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1.
Cell ; 154(4): 801-13, 2013 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-23953112

RESUMEN

During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M phase. These results suggest that cohesin-binding functions as a cellular memory that promotes re-establishment of TF clusters after DNA replication and chromatin condensation.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Proteínas Cromosómicas no Histona/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Motivos de Nucleótidos , Cohesinas
2.
J Cell Sci ; 132(14)2019 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-31217285

RESUMEN

Growth factor-induced signal transduction pathways are tightly regulated at multiple points intracellularly, but how cells monitor levels of extracellular ligand and translate this information into appropriate downstream responses remains unclear. Understanding signalling dynamics is thus a key challenge in determining how cells respond to external cues. Here, we demonstrate that different TGF-ß family ligands, namely activin A and BMP4, signal with distinct dynamics, which differ profoundly from those of TGF-ß itself. The signalling dynamics are driven by differences in the localisation and internalisation of receptors for each ligand, which in turn determine the capability of cells to monitor levels of extracellular ligand. By using mathematical modelling, we demonstrate that the distinct receptor behaviours and signalling dynamics observed may be primarily driven by differences in ligand-receptor affinity. Furthermore, our results provide a clear rationale for the different mechanisms of pathway regulation found in vivo for each of these growth factors.


Asunto(s)
Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Activinas/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Ligandos , Ratones , Modelos Biológicos , Células 3T3 NIH , Biosíntesis de Proteínas , Proteínas Smad/metabolismo
3.
Mol Syst Biol ; 15(8): e9059, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31464368

RESUMEN

Haapaniemi et al address the issues raised by Brown et al and discuss several differences between the analyses performed by the two groups.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Sistemas CRISPR-Cas , Daño del ADN , Proteína p53 Supresora de Tumor
4.
Nat Rev Mol Cell Biol ; 8(12): 970-82, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18000526

RESUMEN

Ligands of the transforming growth factor-beta (TGFbeta) superfamily of growth factors initiate signal transduction through a bewildering complexity of ligand-receptor interactions. Signalling then converges to nuclear accumulation of transcriptionally active SMAD complexes and gives rise to a plethora of specific functional responses in both embryos and adult organisms. Current research is focused on the mechanisms that regulate SMAD activity to evoke cell-type-specific and context-dependent transcriptional programmes. An equally important challenge is understanding the functional role of signal strength and duration. How are these quantitative aspects of the extracellular signal regulated? How are they then sensed and interpreted, and how do they affect responses?


Asunto(s)
Transducción de Señal , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/metabolismo , Animales , Humanos , Proteínas Smad/química
5.
Mol Syst Biol ; 13(10): 945, 2017 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-28993443

RESUMEN

Loss-of-function screening by CRISPR/Cas9 gene knockout with pooled, lentiviral guide libraries is a widely applicable method for systematic identification of genes contributing to diverse cellular phenotypes. Here, Random Sequence Labels (RSLs) are incorporated into the guide library, which act as unique molecular identifiers (UMIs) to allow massively parallel lineage tracing and lineage dropout screening. RSLs greatly improve the reproducibility of results by increasing both the precision and the accuracy of screens. They reduce the number of cells needed to reach a set statistical power, or allow a more robust screen using the same number of cells.


Asunto(s)
Técnicas de Inactivación de Genes , Biología de Sistemas/métodos , Sistemas CRISPR-Cas , Línea Celular , Biblioteca de Genes , Células HEK293 , Humanos
6.
EJNMMI Radiopharm Chem ; 9(1): 32, 2024 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-38637347

RESUMEN

BACKGROUND: Megalin (LRP2 receptor) mediates the endocytosis of radiolabeled peptides into proximal tubular kidney cells, which may cause nephrotoxicity due to the accumulation of a radioactive tracer. The study aimed to develop a cellular model of human kidney HK2 cells with LRP2 knockout (KO) using CRISPR/Cas9 technique. This model was employed for the determination of the megalin-mediated accumulation of 68Ga- and 99mTc-labeled 15-mer peptide developed to target the vascular endothelial growth factor (VEGF) receptor in oncology radiodiagnostics. RESULTS: The gene editing in the LRP2 KO model was verified by testing two well-known megalin ligands when higher viability of KO cells was observed after gentamicin treatment at cytotoxic concentrations and lower FITC-albumin internalization by the KO cells was detected in accumulation studies. Fluorescent-activated cell sorting was used to separate genetically modified LRP2 KO cell subpopulations. Moreover, flow cytometry with a specific antibody against megalin confirmed LRP2 knockout. The verified KO model identified both 68Ga- and 99mTc-radiolabeled 15-mer peptides as megalin ligands in accumulation studies. We found that both radiolabeled 15-mers enter LRP2 KO HK2 cells to a lesser extent compared to parent cells. Differences in megalin-mediated cellular uptake depending on the radiolabeling were not observed. Using biomolecular docking, the interaction site of the 15-mer with megalin was also described. CONCLUSION: The CRISPR/Cas9 knockout of LRP2 in human kidney HK2 cells is an effective approach for the determination of radiopeptide internalization mediated by megalin. This in vitro method provided direct molecular evidence for the cellular uptake of radiolabeled anti-VEGFR 15-mer peptides via megalin.

