RESUMO
Hi-C reads, which represent ligation events between different regions of the genome, must be processed into matrices of interaction frequencies for downstream analysis. Here, I describe a procedure for mapping Hi-C reads to the genome and conversion of mapped reads into the HOMER tag directory format and interaction matrix format for visualization with Juicebox. The method is demonstrated for the mouse composite X chromosome in which reads from the active and inactive X chromosomes are combined after mock DMSO treatment or targeted degradation of cohesin.
Assuntos
Cromossomo X , Animais , Cromossomo X/genética , Camundongos , Software , Coesinas , Mapeamento Cromossômico/métodos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Biologia Computacional/métodosRESUMO
Cohesin is a protein complex that plays a key role in regulating chromosome structure and gene expression. While next-generation sequencing technologies have provided extensive information on various aspects of cohesin, integrating and exploring the vast datasets associated with cohesin are not straightforward. CohesinDB ( https://cohesindb.iqb.u-tokyo.ac.jp ) offers a web-based interface for browsing, searching, analyzing, visualizing, and downloading comprehensive multiomics cohesin information in human cells. In this protocol, we introduce how to utilize CohesinDB to facilitate research on transcriptional regulation and chromatin organization.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Coesinas , Navegador , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Software , Biologia Computacional/métodos , Genômica/métodos , Bases de Dados Genéticas , Cromatina/metabolismo , Cromatina/genética , Internet , MultiômicaRESUMO
Spt5 is a well-conserved factor that manipulates multiple stages of transcription from promoter-proximal pausing (PPP) to termination. Recent studies have revealed an unexpected increase of antisense transcripts near promoters in cells expressing mutant Spt5. Here, we identify Spt5p-restricted intragenic antisense transcripts and their close relationship with sense transcription in yeast. We confirm that Spt5 CTR phosphorylation is also important to retain Spt5's facility to regulate antisense transcription. The genes whose antisense transcription is strongly suppressed by Spt5p share strong endogenous sense transcription and weak antisense transcription, and this pattern is conserved in humans. Mechanistically, we found that Spt5p depletion increased histone acetylation to initiate intragenic antisense transcription by altering chromatin structure. We additionally identified termination factors that appear to be involved in the ability of Spt5p to restrict antisense transcription. By unveiling a new role of Spt5 in finely balancing the bidirectionality of transcription, we demonstrate that Spt5-mediated suppression of DSIF complex regulated-unstable transcripts (DUTs) is essential to sustain the accurate transcription by RNA polymerase II.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Transcrição Gênica , Fatores de Elongação da Transcrição , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Humanos , Regulação Fúngica da Expressão Gênica , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fosforilação , RNA Antissenso/genética , RNA Antissenso/metabolismo , Histonas/metabolismo , Regiões Promotoras Genéticas , Cromatina/metabolismo , Cromatina/genéticaRESUMO
OBJECTIVE: We aimed to explore the role of structural maintenance of chromosomes 4 (SMC4) in malignant progression and immunology of colon adenocarcinoma (COAD). METHODS: The expression, genetic and protein features, and immune cell infiltration of SMC4 in pan-cancer were provided by public databases and websites. The protein expression of SMC4 in COAD tissues was screened by immunohistochemical assay. Si-RNA-mediated transfection was performed in COAD cells and the proliferation viability was measured using MTT, colony formation and EdU assays. Cell autophagy was detected by AO staining, western blots, and immunofluorescence staining. The migratory ability was determined using scratch and transwell assays. The expression of epithelial-to-mesenchymal transition (EMT) markers and transcriptional factors were detected using western blots. RESULTS: The expression of SMC4 was upregulated in pan-cancer and had relationships with prognosis, TMB, and MSI of cancer patients. Particularly, SMC4 protein was highly expressed in COAD tissues and correlated with poor prognosis of patients. Depletion of SMC4 inhibited cell proliferation, induced autophagy, and decreased migration through EMT progression in COAD cells. In addition, SMC4 was associated with infiltration of neutrophils, M2 macrophages, and CD4 + T cells in COAD, and had positive association with M2 macrophage markers and immune checkpoints. CONCLUSION: SMC4 was correlated with patients' poor prognosis, proliferation, metastasis, and immune cell infiltrates, and might function as a potential diagnosis and prognostic biomarker in COAD.
