Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 31
Filtrar
1.
Cell ; 187(1): 110-129.e31, 2024 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-38181737

RESUMO

X chromosome inactivation (XCI) serves as a paradigm for RNA-mediated regulation of gene expression, wherein the long non-coding RNA XIST spreads across the X chromosome in cis to mediate gene silencing chromosome-wide. In female naive human pluripotent stem cells (hPSCs), XIST is in a dispersed configuration, and XCI does not occur, raising questions about XIST's function. We found that XIST spreads across the X chromosome and induces dampening of X-linked gene expression in naive hPSCs. Surprisingly, XIST also targets specific autosomal regions, where it induces repressive chromatin changes and gene expression dampening. Thereby, XIST equalizes X-linked gene dosage between male and female cells while inducing differences in autosomes. The dispersed Xist configuration and autosomal localization also occur transiently during XCI initiation in mouse PSCs. Together, our study identifies XIST as the regulator of X chromosome dampening, uncovers an evolutionarily conserved trans-acting role of XIST/Xist, and reveals a correlation between XIST/Xist dispersal and autosomal targeting.


Assuntos
Genes Ligados ao Cromossomo X , RNA Longo não Codificante , Cromossomo X , Animais , Feminino , Humanos , Masculino , Camundongos , Inativação Gênica , RNA Longo não Codificante/genética , Cromossomo X/genética , Células-Tronco Pluripotentes/metabolismo
2.
Cell ; 184(25): 6174-6192.e32, 2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34813726

RESUMO

The lncRNA Xist forms ∼50 diffraction-limited foci to transcriptionally silence one X chromosome. How this small number of RNA foci and interacting proteins regulate a much larger number of X-linked genes is unknown. We show that Xist foci are locally confined, contain ∼2 RNA molecules, and nucleate supramolecular complexes (SMACs) that include many copies of the critical silencing protein SPEN. Aggregation and exchange of SMAC proteins generate local protein gradients that regulate broad, proximal chromatin regions. Partitioning of numerous SPEN molecules into SMACs is mediated by their intrinsically disordered regions and essential for transcriptional repression. Polycomb deposition via SMACs induces chromatin compaction and the increase in SMACs density around genes, which propagates silencing across the X chromosome. Our findings introduce a mechanism for functional nuclear compartmentalization whereby crowding of transcriptional and architectural regulators enables the silencing of many target genes by few RNA molecules.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Mitocondriais/metabolismo , RNA Longo não Codificante/metabolismo , Cromossomo X/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias , Fibroblastos , Inativação Gênica , Humanos , Camundongos , Ligação Proteica , Inativação do Cromossomo X
4.
Cell ; 161(2): 307-18, 2015 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-25843630

RESUMO

Protein-DNA binding is mediated by the recognition of the chemical signatures of the DNA bases and the 3D shape of the DNA molecule. Because DNA shape is a consequence of sequence, it is difficult to dissociate these modes of recognition. Here, we tease them apart in the context of Hox-DNA binding by mutating residues that, in a co-crystal structure, only recognize DNA shape. Complexes made with these mutants lose the preference to bind sequences with specific DNA shape features. Introducing shape-recognizing residues from one Hox protein to another swapped binding specificities in vitro and gene regulation in vivo. Statistical machine learning revealed that the accuracy of binding specificity predictions improves by adding shape features to a model that only depends on sequence, and feature selection identified shape features important for recognition. Thus, shape readout is a direct and independent component of binding site selection by Hox proteins.


Assuntos
DNA/química , DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Alinhamento de Sequência
5.
Cell ; 147(6): 1270-82, 2011 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-22153072

RESUMO

Members of transcription factor families typically have similar DNA binding specificities yet execute unique functions in vivo. Transcription factors often bind DNA as multiprotein complexes, raising the possibility that complex formation might modify their DNA binding specificities. To test this hypothesis, we developed an experimental and computational platform, SELEX-seq, that can be used to determine the relative affinities to any DNA sequence for any transcription factor complex. Applying this method to all eight Drosophila Hox proteins, we show that they obtain novel recognition properties when they bind DNA with the dimeric cofactor Extradenticle-Homothorax (Exd). Exd-Hox specificities group into three main classes that obey Hox gene collinearity rules and DNA structure predictions suggest that anterior and posterior Hox proteins prefer DNA sequences with distinct minor groove topographies. Together, these data suggest that emergent DNA recognition properties revealed by interactions with cofactors contribute to transcription factor specificities in vivo.


