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1.
Nat Commun ; 10(1): 2908, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266948

RESUMO

Cohesin and CTCF are master regulators of genome topology. How these ubiquitous proteins contribute to cell-type specific genome structure is poorly understood. Here, we explore quantitative aspects of topologically associated domains (TAD) between pluripotent embryonic stem cells (ESC) and lineage-committed cells. ESCs exhibit permissive topological configurations which manifest themselves as increased inter- TAD interactions, weaker intra-TAD interactions, and a unique intra-TAD connectivity whereby one border makes pervasive interactions throughout the domain. Such 'stripe' domains are associated with both poised and active chromatin landscapes and transcription is not a key determinant of their structure. By tracking the developmental dynamics of stripe domains, we show that stripe formation is linked to the functional state of the cell through cohesin loading at lineage-specific enhancers and developmental control of CTCF binding site occupancy. We propose that the unique topological configuration of stripe domains represents a permissive landscape facilitating both productive and opportunistic gene regulation and is important for cellular identity.


Assuntos
Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Elementos Facilitadores Genéticos , Células-Tronco Pluripotentes/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Pluripotentes/química , Ligação Proteica , Domínios Proteicos , Especificidade da Espécie
2.
Gene ; 710: 265-272, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31200085

RESUMO

Patients with leukocyte adhesion deficiency type 1 (LAD1) suffer from life-threatening bacterial infections due to mutations in the common ß2 integrin subunit (CD18/ITGB2 gene). We tested different fragments of the ubiquitous chromatin opening element (UCOE) from the human HNRPA2B1-CBX3 locus for their efficiency in driving the human CD18 gene expression and compared it with that of an elongation factor 1 alpha promoter (EF1αL, 1169 bp; EF1αS 248 bp) and a murine stem cell virus (MSCV) promoter within the context of the same lentiviral vector backbone. These vectors were tested in vitro for the human CD18 gene expression on the surface of CD34+ hematopoietic stem cells (HSCs) isolated from both moderate and severe LAD1 patients. Among the promoters tested in the patients' CD34+ HSCs, only U631 bp, U652 bp, U1262 bp, 5' 2.2 kb A2UCOE and EF1αS resulted in higher percentage of CD18+CD34+ cells comparable to that of the MSCV promoter. The U655 bp, U723 bp, U1296 bp, U2598 bp and EF1αL promoters resulted in comparatively lower numbers of CD18+CD34+ cells. This study would be useful in investigating the human CD18 gene expression in an ex vivo experiment to demonstrate the phenotypic correction of LAD1 in a pre-clinical model.


Assuntos
Antígenos CD18/genética , Proteínas Cromossômicas não Histona/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Lentivirus/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Fator 1 de Elongação de Peptídeos/genética , Terapia Genética , Vetores Genéticos/genética , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Síndrome da Aderência Leucocítica Deficitária/terapia , Regiões Promotoras Genéticas , Elementos Reguladores de Transcrição , Transdução Genética
3.
Folia Histochem Cytobiol ; 57(2): 64-73, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31246264

RESUMO

INTRODUCTION: This study endeavors to analyze the effects of miR-1204 on the expression of DEK oncogene in non-small cell lung cancer (NSCLC) cell lines and to study the molecular mechanisms of these effects. MATERIAL AND METHODS: The miR-1204 mimics and inhibitors were transfected into the (A549 and SPC) NSCLC cells. Then the mRNA levels, cell viability, apoptosis rate, morphology and caspase activity were determined. The expression of apoptosis-related proteins Bcl-2 and Bax was also analyzed. RESULTS: In NSCLC cell lines (A549 and SPC), DEK mRNA levels were down-regulated in miR-1204 overex-pression group. In miR-1204 inhibition group, the expression of DEK mRNA showed an opposite trend. The overexpression of miR-1204 increases the apoptosis rate in NSCLC cells. The Bcl-2 levels in the miR-1204 over-expression group were decreased, while the Bax level was increased. In the miR-1204 inhibition group, expression of Bcl-2 and Bax showed opposite trends. Cell staining revealed cell's morphological changes; the apoptosis in the miR-1204 overexpression group revealed significant morphological features, such as brighter nuclei and nu-clear condensation. Results indicated a typical characteristic of apoptosis in the miR-1204 overexpression group. Caspase-9 and Caspase-3 were involved in the apoptosis pathway, which was mediated by miR-1204 and DEK. CONCLUSIONS: The miR-1204 induces apoptosis of NSCLC cells by inhibiting the expression of DEK. The mech-anism of apoptosis involves down-regulation of Bcl-2 and up-regulation of Bax expression. Moreover, the apoptosis was mediated by mitochondria-related caspase 9/3 pathway.