Assuntos
Adenocarcinoma , Biomarcadores Tumorais , Proteínas de Ciclo Celular , Movimento Celular , Proliferação de Células , Neoplasias do Colo , Transição Epitelial-Mesenquimal , Humanos , Neoplasias do Colo/patologia , Neoplasias do Colo/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Prognóstico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/diagnóstico , Adenocarcinoma/imunologia , Adenocarcinoma/metabolismo , Transição Epitelial-Mesenquimal/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Autofagia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Masculino , Feminino , Regulação para Cima , Adenosina TrifosfatasesRESUMO
This study sheds light on the pivotal role of the oncoprotein DEK in B-cell lymphoma. We reveal DEK expression correlates with increased tumor proliferation and inferior overall survival in cases diagnosed with low-grade B-cell lymphoma (LGBCL). We also found significant correlation between DEK expression and copy number alterations in LGBCL tumors, highlighting a novel mechanism of LGBCL pathogenesis that warrants additional exploration. To interrogate the mechanistic role of DEK in B-cell lymphoma, we generated a DEK knockout cell line model, which demonstrated DEK depletion caused reduced proliferation and altered expression of key cell cycle and apoptosis-related proteins, including Bcl-2, Bcl-xL, and p53. Notably, DEK depleted cells showed increased sensitivity to apoptosis-inducing agents, including venetoclax and staurosporine, which underscores the therapeutic potential of targeting DEK in B-cell lymphomas. Overall, our study contributes to a better understanding of DEK's role as an oncoprotein in B-cell lymphomas, highlighting its potential as both a promising therapeutic target and a novel biomarker for aggressive LGBCL. Further research elucidating the molecular mechanisms underlying DEK-mediated tumorigenesis could pave the way for improved treatment strategies and better clinical outcomes for patients with B-cell lymphoma.
Assuntos
Proliferação de Células , Proteínas Cromossômicas não Histona , Linfoma de Células B , Proteínas Oncogênicas , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Humanos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Linfoma de Células B/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/patologia , Linhagem Celular Tumoral , Linfócitos B/metabolismo , Linfócitos B/patologia , Apoptose , Feminino , Regulação Neoplásica da Expressão Gênica , Masculino , Gradação de TumoresRESUMO
Pathogenic variants in the cohesin genes, NIPBL and SMC1A, both cause Cornelia de Lange syndrome (CdLS), a rare genetic disorder associated with developmental delay and intellectual disability. This study aimed to compare sleep behaviors across individuals with CdLS caused by a variant in NIPBL or SMC1A, and identify relationships between sleep and behavior functioning. A total of 31 caregivers of individuals with a variant in NIPBL (N = 22) or SMC1A (N = 9) completed questionnaires regarding their child's sleep behaviors, behavior regulation, attention, and autistic features (repetitive behaviors and social communication difficulties) as part of the Coordination of Rare Diseases (CoRDS) registry. Findings showed a trend of increased behavior regulation difficulties and repetitive behaviors in the NIPBL compared to the SMC1A participants. Both groups presented with a similar degree of attention, social communication, and sleep challenges. In the whole sample, sleep disturbance was strongly correlated with more behavior regulation difficulties, a relationship that was more robust in the NIPBL sample. In brief, study results support our prior observations of greater behavior difficulties among those with a variant in NIPBL as compared to SMC1A. Preliminary findings point to unique associations between sleep and behavior regulation in the NIPBL group, suggesting sleep interventions may yield differential effects on behavior management across variants.