Chromatin potentiates transcription

Abstract

Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription.RNA polymerase II | PHO5 | Saccharomyces cerevisiae A ssembly of purified histones on promoter DNA interferes with the initiation of transcription by RNA polymerase II and general transcription factors (GTFs) in vitro (1) and in vivo (2). Factors that relieve inhibition by histones have been identified by transcribing chromatin reconstituted with purified histones and genetic analysis. These factors include ATP-dependent chromatin remodelers (3, 4), histone-modifying enzymes (5-7), FACT (8), and TFIIS (9). Although informative, these studies are incomplete, because chromatin reconstituted with purified histones differs from chromatin assembled in vivo. Reconstituted chromatin lacks the patterns of histone modification, histone variants, and nonhistone proteins shown to play important roles in transcription in vivo. Nucleosome positioning, also important for transcription in vivo, cannot be accurately reconstituted in vitro. We have, therefore, investigated chromatin assembled in vivo as a template for transcription in vitro. ResultsPHO5 chromatin in the transcriptionally repressed state was excised from yeast chromosomes in circular form by recombination and purified by affinity chromatography as described ( Fig. S1 A and B) (10, 11). PHO5 chromatin isolated in this way was indistinguishable from chromatin at the chromosomal locus on the basis of digestion with specific and nonspecific endonucleases ( Fig. S1 C and D) (10,12). Because the chromatin was derived from a single copy gene, very small quantities were obtained: on the order of 10 fmol/L cell culture. At this low level, transcription cannot be detected directly by radioisotope incorporation or fluorescence, and therefore, we turned to RT-PCR. Two sets of primers were used: one to amplify the region downstream of the transcription start sites (TSSs) and detect all transcripts ("downstream" primer pair) and one to amplify any signal from cryptic transcripts originating upstream and reading through the promoter ("upstream" primer pair) (Fig. 1A and Fig. S2A). Subtraction of the upstream signal from the downstream signal revealed the level of p...

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