4 (BRD4) microRNA was assessed using qRT-PCR testing. The content of BRD4 was detected by Western blotting. Cell migration was evaluated by conducting a wound healing assay. Cell proliferation was determined using an EdU assay. Apoptosis was detected by the TUNEL assay. The contents of interferon gamma (IFN-γ), IL-6, IL-8 and IL-17 were detected by ELISA. BRD4 was up-regulated in psoriatic skin tissues and in the IL-17A group compared to the healthy adult human skin tissues and the control group. Silencing BRD4 inhibited cell migration, proliferation and inflammatory response but induced apoptosis in IL-17A-treated HaCaT cells. Conversely, BRD4 over-expression promoted cell migration, proliferation and inflammatory response but suppressed apoptosis in IL-17A-treated HaCaT cells. Gallic acid repressed cell migration, proliferation and inflammatory response but indu-ced apoptosis in HaCaT cells transfected with IL-17A by down-regulating BRD4. Gallic acid represses cell migration, proliferation and inflammatory response but induces apoptosis in IL-17A-transfected HaCaT cells by down-regulating BRD4.
- MeSH
- apoptóza * účinky léků MeSH
- buněčné linie keratinocytů HaCaT MeSH
- buněčné linie MeSH
- dospělí MeSH
- interleukin-17 metabolismus MeSH
- jaderné proteiny metabolismus genetika MeSH
- keratinocyty * účinky léků metabolismus MeSH
- kyselina gallová * farmakologie MeSH
- lidé MeSH
- mikro RNA genetika metabolismus MeSH
- pohyb buněk * účinky léků MeSH
- proliferace buněk * účinky léků MeSH
- proteiny buněčného cyklu * metabolismus genetika MeSH
- proteiny obsahující bromodoménu MeSH
- psoriáza * metabolismus patologie farmakoterapie MeSH
- regulace genové exprese účinky léků MeSH
- transkripční faktory * metabolismus MeSH
- zánět * patologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Disruption of cell division cycle associated 7 (CDCA7) has been linked to aberrant DNA hypomethylation, but the impact of DNA methylation loss on transcription has not been investigated. Here, we show that CDCA7 is critical for maintaining global DNA methylation levels across multiple tissues in vivo. A pathogenic Cdca7 missense variant leads to the formation of large, aberrantly hypomethylated domains overlapping with the B genomic compartment but without affecting the deposition of H3K9 trimethylation (H3K9me3). CDCA7-associated aberrant DNA hypomethylation translated to localized, tissue-specific transcriptional dysregulation that affected large gene clusters. In the brain, we identify CDCA7 as a transcriptional repressor and epigenetic regulator of clustered protocadherin isoform choice. Increased protocadherin isoform expression frequency is accompanied by DNA methylation loss, gain of H3K4 trimethylation (H3K4me3), and increased binding of the transcriptional regulator CCCTC-binding factor (CTCF). Overall, our in vivo work identifies a key role for CDCA7 in safeguarding tissue-specific expression of gene clusters via the DNA methylation pathway.
- MeSH
- DNA MeSH
- jaderné proteiny * metabolismus MeSH
- metylace DNA MeSH
- myši MeSH
- protein - isoformy genetika MeSH
- proteiny buněčného cyklu * metabolismus MeSH
- represorové proteiny genetika MeSH
- transkripční faktory genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The human homologues of murine double minute 2 (MDM2) and 4 (MDM4) negatively regulate p53 tumour suppressor activity and are reported to be frequently overexpressed in human malignancies, prompting clinical trials with drugs that prevent interactions between MDM2/MDM4 and p53. Bone marrow samples from 111 patients with acute myeloblastic leukaemia, myelodysplastic syndrome or chronic myelomonocytic leukaemia were examined for protein (fluorescence-activated cell sorting) and messenger RNA (mRNA) expression (quantitative polymerase chain reaction) of MDM2, MDM4 and tumour protein p53 (TP53). Low protein expression of MDM2 and MDM4 was observed in immature cells from patients with excess of marrow blasts (>5%) compared with CD34+ /CD45low cells from healthy donors and patients without excess of marrow blasts (<5%). The mRNA levels were indistinguishable in all samples examined regardless of disease status or blast levels. Low MDM2 and MDM4 protein expression were correlated with poor survival. These data show a poor correlation between mRNA and protein expression levels, suggesting that quantitative flow cytometry analysis of protein expression levels should be used to predict and validate the efficacy of MDM2 and MDM4 inhibitors. These findings show that advanced disease is associated with reduced MDM2 and MDM4 protein expression and indicate that the utility of MDM2 and MDM4 inhibitors may have to be reconsidered in the treatment of advanced myeloid malignancies.
