RESUMO
For accurate mitotic cell division, replicated chromatin must be assembled into chromosomes and faithfully segregated into daughter cells. While protein factors like condensin play key roles in this process, it is unclear how chromosome assembly proceeds as molecular events of nucleosomes in living cells and how condensins act on nucleosomes to organize chromosomes. To approach these questions, we investigate nucleosome behavior during mitosis of living human cells using single-nucleosome tracking, combined with rapid-protein depletion technology and computational modeling. Our results show that local nucleosome motion becomes increasingly constrained during mitotic chromosome assembly, which is functionally distinct from condensed apoptotic chromatin. Condensins act as molecular crosslinkers, locally constraining nucleosomes to organize chromosomes. Additionally, nucleosome-nucleosome interactions via histone tails constrain and compact whole chromosomes. Our findings elucidate the physical nature of the chromosome assembly process during mitosis.
Assuntos
Adenosina Trifosfatases , Cromatina , Proteínas de Ligação a DNA , Mitose , Complexos Multiproteicos , Nucleossomos , Humanos , Nucleossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Complexos Multiproteicos/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Células HeLa , Cromossomos Humanos/metabolismo , Cromossomos Humanos/genética , Cromossomos/metabolismoRESUMO
BACKGROUND: Obstructive sleep apnoea (OSA) is a sleep-disordered breathing characterized by intermittent hypoxia (IH) that may cause cognitive dysfunction. However, the impact of IH on molecular processes involved in cognitive function remains unclear. METHODS: C57BL / 6 J mice were exposed to either normoxia (control) or IH for 6 weeks. DNA hydroxymethylation was quantified by hydroxymethylated DNA immunoprecipitation (hMeDIP) sequencing. ten-eleven translocation 1 (Tet1) was knocked down by lentivirus. Specifically, cognitive function was assessed by behavioral experiments, pathological features were assessed by HE staining, the hippocampal DNA hydroxymethylation was examined by DNA dot blot and immunohistochemical staining, while the Wnt signaling pathway and its downstream effects were studied using qRT-PCR, immunofluorescence staining, and Luminex liquid suspension chip analysis. RESULTS: IH mice showed pathological changes and cognitive dysfunction in the hippocampus. Compared with the control group, IH mice exhibited global DNA hydroxylmethylation in the hippocampus, and the expression of three hydroxylmethylases increased significantly. The Wnt signaling pathway was activated, and the mRNA and 5hmC levels of Wnt3a, Ccnd2, and Prickle2 were significantly up-regulated. Further caused downstream neurogenesis abnormalities and neuroinflammatory activation, manifested as increased expression of IBA1 (a marker of microglia), GFAP (a marker of astrocytes), and DCX (a marker of immature neurons), as well as a range of inflammatory cytokines (e.g. TNFa, IL3, IL9, and IL17A). After Tet1 knocked down, the above indicators return to normal. CONCLUSION: Activation of Wnt signaling pathway by hippocampal Tet1 is associated with cognitive dysfunction induced by IH.
Assuntos
Disfunção Cognitiva , Hipocampo , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas , Apneia Obstrutiva do Sono , Via de Sinalização Wnt , Animais , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Via de Sinalização Wnt/fisiologia , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Disfunção Cognitiva/etiologia , Apneia Obstrutiva do Sono/metabolismo , Apneia Obstrutiva do Sono/complicações , Apneia Obstrutiva do Sono/patologia , Masculino , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Metilação de DNA , Proteínas de Ligação a DNARESUMO
Breast Cancer Type 1 Susceptibility Protein (BRCA1) is a tumor-suppressor protein that regulates various cellular pathways, including those that are essential for preserving genome stability. One essential mechanism involves a BRCA1-A complex that is recruited to double-strand breaks (DSBs) by RAP80 before initiating DNA damage repair (DDR). How RAP80 itself is recruited to DNA damage sites, however, is unclear. Here, we demonstrate an intrinsic correlation between a methyltransferase DOT1L-mediated RAP80 methylation and BRCA1-A complex chromatin recruitment that occurs during cancer cell radiotherapy resistance. Mechanistically, DOT1L is quickly recruited onto chromatin and methylates RAP80 at multiple lysines in response to DNA damage. Methylated RAP80 is then indispensable for binding to ubiquitinated H2A and subsequently triggering BRCA1-A complex recruitment onto DSBs. Importantly, DOT1L-catalyzed RAP80 methylation and recruitment of BRCA1 have clinical relevance, as inhibition of DOT1L or RAP80 methylation seems to enhance the radiosensitivity of cancer cells both in vivo and in vitro. These data reveal a crucial role for DOT1L in DDR through initiating recruitment of RAP80 and BRCA1 onto chromatin and underscore a therapeutic strategy based on targeting DOT1L to overcome tumor radiotherapy resistance.