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Síndrome de Cornélia de Lange , Sono , Humanos , Síndrome de Cornélia de Lange/genética , Síndrome de Cornélia de Lange/fisiopatologia , Síndrome de Cornélia de Lange/psicologia , Feminino , Masculino , Proteínas de Ciclo Celular/genética , Criança , Proteínas Cromossômicas não Histona/genética , Sono/genética , Sono/fisiologia , Pré-Escolar , Adolescente , Transtornos do Sono-Vigília/genética , Transtornos do Sono-Vigília/fisiopatologia , Inquéritos e Questionários , Mutação/genética , AdultoRESUMO
Breast cancer is the most frequently diagnosed cancer worldwide, constituting 15% of cases in 2023. The predominant cause of breast cancer-related mortality is metastasis, and a lack of metastasis-targeted therapies perpetuates dismal outcomes for late-stage patients. By using meiotic genetics to study inherited transcriptional network regulation, we have identified, to the best of our knowledge, a new class of "essential expression-restricted" genes as potential candidates for metastasis-targeted therapeutics. Building upon previous work implicating the CCR4-NOT RNA deadenylase complex in metastasis, we demonstrate that RNA-binding proteins NANOS1, PUM2, and CPSF4 also regulate metastatic potential. Using various models and clinical data, we pinpoint Smarcd1 mRNA as a target of all three RNA-BPs. Strikingly, both high and low expression of Smarcd1 correlate with positive clinical outcomes, while intermediate expression significantly reduces the probability of survival. Applying the theory of "essential genes" from evolution, we identify 50 additional genes that require precise expression levels for metastasis to occur. Specifically, small perturbations in Smarcd1 expression significantly reduce metastasis in mouse models and alter splicing programs relevant to the ER+/HER2-enriched breast cancer. Identification subtype-specific essential expression-restricted metastasis modifiers introduces a novel class of genes that, when therapeutically "nudged" in either direction, may significantly improve late-stage breast cancer patients.
Assuntos
Neoplasias da Mama , Regulação Neoplásica da Expressão Gênica , Humanos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Feminino , Camundongos , Metástase Neoplásica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Telomeres, the ends of eukaryotic linear chromosomes, are composed of repeated DNA sequences and specialized proteins, with the conserved telomeric Cdc13/CTC1-Stn1-Ten1 (CST) complex providing chromosome stability via telomere end protection and the regulation of telomerase accessibility. In this study, SIZ1, coding for a SUMO E3 ligase, and TOP2 (a SUMO target for Siz1 and Siz2) were isolated as extragenic suppressors of Saccharomyces cerevisiae CST temperature-sensitive mutants. ten1-sz, stn1-sz and cdc13-sz mutants were isolated next due to being sensitive to intracellular Siz1 dosage. In parallel, strong negative genetic interactions between mutants of CST and septins were identified, with septins being noticeably sumoylated through the action of Siz1. The temperature-sensitive arrest in these new mutants of CST was dependent on the G2/M Mad2-mediated and Bub2-mediated spindle checkpoints as well as on the G2/M Mec1-mediated DNA damage checkpoint. Our data suggest the existence of yet unknown functions of the telomeric Cdc13-Stn1-Ten1 complex associated with mitotic spindle positioning and/or assembly that could be further elucidated by studying these new ten1-sz, stn1-sz and cdc13-sz mutants.
