RESUMO
General transcription factor IIIC subunit 5 (GTF3C5) encodes transcription factor IIIC63 (TFIIIC63). It binds to DNA to recruit another transcription factor, TFIIIB, and RNA polymerase III (Pol III) to mediate the transcription of small noncoding RNAs, such as tRNAs. Here, we report four individuals from three families presenting with a multisystem developmental disorder phenotype with biallelic variants in GTF3C5. The overlapping features include growth retardation, developmental delay, intellectual disability, dental anomalies, cerebellar malformations, delayed bone age, skeletal anomalies, and facial dysmorphism. Using lymphoblastoid cell lines (LCLs) from two affected individuals, we observed a reduction in TFIIIC63 protein levels compared to control LCLs. Genome binding of TFIIIC63 protein is also reduced in LCL from one of the affected individuals. Additionally, approximately 40% of Pol III binding regions exhibited reduction in the level of Pol III occupancy in the mutant genome relative to the control, while approximately 54% of target regions showed comparable levels of Pol III occupancy between the two, indicating partial impairment of Pol III occupancy in the mutant genome. Yeasts with subject-specific variants showed temperature sensitivity and impaired growth, supporting the notion that the identified variants have deleterious effects. gtf3c5 mutant zebrafish showed developmental defects, including a smaller body, head, and eyes. Taken together, our data show that GTF3C5 plays an important role in embryonic development, and that biallelic variants in this gene cause a multisystem developmental disorder. Our study adds GTF3C5-related disorder to the growing list of genetic disorders associated with Pol III transcription machinery.
Assuntos
Deficiências do Desenvolvimento , RNA Polimerase III , Fatores de Transcrição TFIII , Animais , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Alelos , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Deficiência Intelectual/genética , Mutação , Linhagem , Fenótipo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo , Transcrição Gênica , Peixe-Zebra/genéticaRESUMO
AIMS: Aimed to identify a new susceptibility gene associated with primary biliary cholangitis (PBC) in Chinese Han and investigate the possible mechanism of that gene in PBC. METHODS: A total of 466 PBC and 694 healthy controls (HC) were included in our study, and genotyping GTF2I gene variants by Sequenom. CD19 + B cells were isolated for Chromatin immunoprecipitation sequencing (ChIP-seq). Additionally, MEME-ChIP was utilized to perform searches for known motifs and de novo motif discovery. The GTF2I ChIP-seq of hematopoietic cell line (K562) results were obtained from ENCODE (GSE176987, GSE177691). The Genomic HyperBrowser was used to determine overlap and hierarchal clustering between ours and ENCODE datasets. RESULTS: The frequency of the rs117026326 variant T allele was significantly higher in PBC patients than that in HC (20.26% compared with 13.89%, Pc = 1.09E-04). Furthermore, we observed an elevated proportion of GTF2I binding site located in the upstream and 5' UTR of genes in PBC in comparison with HC. Additionally, an in-depth analysis of IL21R region revealed that GTF2I might bind to the IL21R promoter to regulate the expression of the IL21R, with four peaks of GTF2I binding sites, including three increased binding sites in upstream, one increased binding site in 5' UTR. Motif analysis by MEME-ChIP uncovered five significant motifs. A significant overlap between our ChIP and GSE176987, GSE17769 were found by the Genomic HyperBroswer. CONCLUSIONS: Our study confirmed that GTF2I was associated with PBC in Chinese Han. Furthermore, our gene function analysis indicated that IL21R may be the target gene regulated by GTF2I.
Assuntos
Cirrose Hepática Biliar , Fatores de Transcrição TFIII , Fatores de Transcrição TFII , Humanos , Regiões 5' não Traduzidas , China , Sequenciamento de Cromatina por Imunoprecipitação , Cirrose Hepática Biliar/genética , Receptores de Interleucina-21/genética , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFIII/genéticaRESUMO
Gtf2i encodes the general transcription factor II-I (TFII-I), with peak expression during pre-natal and early post-natal brain development stages. Because these stages are critical for proper brain development, we studied at the single-cell level the consequences of Gtf2i's deletion from excitatory neurons, specifically on mitochondria. Here we show that Gtf2i's deletion resulted in abnormal morphology, disrupted mRNA related to mitochondrial fission and fusion, and altered autophagy/mitophagy protein expression. These changes align with elevated reactive oxygen species levels, illuminating Gtf2i's importance in neurons mitochondrial function. Similar mitochondrial issues were demonstrated by Gtf2i heterozygous model, mirroring the human condition in Williams syndrome (WS), and by hemizygous neuronal Gtf2i deletion model, indicating Gtf2i's dosage-sensitive role in mitochondrial regulation. Clinically relevant, we observed altered transcript levels related to mitochondria, hypoxia, and autophagy in frontal cortex tissue from WS individuals. Our study reveals mitochondrial and autophagy-related deficits shedding light on WS and other Gtf2i-related disorders.