7.
Cancers (Basel) ; 16(13)2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-39001501

RESUMEN

The recurrence of diffuse large B-cell lymphoma (DLBCL) has been observed in 40% of cases. The standard of care for refractory/relapsed DLBCL (RR-DLBCL) is platinum-based treatment prior to autologous stem cell transplantation; however, the prognosis for RR-DLBCL patients remains poor. Thus, to identify genes affecting the cisplatin response in DLBCL, cisplatin-based whole-genome CRISPR-Cas9 knockout screens were performed in this study. We discovered DNA damage response (DDR) pathways as enriched among identified sensitizing CRISPR-mediated gene knockouts. In line, the knockout of the nucleotide excision repair genes XPA and ERCC6 sensitized DLBCL cells to platinum drugs irrespective of proliferation rate, thus documenting DDR as essential for cisplatin sensitivity in DLBCL. Functional analysis revealed that the loss of XPA and ERCC6 increased DNA damage levels and altered cell cycle distribution. Interestingly, we also identified BTK, which is involved in B-cell receptor signaling, to affect cisplatin response. The knockout of BTK increased cisplatin sensitivity in DLBCL cells, and combinatory drug screens revealed a synergistic effect of the BTK inhibitor, ibrutinib, with platinum drugs at low concentrations. Applying local and external DLBCL cohorts, we addressed the clinical relevance of the genes identified in the CRISPR screens. BTK was among the most frequently mutated genes with a frequency of 3-5%, and XPA and ERCC6 were also mutated, albeit at lower frequencies. Furthermore, 27-54% of diagnostic DLBCL samples had mutations in pathways that can sensitize cells to cisplatin. In conclusion, this study shows that XPA and ERCC6, in addition to BTK, are essential for the response to platinum-based drugs in DLBCL.

8.
Biophys J ; 104(12): 2595-606, 2013 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-23790367

RESUMEN

Chromosome bi-orientation at the metaphase spindle is essential for precise segregation of the genetic material. The process is error-prone, and error-correction mechanisms exist to switch misaligned chromosomes to the correct, bi-oriented configuration. Here, we analyze several possible dynamical scenarios to explore how cells might achieve correct bi-orientation in an efficient and robust manner. We first illustrate that tension-mediated feedback between the sister kinetochores can give rise to a bistable switch, which allows robust distinction between a loose attachment with low tension and a strong attachment with high tension. However, this mechanism has difficulties in explaining how bi-orientation is initiated starting from unattached kinetochores. We propose four possible mechanisms to overcome this problem (exploiting molecular noise; allowing an efficient attachment of kinetochores already in the absence of tension; a trial-and-error oscillation; and a stochastic bistable switch), and assess their impact on the bi-orientation process. Based on our results and supported by experimental data, we put forward a trial-and-error oscillation and a stochastic bistable switch as two elegant mechanisms with the potential to promote bi-orientation both efficiently and robustly.


Asunto(s)
Posicionamiento de Cromosoma , Cromosomas/metabolismo , Modelos Genéticos , Animales , Segregación Cromosómica , Humanos , Cinetocoros/metabolismo , Procesos Estocásticos
9.
Nat Commun ; 14(1): 5024, 2023 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-37596278

RESUMEN

A perimetastatic capsule is a strong positive prognostic factor in liver metastases, but its origin remains unclear. Here, we systematically quantify the capsule's extent and cellular composition in 263 patients with colorectal cancer liver metastases to investigate its clinical significance and origin. We show that survival improves proportionally with increasing encapsulation and decreasing tumor-hepatocyte contact. Immunostaining reveals the gradual zonation of the capsule, transitioning from benign-like NGFRhigh stroma at the liver edge to FAPhigh stroma towards the tumor. Encapsulation correlates with decreased tumor viability and preoperative chemotherapy. In mice, chemotherapy and tumor cell ablation induce capsule formation. Our results suggest that encapsulation develops where tumor invasion into the liver plates stalls, representing a reparative process rather than tumor-induced desmoplasia. We propose a model of metastases growth, where the efficient tumor colonization of the liver parenchyma and a reparative liver injury reaction are opposing determinants of metastasis aggressiveness.