Assuntos
Proteína BRCA1 , Reparo do DNA , Chaperonas de Histonas , Histona-Lisina N-Metiltransferase , Humanos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Metilação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Cromatina/metabolismo , Metiltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Animais , Feminino , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Camundongos , Tolerância a Radiação/genética , Metilação de DNARESUMO
Chronic obstructive pulmonary disease is a common chronic lung disease with an ever-increasing incidence. Despite years of drug research and approvals, we are still not able to halt progress or restore normal lung function. Our previous studies have demonstrated that liver growth factor-LGF has an effect on the repair of the affected tissue in a mouse model of cigarette smoke exposure, but by what pathways it achieves this is unknown. The present study aimed to identify differentially expressed genes between emphysematous mice treated with LGF to identify potential therapeutic targets for the treatment of pulmonary emphysema. The emphysema mouse model was induced by prolonged exposure to cigarette smoke. To determine the gene expression profile of the lung in smokers treated or not with LGF, lung messenger RNA gene expression was assessed with the Agilent Array platform. We carried out differentially expressed gene analysis, functional enrichment and validated in treated mouse lung samples. The treated group significantly improved lung function (~35%) and emphysema level (~20%), consistent with our previous published studies. Microarray analysis demonstrated 290 differentially expressed genes in total (2.0-fold over or lower expressed). Injury repair-associated genes and pathways were further enhanced in the lung of LGF treated mice. The expression trends of two genes (Zscan2 and Bag6) were different in emphysematous lungs treated with LGF compared to untreated lungs. Therefore, Zscan2 and Bag6 genes could play a role in regulating inflammation and the immune response in the lung that undergoes partial lung regeneration. However, further studies are necessary to demonstrate this causal relationship.
Assuntos
Modelos Animais de Doenças , Pulmão , Doença Pulmonar Obstrutiva Crônica , Fatores de Transcrição , Animais , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Camundongos , Pulmão/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Enfisema Pulmonar/genética , Enfisema Pulmonar/metabolismo , Perfilação da Expressão Gênica , Camundongos Endogâmicos C57BL , Masculino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
The cellular origin of clear cell ovarian carcinoma (CCOC), a major histological subtype of ovarian carcinoma remains elusive. Here, we explored the candidate cellular origin and identify molecular subtypes using integrated genomic/epigenomic analysis. We performed whole exome-sequencing, microarray, and DNA methylation array in 78 CCOC samples according to the original diagnosis. The findings revealed that ARID1A and/or PIK3CA mutations were mutually exclusive with DNA repair related genes, including TP53, BRCA1, and ATM. Clustering of CCOC and other ovarian carcinomas (n = 270) with normal tissues from the fallopian tube, ovarian surface epithelium, endometrial epithelium, and pelvic peritoneum mesothelium (PPM) in a methylation array showed that major CCOC subtypes (with ARID1A and/or PIK3CA mutations) were associated with the PPM-lile cluster (n = 64). This cluster was sub-divided into three clusters: (1) mismatch repair (MMR) deficient with tumor mutational burden-high (n = 2), (2) alteration of ARID1A (n = 51), and (3) ARID1A wild-type (n = 11). The remaining samples (n = 14) were subdivided into (4) ovarian surface epithelium-like (n = 11) and (5) fallopian tube-like (considered as high-grade serous histotype; n = 3). Among these, subtypes (1-3) and others (4 and 5) were found to be associated with immunoreactive signatures and epithelial-mesenchymal transition, respectively. These results contribute to the stratification of CCOC into biological subtypes.