Assuntos
Fatores de Transcrição TFIII , Síndrome de Williams , Humanos , Autofagia/genética , Heterozigoto , Neurônios/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo , Síndrome de Williams/genética , Síndrome de Williams/metabolismoRESUMO
Copy number variations at 7q11.23 cause neurodevelopmental disorders with shared and opposite manifestations. Deletion causes Williams-Beuren syndrome featuring hypersociability, while duplication causes 7q11.23 microduplication syndrome (7Dup), frequently exhibiting autism spectrum disorder (ASD). Converging evidence indicates GTF2I as key mediator of the cognitive-behavioral phenotypes, yet its role in cortical development and behavioral hallmarks remains largely unknown. We integrated proteomic and transcriptomic profiling of patient-derived cortical organoids, including longitudinally at single-cell resolution, to dissect 7q11.23 dosage-dependent and GTF2I-specific disease mechanisms. We observed dosage-dependent impaired dynamics of neural progenitor proliferation, transcriptional imbalances, and highly specific alterations in neuronal output, leading to precocious excitatory neuron production in 7Dup, which was rescued by restoring physiological GTF2I levels. Transgenic mice with Gtf2i duplication recapitulated progenitor proliferation and neuronal differentiation defects alongside ASD-like behaviors. Consistently, inhibition of lysine demethylase 1 (LSD1), a GTF2I effector, was sufficient to rescue ASD-like phenotypes in transgenic mice, establishing GTF2I-LSD1 axis as a molecular pathway amenable to therapeutic intervention in ASD.
Assuntos
Transtorno do Espectro Autista , Fatores de Transcrição TFIII , Fatores de Transcrição TFII , Camundongos , Animais , Humanos , Transtorno do Espectro Autista/genética , Variações do Número de Cópias de DNA , Proteômica , Comportamento Social , Fenótipo , Camundongos Transgênicos , Diferenciação Celular/genética , Histona Desmetilases/genética , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFII/genéticaRESUMO
In vertebrates, SMARCAD1 participates in transcriptional regulation, heterochromatin maintenance, DNA repair, and replication. The molecular basis underlying its involvement in these processes is not well understood. We identified the RNA polymerase III general transcription factor TFIIIC as an interaction partner of native SMARCAD1 in mouse and human models using endogenous co-immunoprecipitations. TFIIIC has dual functionality, acting as a general transcription factor and as a genome organizer separating chromatin domains. We found that its partnership with SMARCAD1 is conserved across different mammalian cell types, from somatic to pluripotent cells. Using purified proteins, we confirmed that their interaction is direct. A gene expression analysis suggested that SMARCAD1 is dispensable for TFIIIC function as an RNA polymerase III transcription factor in mouse ESCs. The distribution of TFIIIC and SMARCAD1 in the ESC genome is distinct, and unlike in yeast, SMARCAD1 is not enriched at active tRNA genes. Further analysis of SMARCAD1-binding partners in pluripotent and differentiated mammalian cells reveals that SMARCAD1 associates with several factors that have key regulatory roles in chromatin organization, such as cohesin, laminB, and DDX5. Together, our work suggests for the first time that the SMARCAD1 enzyme participates in genome organization in mammalian nuclei through interactions with architectural proteins.