Asunto(s)
Neoplasias Hepáticas , Animales , Ratones , Hepatocitos , Agresión , Relevancia Clínica
10.
J Biol Chem ; 286(9): 7648-60, 2011 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-21177872

RESUMEN

Factor-inhibiting hypoxia-inducible factor (FIH) catalyzes the ß-hydroxylation of an asparagine residue in the C-terminal transcriptional activation domain of the hypoxia inducible factor (HIF), a modification that negatively regulates HIF transcriptional activity. FIH also catalyzes the hydroxylation of highly conserved Asn residues within the ubiquitous ankyrin repeat domain (ARD)-containing proteins. Hydroxylation has been shown to stabilize localized regions of the ARD fold in the case of a three-repeat consensus ankyrin protein, but this phenomenon has not been demonstrated for the extensive naturally occurring ARDs. Here we report that the cytoskeletal ankyrin family are substrates for FIH-catalyzed hydroxylations. We show that the ARD of ankyrinR is multiply hydroxylated by FIH both in vitro and in endogenous proteins purified from human and mouse erythrocytes. Hydroxylation of the D34 region of ankyrinR ARD (ankyrin repeats 13-24) increases its conformational stability and leads to a reduction in its interaction with the cytoplasmic domain of band 3 (CDB3), demonstrating the potential for FIH-catalyzed hydroxylation to modulate protein-protein interactions. Unexpectedly we found that aspartate residues in ankyrinR and ankyrinB are hydroxylated and that FIH-catalyzed aspartate hydroxylation also occurs in other naturally occurring AR sequences. The crystal structure of an FIH variant in complex with an Asp-substrate peptide together with NMR analyses of the hydroxylation product identifies the 3S regio- and stereoselectivity of the FIH-catalyzed Asp hydroxylation, revealing a previously unprecedented posttranslational modification.


Asunto(s)
Ancirinas/metabolismo , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Citoesqueleto/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Ancirinas/química , Ancirinas/genética , Dominio Catalítico , Cristalografía , Células HEK293 , Humanos , Hidroxilación/fisiología , Oxigenasas de Función Mixta , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Transducción de Señal/fisiología
11.
Front Oncol ; 12: 852980, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35530310

RESUMEN

Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we extend previous findings by showing that the dienone compound b-AP15 inhibits proteasomal degradation of long-lived proteins. We show that exposure to b-AP15 results in increased association of the chaperones VCP/p97/Cdc48 and BAG6 with proteasomes. Comparisons between the gene expression profile generated by b-AP15 to those elicited by siRNA showed that knock-down of the proteasome-associated deubiquitinase (DUB) USP14 is the closest related to drug response. USP14 is a validated target for b-AP15 and we show that b-AP15 binds covalently to two cysteines, Cys203 and Cys257, in the ubiquitin-binding pocket of the enzyme. Consistent with this, deletion of USP14 resulted in decreased sensitivity to b-AP15. Targeting of USP14 was, however, found to not fully account for the observed proteasome inhibition. In search for additional targets, we utilized genome-wide CRISPR/Cas9 library screening and Proteome Integral Solubility Alteration (PISA) to identify mechanistically essential genes and b-AP15 interacting proteins respectively. Deletion of genes encoding mitochondrial proteins decreased the sensitivity to b-AP15, suggesting that mitochondrial dysfunction is coupled to cell death induced by b-AP15. Enzymes known to be involved in Phase II detoxification such as aldo-ketoreductases and glutathione-S-transferases were identified as b-AP15-targets using PISA. The finding that different exploratory approaches yielded different results may be explained in terms of a "target" not necessarily connected to the "mechanism of action" thus highlighting the importance of a holistic approach in the identification of drug targets. We conclude that b-AP15, and likely also other dienone compounds of the same class, affect protein degradation and proteasome function at more than one level.

12.
Proc Natl Acad Sci U S A ; 105(18): 6608-13, 2008 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-18443295

RESUMEN

TGF-beta-induced Smad signal transduction from the membrane into the nucleus is not linear and unidirectional, but rather a dynamic network that couples Smad phosphorylation and dephosphorylation through continuous nucleocytoplasmic shuttling of Smads. To understand the quantitative behavior of this network, we have developed a tightly constrained computational model, exploiting the interplay between mathematical modeling and experimental strategies. The model simultaneously reproduces four distinct datasets with excellent accuracy and provides mechanistic insights into how the network operates. We use the model to make predictions about the outcome of fluorescence recovery after photobleaching experiments and the behavior of a functionally impaired Smad2 mutant, which we then verify experimentally. Successful model performance strongly supports the hypothesis of a dynamic maintenance of Smad nuclear accumulation during active signaling. The presented work establishes Smad nucleocytoplasmic shuttling as a dynamic network that flexibly transmits quantitative features of the extracellular TGF-beta signal, such as its duration and intensity, into the nucleus.


