RESUMEN
Differentiated cells are epigenetically stable, but can be reprogrammed to pluripotency by expression of the OSKM transcription factors. Despite significant effort, relatively little is known about the cellular requirements for reprogramming and how they affect the properties of induced pluripotent stem cells. We have performed high-content screening with small interfering RNAs targeting 300 chromatin-associated factors and extracted colony-level quantitative features. This revealed five morphological phenotypes in early reprogramming, including one displaying large round colonies exhibiting an early block of reprogramming. Using RNA sequencing, we identified transcriptional changes associated with these phenotypes. Furthermore, double knockdown epistasis experiments revealed that BRCA1, BARD1, and WDR5 functionally interact and are required for the DNA damage response. In addition, the mesenchymal-to-epithelial transition is affected in Brca1, Bard1, and Wdr5 knockdowns. Our data provide a resource of chromatin-associated factors in early reprogramming and underline colony morphology as an important high-dimensional readout for reprogramming quality.
Asunto(s)
Proteína BRCA1/genética , Reprogramación Celular/genética , Daño del ADN , Transición Epitelial-Mesenquimal/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Proteína BRCA1/metabolismo , Cromatina/genética , Cromatina/metabolismo , Reparación del ADN , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Fenotipo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Environmental stimuli often lead to heterogeneous cellular responses and transcriptional output. We developed single-cell RNA and Immunodetection (RAID) to allow combined analysis of the transcriptome and intracellular (phospho-)proteins from fixed single cells. RAID successfully recapitulated differentiation-state changes at the protein and mRNA level in human keratinocytes. Furthermore, we show that differentiated keratinocytes that retain high phosphorylated FAK levels, a feature associated with stem cells, also express a selection of stem cell associated transcripts. Our data demonstrates that RAID allows investigation of heterogeneous cellular responses to environmental signals at the mRNA and phospho-proteome level.
Asunto(s)
Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Queratinocitos/citología , Análisis de la Célula Individual/métodos , Diferenciación Celular , Células Cultivadas , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Humanos , Queratinocitos/química , Fosforilación , Proteómica/métodos , Quinazolinas/farmacología , Fijación del Tejido , Tirfostinos/farmacologíaRESUMEN
Cell-based small molecule screening is an effective strategy leading to new medicines. Scientists in the pharmaceutical industry as well as in academia have made tremendous progress in developing both large-scale and smaller-scale screening assays. However, an accessible and universal technology for measuring large numbers of molecular and cellular phenotypes in many samples in parallel is not available. Here we present the immuno-detection by sequencing (ID-seq) technology that combines antibody-based protein detection and DNA-sequencing via DNA-tagged antibodies. We use ID-seq to simultaneously measure 70 (phospho-)proteins in primary human epidermal stem cells to screen the effects of ~300 kinase inhibitor probes to characterise the role of 225 kinases. The results show an association between decreased mTOR signalling and increased differentiation and uncover 13 kinases potentially regulating epidermal renewal through distinct mechanisms. Taken together, our work establishes ID-seq as a flexible solution for large-scale high-dimensional phenotyping in fixed cell populations.
Asunto(s)
Anticuerpos/metabolismo , Inmunoensayo/métodos , Proteoma/metabolismo , Proteómica/métodos , Análisis de Secuencia de ADN/métodos , Anticuerpos/inmunología , Diferenciación Celular/genética , Células Cultivadas , Células Epidérmicas/citología , Perfilación de la Expresión Génica , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Fenotipo , Proteoma/genética , Proteoma/inmunología , Transducción de Señal/genética , Células Madre/metabolismoRESUMEN
Wnt/ß-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multimeric Wnt-FZD assemblies via intramembrane interactions. Subsequently, these Wnt-FZD-TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions.
Asunto(s)
Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vía de Señalización Wnt/fisiología , Proteína Wnt3A/metabolismo , Animales , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteínas de la Membrana/genética , Ratones , Complejos Multiproteicos/genética , Proteínas del Tejido Nervioso/genética , Proteína Wnt3A/genéticaRESUMEN
Immuno-PCR combines specific antibody-based protein detection with the sensitivity of PCR-based quantification through the use of antibody-DNA conjugates. The production of such conjugates depends on the availability of quick and efficient conjugation strategies for the two biomolecules. Here, we present an approach to produce cleavable antibody-DNA conjugates, employing the fast kinetics of the inverse electron-demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO). Our strategy consists of three steps. First, antibodies are functionalized with chemically cleavable NHS-s-s-tetrazine. Subsequently, double-stranded DNA is functionalized with TCO by enzymatic addition of N3-dATP and coupling to trans-Cyclooctene-PEG12-Dibenzocyclooctyne (TCO-PEG12-DBCO). Finally, conjugates are quickly and efficiently obtained by mixing the functionalized antibodies and dsDNA at low molar ratios of 1:2. In addition, introduction of a chemically cleavable disulphide linker facilitates release and sensitive detection of the dsDNA after immuno-staining. We show specific and sensitive protein detection in immuno-PCR for human epidermal stem cell markers, ITGA6 and ITGB1, and the differentiation marker Transglutaminase 1 (TGM1). We anticipate that the production of chemically cleavable antibody-DNA conjugates will provide a solid basis for the development of multiplexed immuno-PCR experiments and immuno-sequencing methodologies.