Assuntos
DNA Helicases , Fatores Genéricos de Transcrição , Fatores de Transcrição TFIII , Animais , Humanos , Camundongos , Cromatina/genética , DNA Helicases/genética , Mamíferos , RNA Polimerase III , Fatores de Transcrição TFIII/genéticaRESUMO
Transcription factor (TF) IIIC recruits RNA polymerase (Pol) III to most of its target genes. Recognition of intragenic A- and B-box motifs in transfer RNA (tRNA) genes by TFIIIC modules τA and τB is the first critical step for tRNA synthesis but is mechanistically poorly understood. Here, we report cryo-electron microscopy structures of the six-subunit human TFIIIC complex unbound and bound to a tRNA gene. The τB module recognizes the B-box via DNA shape and sequence readout through the assembly of multiple winged-helix domains. TFIIIC220 forms an integral part of both τA and τB connecting the two subcomplexes via a ~550-amino acid residue flexible linker. Our data provide a structural mechanism by which high-affinity B-box recognition anchors TFIIIC to promoter DNA and permits scanning for low-affinity A-boxes and TFIIIB for Pol III activation.
Assuntos
Fatores de Transcrição TFIII , Humanos , Microscopia Crioeletrônica , Fatores de Transcrição TFIII/genética , Transcrição Gênica , DNA/metabolismo , RNA de Transferência/genéticaRESUMO
The TFIIF-like Rpc53/Rpc37 heterodimer of RNA polymerase (pol) III is involved in various stages of transcription. The C-terminal region of Rpc53 dimerizes with Rpc37 to anchor on the lobe domain of the pol III cleft. However, structural and functional features of the Rpc53 N-terminal region had not been characterized previously. Here, we conducted site-directed alanine replacement mutagenesis on the Rpc53 N-terminus, generating yeast strains that exhibited a cold-sensitive growth defect and severely compromised pol III transcriptional activity. Circular dichroism and NMR spectroscopy revealed a highly disordered 57-amino acid polypeptide in the Rpc53 N-terminus. This polypeptide is a versatile protein-binding module displaying nanomolar-level binding affinities for Rpc37 and the Tfc4 subunit of the transcription initiation factor TFIIIC. Accordingly, we denote this Rpc53 N-terminus polypeptide as the TFIIIC-binding region or CBR. Alanine replacements in the CBR significantly reduced its binding affinity for Tfc4, highlighting its functional importance to cell growth and transcription in vitro. Our study reveals the functional basis for Rpc53's CBR in assembly of the pol III transcription initiation complex.
Assuntos
RNA Polimerase III , Fatores de Transcrição TFIII , RNA Polimerase III/metabolismo , Transcrição Gênica , Fatores de Transcrição TFIII/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Peptídeos/metabolismoRESUMO
BACKGROUND: Thymomas belong to rare tumors giving rise to thymic epithelial tissue. There is a classification of several forms of thymoma: A, AB, B1, B2, B3, thymic carcinoma (TC) and thymic neuroendocrine thymoma. In this meta-analysis study, we have focused on thymoma using articles based on the disease's next-generation sequencing (NGS) genomic profiling. MATERIALS AND METHODS: We conducted a systematic review and meta-analysis of the prevalence of studies that discovered the genes and variants occurring in the less aggressive forms of the thymic epithelial tumors. Studies published before 12th December 2022 were identified through PubMed, Web of Science (WoS), and SCOPUS databases. Two reviewers have searched for the bases and selected the articles for the final analysis, based on well-defined exclusion and inclusion criteria. RESULTS: Finally, 12 publications were included in the qualitative as well as quantitative analysis. The three genes, GTF2I, TP53, and HRAS, emerged as disease-significant in the observed studies. The Odds Ratio for all three extracted genes GTF2I (OR = 1.58, CI [1.51, 1.66] p < 0.00001), TP53 (OR = 1.36, CI [1.12, 1.65], p < 0.002), and HRAS (OR = 1.02, CI [1.00, 1.04], p < 0.001). CONCLUSIONS: According to obtained data, we noticed that the GTF2I gene exhibits a significant prevalence in the cohort of observed thymoma patients. Moreover, analyzing published articles NGS has suggested GTF2I, TP53, and HRAS genes as the most frequently mutated genes in thymoma that have pathogenic single nucleotide variants (SNV) and Insertion/Deletion (InDel), which contribute to disease development and progression. These variants could be valuable biomarkers and target points specific to thymoma.