Asunto(s)
Modelos Biológicos , Transducción de Señal , Proteína Smad2/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Sustitución de Aminoácidos/efectos de los fármacos , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Humanos , Cinética , Proteínas Mutantes/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/farmacología
13.
Elife ; 102021 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-34898428

RESUMEN

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein-Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.


Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Células Cultivadas/fisiología , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Edición Génica/métodos , Reparación del ADN/genética , Reparación del ADN/fisiología , Humanos
14.
Comput Struct Biotechnol J ; 18: 2237-2246, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32952937

RESUMEN

Over the last decade Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has been developed into a potent molecular biology tool used to rapidly modify genes or their expression in a multitude of ways. In parallel, CRISPR-based screening approaches have been developed as powerful discovery platforms for dissecting the genetic basis of cellular behavior, as well as for drug target discovery. CRISPR screens can be designed in numerous ways. Here, we give a brief background to CRISPR screens and discuss the pros and cons of different design approaches, including unbiased genome-wide screens that target all known genes, as well as hypothesis-driven custom screens in which selected subsets of genes are targeted (Fig. 1). We provide several suggestions for how a custom screen can be designed, which could broadly serve as inspiration for any experiment that includes candidate gene selection. Finally, we discuss how results from CRISPR screens could be translated into drug development, as well as future trends we foresee in the rapidly evolving CRISPR screen field.

15.
Mol Cell Biol ; 25(22): 9845-58, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16260601

RESUMEN

Upon transforming growth factor beta (TGF-beta) stimulation, Smads accumulate in the nucleus, where they regulate gene expression. Using fluorescence perturbation experiments on Smad2 and Smad4 fused to either enhanced green fluorescent protein or photoactivatable green fluorescent protein, we have studied the kinetics of Smad nucleocytoplasmic shuttling in a quantitative manner in vivo. We have obtained rate constants for import and export of Smad2 and show that the cytoplasmic localization of Smad2 in uninduced cells reflects its nuclear export being more rapid than import. We find that TGF-beta-induced nuclear accumulation of Smad2 is caused by a pronounced drop in the export rate of Smad2 from the nucleus, which is associated with a strong decrease in nuclear mobility of Smad2 and Smad4. TGF-beta-induced nuclear accumulation involves neither a release from cytoplasmic retention nor an increase in Smad2 import rate. Hence, TGF-beta-dependent nuclear accumulation of Smad2 is caused exclusively by selective nuclear trapping of phosphorylated, complexed Smad2. The proposed mechanism reconciles signal-dependent nuclear accumulation of Smad2 with its continuous nucleocytoplasmic cycling properties.


Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Smad/fisiología , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Transporte Biológico , Línea Celular Tumoral , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Cinética , Luz , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Fosforilación , Plásmidos/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta2 , Xenopus
16.
Nat Med ; 24(7): 927-930, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29892067

RESUMEN

Here, we report that genome editing by CRISPR-Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR-Cas9.


Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Daño del ADN , Edición Génica , Proteína p53 Supresora de Tumor/metabolismo , Ciclo Celular , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células HEK293 , Humanos , Ribonucleoproteínas/metabolismo
17.
Nat Commun ; 9(1): 3664, 2018 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-30202008

RESUMEN

Point mutations in cancer have been extensively studied but chromosomal gains and losses have been more challenging to interpret due to their unspecific nature. Here we examine high-resolution allelic imbalance (AI) landscape in 1699 colorectal cancers, 256 of which have been whole-genome sequenced (WGSed). The imbalances pinpoint 38 genes as plausible AI targets based on previous knowledge. Unbiased CRISPR-Cas9 knockout and activation screens identified in total 79 genes within AI peaks regulating cell growth. Genetic and functional data implicate loss of TP53 as a sufficient driver of AI. The WGS highlights an influence of copy number aberrations on the rate of detected somatic point mutations. Importantly, the data reveal several associations between AI target genes, suggesting a role for a network of lineage-determining transcription factors in colorectal tumorigenesis. Overall, the results unravel the contribution of AI in colorectal cancer and provide a plausible explanation why so few genes are commonly affected by point mutations in cancers.


