RESUMEN
We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of "elongating" RNAPII, Prep1, beta-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes.
Asunto(s)
Actinas/metabolismo , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacología , Actinas/genética , Antineoplásicos/farmacología , Línea Celular Tumoral , Citocalasina D/farmacología , Proteínas de Unión al ADN , Regulación hacia Abajo , Proteínas de Homeodominio/metabolismo , Humanos , Immunoblotting , Mutación , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Factores de Transcripción de Octámeros/genética , Factores de Transcripción de Octámeros/metabolismo , Factor de Empalme Asociado a PTB , Polímeros/metabolismo , Interferencia de ARN , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
OBJECTIVE: Prep1 and Pbx2 are the main homeodomain DNA-binding proteins of the TALE (three amino acid loop extension) family expressed in the thymus. We previously reported reduced Pbx2 expression and defective thymocyte maturation in Prep1 hypomorphic mice. To further investigate the role of this homeodomain DNA-binding protein in T cell development, we generated transgenic mice expressing the N-terminal fragment of Pbx1 (Pbx1NT) under the control of the Lck proximal promoter. PRINCIPAL FINDINGS: Pbx1NT causes Prep1 cytosolic sequestration, abolishes Prep1-dependent DNA-binding activity and results in reduced Pbx2 expression in developing thymocytes. Transgenic thymi reveal increased numbers of CD4(-) CD8(-) CD44(-) (DN3 and DN4) thymocytes, due to a higher frequency of DN2 and DN4 Pbx1NT thymocytes in the S phase. Transgenic thymocytes however do not accumulate at later stages, as revealed by a normal representation of CD4/CD8 double positive and single positive thymocytes, due to a higher rate of apoptotic cell death of DN4 Pbx1NT thymocytes. CONCLUSION: The results obtained by genetic (Prep1 hypomorphic) and functional (Pbx1NT transgenic) inactivation of Prep1 support nonredundant roles for this homeodomain protein during different stages of T cell development.