Asunto(s)
Anticuerpos/metabolismo , ADN/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Proteínas/análisis , Anticuerpos/química , ADN/genética , Humanos , Sensibilidad y EspecificidadRESUMEN
Wnt/ß-catenin signalling controls development and adult tissue homeostasis and causes cancer when inappropriately activated. In unstimulated cells, an Axin1-centred multi-protein complex phosphorylates the transcriptional co-activator ß-catenin, marking it for degradation. Wnt signalling antagonizes ß-catenin proteolysis, leading to its accumulation and target gene expression. How Wnt stimulation alters the size distribution, composition and activity of endogenous Axin1 complexes remains poorly understood. Here, we employed two-dimensional blue native/SDS-PAGE to analyse endogenous Axin1 and ß-catenin complexes during Wnt signalling. We show that the size range of Axin1 complexes is conserved between species and remains largely unaffected by Wnt stimulation. We detect a striking Wnt-dependent, cytosolic accumulation of both non-phosphorylated and phosphorylated ß-catenin within a 450 kDa Axin1-based complex and in a distinct, Axin1-free complex of 200 kDa. These results argue that during Wnt stimulation, phosphorylated ß-catenin is released from the Axin1 complex but fails to undergo immediate degradation. Importantly, in APC-mutant cancer cells, the distribution of Axin1 and ß-catenin complexes strongly resembles that of Wnt-stimulated cells. Our findings argue that Wnt signals and APC mutations interfere with the turnover of phosphorylated ß-catenin. Furthermore, our results suggest that the accumulation of small-sized ß-catenin complexes may serve as an indicator of Wnt pathway activity in primary cancer cells.
Asunto(s)
Proteína Axina/metabolismo , Citoplasma/metabolismo , Procesamiento Proteico-Postraduccional , Vía de Señalización Wnt , beta Catenina/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Fosforilación , Unión Proteica , ProteolisisRESUMEN
BACKGROUND: Pectins are diverse and very complex biomolecules and their structure depends on the plant species and tissue. It was previously shown that derivatives of pectic polymers and oligosaccharides from pectins have positive effects on human health. To obtain specific pectic oligosaccharides, highly defined enzymatic mixes are required. Filamentous fungi are specialized in plant cell wall degradation and some produce a broad range of pectinases. They may therefore shed light on the enzyme mixes needed for partial hydrolysis. RESULTS: The growth profiles of 12 fungi on four pectins and four structural elements of pectins show that the presence/absence of pectinolytic genes in the fungal genome clearly correlates with their ability to degrade pectins. However, this correlation is less clear when we zoom in to the pectic structural elements. CONCLUSIONS: This study highlights the complexity of the mechanisms involved in fungal degradation of complex carbon sources such as pectins. Mining genomes and comparative genomics are promising first steps towards the production of specific pectinolytic fractions.
Asunto(s)
Hongos/enzimología , Hongos/metabolismo , Pectinas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hongos/genética , Hongos/crecimiento & desarrollo , Genoma Fúngico/genética , Poligalacturonasa/genética , Poligalacturonasa/metabolismo , Trichoderma/enzimología , Trichoderma/genética , Trichoderma/crecimiento & desarrollo , Trichoderma/metabolismoRESUMEN
Degradation of cytosolic ß-catenin by the APC/Axin1 destruction complex represents the key regulated step of the Wnt pathway. It is incompletely understood how the Axin1 complex exerts its Wnt-regulated function. Here, we examine the mechanism of Wnt signaling under endogenous levels of the Axin1 complex. Our results demonstrate that ß-catenin is not only phosphorylated inside the Axin1 complex, but also ubiquinated and degraded via the proteasome, all within an intact Axin1 complex. In disagreement with current views, we find neither a disassembly of the complex nor an inhibition of phosphorylation of Axin1-bound ß-catenin upon Wnt signaling. Similar observations are made in primary intestinal epithelium and in colorectal cancer cell lines carrying activating Wnt pathway mutations. Wnt signaling suppresses ß-catenin ubiquitination normally occurring within the complex, leading to complex saturation by accumulated phospho-ß-catenin. Subsequently, newly synthesized ß-catenin can accumulate in a free cytosolic form and engage nuclear TCF transcription factors.
Asunto(s)
Proteína Axina/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Datos de Secuencia Molecular , Mutación , Péptidos/análisis , Péptidos/química , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , beta Catenina/genéticaRESUMEN
SCF(ßTrCP) is the ubiquitin ligase for a wide variety of substrates and functions in many cellular processes. ßTrCP, the substrate binding factor of the SCF complex, has two isoforms, produced from different genes, and several splice variants. Despite a certain level of redundancy, knock-out studies show different phenotypes indicating different preferential substrates for the two isoforms. However, until now functional differences between ßTrCP1 and 2 were not studied at the endogenous protein level. We generated isoform-specific antibodies against ßTrCP to characterise endogenous ßTrCP isoforms and splice variants. We show that endogenous ßTrCP1 and 2 localise to both nucleus and cytosol. Interestingly, we find that one splice variant of ßTrCP2 localises exclusively to the nucleus and another only to the cytosol. In addition, we show that the substrate binding domain of ßTrCP is the dominant localisation determinant.