Assuntos
Adenocarcinoma de Células Claras , Metilação de DNA , Proteínas de Ligação a DNA , Mutação , Neoplasias Ovarianas , Fatores de Transcrição , Humanos , Feminino , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/patologia , Genômica/métodos , Classe I de Fosfatidilinositol 3-Quinases/genética , Epigenômica/métodos , Sequenciamento do Exoma , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder with minimally effective treatment options. An important hurdle in ALS drug development is the non-invasive therapeutic access to the motor cortex currently limited by the presence of the blood-brain barrier (BBB). Focused ultrasound and microbubble (FUS+ MB) treatment is an emerging technology that was successfully used in ALS patients to temporarily open the cortical BBB. However, FUS+ MB-mediated drug delivery across ALS patients' BBB has not yet been reported. Similarly, the effects of FUS+ MB on human ALS BBB cells remain unexplored. METHODS: Here we established the first FUS+ MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+ MB bioeffects in vitro. RESULTS: Generated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including reduced BBB integrity and permeability, and TDP-43 proteinopathy. The results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with no adverse cellular effects of FUS+ MB as reflected by lactate dehydrogenase (LDH) release viability assay and the lack of visible monolayer damage or morphology change in FUS+ MB treated cells. This was accompanied by the molecular bioeffects of FUS+ MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+ MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+ MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model. CONCLUSIONS: Together, this study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+ MB and provides a fully-human platform for FUS+ MB-mediated drug delivery screening on an ALS BBB in vitro model.
Assuntos
Esclerose Lateral Amiotrófica , Barreira Hematoencefálica , Proteínas de Ligação a DNA , Microbolhas , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Humanos , Proteínas de Ligação a DNA/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Células Endoteliais/metabolismo , Anticorpos/administração & dosagem , Ondas Ultrassônicas , Células CultivadasRESUMO
DNA double-strand breaks (DSBs) must be repaired to ensure cell survival and genomic integrity. In yeast, the Mre11-Rad50-Xrs2 complex (MRX) collaborates with Sae2 to initiate DSB repair. Sae2 stimulates two MRX nuclease activities, endonuclease and 3'-5' exonuclease. However, how Sae2 controls the two nuclease activities remains enigmatic. Using a combined genetic and biochemical approach, we identified a separation-of-function rad50 mutation, rad50-C47, that causes a defect in Sae2-dependent MRX 3'-5' exonuclease activity, but not endonuclease activity. We found that both the endo- and 3'-5' exonuclease activities are essential to release Spo11 from DNA ends, whereas only the endonuclease activity is required for hairpin removal. We also uncovered that MRX-Sae2 endonuclease introduces a cleavage at defined distances from the Spo11-blocked end with gradually decreasing efficiency. Our findings demonstrate that Sae2 stimulates the MRX endo- and exonuclease activities via Rad50 by different mechanisms, ensuring diverse actions of MRX-Sae2 nuclease at DNA ends.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Endodesoxirribonucleases , Endonucleases , Exodesoxirribonucleases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Endonucleases/metabolismo , Endonucleases/genética , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Mutação , Reparo do DNA , DNA Fúngico/metabolismo , DNA Fúngico/genéticaRESUMO
BACKGROUND: Inflammatory diseases are often initiated by the activation of inflammasomes triggered by pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs), which mediate pyroptosis. Although pyroptosis resulting from aberrant inflammasome triggering in thyroid follicular cells (TFCs) has been observed in Hashimoto's thyroiditis (HT) patients, the underlying mechanisms remain largely unknown. Given the extensive involvement of protein ubiquitination and deubiquitination in inflammatory diseases, we aimed to investigate how deubiquitinating enzymes regulate thyroid follicular cell pyroptosis and HT pathogenesis. METHODS: Our study specifically investigated the role of Ubiquitin-specific peptidase 1 (USP1), a deubiquitinase (DUB), in regulating the inflammasome components NLRP3 and AIM2, which are crucial in pyroptosis. We conducted a series of experiments to elucidate the function of USP1 in promoting pyroptosis associated with inflammasomes and the progression of HT. These experiments involved techniques such as USP1 knockdown or inhibition, measurement of key pyroptosis indicators including caspase-1, caspase-1 p20, and GSDMD-N, and examination of the effects of USP1 abrogation on HT using a mouse model. Furthermore, we explored the impact of USP1 on NLRP3 transcription and its potential interaction with p65 nuclear transportation. RESULTS: Our findings provide compelling evidence indicating that USP1 plays a pivotal role in promoting inflammasome-mediated pyroptosis and HT progression by stabilizing NLRP3 and AIM2 through deubiquitination. Furthermore, we discovered that USP1 modulates the transcription of NLRP3 by facilitating p65 nuclear transportation. Knockdown or inhibition of USP1 resulted in weakened cell pyroptosis, as evidenced by reduced levels of caspase-1 p20 and GSDMD-N, which could be restored upon AIM2 overexpression. Remarkably, USP1 abrogation significantly ameliorated HT in the mice model, likely to that treating mice with pyroptosis inhibitors VX-765 and disulfiram. CONCLUSIONS: Our study highlights a regulatory mechanism of USP1 on inflammasome activation and pyroptosis in TFCs during HT pathogenesis. These findings expand our understanding of HT and suggest that inhibiting USP1 may be a potential treatment strategy for managing HT.