Assuntos
Apoptose/fisiologia , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas Cromossômicas não Histona/genética , Neoplasias Pulmonares/genética , MicroRNAs/fisiologia , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Mensageiro/fisiologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2/metabolismo
4.
Nat Cell Biol ; 21(6): 743-754, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160708

RESUMO

Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A binding sites are identified within the megabase-long, repetitive α-satellite DNAs at each centromere. CENP-A is shown in early G1 to be assembled into nucleosomes within each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. DNA replication is demonstrated to remove ectopically loaded, non-centromeric CENP-A. In contrast, tethering of centromeric CENP-A to the sites of DNA replication through the constitutive centromere associated network (CCAN) is shown to enable precise reloading of centromere-bound CENP-A onto the same DNA sequences as in its initial prereplication loading. Thus, DNA replication acts as an error correction mechanism for maintaining centromere identity through its removal of non-centromeric CENP-A coupled with CCAN-mediated retention and precise reloading of centromeric CENP-A.


Assuntos
Proteína Centromérica A/genética , Centrômero/genética , Cromossomos Humanos/genética , Replicação do DNA/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Fase G1/genética , Células HeLa , Histonas/genética , Humanos , Nucleossomos/genética
5.
Photochem Photobiol Sci ; 18(7): 1675-1684, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31218318

RESUMO

UV-B exposure of plants regulates expression of numerous genes concerned with various responses. Sudden exposure of non-acclimated plants to high fluence rate, short wavelength UV-B induces expression via stress-related signaling pathways that are not specific to the UV-B stimulus, whereas low fluence rates of UV-B can regulate expression via the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8). However, there is little information about whether non-stressful, low fluence rate UV-B treatments can activate gene expression independently of UVR8. Here, transcriptomic analysis of wild-type and uvr8 mutant Arabidopsis exposed to low fluence rate UV-B showed that numerous genes were regulated independently of UVR8. Moreover, nearly all of these genes were distinct to those induced by stress treatments. A small number of genes were expressed at all UV-B fluence rates employed and may be concerned with activation of eustress responses that facilitate acclimation to changing conditions. Expression of the gene encoding the transcription factor ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 13 (ANAC13) was studied to characterise a low fluence rate, UVR8-independent response. ANAC13 is induced by as little as 0.1 µmol m-2 s-1 UV-B and its regulation is independent of components of the canonical UVR8 signaling pathway COP1 and HY5/HYH. Furthermore, UV-B induced expression of ANAC13 is independent of the photoreceptors CRY1, CRY2, PHOT1 and PHOT2 and phytochromes A, B, D and E. ANAC13 expression is induced over a range of UV-B wavelengths at low doses, with maximum response at 310 nm. This study provides a basis for further investigation of UVR8 and stress independent, low fluence rate UV-B signaling pathway(s).


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Raios Ultravioleta , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Cromossômicas não Histona/genética , Criptocromos/genética , Criptocromos/metabolismo , Transdução de Sinais/efeitos da radiação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
6.
Cancer Res ; 79(11): 2808-2809, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31160308

RESUMO

Malignant rhabdoid tumors (MRT) are rare but deadly pediatric tumors characterized by mutations in the SMARCB1/SNF5/INI1/BAF47 gene. Currently, there are no targeted therapies for MRTs. In a previous issue of Cancer Research, Howard and colleagues utilize the power of genome-wide RNAi and CRISPR screening to identify MDM2 and MDM4 as potential drug targets for MRTs. Most MRTs retain an intact p53 pathway and the authors show that these cells are particularly sensitive to MDM2 and MDM4 inhibition due to SMARCB1's role in regulating p53-depedent apoptotic genes. This discovery suggests potential clinical trials of MDM2 inhibitors in patients with MRT.See related article by Howard and colleagues; Cancer Res 79(9):2404-14.


