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1.
J Cell Sci ; 136(7)2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36897575

RESUMO

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Sulfatases/genética , Sulfatases/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismo
2.
Mol Cell ; 67(6): 1013-1025.e9, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28867293

RESUMO

In response to stresses, cells often halt normal cellular processes, yet stress-specific pathways must bypass such inhibition to generate effective responses. We investigated how cells redistribute global transcriptional activity in response to DNA damage. We show that an oscillatory increase of p53 levels in response to double-strand breaks drives a counter-oscillatory decrease of MYC levels. Using RNA sequencing (RNA-seq) of newly synthesized transcripts, we found that p53-mediated reduction of MYC suppressed general transcription, with the most highly expressed transcripts reduced to a greater extent. In contrast, upregulation of p53 targets was relatively unaffected by MYC suppression. Reducing MYC during the DNA damage response was important for cell-fate regulation, as counteracting MYC repression reduced cell-cycle arrest and elevated apoptosis. Our study shows that global inhibition with specific activation of transcriptional pathways is important for the proper response to DNA damage; this mechanism may be a general principle used in many stress responses.


Assuntos
Neoplasias da Mama/genética , Quebras de DNA de Cadeia Dupla , Proteínas Proto-Oncogênicas c-myc/genética , Transcrição Gênica , Transcriptoma , Proteína Supressora de Tumor p53/genética , Apoptose , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas , Pontos de Checagem do Ciclo Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/metabolismo
3.
Mol Syst Biol ; 15(9): e8685, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31556489

RESUMO

The p53 tumor suppressor regulates distinct responses to cellular stresses. Although different stresses generate different p53 dynamics, the mechanisms by which cells decode p53 dynamics to differentially regulate target genes are not well understood. Here, we determined in individual cells how canonical p53 target gene promoters vary in responsiveness to features of p53 dynamics. Employing a chemical perturbation approach, we independently modulated p53 pulse amplitude, duration, or frequency, and we then monitored p53 levels and target promoter activation in individual cells. We identified distinct signal processing features-thresholding in response to amplitude modulation, a refractory period in response to duration modulation, and dynamic filtering in response to frequency modulation. We then showed that the signal processing features not only affect p53 target promoter activation, they also affect p53 regulation and downstream cellular functions. Our study shows how different promoters can differentially decode features of p53 dynamics to generate distinct responses, providing insight into how perturbing p53 dynamics can be used to generate distinct cell fates.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteína Supressora de Tumor p53 , Biologia Computacional , Células HEK293 , Humanos , Células MCF-7 , Técnicas Analíticas Microfluídicas , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/farmacologia
4.
Sci Rep ; 9(1): 5976, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979970

RESUMO

Synthetic biological circuits that can generate outputs with distinct expression dynamics are useful for a variety of biomedical and industrial applications. We present a method to control output dynamics by altering output mRNA decay rates. Using oscillatory expression of the transcription factor p53 as the circuit regulator, we use two approaches for controlling target gene transcript degradation rates based on the output gene's 3'-untranslated region (3'-UTR): introduction of copies of destabilizing AU-rich elements into the 3'-UTR or swapping in naturally occurring 3'-UTRs conferring different transcript stabilities. As a proof of principle, we apply both methods to control the expression dynamics of a fluorescent protein and visualize the circuit output dynamics in single living cells. We then use the naturally occurring 3'-UTR approach to restore apoptosis in a tunable manner in a cancer cell line deficient for caspase-3 expression. Our method can be readily adapted to regulate multiple outputs each with different expression dynamics under the control of a single naturally occurring or synthetically constructed biological oscillator.


Assuntos
Regiões 3' não Traduzidas/genética , Regulação da Expressão Gênica , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Apoptose/fisiologia , Caspase 3/deficiência , Caspase 3/genética , Linhagem Celular Tumoral , Engenharia Genética/métodos , Humanos , Proteínas Luminescentes/metabolismo , Periodicidade , Estudo de Prova de Conceito , Estabilidade de RNA/fisiologia , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/genética
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