Assuntos
Doença de Hashimoto , Inflamassomos , Piroptose , Proteases Específicas de Ubiquitina , Animais , Inflamassomos/metabolismo , Camundongos , Doença de Hashimoto/metabolismo , Doença de Hashimoto/patologia , Humanos , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Progressão da Doença , Modelos Animais de Doenças , Glândula Tireoide/metabolismo , Glândula Tireoide/patologiaRESUMO
Human Hippo signaling pathway is an evolutionarily conserved regulator network that controls organ development and has been implicated in various cancers. Transcriptional enhanced associate domain-4 (TEAD4) is the final nuclear effector of Hippo pathway, which is activated by Yes-associated protein (YAP) through binding to two separated YAP regions of α1-helix and Ω-loop. Previous efforts have all been addressed on deriving peptide inhibitors from the YAP to target TEAD4. Instead, we herein attempted to rationally design a so-called 'YAP helixα1-trap' based on the TEAD4 to target YAP by using dynamics simulation and energetics analysis as well as experimental assays at molecular and cellular levels. The trap represents a native double-stranded helical hairpin covering a specific YAP-binding site on TEAD4 surface, which is expected to form a three-helix bundle with the α1-helical region of YAP, thus competitively disrupting TEAD4-YAP interaction. The hairpin was further stapled by a disulfide bridge across its two helical arms. Circular dichroism characterized that the stapling can effectively constrain the trap into a native-like structured conformation in free state, thus largely minimizing the entropy penalty upon its binding to YAP. Affinity assays revealed that the stapling can considerably improve the trap binding potency to YAP α1-helix by up to 8.5-fold at molecular level, which also exhibited a good tumor-suppressing effect at cellular level if fused with TAT cell permeation sequence. In this respect, it is considered that the YAP helixα1-trap-mediated blockade of Hippo pathway may be a new and promising therapeutic strategy against cancers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos , Proteínas de Ligação a DNA , Simulação de Dinâmica Molecular , Proteínas Musculares , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Proteínas de Sinalização YAP , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Humanos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dissulfetos/química , Dissulfetos/farmacologia , Ligação Proteica , Sítios de Ligação , Linhagem Celular Tumoral , Desenho Assistido por Computador , Desenho de FármacosRESUMO
BACKGROUND: Antitumor effect of chimeric antigen receptor (CAR)-T cells against solid tumors is limited due to various factors, such as low infiltration rate, poor expansion capacity, and exhaustion of T cells within the tumor. NR4A transcription factors have been shown to play important roles in T-cell exhaustion in mice. However, the precise contribution of each NR4a factor to human T-cell differentiation remains to be clarified. METHODS: In this study, we deleted NR4A family factors, NR4A1, NR4A2, and NR4A3, in human CAR-T cells recognizing human epidermal growth factor receptor type 2 (HER2) by using the CRISPR/Cas9 system. We induced T-cell exhaustion in these cells in vitro through repeated co-culturing of CAR-T cells with Her2+A549 lung adenocarcinoma cells and evaluated cell surface markers such as memory and exhaustion phenotypes, proliferative capacity, cytokine production and metabolic activity. We validated the antitumor toxicity of NR4A1/2/3 triple knockout (TKO) CAR-T cells in vivo by transferring CAR-T cells into A549 tumor-bearing immunodeficient mice. RESULTS: Human NR4A-TKO CAR-T cells were resistant against exhaustion induced by repeated antigen stimulation in vitro, and maintained higher tumor-killing activity both in vitro and in vivo compared with control CAR-T cells. A comparison of the effectiveness of NR4A single, double, and TKOs demonstrated that triple KO was the most effective in avoiding exhaustion. Furthermore, a strong enhancement of antitumor effects by NR4A TKO was also observed in T cells from various donors including aged persons. Mechanistically, NR4A TKO CAR-T cells showed enhanced mitochondrial oxidative phosphorylation, therefore could persist for longer periods within the tumors. CONCLUSIONS: NR4A factors regulate CAR-T cell persistence and stemness through mitochondrial gene expression, therefore NR4A is a highly promising target for the generation of superior CAR-T cells against solid tumors.