Assuntos
DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de Homeodomínio/metabolismo , Multimerização Proteica , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/química , Técnicas Genéticas , Proteínas de Homeodomínio/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Fatores de Transcrição/química
6.
Mol Cell ; 72(3): 444-456.e7, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30401431

RESUMO

Skin sun exposure induces two protection programs: stress responses and pigmentation, the former within minutes and the latter only hours afterward. Although serving the same physiological purpose, it is not known whether and how these programs are coordinated. Here, we report that UVB exposure every other day induces significantly more skin pigmentation than the higher frequency of daily exposure, without an associated increase in stress responses. Using mathematical modeling and empirical studies, we show that the melanocyte master regulator, MITF, serves to synchronize stress responses and pigmentation and, furthermore, functions as a UV-protection timer via damped oscillatory dynamics, thereby conferring a trade-off between the two programs. MITF oscillations are controlled by multiple negative regulatory loops, one at the transcriptional level involving HIF1α and another post-transcriptional loop involving microRNA-148a. These findings support trait linkage between the two skin protection programs, which, we speculate, arose during furless skin evolution to minimize skin damage.


Assuntos
Fator de Transcrição Associado à Microftalmia/metabolismo , Pele/metabolismo , Pele/efeitos da radiação , Animais , Linhagem Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Masculino , Melanócitos/fisiologia , Melanócitos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/fisiologia , Fator de Transcrição Associado à Microftalmia/efeitos da radiação , Cultura Primária de Células , Pigmentação da Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos
7.
Mediators Inflamm ; 2019: 3451461, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148944

RESUMO

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine, secreted by macrophages and other immune cells to attenuate inflammation. Autocrine type I interferons (IFNs) largely mediate the delayed expression of IL-10 by LPS-stimulated macrophages. We have previously shown that IL-10 is synergistically expressed in macrophages following a costimulus of a TLR agonist and cAMP. We now show that the cAMP pathway directly upregulates IL-10 transcription and plays an important permissive and synergistic role in early, but not late, LPS-stimulated IL-10 mRNA and protein expression in mouse macrophages and in a mouse septic shock model. Our results suggest that the loss of synergism is not due to desensitization of the cAMP inducing signal, and it is not mediated by a positive crosstalk between the cAMP and type I IFN pathways. First, cAMP elevation in LPS-treated cells decreased the secretion of type I IFN. Second, autocrine/paracrine type I IFNs induce IL-10 promoter reporter activity only additively, but not synergistically, with the cAMP pathway. IL-10 promoter reporter activity was synergistically induced by cAMP elevation in macrophages stimulated by an agonist of either TLR4, TLR2/6, or TLR7, receptors which signal via MyD88, but not by an agonist of TLR3 which signals independently of MyD88. Moreover, MyD88 knockout largely reduced the synergistic IL-10 expression, indicating that MyD88 is required for the synergism displayed by LPS with cAMP. This report delineates the temporal regulation of early cAMP-accelerated vs. late type I IFN-dependent IL-10 transcription in LPS-stimulated murine macrophages that can limit inflammation at its onset.


