ABSTRACT
We have examined the effect of HMG17 on transcription by RNA polymerase II by the assembly and analysis of HMG17-containing chromatin templates consisting of regularly spaced nucleosomal arrays. Structural analysis of the chromatin indicated that HMG17 is incorporated into chromatin in a physiological manner with the full complement of core histones. The transcriptional studies revealed that HMG17 stimulates transcription in conjunction with the sequence-specific activator GAL4-VP16. This effect was observed with chromatin, but not with non-nucleosomal templates, and required the presence of HMG17 during chromatin assembly. The incorporation of HMG17 into chromatin resulted in a 7- to 40-fold stimulation of GAL4-VP16-activated transcription to levels that were comparable to those observed with histone-free DNA templates. In contrast, transcription from HMG17-containing chromatin was not detectable in the absence of GAL4-VP16 or with a GAL4 derivative [GAL4(1-147)] lacking the VP16 activation domain. Finally, the incorporation of HMG17 into chromatin was found to increase the efficiency of transcription initiation, but not the extent of transcriptional elongation. Thus, HMG17 is a chromatin-specific transcriptional coactivator that increases the efficiency of initiation of transcription by RNA polymerase II.
Subject(s)
Chromatin/metabolism , High Mobility Group Proteins/metabolism , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins , Trans-Activators/metabolism , Transcription Factors , Transcription, Genetic , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA/metabolism , DNA-Binding Proteins , Drosophila/embryology , Embryo, Nonmammalian/metabolism , Fungal Proteins/metabolism , Herpes Simplex Virus Protein Vmw65/metabolism , High Mobility Group Proteins/biosynthesis , High Mobility Group Proteins/isolation & purification , Histones/metabolism , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Templates, Genetic , Thymus Gland/metabolismABSTRACT
Promoter- and enhancer-binding factors appear to function by facilitating the transcription reaction as well as by counteracting chromatin-mediated repression (antirepression). We have examined the mechanism by which a hybrid activator, GAL4-VP16, is able to counteract histone H1-mediated repression by using both H1-DNA complexes and reconstituted H1-containing chromatin templates. The GAL4 DNA binding domain alone was sufficient to disrupt local H1-DNA interactions, but a transcriptional region was additionally necessary for antirepression. GAL4-VP16-mediated antirepression required an auxiliary factor, denoted as a co-antirepressor, which was partially purified from Drosophila embryos. We have found that the co-antirepressor activity was sensitive to digestion with RNase A. Moreover, total RNA from Drosophila embryos could partially substitute for the co-antirepressor fraction, which indicated that the co-antirepressor may function as a histone acceptor ("histone sink"). These findings suggest a model for gene activation in which sequence-specific transcription factors disrupt H1-DNA interactions at the promoter to facilitate transfer of H1 to a histone acceptor, which then allows access of the basal transcription factors to the DNA template.