Assuntos
Imunoterapia Adotiva , Mitocôndrias , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , Mitocôndrias/metabolismo , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Receptores dos Hormônios Tireóideos/metabolismo , Receptores dos Hormônios Tireóideos/genética , Neoplasias/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Feminino , Proteínas de Ligação a DNA , Receptores de EsteroidesRESUMO
Heterochromatin de-condensation in companion gametic cells is conserved in both plants and animals. In plants, microspore undergoes asymmetric pollen mitosis (PMI) to produce a vegetative cell (VC) and a generative cell (GC). Subsequently, the GC undergoes pollen mitosis (PMII) to produce two sperm cells (SC). Consistent with heterochromatin de-condensation in the VC, H3K9me2, a heterochromatin mark, is barely detected in VC. However, how H3K9me2 is differentially regulated during pollen mitosis remains unclear. Here, we show that H3K9me2 is gradually evicted from the VC since PMI but remain unchanged in the GC and SC. ARID1, a pollen-specific transcription factor that facilitates PMII, promotes H3K9me2 maintenance in the GC/SC but slows down its eviction in the VC. The genomic targets of ARID1 mostly overlaps with H3K9me2 loci, and ARID1 recruits H3K9 methyltransferase SUVH6. Our results uncover that differential pattern of H3K9me2 between two cell types is regulated by ARID1 during pollen mitosis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Histonas , Mitose , Pólen , Fatores de Transcrição , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Histonas/genética , Metilação , Pólen/metabolismo , Pólen/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
DSS1, essential for BRCA2-RAD51 dependent homologous recombination (HR), associates with the helical domain (HD) and OB fold 1 (OB1) of the BRCA2 DSS1/DNA-binding domain (DBD) which is frequently targeted by cancer-associated pathogenic variants. Herein, we reveal robust ss/dsDNA binding abilities in HD-OB1 subdomains and find that DSS1 shuts down HD-OB1's DNA binding to enable ssDNA targeting of the BRCA2-RAD51 complex. We show that C-terminal helix mutations of DSS1, including the cancer-associated R57Q mutation, disrupt this DSS1 regulation and permit dsDNA binding of HD-OB1/BRCA2-DBD. Importantly, these DSS1 mutations impair BRCA2/RAD51 ssDNA loading and focus formation and cause decreased HR efficiency, destabilization of stalled forks and R-loop accumulation, and hypersensitize cells to DNA-damaging agents. We propose that DSS1 restrains the intrinsic dsDNA binding of BRCA2-DBD to ensure BRCA2/RAD51 targeting to ssDNA, thereby promoting optimal execution of HR, and potentially replication fork protection and R-loop suppression.
Assuntos
Proteína BRCA2 , Replicação do DNA , DNA de Cadeia Simples , DNA , Recombinação Homóloga , Mutação , Rad51 Recombinase , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/química , Humanos , DNA/metabolismo , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Homeostase , Ligação Proteica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Domínios Proteicos , Linhagem Celular Tumoral , Dano ao DNA , Complexo de Endopeptidases do ProteassomaRESUMO
OBJECTIVES: The present study used single-cell RNA sequencing (scRNA-seq) to characterize the cellular composition of ovarian carcinosarcoma (OCS) and identify its molecular characteristics. METHODS: scRNA-seq was performed in resected primary OCS for an in-depth analysis of tumor cells and the tumor microenvironment. Immunohistochemistry staining was used for validation. The scRNA-seq data of OCS were compared with those of high-grade serous ovarian carcinoma (HGSOC) tumors and other OCS tumors. RESULTS: Both malignant epithelial and malignant mesenchymal cells were observed in the OCS patient of this study. We identified four epithelial cell subclusters with different biological roles. Among them, epithelial subcluster 4 presented high levels of breast cancer type 1 susceptibility protein homolog (BRCA1) and DNA topoisomerase 2-α (TOP2A) expression and was related to drug resistance and cell cycle. We analyzed the interaction between epithelial and mesenchymal cells and found that fibroblast growth factor (FGF) and pleiotrophin (PTN) signalings were the main pathways contributing to communication between these cells. Moreover, we compared the malignant epithelial and mesenchymal cells of this OCS tumor with our previous published HGSOC scRNA-seq data and OCS data. All the epithelial subclusters in the OCS tumor could be found in the HGSOC samples. Notably, the mesenchymal subcluster C14 exhibited specific expression patterns in the OCS tumor, characterized by elevated expression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1), collagen type XXIII α1 chain (COL23A1), cholecystokinin (CCK), bone morphogenetic protein 7 (BMP7), PTN, Wnt inhibitory factor 1 (WIF1), and insulin-like growth factor 2 (IGF2). Moreover, this subcluster showed distinct characteristics when compared with both another previously published OCS tumor and normal ovarian tissue. CONCLUSIONS: This study provides the single-cell transcriptomics signature of human OCS, which constitutes a new resource for elucidating OCS diversity.