Assuntos
Tumor Rabdoide/genética , Criança , Proteínas Cromossômicas não Histona/genética , Humanos , Mutação , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína SMARCB1/genética
7.
Nat Commun ; 10(1): 2343, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138803

RESUMO

Despite the conserved essential function of centromeres, centromeric DNA itself is not conserved. The histone-H3 variant, CENP-A, is the epigenetic mark that specifies centromere identity. Paradoxically, CENP-A normally assembles on particular sequences at specific genomic locations. To gain insight into the specification of complex centromeres, here we take an evolutionary approach, fully assembling genomes and centromeres of related fission yeasts. Centromere domain organization, but not sequence, is conserved between Schizosaccharomyces pombe, S. octosporus and S. cryophilus with a central CENP-ACnp1 domain flanked by heterochromatic outer-repeat regions. Conserved syntenic clusters of tRNA genes and 5S rRNA genes occur across the centromeres of S. octosporus and S. cryophilus, suggesting conserved function. Interestingly, nonhomologous centromere central-core sequences from S. octosporus and S. cryophilus are recognized in S. pombe, resulting in cross-species establishment of CENP-ACnp1 chromatin and functional kinetochores. Therefore, despite the lack of sequence conservation, Schizosaccharomyces centromere DNA possesses intrinsic conserved properties that promote assembly of CENP-A chromatin.


Assuntos
Centrômero/genética , Montagem e Desmontagem da Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Sequência Conservada , Epigênese Genética , Histonas , Cinetocoros , RNA Ribossômico 5S , RNA de Transferência , Proteínas de Schizosaccharomyces pombe/metabolismo , Sintenia
8.
Life Sci ; 229: 225-232, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31085244

RESUMO

AIMS: Cellular senescence is a well-known cancer prevention mechanism, inducing cancer cells to senescence can enhance cancer immunotherapy. However, how cellular senescence is regulated is not fully understood. Dynamic chromatin changes have been discovered during cellular senescence, while the causality remains elusive. BAZ1A, a gene coding the accessory subunit of ATP-dependent chromatin remodeling complex, showed decreased expression in multiple cellular senescence models. We aim to investigate the functional role of BAZ1A in regulating senescence in cancer and normal cells. MATERIALS AND METHODS: Knockdown of BAZ1A was performed via lentivirus mediated short hairpin RNA (shRNA) in various cancer cell lines (A549 and U2OS) and normal cells (HUVEC, NIH3T3 and MEF). A series of senescence-associated phenotypes were quantified by CCK-8 assay, SA-ß-Gal staining and EdU incorporation assay, etc. KEY FINDINGS: Knockdown (KD) of BAZ1A induced series of senescence-associated phenotypes in both cancer and normal cells. BAZ1A-KD caused the upregulated expression of SMAD3, which in turn activated the transcription of p21 coding gene CDKN1A and resulted in senescence-associated phenotypes in human cancer cells (A549 and U2OS). SIGNIFICANCE: Our results revealed chromatin remodeling modulator BAZ1A acting as a novel regulator of cellular senescence in both normal and cancer cells, indicating a new target for potential cancer treatment.


Assuntos
Neoplasias Ósseas/patologia , Senescência Celular , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Osteossarcoma/patologia , Fatores de Transcrição/metabolismo , Células A549 , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Células NIH 3T3 , Osteossarcoma/genética , Osteossarcoma/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética
9.
Cancer Sci ; 110(7): 2133-2144, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31066149

