Evidence for eviction and rapid deposition of histones upon transcriptional elongation by RNA polymerase II

Abstract

Biochemical experiments indicate that transcriptional elongation by RNA polymerase II (Pol II) is inhibited by nucleosomes and hence requires chromatin-modifying activities. Here, we examine the fate of histones upon passage of elongating Pol II in vivo. Histone density throughout the entire Saccharomyces cerevisiae GAL10 coding region is inversely correlated with Pol II association and transcriptional activity, suggesting that the elongating Pol II machinery efficiently evicts core histones from the DNA. Furthermore, new histones appear to be deposited onto DNA less than 1 min after passage of Pol II. Transcription-dependent deposition of histones requires the FACT complex that travels with elongating Pol II. Our results suggest that Pol II transcription generates a highly dynamic equilibrium of histone eviction and histone deposition and that there is significant histone exchange throughout most of the yeast genome within a single cell cycle.

FIG. 1.

GAL activation causes reduced histone density at promoters and coding regions. (A) Densities of histones H2B and H3 at the indicated promoter and coding regions in a strain expressing FLAG-tagged H2B grown in YP medium containing either 2% galactose or glucose were normalized to an open reading frame (ORF)-free internal control region, and the highest level was arbitrarily set to 1. (B) Densities of histones H4 and H3 in a strain expressing Myc-tagged H4 were analyzed in a similar manner.

FIG. 2.

Inverse correlation between histone and Pol II occupancy. (A) Pol II and (B) H2B density at the indicated promoter and coding regions in cells expressing FLAG-tagged H2B and grown in YP medium containing the indicated mixtures of glucose and galactose. (C) Pol II and (D) H3 density in wild-type and kin28-ts16 cells grown in YP medium containing 2% galactose and shifted to 37°C for 2 h. ORF, open reading frame.

FIG. 3.

Core histones are deposited upon cessation of transcription. Shown are densities of Pol II and histones H2B, H3, and H4 at the indicated promoter and coding regions in galactose-grown cells (zero time point) shifted into glucose medium for the indicated time (in minutes). Histone densities were normalized to an open reading frame (ORF)-free control region, and the zero time point was arbitrarily defined. Standard deviations (not shown) were generally less than 15% of the means.

FIG. 4.

Histone H3 deposition tracks with clearance of Pol II. Galactose-grown cells containing GAL1-YLR454 were shifted into glucose medium for the indicated time (in minutes) and examined for (A) Pol II density (8-min time point arbitrarily set to 1) and (B) H3 density (0-min time point arbitrarily set to 1) at the indicated regions within the 8-kb YLR454 coding region. Standard deviations (not shown) were generally less than 15% of the means. ORF, open reading frame.

FIG. 5.

Spt16 is important for transcription-associated histone deposition. (A) Pol II and (B) H3 density at the indicated promoter and coding regions in wild-type and spt16-197 cells grown in YP medium containing 2% galactose and shifted to 37°C for 1 h. ORF, open reading frame.