RESUMO
Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, we identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Histona Desmetilases/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Neuroglia/metabolismo , Receptores Notch/genética , Animais , Evolução Biológica , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Histona Desmetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Neuroglia/citologia , Ligação Proteica , Domínios Proteicos , Receptores Notch/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Medulloblastoma is the most common malignant brain tumor in children. Therapeutic approaches to medulloblastoma (combination of surgery, radiotherapy, and chemotherapy) have led to significant improvements, but these are achieved at a high cost to quality of life. Alternative therapeutic approaches are needed. Genetic mutations leading to the activation of the Hedgehog pathway drive tumorigenesis in ~30% of medulloblastoma. In a yeast two-hybrid proteomic screen, we discovered a novel interaction between GLI1, a key transcription factor for the mediation of Hedgehog signals, and PIN1, a peptidylprolyl cis/trans isomerase that regulates the postphosphorylation fate of its targets. The GLI1/PIN1 interaction was validated by reciprocal pulldowns using epitope-tagged proteins in HEK293T cells as well as by co-immunoprecipiations of the endogenous proteins in a medulloblastoma cell line. Our results support a molecular model in which PIN1 promotes GLI1 protein abundance, thus contributing to the positive regulation of Hedgehog signals. Most importantly, in vivo functional analyses of Pin1 in the GFAP-tTA;TRE-SmoA1 mouse model of Hedgehog-driven medulloblastoma demonstrate that the loss of Pin1 impairs tumor development and dramatically increases survival. In summary, the discovery of the GLI1/PIN1 interaction uncovers PIN1 as a novel therapeutic target in Hedgehog-driven medulloblastoma tumorigenesis.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/deficiência , Animais , Linhagem Celular Tumoral , Neoplasias Cerebelares/mortalidade , Neoplasias Cerebelares/patologia , Modelos Animais de Doenças , Humanos , Meduloblastoma/mortalidade , Meduloblastoma/patologia , Camundongos , Camundongos Transgênicos , Prognóstico , Ligação Proteica , Mapeamento de Interação de Proteínas , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
Quantitative traits are often influenced by many loci with small effects. Identifying most of these loci and resolving them to specific genes or genetic variants is challenging. Yet, achieving such a detailed understanding of quantitative traits is important, as it can improve our knowledge of the genetic and molecular basis of heritable phenotypic variation. In this study, we use a genetic mapping strategy that involves recurrent backcrossing with phenotypic selection to obtain new insights into an ecologically, industrially, and medically relevant quantitative trait-tolerance of oxidative stress, as measured based on resistance to hydrogen peroxide. We examine the genetic basis of hydrogen peroxide resistance in three related yeast crosses and detect 64 distinct genomic loci that likely influence the trait. By precisely resolving or cloning a number of these loci, we demonstrate that a broad spectrum of cellular processes contribute to hydrogen peroxide resistance, including DNA repair, scavenging of reactive oxygen species, stress-induced MAPK signaling, translation, and water transport. Consistent with the complex genetic and molecular basis of hydrogen peroxide resistance, we show two examples where multiple distinct causal genetic variants underlie what appears to be a single locus. Our results improve understanding of the genetic and molecular basis of a highly complex, model quantitative trait.