RESUMO

Lymphoid-specific helicase (LSH) is overexpressed in tumor tissues and its overexpression is associated with poor prognosis in several cancers. However, the role and molecular mechanism of LSH in hepatocellular carcinoma (HCC) remains largely unknown. Herein, we report that LSH was overexpressed in tumor tissues of HCC, and overexpression of LSH was associated with poor prognosis from a public HCC database, and validated by clinical samples from our department. Ectopic LSH expression promoted the growth of HCC cells in vivo and in vitro. Mechanistically, LSH overexpression promoted tumor growth by activating transcription of centromere protein F (CENPF). Clinically, overexpression of LSH and/or CENPF correlated with shorter overall survival and higher cumulative recurrence rates of HCC. In conclusion, LSH promotes tumor growth of HCC through transcriptional regulation of CENPF expression. Therefore, LSH may be a novel predictor for prognosis and a potential therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Proteínas Cromossômicas não Histona/genética , DNA Helicases/metabolismo , Neoplasias Hepáticas/patologia , Proteínas dos Microfilamentos/genética , Regulação para Cima , Idoso , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Transplante de Neoplasias , Prognóstico , Análise de Sequência de RNA , Análise de Sobrevida , Análise Serial de Tecidos , Ativação Transcricional
10.
Mol Med Rep ; 19(5): 4205-4212, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30942427

RESUMO

CBX3, namely chromobox protein homolog 3, a member of the heterochomatin protein 1 (HP1) family, has been shown to be associated with the tumorigenesis of various types of cancer. The aim of the present study was to assess the biological role and the clinicopathological importance of CBX3 in osteosarcoma. The Oncomine database was utilized to determine the CBX3 expression in sarcoma patients. A retrospective cohort study was conducted to evaluate the prognostic value of CBX3 expression. In addition, correlations between the clinicopathological features of the osteosarcoma patients and CBX3 expression were assessed and involved recurrence, distant metastasis, lymph node metastasis, response to chemotherapy, pathological differentiation, clinical stage, anatomic location, tumor size and age. To investigate the function of CBX3 in osteosarcoma, a small interfering RNA for CBX3 was designed and this was used for the transfection of osteosarcoma MG63 cells. Then, the effects of CBX3 on proliferation, cell cycle distribution and apoptosis of osteosarcoma cells were investigated via CCK­8 assay and cell cycle assay and cell apoptosis analysis, respectively. Based on our findings, upregulation of CBX3 expression was noted both in osteosarcoma and also other sarcoma types, which included pleomorphic liposarcoma, myxofibrosarcoma, myxoid/round cell liposarcoma and dedifferentiated liposarcoma. In addition, based on the retrospective cohort study, CBX3 expression was associated with the disease­free survival (DFS) and overall survival (OS) of the osteosarcoma patients and a large tumor size, high distant metastasis rate and high clinical stage rate. In addition, the proliferation ability was blocked by the knockdown of CBX3 through the application of CBX3 siRNA, and CBX3 knockdown also led to increased apoptosis and cell cycle arrest at G0 and G1 phases in osteosarcoma cells. CBX3 is highly expressed in human osteosarcoma tissues. Meanwhile, high CBX3 is a predictor of the poor prognosis of osteosarcoma patients. To conclude, the growth of osteosarcoma can be promoted by CBX3, which may be used as an independent potential prognostic biomarker for patients suffering from osteosarcoma.


Assuntos
Neoplasias Ósseas/genética , Transformação Celular Neoplásica/genética , Proteínas Cromossômicas não Histona/genética , Expressão Gênica , Osteossarcoma/genética , Adolescente , Apoptose/genética , Biomarcadores Tumorais , Neoplasias Ósseas/patologia , Neoplasias Ósseas/terapia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Criança , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Osteossarcoma/patologia , Osteossarcoma/terapia , Prognóstico , RNA Interferente Pequeno , Resultado do Tratamento , Carga Tumoral , Adulto Jovem
11.
Epigenetics Chromatin ; 12(1): 21, 2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940194