Assuntos
Proteínas de Ciclo Celular , Dano ao DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fuso Acromático , Proteínas de Ligação a Telômeros , Telômero , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Telômero/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteínas de Ligação a Telômeros/genética , Fuso Acromático/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Mutação/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genéticaRESUMO
BACKGROUND: Colon cancer (CC) is a highly prevalent malignancy that contributes significantly to global morbidity and mortality. The polycomb group ring finger 2 (PCGF2) has been identified as a relevant factor influencing the outcomes of CC. At the same time, the centromere-associated protein E (CENPE) is implicated in promoting carcinogenesis and adversely affecting the survival of tumor patients. The primary objective of this study was to elucidate the precise impact of PCGF2 on CC and unravel the underlying mechanisms associated with CENPE. METHODS: Human normal colon epithelial cells and CC cells were utilized to investigate the differential expression of PCGF2 and CENPE. CC cell line LOVO was exploited and transfected for PCGF2 regulation. Subsequently, cell viability and proliferation were assessed using the cell counting kit 8 (CCK-8) and colony forming assay. Cell viability and proliferation were assessed using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, while cell migration and invasion capabilities were determined using the transwell assay, and mRNA levels of cell cycle-related genes were measured for evaluating cell cycle activation. In addition, mice were used for in vivo experiments to investigate the progression of CC cells with different levels of PCGF2. Moreover, GSK-923295 was used to inhibit CENPE, followed by the evaluation of cell progression. RESULTS: PCGF2 and CENPE were upregulated in CC cell lines (p < 0.001), and upregulation/downregulation of PCGF2 led to the upregulation and downregulation of CENPE (p < 0.001). The upregulation/downregulation of PCGF2 led to an increase/decrease in viability, proliferation, migration, and invasion while suppressing/enhancing apoptosis in LOVO cells (p < 0.001), promoting cell progression. The tumor progression of LOVO cells with PCGF2 knockdown was slower (p < 0.001). The PCGF2-promoting LOVO cell progression was disrupted when CENPE was inhibited, presented by the reversely decreased viability, proliferation, migration, invasion, and cell cycle activation, and increased apoptosis (p < 0.001). CONCLUSION: PCGF2 promotes CC cell progression by upregulating CENPE, providing PCGF2 inhibition and CENPE inhibition as potential therapeutic targets for treating CC.
Assuntos
Proliferação de Células , Neoplasias do Colo , Regulação Neoplásica da Expressão Gênica , Regulação para Cima , Humanos , Neoplasias do Colo/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Animais , Camundongos , Linhagem Celular Tumoral , Proliferação de Células/genética , Movimento Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Camundongos Nus , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/genética , Apoptose/genética , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos dos fármacosRESUMO
The in vivo three-dimensional genomic architecture of adult mature neurons at homeostasis and after medically relevant perturbations such as axonal injury remains elusive. Here, we address this knowledge gap by mapping the three-dimensional chromatin architecture and gene expression program at homeostasis and after sciatic nerve injury in wild-type and cohesin-deficient mouse sensory dorsal root ganglia neurons via combinatorial Hi-C, promoter-capture Hi-C, CUT&Tag for H3K27ac and RNA-seq. We find that genes involved in axonal regeneration form long-range, complex chromatin loops, and that cohesin is required for the full induction of the regenerative transcriptional program. Importantly, loss of cohesin results in disruption of chromatin architecture and severely impaired nerve regeneration. Complex enhancer-promoter loops are also enriched in the human fetal cortical plate, where the axonal growth potential is highest, and are lost in mature adult neurons. Together, these data provide an original three-dimensional chromatin map of adult sensory neurons in vivo and demonstrate a role for cohesin-dependent long-range promoter interactions in nerve regeneration.
Assuntos
Axônios , Cromatina , Coesinas , Regeneração Nervosa , Regiões Promotoras Genéticas , Células Receptoras Sensoriais , Animais , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Camundongos , Regiões Promotoras Genéticas/genética , Cromatina/metabolismo , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Axônios/metabolismo , Axônios/fisiologia , Humanos , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Elementos Facilitadores Genéticos/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Nervo Isquiático/metabolismoRESUMO
Despite their prevalent cancer implications, the in vivo dynamics of SWI/SNF chromatin remodelers and how misregulation of such dynamics underpins cancer remain poorly understood. Using live-cell single-molecule tracking, we quantify the intranuclear diffusion and chromatin-binding of three key subunits common to all major human SWI/SNF remodeler complexes (BAF57, BAF155 and BRG1), and resolve two temporally distinct stable binding modes for the fully assembled complex. Super-resolved density mapping reveals heterogeneous, nanoscale remodeler binding "hotspots" across the nucleoplasm where multiple binding events (especially longer-lived ones) preferentially cluster. Importantly, we uncover distinct roles of the bromodomain in modulating chromatin binding/targeting in a DNA-accessibility-dependent manner, pointing to a model where successive longer-lived binding within "hotspots" leads to sustained productive remodeling. Finally, systematic comparison of six common BRG1 mutants implicated in various cancers unveils alterations in chromatin-binding dynamics unique to each mutant, shedding insight into a multi-modal landscape regulating the spatio-temporal organizational dynamics of SWI/SNF remodelers.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina , Proteínas Cromossômicas não Histona , DNA Helicases , Neoplasias , Proteínas Nucleares , Imagem Individual de Molécula , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Imagem Individual de Molécula/métodos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Cromatina/metabolismo , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ligação Proteica , Mutação , Linhagem Celular Tumoral , Domínios Proteicos , Adenosina TrifosfatasesRESUMO