Assuntos
Neoplasias Epiteliais e Glandulares , Timoma , Neoplasias do Timo , Fatores de Transcrição TFIII , Humanos , Timoma/genética , Timoma/patologia , Neoplasias do Timo/genética , Neoplasias do Timo/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Fatores de Transcrição TFIII/genéticaRESUMO
The ubiquitously expressed transcription factor TFII-I is a multifunctional protein with pleiotropic roles in gene regulation. TFII-I associated polymorphisms are implicated in Sjögren's syndrome and Lupus in humans and, germline deletion of the Gtf2i gene in mice leads to embryonic lethality. Here we report a unique role for TFII-I in homeostasis of innate properties of B lymphocytes. Loss of Gtf2i in murine B lineage cells leads to an alteration in transcriptome, chromatin landscape and associated transcription factor binding sites, which exhibits myeloid-like features and coincides with enhanced sensitivity to LPS induced gene expression. TFII-I deficient B cells also show increased switching to IgG3, a phenotype associated with inflammation. These results demonstrate a role for TFII-I in maintaining immune homeostasis and provide clues for GTF2I polymorphisms associated with B cell dominated autoimmune diseases in humans.
Assuntos
Síndrome de Sjogren , Fatores de Transcrição TFIII , Fatores de Transcrição TFII , Humanos , Camundongos , Animais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina , Ligação Proteica , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismoRESUMO
AIM: Recent study has revealed frequent GTF2I mutation in thymomas, with the frequency being highest in types A and AB, followed by B1, B2, B3 and thymic carcinoma. This has led to the conclusion that GTF2I mutation correlates with more indolent histology subtype and better prognosis. In our study, the GTF2I mutation was tested in thymic epithelial tumours to investigate the relation between the mutation status and histology subtype. METHODS: The GTF2I mutation was tested in 111 thymic epithelial tumours by Sanger sequencing. Correlations between GTF2I mutation status and clinicopathological parameters were evaluated. RESULTS: There were 16 cases of type A, including atypical type, 37 type AB, 13 B1, 23 B2, 9 B3, 6 micronodular type, 2 metaplastic type and 5 thymic carcinomas. GTF2I mutation was seen in 78.6% of type A and 83.9% of type AB, while it was not expressed in type B, metaplastic type or thymic carcinoma (p<0.001). 75% of micronodular type also showed the mutation. Both thymoma histotype and stage were significantly associated with GTF2I mutation by univariate analysis. The presence of GTF2I mutation showed a trend towards a favourable prognosis, but this is likely due to their strong association with more indolent histologic subtypes (types A and AB). CONCLUSIONS: GTF2I mutation appears unique in type A and AB thymomas, including those with atypical features and micronodular type, all of which share spindle cell morphology, indicating they represent a group biologically distinct from type B thymomas.
Assuntos
Neoplasias Epiteliais e Glandulares , Timoma , Neoplasias do Timo , Fatores de Transcrição TFIII , Fatores de Transcrição TFII , Humanos , Timoma/genética , Neoplasias do Timo/patologia , Mutação , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFII/genéticaRESUMO
Autism spectrum disorder (ASD) is an early onset, developmental disorder whose genetic cause is heterogeneous and complex. In total, 70% of ASD cases are due to an unknown etiology. Among the monogenic causes of ASD, fragile X syndrome (FXS) accounts for 2-4% of ASD cases, and 60% of individuals with FXS present with ASD. Epigenetic changes, specifically DNA methylation, which modulates gene expression levels, play a significant role in the pathogenesis of both disorders. Thus, in this study, using the Human Methylation EPIC Bead Chip, we examined the global DNA methylation profiles of biological samples derived from 57 age-matched male participants (2-6 years old), including 23 subjects with ASD, 23 subjects with FXS with ASD (FXSA) and 11 typical developing (TD) children. After controlling for technical variation and white blood cell composition, using the conservatory threshold of the false discovery rate (FDR ≤ 0.05), in the three comparison groups, TD vs. AD, TD vs. FXSA and ASD vs. FXSA, we identified 156, 79 and 3100 differentially methylated sites (DMS), and 14, 13 and 263 differential methylation regions (DMRs). Interestingly, several genes differentially methylated among the three groups were among those listed in the SFARI Gene database, including the PAK2, GTF2I and FOXP1 genes important for brain development. Further, enrichment analyses identified pathways involved in several functions, including synaptic plasticity. Our preliminary study identified a significant role of altered DNA methylation in the pathology of ASD and FXS, suggesting that the characterization of a DNA methylation signature may help to unravel the pathogenicity of FXS and ASD and may help the development of an improved diagnostic classification of children with ASD and FXSA. In addition, it may pave the way for developing therapeutic interventions that could reverse the altered methylome profile in children with neurodevelopmental disorders.