- MeSH
- akutní myeloidní leukemie * genetika MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- myelodysplastické syndromy * genetika MeSH
- myši MeSH
- nádorový supresorový protein p53 genetika MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- protoonkogenní proteiny c-mdm2 genetika metabolismus MeSH
- protoonkogenní proteiny genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although many efforts have been made to improve management strategies and diagnostic methods in the past several decades, the prevention of anastomotic complications, such as anastomotic leaks and strictures, remain a major clinical challenge. Therefore, new molecular pathways need to be identified that regulate anastomotic healing, and to design new treatments for patients after anastomosis to reduce the occurrence of complications. Rabbits were treated with a MST1/2 inhibitor XMU-XP-1, a Chinese medicine formula Shenhuang plaster (SHP) or a control vehicle immediately after surgery. The anastomotic burst pressure, collagen deposition, and hydroxyproline concentration were evaluated at 3 and 7 days after the surgery, and qRT-PCR and western-blot analyses were used to characterize mRNA and protein expression levels. Both XMU-XP-1 and SHP significantly increased anastomotic burst pressure, collagen deposition, and the concentration of hydroxyproline in intestinal anastomotic tissue at postoperative day 7 (POD 7). Importantly, SHP could induce TGF-β1 expression, which activated its downstream target Smad-2 to activate the TGF-β1 signaling pathway. Moreover, SHP reduced the phosphorylation level of YAP and increased its active form, and treatment with verteporfin, a YAP-TEAD complex inhibitor, significantly suppressed the effects induced by SHP during anastomotic tissue healing. This study demonstrated that activation of the Hippo-YAP pathway enhances anastomotic healing, and that SHP enhances both the TGF-β1/Smad and YAP signaling pathways to promote rabbit anastomotic healing after surgery. These results suggest that SHP could be used to treat patients who underwent anastomosis to prevent the occurrence of anastomotic complications.
- MeSH
- anastomóza chirurgická MeSH
- hydroxyprolin farmakologie MeSH
- kolagen farmakologie MeSH
- králíci MeSH
- Lagomorpha * metabolismus MeSH
- lidé MeSH
- proteiny buněčného cyklu metabolismus farmakologie MeSH
- signální transdukce MeSH
- transformující růstový faktor beta * metabolismus farmakologie MeSH
- transformující růstový faktor beta1 farmakologie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
An elevated frequency of DNA replication defects is associated with diabetes and cancer. However, data linking these nuclear perturbations to the onset or progression of organ complications remained unexplored. Here, we report that RAGE (Receptor for Advanced Glycated Endproducts), previously believed to be an extracellular receptor, upon metabolic stress localizes to the damaged forks. There it interacts and stabilizes the minichromosome-maintenance (Mcm2-7) complex. Accordingly, RAGE deficiency leads to slowed fork progression, premature fork collapse, hypersensitivity to replication stress agents and reduction of viability, which was reversed by the reconstitution of RAGE. This was marked by the 53BP1/OPT-domain expression and the presence of micronuclei, premature loss-of-ciliated zones, increased incidences of tubular-karyomegaly, and finally, interstitial fibrosis. More importantly, the RAGE-Mcm2 axis was selectively compromised in cells expressing micronuclei in human biopsies and mouse models of diabetic nephropathy and cancer. Thus, the functional RAGE-Mcm2/7 axis is critical in handling replication stress in vitro and human disease.
- MeSH
- diabetes mellitus * MeSH
- lidé MeSH
- MCM komplex, komponenta 2 genetika MeSH
- MCM proteiny metabolismus MeSH
- myši MeSH
- nádory * MeSH
- proteiny buněčného cyklu metabolismus MeSH
- replikace DNA genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The MRE11, RAD50, and NBN genes encode for the nuclear MRN protein complex, which senses the DNA double strand breaks and initiates the DNA repair. The MRN complex also participates in the activation of ATM kinase, which coordinates DNA repair with the p53-dependent cell cycle checkpoint arrest. Carriers of homozygous germline pathogenic variants in the MRN complex genes or compound heterozygotes develop phenotypically distinct rare autosomal recessive syndromes characterized by chromosomal instability and neurological symptoms. Heterozygous germline alterations in the MRN complex genes have been associated with a poorly-specified predisposition to various cancer types. Somatic alterations in the MRN complex genes may represent valuable predictive and prognostic biomarkers in cancer patients. MRN complex genes have been targeted in several next-generation sequencing panels for cancer and neurological disorders, but interpretation of the identified alterations is challenging due to the complexity of MRN complex function in the DNA damage response. In this review, we outline the structural characteristics of the MRE11, RAD50 and NBN proteins, the assembly and functions of the MRN complex from the perspective of clinical interpretation of germline and somatic alterations in the MRE11, RAD50 and NBN genes.
- MeSH
- ATM protein genetika metabolismus MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- enzymy opravy DNA genetika metabolismus MeSH
- homologní protein MRE11 genetika metabolismus MeSH
- hydrolasy působící na anhydridy kyselin genetika metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- lidé MeSH
- nádorové supresorové proteiny * genetika MeSH
- oprava DNA genetika MeSH
- proteiny buněčného cyklu * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Receiving complete and undamaged genetic information is vital for the survival of daughter cells after chromosome segregation. The most critical steps in this process are accurate DNA replication during S phase and a faithful chromosome segregation during anaphase. Any errors in DNA replication or chromosome segregation have dire consequences, since cells arising after division might have either changed or incomplete genetic information. Accurate chromosome segregation during anaphase requires a protein complex called cohesin, which holds together sister chromatids. This complex unifies sister chromatids from their synthesis during S phase, until separation in anaphase. Upon entry into mitosis, the spindle apparatus is assembled, which eventually engages kinetochores of all chromosomes. Additionally, when kinetochores of sister chromatids assume amphitelic attachment to the spindle microtubules, cells are finally ready for the separation of sister chromatids. This is achieved by the enzymatic cleavage of cohesin subunits Scc1 or Rec8 by an enzyme called Separase. After cohesin cleavage, sister chromatids remain attached to the spindle apparatus and their poleward movement on the spindle is initiated. The removal of cohesion between sister chromatids is an irreversible step and therefore it must be synchronized with assembly of the spindle apparatus, since precocious separation of sister chromatids might lead into aneuploidy and tumorigenesis. In this review, we focus on recent discoveries concerning the regulation of Separase activity during the cell cycle.