Assuntos
Interferon Tipo I/metabolismo , Interleucina-10/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Células RAW 264.7 , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos
8.
Genome Res ; 25(9): 1268-80, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26160164

RESUMO

Transcriptional regulation requires the binding of transcription factors (TFs) to short sequence-specific DNA motifs, usually located at the gene regulatory regions. Interestingly, based on a vast amount of data accumulated from genomic assays, it has been shown that only a small fraction of all potential binding sites containing the consensus motif of a given TF actually bind the protein. Recent in vitro binding assays, which exclude the effects of the cellular environment, also demonstrate selective TF binding. An intriguing conjecture is that the surroundings of cognate binding sites have unique characteristics that distinguish them from other sequences containing a similar motif that are not bound by the TF. To test this hypothesis, we conducted a comprehensive analysis of the sequence and DNA shape features surrounding the core-binding sites of 239 and 56 TFs extracted from in vitro HT-SELEX binding assays and in vivo ChIP-seq data, respectively. Comparing the nucleotide content of the regions around the TF-bound sites to the counterpart unbound regions containing the same consensus motifs revealed significant differences that extend far beyond the core-binding site. Specifically, the environment of the bound motifs demonstrated unique sequence compositions, DNA shape features, and overall high similarity to the core-binding motif. Notably, the regions around the binding sites of TFs that belong to the same TF families exhibited similar features, with high agreement between the in vitro and in vivo data sets. We propose that these unique features assist in guiding TFs to their cognate binding sites.


Assuntos
Sítios de Ligação , Motivos de Nucleotídeos , Fatores de Transcrição/metabolismo , Animais , Composição de Bases , Sequência de Bases , Biologia Computacional/métodos , Regulação da Expressão Gênica , Genômica/métodos , Humanos , Elementos Reguladores de Transcrição , Sequências Reguladoras de Ácido Nucleico , Técnica de Seleção de Aptâmeros , Transcrição Gênica
9.
Dig Dis Sci ; 63(12): 3382-3397, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30196390

RESUMO

BACKGROUND AND AIMS: Concanavalin A is known to activate T cells and to cause liver injury and hepatitis, mediated in part by secretion of TNFα from macrophages. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been shown to prevent tissue damage in various animal models of inflammation. The objectives of this study were to evaluate the efficacy and mechanism of the PARP-1 inhibitor 3-aminobenzamide (3-AB) in preventing concanavalin A-induced liver damage. METHODS: We tested the in vivo effects of 3-AB on concanavalin A-treated mice, its effects on lipopolysaccharide (LPS)-stimulated macrophages in culture, and its ability to act as a scavenger in in vitro assays. RESULTS: 3-AB markedly reduced inflammation, oxidative stress, and liver tissue damage in concanavalin A-treated mice. In LPS-stimulated RAW264.7 macrophages, 3-AB inhibited NFκB transcriptional activity and subsequent expression of TNFα and iNOS and blocked NO production. In vitro, 3-AB acted as a hydrogen peroxide scavenger. The ROS scavenger N-acetylcysteine (NAC) and the ROS formation inhibitor diphenyleneiodonium (DPI) also inhibited TNFα expression in stimulated macrophages, but unlike 3-AB, NAC and DPI were unable to abolish NFκB activity. PARP-1 knockout failed to affect NFκB and TNFα suppression by 3-AB in stimulated macrophages. CONCLUSIONS: Our results suggest that 3-AB has a therapeutic effect on concanavalin A-induced liver injury by inhibiting expression of the key pro-inflammatory cytokine TNFα, via PARP-1-independent NFκB suppression and via an NFκB-independent anti-oxidative mechanism.


Assuntos
Benzamidas/farmacologia , Hepatite , Macrófagos , Doença Aguda , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Células Cultivadas , Concanavalina A/farmacologia , Modelos Animais de Doenças , Hepatite/metabolismo , Hepatite/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Mitógenos/farmacologia , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismo
10.
Bioessays ; 38(7): 605-12, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27192961

RESUMO

Transcription factors (TFs) have to find their binding sites, which are distributed throughout the genome. Facilitated diffusion is currently the most widely accepted model for this search process. Based on this model the TF alternates between one-dimensional sliding along the DNA, and three-dimensional bulk diffusion. In this view, the non-specific associations between the proteins and the DNA play a major role in the search dynamics. However, little is known about how the DNA properties around the motif contribute to the search. Accumulating evidence showing that TF binding sites are embedded within a unique environment, specific to each TF, leads to the hypothesis that the search process is facilitated by favorable DNA features that help to improve the search efficiency. Here, we review the field and present the hypothesis that TF-DNA recognition is dictated not only by the motif, but is also influenced by the environment in which the motif resides.