The establishment of long-lasting immunity against pathogens is facilitated by the germinal center (GC) reaction, during which B cells increase their antibody affinity and differentiate into antibody-secreting cells (ASC) and memory cells. These events involve modifications in chromatin packaging that orchestrate the profound restructuring of gene expression networks that determine cell fate. While several chromatin remodelers were implicated in lymphocyte functions, less is known about SMARCA5. Here, using ribosomal pull-down for analyzing translated genes in GC B cells, coupled with functional experiments in mice, we identified SMARCA5 as a key chromatin remodeler in B cells. While the naive B cell compartment remained unaffected following conditional depletion of Smarca5, effective proliferation during B cell activation, immunoglobulin class switching, and as a result GC formation and ASC differentiation were impaired. Single-cell multiomic sequencing analyses revealed that SMARCA5 is crucial for facilitating the transcriptional modifications and genomic accessibility of genes that support B cell activation and differentiation. These findings offer novel insights into the functions of SMARCA5, which can be targeted in various human pathologies.
Assuntos
Linfócitos B , Diferenciação Celular , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona , Centro Germinativo , Animais , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Linfócitos B/metabolismo , Linfócitos B/imunologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Camundongos , Camundongos Endogâmicos C57BL , Ativação Linfocitária/imunologia , Switching de Imunoglobulina/genética , Adenosina TrifosfatasesRESUMO
In this issue of Molecular Cell, Sahu et al.1 find that shielding heterochromatin from SWI/SNF chromatin remodelers is essential to maintain and epigenetically propagate pre-existing heterochromatin domains, whereas SWI/SNF action protects facultative heterochromatic regions from premature silencing.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona , Heterocromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Epigênese Genética , Inativação Gênica , Heterocromatina/metabolismo , Heterocromatina/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaAssuntos
Tumor Rabdoide , Fatores de Transcrição , Humanos , Tumor Rabdoide/genética , Tumor Rabdoide/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Mutação , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
Beyond its essential roles in ensuring faithful chromosome segregation and genomic stability, the human Smc5/6 complex acts as an antiviral factor. It binds to and impedes the transcription of extrachromosomal DNA templates; an ability which is lost upon integration of the DNA into the chromosome. How the complex distinguishes among different DNA templates is unknown. Here we show that, in human cells, Smc5/6 preferentially binds to circular rather than linear extrachromosomal DNA. We further demonstrate that the transcriptional process, per se, and particularly the accumulation of DNA secondary structures known to be substrates for topoisomerases, is responsible for Smc5/6 recruitment. More specifically, we find that in vivo Smc5/6 binds to positively supercoiled DNA. Those findings, in conjunction with our genome-wide Smc5/6 binding analysis showing that Smc5/6 localizes at few but highly transcribed chromosome loci, not only unveil a previously unforeseen role of Smc5/6 in DNA topology management during transcription but highlight the significance of sensing DNA topology as an antiviral defense mechanism.
Assuntos
Proteínas de Ciclo Celular , DNA Super-Helicoidal , Transcrição Gênica , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/genética , Ligação Proteica , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA/metabolismo , DNA/genética , Conformação de Ácido Nucleico , DNA Circular/metabolismo , DNA Circular/genéticaRESUMO
Chromatin remodelling complexes (CRC) are ATP-dependent molecular machines important for the dynamic organization of nucleosomes along eukaryotic DNA. CRCs SWI/SNF, RSC and INO80 can move positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions which facilitate access of general transcription factors. This function is strongly supported by transcriptional activators being able to interact with subunits of various CRCs. In this work we show that SWI/SNF subunits Swi1, Swi2, Snf5 and Snf6 can bind to activation domains of Ino2 required for expression of phospholipid biosynthetic genes in yeast. We identify an activator binding domain (ABD) of ATPase Swi2 and show that this ABD is functionally dispensable, presumably because ABDs of other SWI/SNF subunits can compensate for the loss. In contrast, mutational characterization of the ABD of the Swi2-related ATPase Sth1 revealed that some conserved basic and hydrophobic amino acids within this domain are essential for the function of Sth1. While ABDs of Swi2 and Sth1 define separate functional protein domains, mapping of an ABD within ATPase Ino80 showed co-localization with its HSA domain also required for binding actin-related proteins. Comparative interaction studies finally demonstrated that several unrelated activators each exhibit a specific binding pattern with ABDs of Swi2, Sth1 and Ino80.