Asunto(s)
Desequilibrio Alélico , Neoplasias Colorrectales/genética , Predisposición Genética a la Enfermedad , Sistemas CRISPR-Cas , Aberraciones Cromosómicas , Cromosomas Humanos Par 8 , Neoplasias Colorrectales/patología , Variaciones en el Número de Copia de ADN , Dinamarca , Perfilación de la Expresión Génica , Genómica , Genotipo , Humanos , Pérdida de Heterocigocidad , Repeticiones de Microsatélite , Fenotipo , Mutación Puntual , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN Interferente Pequeño/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Secuenciación Completa del Genoma
18.
Nat Commun ; 6: 10050, 2015 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-26632596

RESUMEN

The mammalian cell cycle is controlled by the E2F family of transcription factors. Typical E2Fs bind to DNA as heterodimers with the related dimerization partner (DP) proteins, whereas the atypical E2Fs, E2F7 and E2F8 contain two DNA-binding domains (DBDs) and act as repressors. To understand the mechanism of repression, we have resolved the structure of E2F8 in complex with DNA at atomic resolution. We find that the first and second DBDs of E2F8 resemble the DBDs of typical E2F and DP proteins, respectively. Using molecular dynamics simulations, biochemical affinity measurements and chromatin immunoprecipitation, we further show that both atypical and typical E2Fs bind to similar DNA sequences in vitro and in vivo. Our results represent the first crystal structure of an E2F protein with two DBDs, and reveal the mechanism by which atypical E2Fs can repress canonical E2F target genes and exert their negative influence on cell cycle progression.


Asunto(s)
Proteínas de Unión al ADN/química , ADN/metabolismo , Factores de Transcripción E2F/química , Familia de Multigenes , Cristalografía por Rayos X , ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Humanos , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Especificidad de la Especie
19.
Sci Signal ; 7(344): lc2, 2014 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-25249656

RESUMEN

Using an integrative experimental and computational modeling approach to dissect the signaling dynamics of the transforming growth factor-ß to Smad (TGF-ß/Smad) pathway, we discovered that previous exposure to ligand desensitizes cells, rendering them refractory to further acute TGF-ß stimulation. We demonstrated that this refractory behavior, which also explains signal attenuation, is caused by the fast depletion from the cell surface of signaling-competent receptors upon TGF-ß binding and their slow replenishment, which is the rate-limiting step for regaining full competence for acute ligand induction. In their Comment, Warmflash and colleagues suggest that receptor dynamics do not necessarily reflect the dynamics of TGF-ß target gene transcription. We argue that to understand receptor dynamics, phosphorylated Smad2 abundance is the optimal readout, because this directly reflects receptor activity. Target gene transcription, in contrast, is influenced by many other factors in addition to nuclear abundance of activated Smad complexes and is thus a poor readout for receptor dynamics. Warmflash et al. also claim that our results are inconsistent with parts of the literature, in particular with data published by Zi et al. (Mol. Syst. Biol. 7, 492, 2011) and by Sorre et al. (Dev. Cell 20, 334, 2014). However, we show with our mathematical model that our results are consistent with the data in question.


Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Humanos
20.
Sci Signal ; 6(305): ra106, 2013 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-24327760

RESUMEN

Understanding the complex dynamics of growth factor signaling requires both mechanistic and kinetic information. Although signaling dynamics have been studied for pathways downstream of receptor tyrosine kinases and G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors, they have not been investigated for the transforming growth factor-ß (TGF-ß) superfamily pathways. Using an integrative experimental and mathematical modeling approach, we dissected the dynamic behavior of the TGF-ß to Smad pathway, which is mediated by type I and type II receptor serine/threonine kinases, in response to acute, chronic, and repeated ligand stimulations. TGF-ß exposure produced a transient response that attenuated over time, resulting in desensitized cells that were refractory to further acute stimulation. This loss of signaling competence depended on ligand binding, but not on receptor activity, and was restored only after the ligand had been depleted. Furthermore, TGF-ß binding triggered the rapid depletion of signaling-competent receptors from the cell surface, with the type I and type II receptors exhibiting different degradation and trafficking kinetics. A computational model of TGF-ß signal transduction from the membrane to the nucleus that incorporates our experimental findings predicts that autocrine signaling, such as that associated with tumorigenesis, severely compromises the TGF-ß response, which we confirmed experimentally. Thus, we have shown that the long-term signaling behavior of the TGF-ß pathway is determined by receptor dynamics, does not require TGF-ß-induced gene expression, and influences context-dependent responses in vivo.


Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Western Blotting , Línea Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Cinética , Ligandos , Modelos Biológicos , Fosforilación , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta1/farmacología
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