Assuntos
DNA/metabolismo , Modelos Químicos , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , DNA/química , Difusão , Humanos , Modelos Genéticos , Ligação Proteica
11.
Nucleic Acids Res ; 42(1): 430-41, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24078250

RESUMO

Protein-DNA recognition is a critical component of gene regulatory processes but the underlying molecular mechanisms are not yet completely understood. Whereas the DNA binding preferences of transcription factors (TFs) are commonly described using nucleotide sequences, the 3D DNA structure is recognized by proteins and is crucial for achieving binding specificity. However, the ability to analyze DNA shape in a high-throughput manner made it only recently feasible to integrate structural information into studies of protein-DNA binding. Here we focused on the homeodomain family of TFs and analyzed the DNA shape of thousands of their DNA binding sites, investigating the covariation between the protein sequence and the sequence and shape of their DNA targets. We found distinct homeodomain regions that were more correlated with either the nucleotide sequence or the DNA shape of their preferred binding sites, demonstrating different readout mechanisms through which homeodomains attain DNA binding specificity. We identified specific homeodomain residues that likely play key roles in DNA recognition via shape readout. Finally, we showed that adding DNA shape information when characterizing binding sites improved the prediction accuracy of homeodomain binding specificities. Taken together, our findings indicate that DNA shape information can generally provide new mechanistic insights into TF binding.


Assuntos
DNA/química , Proteínas de Homeodomínio/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Homeodomínio/metabolismo , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Fatores de Transcrição/metabolismo
12.
Nucleic Acids Res ; 42(Database issue): D148-55, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24214955

RESUMO

Transcription factor binding sites (TFBSs) are most commonly characterized by the nucleotide preferences at each position of the DNA target. Whereas these sequence motifs are quite accurate descriptions of DNA binding specificities of transcription factors (TFs), proteins recognize DNA as a three-dimensional object. DNA structural features refine the description of TF binding specificities and provide mechanistic insights into protein-DNA recognition. Existing motif databases contain extensive nucleotide sequences identified in binding experiments based on their selection by a TF. To utilize DNA shape information when analysing the DNA binding specificities of TFs, we developed a new tool, the TFBSshape database (available at http://rohslab.cmb.usc.edu/TFBSshape/), for calculating DNA structural features from nucleotide sequences provided by motif databases. The TFBSshape database can be used to generate heat maps and quantitative data for DNA structural features (i.e., minor groove width, roll, propeller twist and helix twist) for 739 TF datasets from 23 different species derived from the motif databases JASPAR and UniPROBE. As demonstrated for the basic helix-loop-helix and homeodomain TF families, our TFBSshape database can be used to compare, qualitatively and quantitatively, the DNA binding specificities of closely related TFs and, thus, uncover differential DNA binding specificities that are not apparent from nucleotide sequence alone.


Assuntos
DNA/química , Bases de Dados Genéticas , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Proteínas de Homeodomínio/metabolismo , Humanos , Internet , Camundongos , Conformação de Ácido Nucleico , Motivos de Nucleotídeos
13.
Nucleic Acids Res ; 41(Web Server issue): W56-62, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23703209

RESUMO

We present a method and web server for predicting DNA structural features in a high-throughput (HT) manner for massive sequence data. This approach provides the framework for the integration of DNA sequence and shape analyses in genome-wide studies. The HT methodology uses a sliding-window approach to mine DNA structural information obtained from Monte Carlo simulations. It requires only nucleotide sequence as input and instantly predicts multiple structural features of DNA (minor groove width, roll, propeller twist and helix twist). The results of rigorous validations of the HT predictions based on DNA structures solved by X-ray crystallography and NMR spectroscopy, hydroxyl radical cleavage data, statistical analysis and cross-validation, and molecular dynamics simulations provide strong confidence in this approach. The DNAshape web server is freely available at http://rohslab.cmb.usc.edu/DNAshape/.