Assuntos
Adenosina Trifosfatases , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA , Ligação Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Transcrição , Ativação Transcricional , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação Fúngica da Expressão Gênica , Domínios Proteicos , Proteínas Nucleares , Proteínas de Ciclo Celular , Fatores de Transcrição Hélice-Alça-Hélice BásicosRESUMO
Structural Maintenance of Chromosomes (SMC) complexes are an evolutionary conserved protein family. In most eukaryotes, three SMC complexes have been characterized, as follows: cohesin, condensin, and SMC5/6 complexes. These complexes are involved in a plethora of functions, and defects in SMC genes can lead to an increased risk of chromosomal abnormalities, infertility, and cancer. To investigate the evolution of SMC complex genes in mammals, we analyzed their selective patterns in an extended phylogeny. Signals of positive selection were identified for condensin NCAPG, for two SMC5/6 complex genes (SMC5 and NSMCE4A), and for all cohesin genes with almost exclusive meiotic expression (RAD21L1, REC8, SMC1B, and STAG3). For the latter, evolutionary rates correlate with expression during female meiosis, and most positively selected sites fall in intrinsically disordered regions (IDRs). Our results support growing evidence that IDRs are fast evolving, and that they most likely contribute to adaptation through modulation of phase separation. We suggest that the natural selection signals identified in SMC complexes may be the result of different selective pressures: a host-pathogen arms race in the condensin and SMC5/6 complexes, and an intragenomic conflict for meiotic cohesin genes that is similar to that described for centromeres and telomeres.
Assuntos
Adenosina Trifosfatases , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Coesinas , Proteínas de Ligação a DNA , Evolução Molecular , Complexos Multiproteicos , Seleção Genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Complexos Multiproteicos/genética , Animais , Adenosina Trifosfatases/genética , Humanos , Proteínas de Ligação a DNA/genética , Meiose/genética , FilogeniaRESUMO
PURPOSE: DEK::AFF2 fusion-associated squamous cell carcinoma (DEK::AFF2 SCC), also reported in the literature as low-grade papillary sinonasal (Schneiderian) carcinoma (LGPSC), is a rare, primarily bland-appearing, but locally aggressive neoplasm. Morphologically, these tumors can closely resemble sinonasal papilloma (SP), especially on small or limited biopsy, often leading to misdiagnosis. DEK::AFF2 SCC is devoid of the underlying mutually exclusive EGFR or KRAS driver mutations of SP, suggesting it may represent a distinct unique entity. METHODS: In this study, we conducted a retrospective search of "unusual" SP reported either as atypical, dysplastic, or suspicious for malignant transformation at our institution in the last 13 years (2010-2023), to identify potential cases of DEK::AFF2 SCC. RESULTS: Of the 201 SP cases during this time period, 30 "unusual" SP cases were identified. On morphologic review of these 30 cases, 6 were worrisome for DEK::AFF2 SCC and were selected for AFF2 immunohistochemical stain (IHC), of which 3 cases were positive. All 3 AFF2 IHC positive cases were also positive for DEK::AFF2 fusion by fluorescence in situ hybridization (FISH), thereby, confirming IHC results. CONCLUSIONS: This study highlights that AFF2 IHC can be an invaluable surrogate marker to FISH in identifying DEK::AFF2 SCC in challenging cases to avoid misdiagnosis. Detailed clinical and pathologic data were collected to gain a better understanding of this emerging challenging entity. A literature review was performed to enrich our knowledge of DEK::AFF2 SCC.
