Chromatin structure and expression of a gene transcribed by RNA polymerase III are independent of H2A.Z deposition

Abstract

The genes transcribed by RNA polymerase III (Pol III) generally have intragenic promoter elements. One of them, the yeast U6 snRNA (SNR6) gene is activated in vitro by a positioned nucleosome between its intragenic box A and extragenic, downstream box B separated by approximately 200 bp. We demonstrate here that the in vivo chromatin structure of the gene region is characterized by the presence of an array of positioned nucleosomes, with only one of them in the 5' end of the gene having a regulatory role. A positioned nucleosome present between boxes A and B in vivo does not move when the gene is repressed due to nutritional deprivation. In contrast, the upstream nucleosome which covers the TATA box under repressed conditions is shifted approximately 50 bp further upstream by the ATP-dependent chromatin remodeler RSC upon activation. It is marked with the histone variant H2A.Z and H4K16 acetylation in active state. In the absence of H2A.Z, the chromatin structure of the gene does not change, suggesting that H2A.Z is not required for establishing the active chromatin structure. These results show that the chromatin structure directly participates in regulation of a Pol III-transcribed gene under different states of its activity in vivo.

FIG. 1.

Yeast SNR6 is nucleosomal in vivo. (A) IEL analysis of the chromatin structure in strain UKY403 and its isogenic strain MHY308. The cells grown in YEP-galactose medium to an A₆₀₀ of 0.7 were shifted to YEP medium containing glucose and harvested after 3 h. Gray ovals denote the nucleosomal size protections. The dot denotes the single MNase cut between boxes A and B in the naked DNA. The short bar marks the exposed region around the TATA box in the chromatin. (B) Nucleosome mapping on SNR6 by the ChIP assay. Chromatin with Flag-tagged histone H2B was immunoprecipitated after MNase digestion using FLAG-M2 agarose and analyzed by real-time PCR for different regions of SNR6. The upper panel shows the positions of different amplicons on the gene region. Numbers refer to the transcription initiation site (+1). Occupancies were calculated as relative enrichment over the TEL VIR region, and the averages from three independent experiments, with error bars, are shown.

FIG. 2.

Chromatin structure of SNR6 under repression. Nutrient deprivation in 0.15× YEP medium lacking glucose was used to repress Pol III transcription. Occupancies were calculated as relative enrichment over the TEL VIR region, and the averages from three independent experiments, with error bars, are shown. (A) ChIP assays showing occupancy of the HA-tagged RPC160 over SNR6 in wild-type cells without (active) or with repression for 40 min (repressed). (B) Progressive increase in occupancy of the FLAG-H2B over TATA-A box region under repressed condition. (C) In vivo IEL analysis of chromatin before (lane 1) and after shifting the cells to 0.15× YEP medium for 1 h (lanes 2 to 4). Lane A, control sample under active state without repression. Gray ovals denote the position of a nucleosomal protection region. Arrowheads with numbers indicate the positions of MNase cut sites.

FIG. 3.

Enrichment of the upstream nucleosome with the histone variant H2A.Z. Occupancies were calculated as relative enrichment over the TEL VIR region. Results from ChIP assays showing averages from three independent experiments with error bars are shown. (A) Occupancy of the FLAG-H2B over upstream, TATA-A box, and A-B box regions under active or repressed state. (B) Occupancy of the FLAG-H2A over upstream, TATA-A box, and A-B box region in YEP medium with glucose or after 1 h of repression. (C) Occupancy of Htz1-FLAG on the upstream and A-B box regions of SNR6 after 1 h of repression.

FIG. 4.

Deposition of Htz1 in the upstream nucleosome does not affect SNR6 expression. Occupancies were calculated as relative enrichment over the control region, and the averages from three independent experiments with error bars are shown in each of the panels. (A) Effect of repression and Sas2 deletion on H4K16 acetylation levels in the upstream nucleosome with respect to the TEL VIR region. (B) Occupancy of Sas2-TAP over SNR6 relative to the TEL VIR region decreases under repression. For both panels A and B, values are compared with the occupancy over the A-B box region. (C) Swr1-TAP occupancy over SNR6 relative to the TEL VIR region. (D) Htz1-TAP occupancy in wild-type, swr1Δ, and sas2Δ cells compared to the Cox3 region. (E) Time course analysis of the effect of repression on the U6 RNA level in the absence of Htz1. Total RNA was isolated from wild-type and htz1Δ cells and reverse transcribed with radioactively labeled primers specific for U6 snRNA and Pol II-transcribed U4 snRNA. (F) In vivo IEL analysis of chromatin before (lane 3) and after shifting the htz1Δ cells to 0.15× YEP medium for 1 h (lanes 4 and 5). Control (lanes 1 and 2) shows chromatin of wild-type cells under repression. Gray oval denotes the position of a nucleosomal protection region, while the bar shows the exposed region around the TATA box under the active state in lane 3. Arrowheads mark the positions of the MNase cut sites.

FIG. 5.

Chromatin structure of SNR6 in RSC4-Δ4 mutants. (A) The strain MW4019 differs from MW3993 in having the RSC subunit Sth1 as Myc tagged. IEL analysis shows the shift of an upstream nucleosome with an RSC4 mutation. Yeast cells were grown in YEP medium containing glucose to an A₆₀₀ of 0.7. Gray ovals denote the nucleosomal size protections, and the bar marks the exposed region in lanes 1 and 2. The dot denotes a cut by MNase at the bp −70 position, and the asterisk denotes the new cut site spanning the bp −123 to −178 position. (B) Effect of repression on the occupancy of the Myc-tagged RSC subunits, RSC2 (RSC4 wild type), and Sth1 (RSC4 mutant MW4019).

FIG. 6.

Changes in the chromatin structure of SNR6 under active or repressed conditions. For clarity, Pol III transcription machinery, which remains on the gene even under repression, is omitted. The bar denotes the DNA region exposed under these conditions. The position of the nucleosome between the A and B boxes (dark gray oval) does not change while the upstream nucleosome (shown in light gray) moves. When the gene is activated, RSC, SWR1, and SAS complexes are recruited to the gene. RSC slides one nucleosome to a position upstream of the TATA box, and H2A in this nucleosome is exchanged with H2A.Z by the SWR1 complex. The SAS complex acetylates H4K16. When the gene is repressed, RSC, SAS, and H2A.Z are lost, and this nucleosome goes back to cover the TATA box and transcription start site.