Assuntos
DNA/química , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Software , Genômica , Internet , Conformação de Ácido Nucleico
14.
Proc Natl Acad Sci U S A ; 109(42): E2875-84, 2012 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-23027969

RESUMO

Although the protooncogene c-Jun plays a critical role in cell proliferation, cell death, and malignant transformation, DNA microarray screens have identified only a few human cancer types with aberrant expression of c-Jun. Here, we show that c-Jun accumulation is robustly elevated in human glioblastoma and that this increase contributes to the malignant properties of the cells. Most importantly, the increase in c-Jun protein accumulation occurs with no corresponding increase in c-Jun mRNA or the half-life of the c-Jun protein but, rather, in the translatability of the transcript. The c-Jun 5'UTR harbors a potent internal ribosomal entry site (IRES) with a virus-like IRES domain that directs cap-independent translation in glioblastoma cells. Accumulation of c-Jun is not dependent on MAPK activity but can be stimulated by a cytoskeleton-dependent pathway. Our findings provide evidence that human c-Jun is an IRES-containing cellular transcript that contributes to cancer development through translational activation. This previously undescribed mechanism of c-Jun regulation might also be relevant to other types of human cancer and offers unique potential targets for therapy.


Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Glioblastoma/metabolismo , Biossíntese de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ribossomos/metabolismo , Animais , Western Blotting , Células Cultivadas , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Luciferases , Biossíntese de Proteínas/genética , Ratos , Ratos Sprague-Dawley
15.
Curr Opin Genet Dev ; 87: 102235, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39053028

RESUMO

To regulate gene expression, the macromolecular components of the mammalian interphase nucleus are spatially organized into a myriad of functional compartments. Over the past decade, increasingly sophisticated genomics, microscopy, and functional approaches have probed this organization in unprecedented detail. These investigations have linked chromatin-associated noncoding RNAs to specific nuclear compartments and uncovered mechanisms by which these RNAs establish such domains. In this review, we focus on the long non-coding RNA Xist and summarize new evidence demonstrating the significance of chromatin reconfiguration in creating the inactive X-chromosome compartment. Differences in chromatin compaction correlate with distinct levels of gene repression on the X-chromosome, potentially explaining how human XIST can induce chromosome-wide dampening and silencing of gene expression at different stages of human development.


Assuntos
Mecanismo Genético de Compensação de Dose , RNA Longo não Codificante , Cromossomo X , Humanos , Animais , Mecanismo Genético de Compensação de Dose/genética , Cromossomo X/genética , RNA Longo não Codificante/genética , Cromatina/genética , Inativação do Cromossomo X/genética , RNA não Traduzido/genética , Mamíferos/genética
16.
J Invest Dermatol ; 143(12): 2494-2506.e4, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37236596

RESUMO

Skin pigmentation is paused after sun exposure; however, the mechanism behind this pausing is unknown. In this study, we found that the UVB-induced DNA repair system, led by the ataxia telangiectasia mutated (ATM) protein kinase, represses MITF transcriptional activity of pigmentation genes while placing MITF in DNA repair mode, thus directly inhibiting pigment production. Phosphoproteomics analysis revealed ATM to be the most significantly enriched pathway among all UVB-induced DNA repair systems. ATM inhibition in mouse or human skin, either genetically or chemically, induces pigmentation. Upon UVB exposure, MITF transcriptional activation is blocked owing to ATM-dependent phosphorylation of MITF on S414, which modifies MITF activity and interactome toward DNA repair, including binding to TRIM28 and RBBP4. Accordingly, MITF genome occupancy is enriched in sites of high DNA damage that are likely repaired. This suggests that ATM harnesses the pigmentation key activator for the necessary rapid, efficient DNA repair, thus optimizing the chances of the cell surviving. Data are available from ProteomeXchange with the identifier PXD041121.