RESUMO

BACKGROUND: Stem cell differentiation involves major chromatin reorganisation, heterochromatin formation and genomic relocalisation of structural proteins, including heterochromatin protein 1 gamma (HP1γ). As the principal reader of the repressive histone marks H3K9me2/3, HP1 plays a key role in numerous processes including heterochromatin formation and maintenance. RESULTS: We find that HP1γ is citrullinated in mouse embryonic stem cells (mESCs) and this diminishes when cells differentiate, indicating that it is a dynamically regulated post-translational modification during stem cell differentiation. Peptidylarginine deiminase 4, a known regulator of pluripotency, citrullinates HP1γ in vitro. This requires R38 and R39 within the HP1γ chromodomain, and the catalytic activity is enhanced by trimethylated H3K9 (H3K9me3) peptides. Mutation of R38 and R39, designed to mimic citrullination, affects HP1γ binding to H3K9me3-containing peptides. Using live-cell single-particle tracking, we demonstrate that R38 and R39 are important for HP1γ binding to chromatin in vivo. Furthermore, their mutation reduces the residence time of HP1γ on chromatin in differentiating mESCs. CONCLUSION: Citrullination is a novel post-translational modification of the structural heterochromatin protein HP1γ in mESCs that is dynamically regulated during mESC differentiation. The citrullinated residues lie within the HP1γ chromodomain and are important for H3K9me3 binding in vitro and chromatin association in vivo.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Citrulinação , Heterocromatina/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Heterocromatina/química , Heterocromatina/genética , Código das Histonas , Histonas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Mutação , Ligação Proteica , Desiminases de Arginina em Proteínas/metabolismo
12.
Dis Markers ; 2019: 2473652, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30956729

RESUMO

Background: Circular RNAs have been implicated in various malignancies and can function as potential biomarkers for cancers. Reportedly, circSMARCA5 was downregulated in hepatocellular carcinoma and glioblastoma multiforme, but upregulated in prostate cancer. The functional roles and clinical significance of circSMARCA5 still remain unknown in the context of gastric cancer (GC). Methods: Expression levels of circSMARCA5 were detected by qRT-PCR. Clinical data including patient basic information, clinicopathological features, and survival data were obtained. The Kaplan-Meier methods, multivariate Cox regression models, and the receiver operating characteristic curve were used to assess the clinical significance of circSMARCA5 in GC. Cell proliferation assays and transwell assays were performed to elucidate the functional roles of circSMARCA5 in GC. Results: The circSMARCA5 level was decreased in GC tissues and cell lines. The low expression level of circSMARCA5 was correlated to poorer overall survival and disease-free survival. Low circSMARCA5 expression was revealed as an independent unfavorable predictive factor for GC. The results indicated that circSMARCA5 had a moderate ability for discrimination between GC patients and controls with an area under the curve of 0.806. Upregulation of circSMARCA5 dampened the proliferation, migration, and invasion of GC cells, whereas circSMARCA5 knockdown promoted GC progression. Discussion: Our results demonstrated that circSMARCA5 was decreased and exerted tumor-suppressive effects in GC. circSMARCA5 can function as a potential biomarker for GC prognosis and diagnosis.


Assuntos
Biomarcadores Tumorais/genética , RNA Mensageiro/genética , RNA/genética , Neoplasias Gástricas/genética , Adenosina Trifosfatases/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA/metabolismo , RNA Mensageiro/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
13.
Nature ; 569(7754): 136-140, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30996347

RESUMO

Chromatin remodelling complexes evict, slide, insert or replace nucleosomes, which represent an intrinsic barrier for access to DNA. These remodellers function in most aspects of genome utilization including transcription-factor binding, DNA replication and repair1,2. Although they are frequently mutated in cancer3, it remains largely unclear how the four mammalian remodeller families (SWI/SNF, ISWI, CHD and INO80) orchestrate the global organization of nucleosomes. Here we generated viable embryonic stem cells that lack SNF2H, the ATPase of ISWI complexes, enabling study of SNF2H cellular function, and contrast it to BRG1, the ATPase of SWI/SNF. Loss of SNF2H decreases nucleosomal phasing and increases linker lengths, providing in vivo evidence for an ISWI function in ruling nucleosomal spacing in mammals. Systematic analysis of transcription-factor binding reveals that these remodelling activities have specific effects on binding of different transcription factors. One group critically depends on BRG1 and contains the transcriptional repressor REST, whereas a non-overlapping set of transcription factors, including the insulator protein CTCF, relies on SNF2H. This selectivity readily explains why chromosomal folding and insulation of topologically associated domains requires SNF2H, but not BRG1. Collectively, this study shows that mammalian ISWI is critical for nucleosomal periodicity and nuclear organization and that transcription factors rely on specific remodelling pathways for correct genomic binding.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Animais , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , DNA Helicases/metabolismo , Células-Tronco Embrionárias/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica
14.
Nat Med ; 25(5): 767-775, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31011208