Assuntos
Carcinossarcoma , DNA Topoisomerases Tipo II , Neoplasias Ovarianas , Análise de Célula Única , Transcriptoma , Humanos , Feminino , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Carcinossarcoma/genética , Carcinossarcoma/metabolismo , Carcinossarcoma/patologia , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Microambiente Tumoral , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Citocinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Análise de Sequência de RNA , Pessoa de Meia-Idade , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/metabolismo , Proteínas de Ligação a Poli-ADP-RiboseRESUMO
The histone-like nucleoid structuring (H-NS) protein is a DNA binding factor, found in gammaproteobacteria, with functional equivalents in diverse microbes. Universally, such proteins are understood to silence transcription of horizontally acquired genes. Here, we identify transposon capture as a major overlooked function of H-NS. Using genome-scale approaches, we show that H-NS bound regions are transposition "hotspots". Since H-NS often interacts with pathogenicity islands, such targeting creates clinically relevant phenotypic diversity. For example, in Acinetobacter baumannii, we identify altered motility, biofilm formation, and interactions with the human immune system. Transposon capture is mediated by the DNA bridging activity of H-NS and, if absent, more ubiquitous transposition results. Consequently, transcribed and essential genes are disrupted. Hence, H-NS directs transposition to favour evolutionary outcomes useful for the host cell.
Assuntos
Acinetobacter baumannii , Proteínas de Bactérias , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA , Elementos de DNA Transponíveis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Humanos , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Genoma Bacteriano , Ilhas GenômicasRESUMO
There are currently no available FDA-cleared biodosimetry tools for rapid and accurate assessment of absorbed radiation dose following a radiation/nuclear incident. Previously we developed a protein biomarker-based FAST-DOSE bioassay system for biodosimetry. The aim of this study was to integrate an ELISA platform with two high-performing FAST-DOSE biomarkers, BAX and DDB2, and to construct machine learning models that employ a multiparametric biomarker strategy for enhancing the accuracy of exposure classification and radiation dose prediction. The bioassay showed 97.92% and 96% accuracy in classifying samples in human and non-human primate (NHP) blood samples exposed ex vivo to 0-5 Gy X-rays, respectively up to 48 h after exposure, and an adequate correlation between reconstructed and actual dose in the human samples (R2 = 0.79, RMSE = 0.80 Gy, and MAE = 0.63 Gy) and NHP (R2 = 0.80, RMSE = 0.78 Gy, and MAE = 0.61 Gy). Biomarker measurements in vivo from four NHPs exposed to a single 2.5 Gy total body dose showed a persistent upregulation in blood samples collected on days 2 and 5 after irradiation. The data indicates that using a combined approach of targeted proteins can increase bioassay sensitivity and provide a more accurate dose prediction.
Assuntos
Biomarcadores , Proteínas de Ligação a DNA , Proteína X Associada a bcl-2 , Animais , Humanos , Biomarcadores/sangue , Proteínas de Ligação a DNA/sangue , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/sangue , Exposição à Radiação/efeitos adversos , Masculino , Radiometria/métodos , Macaca mulatta , Feminino , Aprendizado de Máquina , Doses de RadiaçãoRESUMO
Histone methylation can affect chromosome structure and binding to other proteins, depending on the type of amino acid being modified and the number of methyl groups added, this modification may promote transcription of genes (H3K4me2, H3K4me3, and H3K79me3) or reduce transcription of genes (H3K9me2, H3K9me3, H3K27me2, H3K27me3, and H4K20me3). In addition, advances in tumor immunotherapy have shown that histone methylation as a type of protein post-translational modification is also involved in the proliferation, activation and metabolic reprogramming of immune cells in the tumor microenvironment. These post-translational modifications of proteins play a crucial role in regulating immune escape from tumors and immunotherapy. Lysine methyltransferases are important components of the post-translational histone methylation modification pathway. Lysine methyltransferase 2C (KMT2C), also known as MLL3, is a member of the lysine methyltransferase family, which mediates the methylation modification of histone 3 lysine 4 (H3K4), participates in the methylation of many histone proteins, and regulates a number of signaling pathways such as EMT, p53, Myc, DNA damage repair and other pathways. Studies of KMT2C have found that it is aberrantly expressed in many diseases, mainly tumors and hematological disorders. It can also inhibit the onset and progression of these diseases. Therefore, KMT2C may serve as a promising target for tumor immunotherapy for certain diseases. Here, we provide an overview of the structure of KMT2C, disease mechanisms, and diseases associated with KMT2C, and discuss related challenges.
Assuntos
Neoplasias , Humanos , Neoplasias/imunologia , Neoplasias/terapia , Metilação , Processamento de Proteína Pós-Traducional , Animais , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Microambiente Tumoral/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
Mammalian TIP60 is a multi-functional enzyme with histone acetylation and histone dimer exchange activities. It plays roles in diverse cellular processes including transcription, DNA repair, cell cycle control, and embryonic development. Here we report the cryo-electron microscopy structures of the human TIP60 complex with the core subcomplex and TRRAP module refined to 3.2-Å resolution. The structures show that EP400 acts as a backbone integrating the motor module, the ARP module, and the TRRAP module. The RUVBL1-RUVBL2 hexamer serves as a rigid core for the assembly of EP400 ATPase and YL1 in the motor module. In the ARP module, an ACTL6A-ACTB heterodimer and an extra ACTL6A make hydrophobic contacts with EP400 HSA helix, buttressed by network interactions among DMAP1, EPC1, and EP400. The ARP module stably associates with the motor module but is flexibly tethered to the TRRAP module, exhibiting a unique feature of human TIP60. The architecture of the nucleosome-bound human TIP60 reveals an unengaged nucleosome that is located between the core subcomplex and the TRRAP module. Our work illustrates the molecular architecture of human TIP60 and provides architectural insights into how this complex is bound by the nucleosome.
Assuntos
Microscopia Crioeletrônica , Lisina Acetiltransferase 5 , Humanos , Lisina Acetiltransferase 5/metabolismo , Lisina Acetiltransferase 5/química , Lisina Acetiltransferase 5/genética , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Nucleossomos/química , DNA Helicases/metabolismo , DNA Helicases/química , Modelos Moleculares , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Ligação Proteica , Multimerização Proteica , Proteínas que Contêm Bromodomínio , Proteínas Adaptadoras de Transdução de SinalRESUMO
The integration of extrinsic signaling with cell-intrinsic transcription factors can direct progenitor cells to differentiate into distinct cell fates. In the developing Drosophila eye, differentiation of photoreceptors R1-R7 requires EGFR signaling mediated by the transcription factor Pointed, and our single-cell RNA-Seq analysis shows that the same photoreceptors require the eye-specific transcription factor Glass. We find that ectopic expression of Glass and activation of EGFR signaling synergistically induce neuronal gene expression in the wing disc in a Pointed-dependent manner. Targeted DamID reveals that Glass and Pointed share many binding sites in the genome of developing photoreceptors. Comparison with transcriptomic data shows that Pointed and Glass induce photoreceptor differentiation through intermediate transcription factors, including the redundant homologs Scratch and Scrape, as well as directly activating neuronal effector genes. Our data reveal synergistic activation of a multi-layered transcriptional network as the mechanism by which EGFR signaling induces neuronal identity in Glass-expressing cells.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Drosophila , Drosophila melanogaster , Receptores ErbB , Regulação da Expressão Gênica no Desenvolvimento , Neurônios , Transdução de Sinais , Fatores de Transcrição , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Neurônios/metabolismo , Neurônios/citologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Diferenciação Celular , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/citologia , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Discos Imaginais/metabolismo , Discos Imaginais/citologia , Proteínas do Tecido Nervoso , Proteínas Proto-Oncogênicas , Receptores de Peptídeos de InvertebradosRESUMO
The E-twenty-six variant 1 (ETV1)-dependent transcriptome plays an important role in atrial electrical and structural remodelling and the occurrence of atrial fibrillation (AF), but the underlying mechanism of ETV1 in AF is unclear. In this study, cardiomyocyte-specific ETV1 knockout (ETV1f/fMyHCCre/+, ETV1-CKO) mice were constructed to observe the susceptibility to AF and the underlying mechanism in AF associated with ETV1-CKO mice. AF susceptibility was examined by intraesophageal burst pacing, induction of AF was increased obviously in ETV1-CKO mice than WT mice. Electrophysiology experiments indicated shortened APD50 and APD90, increased incidence of DADs, decreased density of ICa,L in ETV1-CKO mice. There was no difference in VINACT,1/2 and VACT,1/2, but a significantly longer duration of the recovery time after inactivation in the ETV1-CKO mice. The recording of intracellular Ca2+ showed that there was significantly increased in the frequency of calcium spark, Ca2+ transient amplitude, and proportion of SCaEs in ETV1-CKO mice. Reduction of Cav1.2 rather than NCX1 and SERCA2a, increase RyR2, p-RyR2 and CaMKII was reflected in ETV1-CKO group. This study demonstrates that the increase in calcium spark and SCaEs corresponding to Ca2+ transient amplitude may trigger DAD in membrane potential in ETV1-CKO mice, thereby increasing the risk of AF.
