RESUMEN
Chromatin remodeling complexes such as SWI/SNF use the energy of ATP hydrolysis to remodel nucleosomal DNA and increase transcription of nucleosomal templates. Human heat shock factor one (hHSF1) is a tightly regulated activator that stimulates transcriptional initiation and elongation using different portions of its activation domains. Here we demonstrate that hHSF1 associates with BRG1, the ATPase subunit of human SWI/SNF (hSWI/SNF) at endogenous protein concentrations. We also show that hHSF1 activation domains recruit hSWI/SNF to a chromatin template in a purified system. Mutation of hHSF1 residues responsible for activation of transcriptional elongation has the most severe effect on recruitment of SWI/SNF and association of hHSF1 with BRG1, suggesting that recruitment of chromatin remodeling activity might play a role in stimulation of elongation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Sitios de Unión , Cromatina , ADN Helicasas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Células HeLa , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/aislamiento & purificación , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Moldes Genéticos , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificaciónRESUMEN
Transcriptional activators can stimulate multiple steps in the transcription process. We have used GAL4 fusion proteins to characterize the ability of different transcriptional activation domains to stimulate transcriptional elongation on the hsp70 gene in vitro. Stimulation of elongation apparently occurs via a mechanistic pathway different from that of stimulation of initiation: the herpes simplex virus VP16, heat shock factor 1 (HSF1) and amphipathic helix (AH) activation domains all stimulate initiation, but only VP16 and HSF1 stimulate elongation; and mutations in hydrophobic residues of the HSF1 activation domains impair stimulation of elongation but not of initiation, while mutations in adjacent acidic residues impair stimulation of initiation more than of elongation. Experiments in which activators were exchanged between initiation and elongation demonstrate that the elongation function of HSF1 will stimulate RNA polymerase that has initiated and is transcriptionally engaged. Transcriptional activators thus appear to have at least two distinct functions that reside in the same domain, and that act at different times to stimulate initiation and elongation.