RESUMO
MOTIVATION: Unraveling the transcriptional programs that control how cells divide, differentiate, and respond to their environments requires a precise understanding of transcription factors' (TFs) DNA-binding activities. Calling cards (CC) technology uses transposons to capture transient TF binding events at one instant in time and then read them out at a later time. This methodology can also be used to simultaneously measure TF binding and mRNA expression from single-cell CC and to record and integrate TF binding events across time in any cell type of interest without the need for purification. Despite these advantages, there has been a lack of dedicated bioinformatics tools for the detailed analysis of CC data. RESULTS: We introduce Pycallingcards, a comprehensive Python module specifically designed for the analysis of single-cell and bulk CC data across multiple species. Pycallingcards introduces two innovative peak callers, CCcaller and MACCs, enhancing the accuracy and speed of pinpointing TF binding sites from CC data. Pycallingcards offers a fully integrated environment for data visualization, motif finding, and comparative analysis with RNA-seq and ChIP-seq datasets. To illustrate its practical application, we have reanalyzed previously published mouse cortex and glioblastoma datasets. This analysis revealed novel cell-type-specific binding sites and potential sex-linked TF regulators, furthering our understanding of TF binding and gene expression relationships. Thus, Pycallingcards, with its user-friendly design and seamless interface with the Python data science ecosystem, stands as a critical tool for advancing the analysis of TF functions via CC data. AVAILABILITY AND IMPLEMENTATION: Pycallingcards can be accessed on the GitHub repository: https://github.com/The-Mitra-Lab/pycallingcards.
Assuntos
Ecossistema , Fatores de Transcrição , Animais , Camundongos , Imunoprecipitação da Cromatina , Fatores de Transcrição/metabolismo , Sítios de Ligação , Ligação Proteica , Análise de Sequência de DNARESUMO
Pseudohyphal growth is a nutrient-regulated program in which budding yeast form multicellular filaments of elongated and connected cells. Filamentous growth is required for virulence in pathogenic fungi and provides an informative model of stress-responsive signaling. The genetics and regulatory networks modulating pseudohyphal growth have been studied extensively, but little is known regarding the changes in metabolites that enable pseudohyphal filament formation. Inositol signaling molecules are an important class of metabolite messengers encompassing highly phosphorylated and diffusible inositol polyphosphates (InsPs). We report here that the InsP biosynthesis pathway is required for wild-type pseudohyphal growth. Under nitrogen-limiting conditions that can induce filamentation, InsPs exhibit characteristic profiles, distinguishing the InsP7 pyrophosphate isoforms 1PP-InsP5 and 5PP-InsP5. Deletion and overexpression analyses of InsP kinases identify elevated levels of 5PP-InsP5 relative to 1PP-InsP5 in mutants exhibiting hyper-filamentous growth. Overexpression of KCS1, which promotes formation of inositol pyrophosphates, is sufficient to drive pseudohyphal filamentation on medium with normal nitrogen levels. We find that the kinases Snf1p (AMPK), Kss1p, and Fus3p (MAPKs), required for wild-type pseudohyphal growth, are also required for wild-type InsP levels. Deletion analyses of the corresponding kinase genes indicate elevated InsP3 levels and an absence of exaggerated 5PP-InsP5 peaks in trace profiles from snf1Δ/Δ and kss1Δ/Δ mutants exhibiting decreased pseudohyphal filamentation. Elevated 5PP-InsP5:1PP-InsP5 ratios are present in the hyperfilamentous fus3 deletion mutant. Collectively, the data identify the presence of elevated 5PP-InsP5 levels relative to other inositol pyrophosphates as an in vivo marker of hyper-filamentous growth, while providing initial evidence for the regulation of InsP signaling by pseudohyphal growth kinases.
Assuntos
Hifas/metabolismo , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/fisiologia , Ciclo Celular/fisiologia , Regulação Fúngica da Expressão Gênica/genética , Hifas/genética , Fenótipo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Fosfotransferases/metabolismo , Polifosfatos/metabolismo , Isoformas de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genéticaRESUMO
UNLABELLED: TcpP and ToxR coordinately regulate transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP and ToxR are membrane-localized transcription factors, each with a periplasmic domain containing two cysteines. In ToxR, these cysteines form an intramolecular disulfide bond and a cysteine-to-serine substitution affects activity. We determined that the two periplasmic cysteines of TcpP also form an intramolecular disulfide bond. Disruption of this intramolecular disulfide bond by mutation of either cysteine resulted in formation of intermolecular disulfide bonds. Furthermore, disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP. While the decreased stability of TcpP-C207S resulted in a nearly complete loss of toxT activation and cholera toxin (CT) production, the second cysteine mutant, TcpP-C218S, was partially resistant to proteolytic degradation and maintained â¼50% toxT activation capacity. TcpP-C218S was also TcpH independent, since deletion of tcpH did not affect the stability of TcpP-C218S, whereas wild-type TcpP was degraded in the absence of TcpH. Finally, TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, suggesting that the single periplasmic cysteine in TcpH may assist with disulfide bond formation in TcpP by interacting with the periplasmic cysteines of TcpP. Consistent with this finding, a TcpH-C114S mutant was unable to stabilize TcpP and was itself unstable. Our findings demonstrate a periplasmic disulfide bond in TcpP is critical for TcpP stability and virulence gene expression. IMPORTANCE: The Vibrio cholerae transcription factor TcpP, in conjunction with ToxR, regulates transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP is a membrane-localized transcription factor with a periplasmic domain containing two cysteines. We determined that the two periplasmic cysteines of TcpP form an intramolecular disulfide bond and disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP and reduced virulence gene expression. Normally TcpH, another membrane-localized periplasmic protein, protects TcpP from degradation. However, we found that TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, indicating that the periplasmic cysteines of TcpP are required for functional interaction with TcpH and that this interaction is required for both TcpP and TcpH stability.