RESUMO

Anti-tumor immunity is driven by self versus non-self discrimination. Many immunotherapeutic approaches to cancer have taken advantage of tumor neoantigens derived from somatic mutations. Here, we demonstrate that gene fusions are a source of immunogenic neoantigens that can mediate responses to immunotherapy. We identified an exceptional responder with metastatic head and neck cancer who experienced a complete response to immune checkpoint inhibitor therapy, despite a low mutational load and minimal pre-treatment immune infiltration in the tumor. Using whole-genome sequencing and RNA sequencing, we identified a novel gene fusion and demonstrated that it produces a neoantigen that can specifically elicit a host cytotoxic T cell response. In a cohort of head and neck tumors with low mutation burden, minimal immune infiltration and prevalent gene fusions, we also identified gene fusion-derived neoantigens that generate cytotoxic T cell responses. Finally, analyzing additional datasets of fusion-positive cancers, including checkpoint-inhibitor-treated tumors, we found evidence of immune surveillance resulting in negative selective pressure against gene fusion-derived neoantigens. These findings highlight an important class of tumor-specific antigens and have implications for targeting gene fusion events in cancers that would otherwise be less poised for response to immunotherapy, including cancers with low mutational load and minimal immune infiltration.


Assuntos
Antígenos de Neoplasias/genética , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T Citotóxicos/imunologia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/imunologia , Fusão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Fatores de Transcrição NFI/genética , Fatores de Transcrição NFI/imunologia , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Sequenciamento Completo do Genoma
15.
Cell Mol Biol (Noisy-le-grand) ; 65(3): 25-31, 2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30942153

RESUMO

Flowering is a very important developmental stage in the plant life cycle. LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) has been shown to participate in epigenetic silencing of flowering genes. Here, for the first time, we isolated and characterized six CmLHP1 homolog genes from the important day-neutral ornamental Chrysanthemum morifolium cultivar 'Jin budiao'. These homolog genes were most likely generated by whole-genome duplication. Bioinformatic analysis showed that chrysanthemum LHP1 homologs present low similarity to other plant LHP1-like genes. However, three nuclear localization signals and two domains were highly conserved among them. The secondary structures of the CmLHP1 homologs mainly include α-helices and random coils, indicating that the proteins are mixed proteins. Phylogenetic tree analysis indicated that the six CmLHP1 genes constituted a small clade and had the closest relationship with LsLHP1 (Lactuca sativa LHP1). Quantitative RT-PCR analysis showed that the CmLHP1 homologs were expressed in different tissues during the developmental period of chrysanthemum, but they were highly expressed in the buds, especially during the key S1 stage of the inflorescence. Furthermore, the expression patterns of CmLHP1 homologs showed divergence under different photoperiods. Both CmLHP1b and CmLHP1e exhibited photoperiod sensitivity in leaves. Intriguingly, CmLHP1c was insensitive to photoperiod in both the shoot apexes and the leaves. Subcellular localization revealed that the six CmLHP1 proteins were located in the nucleus. These results reveal that CmLHP1 homolog genes could be strong candidates as important regulators of flowering time in chrysanthemum.


Assuntos
Proteínas Cromossômicas não Histona/genética , Chrysanthemum/genética , Clonagem Molecular/métodos , Regulação da Expressão Gênica de Plantas , Homologia de Sequência de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas Cromossômicas não Histona/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Frações Subcelulares/metabolismo
16.
Essays Biochem ; 63(1): 157-165, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30940740

RESUMO

Orchestrating vertebrate genomes require a complex interplay between the linear composition of the genome and its 3D organization inside the nucleus. This requires the function of specialized proteins, able to tune various aspects of genome organization and gene regulation. The CCCTC-binding factor (CTCF) is a DNA binding factor capable of regulating not only the 3D genome organization, but also key aspects of gene expression, including transcription activation and repression, RNA splicing, and enhancer/promoter insulation. A growing body of evidence proposes that CTCF, together with cohesin contributes to DNA loop formation and 3D genome organization. CTCF binding sites are mutation hotspots in cancer, while mutations in CTCF itself lead to intellectual disabilities, emphasizing its importance in disease etiology. In this review we cover various aspects of CTCF function, revealing the polyvalence of this factor as a highly diversified tool for vertebrate genome organization and transcription regulation.


Assuntos
Fator de Ligação a CCCTC/genética , Cromatina/genética , Regulação da Expressão Gênica , Genoma , Animais , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/genética , DNA/metabolismo , Humanos , Mutação , RNA/genética , RNA/metabolismo
17.
Nat Commun ; 10(1): 1673, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975984

RESUMO

Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end-directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics.


Assuntos
Segregação de Cromossomos , Cinesina/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas Cromossômicas não Histona/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Dinâmica não Linear , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Imagem Individual de Molécula , Processos Estocásticos , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/metabolismo
18.
Nat Commun ; 10(1): 1686, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975996

RESUMO

Cohesin is a multiprotein ring that is responsible for cohesion of sister chromatids and formation of DNA loops to regulate gene expression. Genomic analyses have identified that the cohesin subunit STAG2 is frequently inactivated by mutations in cancer. However, the reason STAG2 mutations are selected during tumorigenesis and strategies for therapeutically targeting mutant cancer cells are largely unknown. Here we show that STAG2 is essential for DNA replication fork progression, whereby STAG2 inactivation in non-transformed cells leads to replication fork stalling and collapse with disruption of interaction between the cohesin ring and the replication machinery as well as failure to establish SMC3 acetylation. As a consequence, STAG2 mutation confers synthetic lethality with DNA double-strand break repair genes and increased sensitivity to select cytotoxic chemotherapeutic agents and PARP or ATR inhibitors. These studies identify a critical role for STAG2 in replication fork procession and elucidate a potential therapeutic strategy for cohesin-mutant cancers.


Assuntos
Antígenos Nucleares/metabolismo , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Neoplasias/genética , Mutações Sintéticas Letais , Antígenos Nucleares/genética , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Cromátides/metabolismo , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Inativação de Genes , Humanos , Mutagênese , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo , Reparo de DNA por Recombinação
19.
Nat Commun ; 10(1): 1519, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944321

RESUMO

Hyperdiploidy, i.e. gain of whole chromosomes, is one of the most common genetic features of childhood acute lymphoblastic leukemia (ALL), but its pathogenetic impact is poorly understood. Here, we report a proteogenomic analysis on matched datasets from genomic profiling, RNA-sequencing, and mass spectrometry-based analysis of >8,000 genes and proteins as well as Hi-C of primary patient samples from hyperdiploid and ETV6/RUNX1-positive pediatric ALL. We show that CTCF and cohesin, which are master regulators of chromatin architecture, display low expression in hyperdiploid ALL. In line with this, a general genome-wide dysregulation of gene expression in relation to topologically associating domain (TAD) borders were seen in the hyperdiploid group. Furthermore, Hi-C of a limited number of hyperdiploid childhood ALL cases revealed that 2/4 cases displayed a clear loss of TAD boundary strength and 3/4 showed reduced insulation at TAD borders, with putative leukemogenic effects.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Transcrição Genética , Adolescente , Aneuploidia , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/genética , Criança , Pré-Escolar , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Aberrações Cromossômicas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Lactente , Recém-Nascido , Masculino , Proteogenômica/métodos , Proteoma/genética , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/genética , Análise de Sequência de RNA
20.
Dokl Biochem Biophys ; 484(1): 66-68, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31012017

RESUMO

The PBAF(SWI/SNF) multiprotein complex, which changes the chromatin structure, is widely involved in the regulation of eukaryotic gene expression. A specific component of this complex is the PHF10 protein, which is involved in recruiting this complex to chromatin. We showed that the PHF10 expression in cells of different lines is activated by the c-MYC oncogene. Since PHF10 stimulates cell proliferation, its c-MYC-dependent activation in cancer cells should lead to an increase in their proliferation rate.


Assuntos
Proteínas Cromossômicas não Histona/biossíntese , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/biossíntese , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/genética
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