Role of Nucleosomal Cores and Histone H1 in Regulation of Transcription by RNA Polymerase II

Abstract

The relation between chromatin structure and transcriptional activity was examined by in vitro transcription analysis of chromatin reconstituted in the absence or presence of histone H1. To maintain well-defined template DNA, purified components were used in the reconstitution of chromatin. Reconstitution of nucleosomal cores to an average density of 1 nucleosome per 200 base pairs of DNA resulted in a mild reduction of basal RNA polymerase II transcription to 25 to 50 percent of that obtained with naked DNA templates. This nucleosome-mediated repression was due to nucleosomal cores located at the RNA start site and could not be counteracted by the sequence-specific transcription activators Sp1 and GAL4-VP16. When H1 was incorporated into the chromatin at 0.5 to 1.0 molecule per nucleosome (200 base pairs of DNA), RNA synthesis was reduced to 1 to 4 percent of that observed with chromatin containing only nucleosomal cores, and this H1-mediated repression could be counteracted by the addition of Sp1 or GAL4-VP16 (antirepression). With naked DNA templates, transcription was increased by a factor of 3 and 8 by Sp1 and GAL4-VP-16, respectively (true activation). With H1-repressed chromatin templates, however, the magnitude of transcriptional activation mediated by Sp1 and GAL4-VP16 was 90 and more than 200 times higher, respectively, because of the combined effects of true activation and antirepression. The data provide direct biochemical evidence that support and clarify previously proposed models in which there is depletion or reconfiguration of nucleosomal cores and histone H1 at the promoter regions of active genes.

Dynamics of interaction of RNA polymerase II with nucleosomes. I. Effect of salts

“…How the presence of nucleosomes on chromatin allows its replication and transcription to take place is an important and interesting question. Although both the status of nucleosomes and the role of histones in transcription initiation are becoming more and more clear (Grunstein, 1990b;Laybourn & Kadonaga, 1991;Workman et al, 1991), the status of nucleosomes during transcription elongation remains controversial (Lorch et al, 1988;Grunstein, 1990b;Clark & Felsenfeld, 1991 ;Kornberg & Lorch, 1991;Lee & Garrard, 1991;Thoma, 1991;Felsenfeld, 1992). The answer to this question spans two extremes: nucleosomes either dismantle or remain intact.…”

A New Class of Histone H2A Mutations in Saccharomyces cerevisiae Causes Specific Transcriptional Defects In Vivo

“…A growing body of evidence from both in vivo and in vitro studies has shown that the structure of chromatin influences gene expression (see references 25 and 53 for reviews). Biochemical experiments have shown that histones can repress transcription in vitro (see reference 53 for a review) and that transcriptional activators can overcome this repression (21,41,45,79,81,82). In vivo experiments in Saccharomyces cerevisiae have shown that the loss of transcriptional activators or of activator binding sites can be suppressed by mutations in histone genes (27,30,37,57).…”

Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context

“…Prior to 2001, histone H1 had only been incorporated into chromatin using salt dialysis (14) or S-190 cell extracts from Drosophila (6). While these methods are still widely used today, we feel it is best to take advantage of a recombinant system for assembling chromatin.…”

“…Therefore, the core histone to DNA ratio at which templates are best assembled in the presence of linker histone should be less than the ratio from chromatin assembled in the absence of H1. Topological analysis can be used as indirect assay for incorporation of H1, as binding of the linker histone increases the DNA associated with each nucleosome (14,15).…”

“…Since a nucleosome containing H1 protects 10-20 additional base pairs from micrococcal nuclease digestion (14), increased nucleosome repeat length is used to verify proper addition of linker histone. The repeat length increased from 169 (+/− 6) base pairs in the absence of H1 to 190 (+/− 4) base pairs upon H1 incorporation (see Fig.…”