Assuntos
Fibrilação Atrial , Cálcio , Átrios do Coração , Camundongos Knockout , Miócitos Cardíacos , Fatores de Transcrição , Animais , Miócitos Cardíacos/metabolismo , Camundongos , Fibrilação Atrial/metabolismo , Fibrilação Atrial/genética , Cálcio/metabolismo , Átrios do Coração/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Sinalização do Cálcio , Potenciais de Ação , Potenciais da Membrana , MasculinoRESUMO
The retina is increasingly recognised as a potential source of biomarkers for neurodegenerative diseases. Hallmark protein aggregates in the retinal neuronal tissue could be imaged through light non-invasively. Post-mortem studies have already shown the presence of specific hallmark proteins in Alzheimer's disease, primary tauopathies, synucleinopathies and frontotemporal lobar degeneration. This study aims to assess proteinopathy in a post-mortem cohort with different neurodegenerative diseases and assess the presence of the primary pathology in the retina. Post-mortem eyes were collected in collaboration with the Netherlands Brain Bank from donors with Alzheimer's disease (n = 17), primary tauopathies (n = 8), synucleinopathies (n = 27), frontotemporal lobar degeneration (n = 8), mixed pathology (n = 11), other neurodegenerative diseases (n = 6), and cognitively normal controls (n = 25). Multiple cross sections of the retina and optic nerve tissue were immunostained using antibodies against pTau Ser202/Thr205 (AT8), amyloid-beta (4G8), alpha-synuclein (LB509), pTDP-43 Ser409/410 and p62-lck ligand (p62) and were assessed for the presence of aggregates and inclusions. pTau pathology was observed as a diffuse signal in Alzheimer's disease, primary tauopathies and controls with Alzheimer's disease neuropathological changes. Amyloid-beta was observed in the vessel wall and as cytoplasmic granular deposits in all groups. Alpha-synuclein pathology was observed as Lewy neurites in the retina in synucleinopathies associated with Lewy pathology and as oligodendroglial cytoplasmic inclusions in the optic nerve in multiple system atrophy. Anti-pTDP-43 generally showed typical neuronal cytoplasmic inclusion bodies in cases with frontotemporal lobar degeneration with TDP-43 and also in cases with later stages of limbic-associated TDP-43 encephalopathy. P62 showed inclusion bodies similar to those seen with anti-pTDP-43. Furthermore, pTau and alpha-synuclein pathology were significantly associated with increasing Braak stages for neurofibrillary tangles and Lewy bodies, respectively. Mixed pathology cases in this cohort consisted of cases (n = 6) with high Braak LB stages (> 4) and low or moderate AD pathology, high AD pathology (n = 1, Braak NFT 6, Thal phase 5) with moderate LB pathology, or a combination of low/moderate scores for different pathology scores in the brain (n = 4). There were no cases with advanced co-pathologies. In seven cases with Braak LB ≥ 4, LB pathology was observed in the retina, while tau pathology in the retina in the mixed pathology group (n = 11) could not be observed. From this study, we conclude that the retina reflects the presence of the major hallmark proteins associated with neurodegenerative diseases. Although low or moderate levels of copathology were found in the brains of most cases, the retina primarily manifested protein aggregates associated with the main neurodegenerative disease. These findings indicate that with appropriate retinal imaging techniques, retinal biomarkers have the potential to become highly accurate indicators for diagnosing the major neurodegenerative diseases of the brain.