Assuntos
Proteínas Cromossômicas não Histona , Proteínas Oncogênicas , Proteínas de Ligação a Poli-ADP-Ribose , Humanos , Masculino , Proteínas de Ligação a Poli-ADP-Ribose/genética , Pessoa de Meia-Idade , Feminino , Proteínas Oncogênicas/genética , Proteínas Cromossômicas não Histona/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/diagnóstico , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Idoso , Neoplasias dos Seios Paranasais/patologia , Neoplasias dos Seios Paranasais/genética , Adulto , Estudos Retrospectivos , Proteínas de Fusão Oncogênica/genéticaRESUMO
Neuroepithelial tumors with fusion of PLAGL1 or amplification of PLAGL1/PLAGL2 have recently been described often with ependymoma-like or embryonal histology respectively. To further evaluate emerging entities with PLAG-family genetic alterations, the histologic, molecular, clinical, and imaging features are described for 8 clinical cases encountered at St. Jude (EWSR1-PLAGL1 fusion n = 6; PLAGL1 amplification n = 1; PLAGL2 amplification n = 1). A histologic feature observed on initial resection in a subset (4/6) of supratentorial neuroepithelial tumors with EWSR1-PLAGL1 rearrangement was the presence of concurrent ependymal and ganglionic differentiation. This ranged from prominent clusters of ganglion cells within ependymoma/subependymoma-like areas, to interspersed ganglion cells of low to moderate frequency among otherwise ependymal-like histology, or focal areas with a ganglion cell component. When present, the combination of ependymal-like and ganglionic features within a supratentorial neuroepithelial tumor may raise consideration for an EWSR1-PLAGL1 fusion, and prompt initiation of appropriate molecular testing such as RNA sequencing and methylation profiling. One of the EWSR1-PLAGL1 fusion cases showed subclonal INI1 loss in a region containing small clusters of rhabdoid/embryonal cells, and developed a prominent ganglion cell component on recurrence. As such, EWSR1-PLAGL1 neuroepithelial tumors are a tumor type in which acquired inactivation of SMARCB1 and development of AT/RT features may occur and lead to clinical progression. In contrast, the PLAGL2 and PLAGL1 amplified cases showed either embryonal histology or contained an embryonal component with a significant degree of desmin staining, which could also serve to raise consideration for a PLAG entity when present. Continued compilation of associated clinical data and histopathologic findings will be critical for understanding emerging entities with PLAG-family genetic alterations.
Assuntos
Proteína EWS de Ligação a RNA , Neoplasias Supratentoriais , Fatores de Transcrição , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Adulto Jovem , Diferenciação Celular , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Epêndima/patologia , Rearranjo Gênico/genética , Neoplasias Neuroepiteliomatosas/genética , Neoplasias Neuroepiteliomatosas/patologia , Proteínas de Fusão Oncogênica/genética , Proteína EWS de Ligação a RNA/genética , Neoplasias Supratentoriais/genética , Neoplasias Supratentoriais/patologia , Fatores de Transcrição/genéticaRESUMO
The yeast SWR1 complex catalyzes the exchange of histone H2A/H2B dimers in nucleosomes with Htz1/H2B dimers. We use cryoelectron microscopy to determine the structure of an enzyme-bound hexasome intermediate in the reaction pathway of histone exchange, in which an H2A/H2B dimer has been extracted from a nucleosome prior to the insertion of a dimer comprising Htz1/H2B. The structure reveals a key role for the Swc5 subunit in stabilizing the unwrapping of DNA from the histone core of the hexasome. By engineering a crosslink between an Htz1/H2B dimer and its chaperone protein Chz1, we show that this blocks histone exchange by SWR1 but allows the incoming chaperone-dimer complex to insert into the hexasome. We use this reagent to trap an SWR1/hexasome complex with an incoming Htz1/H2B dimer that shows how the reaction progresses to the next step. Taken together the structures reveal insights into the mechanism of histone exchange by SWR1 complex.