Assuntos
Ataxia Telangiectasia , Humanos , Animais , Camundongos , Pigmentação da Pele/genética , Reparo do DNA , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Transdução de Sinais , Dano ao DNA , Fosforilação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo
17.
Proteins ; 80(2): 482-9, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22086767

RESUMO

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure.


Assuntos
Algoritmos , DNA/metabolismo , Modelos Moleculares , Proteínas/química , Proteínas/metabolismo , RNA/metabolismo , Sítios de Ligação , Biologia Computacional/métodos , Bases de Dados de Proteínas , Proteína 1 de Resposta de Crescimento Precoce/química , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Conformação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Software , Eletricidade Estática , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo
18.
Nucleic Acids Res ; 38(Web Server issue): W281-5, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20501600

RESUMO

Alternative splicing (AS) is a post-transcriptional process considered to be responsible for the huge diversity of proteins in higher eukaryotes. AS events are regulated by different splicing factors (SFs) that bind to sequence elements on the RNA. SFmap is a web server for predicting putative SF binding sites in genomic data (http://sfmap.technion.ac.il). SFmap implements the COS(WR) algorithm, which computes similarity scores for a given regulatory motif based on information derived from its sequence environment and its evolutionary conservation. Input for SFmap is a human genomic sequence or a list of sequences in FASTA format that can either be uploaded from a file or pasted into a window. SFmap searches within a given sequence for significant hits of binding motifs that are either stored in our database or defined by the user. SFmap results are provided both as a text file and as a graphical web interface.


Assuntos
Processamento Alternativo , Proteínas de Ligação a RNA/metabolismo , Software , Algoritmos , Sítios de Ligação , Genoma Humano , Humanos , Internet , Análise de Sequência de RNA , Interface Usuário-Computador
19.
Stem Cell Reports ; 17(6): 1268-1278, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35594860

RESUMO

Human germ cell development is a highly regulated process beginning soon after embryo implantation with the specification of primordial germ cells (PGCs) and ending in adulthood with the differentiation of gametes. Here, we show that fibroblast growth factor receptor 3 (FGFR3) is expressed by human PGCs during the first and second trimester, becoming repressed as PGCs differentiate into primordial oocytes. Using fluorescence-activated cell sorting (FACS) with antibodies that recognize FGFR3 followed by single-cell RNA sequencing, we show that isolating FGFR3-positive cells enriches for human PGCs. Taken together, FGFR3 could be used in future studies as a strategy to identify maturing PGCs in vitro.


Assuntos
Células Germinativas , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos , Diferenciação Celular , Citometria de Fluxo , Humanos , Oócitos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo
20.
Nat Cell Biol ; 22(12): 1436-1446, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33257808

RESUMO

X-chromosome dosage compensation in female placental mammals is achieved by X-chromosome inactivation (XCI). Human pre-implantation embryos are an exception, in which dosage compensation occurs by X-chromosome dampening (XCD). Here, we examined whether XCD extends to human prenatal germ cells given their similarities to naive pluripotent cells. We found that female human primordial germ cells (hPGCs) display reduced X-linked gene expression before entering meiosis. Moreover, in hPGCs, both X chromosomes are active and express the long non-coding RNAs X active coating transcript (XACT) and X inactive specific transcript (XIST)-the master regulator of XCI-which are silenced after entry into meiosis. We find that XACT is a hPGC marker, describe XCD associated with XIST expression in hPGCs and suggest that XCD evolved in humans to regulate X-linked genes in pre-implantation embryos and PGCs. Furthermore, we found a unique mechanism of X-chromosome regulation in human primordial oocytes. Therefore, future studies of human germline development must consider the sexually dimorphic X-chromosome dosage compensation mechanisms in the prenatal germline.


Assuntos
Cromossomos Humanos X/genética , Mecanismo Genético de Compensação de Dose , Células Germinativas/metabolismo , Inativação do Cromossomo X , Blastocisto/citologia , Blastocisto/metabolismo , Células Cultivadas , Desenvolvimento Embrionário , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização in Situ Fluorescente , Masculino , RNA Longo não Codificante/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA