DNA polymerase epsilon, acetylases and remodellers cooperate to form a specialized chromatin structure at a tRNA insulator

Abstract

Insulators bind transcription factors and use chromatin remodellers and modifiers to mediate insulation. In this report, we identified proteins required for the efficient formation and maintenance of a specialized chromatin structure at the yeast tRNA insulator. The histone acetylases, SAS-I and NuA4, functioned in insulation, independently of tRNA and did not participate in the formation of the hypersensitive site at the tRNA. In contrast, DNA polymerase epsilon, functioned with the chromatin remodeller, Rsc, and the histone acetylase, Rtt109, to generate a histone-depleted region at the tRNA insulator. Rsc and Rtt109 were required for efficient binding of TFIIIB to the tRNA insulator, and the bound transcription factor and Rtt109 in turn were required for the binding of Rsc to tRNA. Robust insulation during growth and cell division involves the formation of a hypersensitive site at the insulator during chromatin maturation together with competition between acetylases and deacetylases.

Figure 1a

Chromatin structure at the HMR tRNA. (A) Histone H3 at the tRNA. The histogram represents qChIP data, following sonication, for studying the distribution of histone H3 from a wild-type or an HMR tRNAΔ strain. Values for ΔC_t were derived from the difference in the real-time amplification of equal amounts of immunoprecipitated and input DNA. Standard error values were calculated using data from at least four immunoprecipitation reactions with at least two independently cross-linked chromatin samples. Data for the histogram were derived by normalizing ΔC_t values for the various amplicons against the ΔC_t value for the 7.5 kb Tel6R amplicon (VIII). (B) The histogram represents qChIP data following micrococcal nuclease digestion to study the distribution of histone H3 from a wild-type or an HMR tRNAΔ strain.

Figure 1b

(C, D) Sir3 and H4 Ac-K16 distributions are anti-correlative. A histogram representing qChIP data to study the distribution of Sir3 (B) or H4 Ac-K16 (C). Data for the histogram in (C) were normalized as in Figure 1A, whereas the data for (D) were normalized twice: first to amplicon VIII and then to the histone H3 levels at each amplicon. Owing to this, we are unable to provide the standard error values. (E) H3 Ac-K56 was found at sites of Sir3 occupancy. A histogram representing qChIP data to study the distribution of H3 Ac-K56 around the tRNA from a wild-type or an HMR tRNAΔ strain. Data were normalized as in Figure 1D.

Figure 2

A schematic representation of the two SGA screens and the modified HMR locus.

Figure 3a

Chromatin modifiers and remodellers affected the HMR tRNA boundary. (A) A phenotypic assay for the HMR tRNA boundary. The HMR tRNA boundary mating assay shown with wild-type cells carrying either (I) the wild type boundary, (II) a full deletion of the boundary, (III) a 70-bp tRNA deletion of the boundary or (IV) the 70 bp tRNA with 100 bp flanks. Tenfold serial dilutions of overnight cultures with a starting A₆₀₀ of 1.0 were spotted on a fully supplemented minimal medium (growth control) or minimal medium with the mating tester lawn (mating). (B) The HMR tRNA boundary mating assay shown with isogenic wild-type, eaf3Δ or sas2Δ cells. (C) Differential effects of remodeller mutants on the HMR tRNA boundary. The HMR tRNA boundary mating assay shown with isogenic wild-type, rsc2Δ, isw2Δ or swr1Δ cells. (D) Mutants for DNA polymerase ɛ and, to a significantly lesser degree, Isw2 compromised the HMR tRNA boundary. The HMR tRNA boundary mating assay shown with isogenic wild-type, dpb3Δ, dpb4Δ or dls1Δ cells.

Figure 3b

(E) Mutants in all three subunits of the Rtt109 acetylase complex weakened the HMR tRNA boundary. The HMR tRNA boundary mating assay shown with isogenic wild-type, asf1Δ, rtt109Δ or vps75Δ cells. (F) Mutants in H3 Ac-K56 Histone deacetylases compromised the HMR tRNA boundary. The HMR tRNA boundary mating assay shown with isogenic wild-type or hst3Δ–hst4Δ cells.

Figure 4a

Only some chromatin modifiers and remodellers influenced the histone-depleted HMR tRNA region. (A–C) Histone H3 occupancy at the HMR tRNA boundary was unaltered in sas2Δ, isw2Δ and eaf3Δ mutants. Histograms representing qChIP data to study the distribution of histone H3 from a (A) sas2Δ, (B) isw2Δ or (C) an eaf3Δ strain. The H3 distribution in a wild-type strain from Figure 1A is plotted alongside for ease of comparison.

Figure 4b

(D–F): Histone H3 occupancy at the HMR tRNA increased in rtt109Δ, rsc2Δ and dpb3Δ mutants. Histograms representing qChIP data to study the distribution of histone H3 from (D) an rtt109Δ, (E) an rsc2Δ or (F) a dpb3Δ strain. The H3 distribution in a wild-type strain from Figure 1A is plotted alongside for ease of comparison.

Figure 5

Genetic and molecular pathways to the HMR tRNA boundary. (A) Double mutants for dpb3Δ and rsc2Δ or dpb3Δ and rtt109Δ weakened the HMR tRNA boundary to the same extent as an rsc2 or rtt109 single mutant, respectively. The HMR tRNA boundary mating assay shown with isogenic wild-type, dpb3Δ, rsc2Δ, rsc2Δ dpb3Δ, rtt109Δ or rtt109Δ–dpb3Δ cells carrying either (I) the wild-type boundary or (II) the 70 bp tRNA with 100 bp flanks. (B, C) Histone H3 occupancy at the HMR tRNA boundary in dpb3Δ–rtt109Δ or dpb3Δ–rsc2Δ double mutants was unchanged from that of an rtt109Δ or rsc2Δ single mutant, respectively. Histograms representing qChIP data to study the distribution of histone H3 from a (C) dpb3Δ–rtt109Δ or (D) dpb3Δ–rsc2Δ strain. The H3 distribution in the single mutants from Figure 4D–F is plotted alongside for ease of comparison.

Figure 6

Replication fork pausing at the HMR tRNA and insulation were not linked.

Figure 7

tRNA transcription factors at HMR co-operated with a chromatin remodeller and a modifier to form a boundary. (A) The HMR tRNA was important for Rsc2 localization. Histograms representing qChIP data to study the distribution of Rsc2–TAP centred on the tRNA from a wild-type or an HMR tRNAΔ strain. (B) Rtt109 was necessary for Rsc2 localization. Histograms representing qChIP data to study the distribution of Rsc2–TAP centred on the tRNA from a wild-type or an rtt109Δ strain. (C) Stable recruitment of the tRNA transcription factors required Rsc2 and Rtt109. Histograms representing qChIP data to study the distribution of Bdp1–TAP centred on the tRNA from a wild-type, an rsc2Δ or an rtt109Δ strain.