Assuntos
Transtorno do Espectro Autista , Síndrome do Cromossomo X Frágil , Fatores de Transcrição TFIII , Criança , Humanos , Masculino , Pré-Escolar , Síndrome do Cromossomo X Frágil/genética , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Metilação de DNA/genética , Epigênese Genética/genética , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição Forkhead/genéticaRESUMO
BACKGROUND: General transcription factor IIi (GTF2I) mutations are very common in thymic epithelial tumors (TETs) and are related to a more favorable prognosis in TET patients. However, limited research has been conducted on the role of GTF2I in the tumor immune microenvironment (TIME). Further, long non-coding RNAs (lncRNAs) have been associated with the survival of patients with TETs. Therefore, this study aimed to explore the relationship between GTF2I mutations and TIME and build a new potential signature for predicting tumor recurrence in the TETs. Research data was downloaded from The Cancer Genome Atlas database and the CIBERSORT algorithm was used to evaluate TIME differences between GTF2I mutant and wild-type TETs. Relevant differentially expressed lncRNAs based on differentially expressed immune-related genes were identified to establish lncRNA pairs. We constructed a signature using univariate and multivariate Cox regression analyses. RESULTS: GTF2I is the most commonly mutated gene in TETs, and is associated with an increased number of early-stage pathological types, as well as no history of myasthenia gravis or radiotherapy treatment. In the GTF2I wild-type group, immune score and immune cell infiltrations with M2 macrophages, activated mast cells, neutrophils, plasma, T helper follicular cells, and activated memory CD4 T cells were higher than the GTF2I mutant group. A risk model was built using five lncRNA pairs, and the 1-, 3-, and 5-year area under the curves were 0.782, 0.873, and 0.895, respectively. A higher risk score was related to more advanced histologic type. CONCLUSION: We can define the GTF2I mutant-type TET as an immune stable type and the GTF2I wild-type as an immune stressed type. A signature based on lncRNA pairs was also constructed to effectively predict tumor recurrence.
Assuntos
Neoplasias Epiteliais e Glandulares , RNA Longo não Codificante , Fatores Genéricos de Transcrição , Fatores de Transcrição TFIII , Fatores de Transcrição TFII , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Recidiva Local de Neoplasia/genética , Neoplasias Epiteliais e Glandulares/genética , Prognóstico , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias do Timo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismo , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo , Microambiente TumoralRESUMO
INTRODUCTION: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear. METHODS: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and characterized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1+ thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors. RESULTS: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle-related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial progenitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas. CONCLUSIONS: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.
Assuntos
Neoplasias Epiteliais e Glandulares , Timoma , Neoplasias do Timo , Fatores de Transcrição TFIII , Fatores de Transcrição TFII , Animais , Humanos , Camundongos , Mutação , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Timoma/genética , Timoma/patologia , Neoplasias do Timo/genética , Neoplasias do Timo/patologia , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFIII/genéticaRESUMO
Eukaryotic chromosomes are divided into domains with distinct structural and functional properties, such as differing levels of chromatin compaction and gene transcription. Domains of relatively compact chromatin and minimal transcription are termed heterochromatic, whereas euchromatin is more open and actively transcribed. Insulators separate these domains and maintain their distinct features. Disruption of insulators can cause diseases such as cancer. Many insulators contain tRNA genes (tDNAs), examples of which have been shown to block the spread of activating or silencing activities. This characteristic of specific tDNAs is conserved through evolution, such that human tDNAs can serve as barriers to the spread of silencing in fission yeast. Here we demonstrate that tDNAs from the methylotrophic fungus Pichia pastoris can function effectively as insulators in distantly-related budding yeast. Key to the function of tDNAs as insulators is TFIIIC, a transcription factor that is also required for their expression. TFIIIC binds additional loci besides tDNAs, some of which have insulator activity. Although the mechanistic basis of TFIIIC-based insulation has been studied extensively in yeast, it is largely uncharacterized in metazoa. Utilising publicly-available genome-wide ChIP-seq data, we consider the extent to which mechanisms conserved from yeast to man may suffice to allow efficient insulation by TFIIIC in the more challenging chromatin environments of metazoa and suggest features that may have been acquired during evolution to cope with new challenges. We demonstrate the widespread presence at human tDNAs of USF1, a transcription factor with well-established barrier activity in vertebrates. We predict that tDNA-based insulators in higher organisms have evolved through incorporation of modules, such as binding sites for factors like USF1 and CTCF that are absent from yeasts, thereby strengthening function and providing opportunities for regulation between cell types.
