RESUMEN
The transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) has crucial roles in the control of plasma cell differentiation and in maintaining survival of plasma cells. However, how Blimp-1 ensures the survival of plasma cell malignancy, multiple myeloma (MM), has remained elusive. Here we identified Aiolos, an anti-apoptotic transcription factor of MM cells, as a Blimp-1-interacting protein by mass spectrometry. ChIP coupled with DNA microarray was used to profile the global binding of Aiolos and Blimp-1 to endogenous targets in MM cells, which revealed their co-binding to a large number of genes, including apoptosis-related genes. Accordingly, Blimp-1 and Aiolos regulate similar transcriptomes in MM cells. Analysis of the binding motifs for Blimp-1 and Aiolos uncovered a partial motif that was similar across sites for both proteins. Aiolos promotes the binding of Blimp-1 to target genes and thereby enhances Blimp-1-dependent transcriptional repression. Furthermore, treatment with an anti-MM agent, lenalidomide, caused ubiquitination and proteasomal degradation of Blimp-1, leading to the de-repression of a new Blimp-1 direct target, CULLIN 4A (CUL4A), and reduced Aiolos levels. Accordingly, lenalidomide-induced cell death was partially rescued by reintroduction of Blimp-1 or knockdown of CUL4A. Thus, we demonstrated the functional impacts and underlying mechanisms of the interaction between Aiolos and Blimp-1 in maintaining MM cell survival. We also showed that interruption of Blimp-1/Aiolos regulatory pathways contributes to lenalidomide-mediated anti-MM activity.
Asunto(s)
Apoptosis , Factor de Transcripción Ikaros/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Inhibidores de la Angiogénesis/farmacología , Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Células HEK293 , Humanos , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/inmunología , Lenalidomida , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor 1 de Unión al Dominio 1 de Regulación Positiva/antagonistas & inhibidores , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Talidomida/análogos & derivados , Talidomida/farmacología , Ubiquitinación/efectos de los fármacosRESUMEN
Artificial selection can provide insights into how insecticide resistance mechanisms evolve in populations. The underlying basis of such phenomena can involve complex interactions of multiple genes, and the resolution of this complexity first necessitates confirmation that specific genes are involved in resistance mechanisms. Here, we used a novel approach invoking a constrained RNA sequencing analysis to refine the discovery of specific genes involved in insecticide resistance. Specifically, for gene discovery, an additional constraint was added to the traditional comparisons of susceptible vs. resistant flies by the incorporation of a line in which insecticide susceptibility was 'recovered' within a resistant line by the removal of insecticide stress. In our analysis, the criterion for the classification of any gene as related to insecticide resistance was based on evidence for differential expression in the resistant line as compared with both the susceptible and recovered lines. The incorporation of this additional constraint reduced the number of differentially expressed genes putatively involved in resistance to 464, compared with more than 1000 that had been identified previously using this same species. In addition, our analysis identified several key genes involved in metabolic detoxification processes that showed up-regulated expression. Furthermore, the involvement of acetylcholinesterase, a known target for modification in insecticide resistance, was associated with three key nonsynonymous amino acid substitutions within our data. In conclusion, the incorporation of an additional constraint using a 'recovered' line for gene discovery provides a higher degree of confidence in genes identified to be involved in insecticide resistance phenomena.