Assuntos
Schizosaccharomyces , Fatores de Transcrição TFIII , Animais , Cromatina/genética , Cromossomos , Humanos , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição TFIII/genética , Transcrição GênicaRESUMO
Transcription factors (TFs) bind DNA in a sequence-specific manner and are generally cell type-specific factors and/or developmental master regulators. In contrast, general TFs (GTFs) are part of very large protein complexes and serve for RNA polymerases' recruitment to promoter sequences, generally in a cell type-independent manner. Whereas, several TFs have been proven to serve as anchors for the 3D genome organization, the role of GTFs in genome architecture have not been carefully explored. Here, we used ChIP-seq and Hi-C data to depict the role of TFIIIC, one of the RNA polymerase III GTFs, in 3D genome organization. We find that TFIIIC genome occupancy mainly occurs at specific regions, which largely correspond to Alu elements; other characteristic classes of repetitive elements (REs) such as MIR, FLAM-C and ALR/alpha are also found depending on the cell's developmental origin. The analysis also shows that TFIIIC-enriched regions are involved in cell type-specific DNA looping, which does not depend on colocalization with the master architectural protein CTCF. This work extends previous knowledge on the role of TFIIIC as a bona fide genome organizer whose action participates in cell type-dependent 3D genome looping via binding to REs.
Assuntos
Cromatina/genética , RNA Polimerase III/genética , Fatores de Transcrição TFIII/genética , Fator de Ligação a CCCTC/genética , Células Cultivadas , Sequenciamento de Cromatina por Imunoprecipitação/métodos , DNA/genética , Humanos , Regiões Promotoras Genéticas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Transcrição Gênica/genéticaRESUMO
The disease-initiating molecular events for age-related macular degeneration (AMD), a multifactorial retinal disease affecting many millions of elderly individuals worldwide, are still unknown. Of the over 30 risk and protective loci so far associated with AMD through whole genome-wide association studies (GWAS), the Age-Related Maculopathy Susceptibility 2 (ARMS2) gene locus represents one of the most highly associated risk regions for AMD. A unique insertion/deletion (in/del) sequence located immediately upstream of the High Temperature Requirement A1 (HTRA1) gene in this region confers high risk for AMD. Using electrophoretic mobility shift assay (EMSA), we identified that two Gtf2i-ß/δ transcription factor isoforms bind to the cis-element 5'- ATTAATAACC-3' contained in this in/del sequence. The binding of these transcription factors leads to enhanced upregulation of transcription of the secretory serine protease HTRA1 in transfected cells and AMD patient-derived induced pluripotent stem cells (iPSCs). Overexpression of Htra1 in mice using a CAG-promoter demonstrated increased blood concentration of Htra1 protein, caused upregulation of vascular endothelial growth factor (VEGF), and produced a choroidal neovascularization (CNV)-like phenotype. Finally, a comparison of 478 AMD patients to 481 healthy, age-matched controls from Japan, India, Australia, and the USA showed a statistically increased level of secreted HTRA1 blood concentration in AMD patients compared with age-matched controls. Taken together, these results suggest a common mechanism across ethnicities whereby increased systemic blood circulation of secreted serine protease HTRA1 leads to subsequent degradation of Bruch's membrane and eventual CNV in AMD.
Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Proteínas/genética , Fatores de Transcrição TFII/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Humanos , Mutação INDEL/genética , Degeneração Macular/genética , Degeneração Macular/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Proteínas/metabolismo , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismoRESUMO
The various malformations of the aerodigestive tract collectively known as esophageal atresia/tracheoesophageal fistula (EA/TEF) constitute a rare group of birth defects of largely unknown etiology. Previous studies have identified a small number of rare genetic variants causing syndromes associated with EA/TEF. We performed a pilot exome sequencing study of 45 unrelated simplex trios (probands and parents) with EA/TEF. Thirteen had isolated and 32 had nonisolated EA/TEF; none had a family history of EA/TEF. We identified de novo variants in protein-coding regions, including 19 missense variants predicted to be deleterious (D-mis) and 3 likely gene-disrupting (LGD) variants. Consistent with previous studies of structural birth defects, there is a trend of increased burden of de novo D-mis in cases (1.57-fold increase over the background mutation rate), and the burden is greater in constrained genes (2.55-fold, p = 0.003). There is a frameshift de novo variant in EFTUD2, a known EA/TEF risk gene involved in mRNA splicing. Strikingly, 15 out of 19 de novo D-mis variants are located in genes that are putative target genes of EFTUD2 or SOX2 (another known EA/TEF gene), much greater than expected by chance (3.34-fold, p value = 7.20e-5). We estimated that 33% of patients can be attributed to de novo deleterious variants in known and novel genes. We identified APC2, AMER3, PCDH1, GTF3C1, POLR2B, RAB3GAP2, and ITSN1 as plausible candidate genes in the etiology of EA/TEF. We conclude that further genomic analysis to identify de novo variants will likely identify previously undescribed genetic causes of EA/TEF.
Assuntos
Atresia Esofágica/genética , Frequência do Gene , Fístula Traqueoesofágica/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Adolescente , Adulto , Caderinas/genética , Criança , Pré-Escolar , Proteínas do Citoesqueleto/genética , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Fatores de Alongamento de Peptídeos/genética , Protocaderinas , RNA Polimerase II/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição TFIII/genética , Proteínas Supressoras de Tumor/genética , Proteínas rab3 de Ligação ao GTP/genéticaRESUMO
Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.
Assuntos
Regulação Fúngica da Expressão Gênica , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/genética , Fatores de Transcrição TFIII/ultraestrutura , Animais , Linhagem Celular , Microscopia Crioeletrônica , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos/genética , Insetos , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIIB/genética , Fator de Transcrição TFIIIB/isolamento & purificação , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/isolamento & purificação , Fatores de Transcrição TFIII/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição GenéticaRESUMO
In fission yeast, the inverted repeats IR-L and IR-R function as boundary elements at the edges of a 20-kb silent heterochromatic domain where nucleosomes are methylated at histone H3K9. Each repeat contains a series of B-box motifs physically associated with the architectural TFIIIC complex and with other factors including the replication regulator Sap1 and the Rix1 complex (RIXC). We demonstrate here the activity of these repeats in heterochromatin formation and maintenance. Deletion of the entire IR-R repeat or, to a lesser degree, deletion of just the B boxes impaired the de novo establishment of the heterochromatic domain. Nucleation proceeded normally at the RNA interference (RNAi)-dependent element cenH but subsequent propagation to the rest of the region occurred at reduced rates in the mutants. Once established, heterochromatin was unstable in the mutants. These defects resulted in bistable populations of cells occupying alternate "on" and "off" epigenetic states. Deleting IR-L in combination with IR-R synergistically tipped the balance toward the derepressed state, revealing a concerted action of the two boundaries at a distance. The nuclear rim protein Amo1 has been proposed to tether the mating-type region and its boundaries to the nuclear envelope, where Amo1 mutants displayed milder phenotypes than boundary mutants. Thus, the boundaries might facilitate heterochromatin propagation and maintenance in ways other than just through Amo1, perhaps by constraining a looped domain through pairing.
Assuntos
Proteínas de Ligação a DNA/genética , Heterocromatina/metabolismo , Sequências Repetidas Invertidas/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica/fisiologia , Heterocromatina/genética , Histonas/metabolismo , Metilação , Proteínas Nucleares/metabolismo , Interferência de RNA/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismoRESUMO
The ChIP-exo assay precisely delineates protein-DNA crosslinking patterns by combining chromatin immunoprecipitation with 5' to 3' exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein-DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between its subunits. Here, we present a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of coordinately bound regulatory regions, and which detects and quantifies protein-DNA crosslinking events within the aligned profiles. By producing consistent measurements of protein-DNA crosslinking strengths across multiple proteins, our approach enables characterization of relative spatial organization within a regulatory complex. Applying our approach to collections of ChIP-exo data, we demonstrate that it can recover aspects of regulatory complex spatial organization at yeast ribosomal protein genes and yeast tRNA genes. We also demonstrate the ability to quantify changes in protein-DNA complex organization across conditions by applying our approach to analyze Drosophila Pol II transcriptional components. Our results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein-DNA complexes.