Intergenic transcription causes repression by directing nucleosome assembly

Abstract

Transcription of non-protein-coding DNA (ncDNA) and its noncoding RNA (ncRNA) products are beginning to emerge as key regulators of gene expression. We previously identified a regulatory system in Saccharomyces cerevisiae whereby transcription of intergenic ncDNA (SRG1) represses transcription of an adjacent protein-coding gene (SER3) through transcription interference. We now provide evidence that SRG1 transcription causes repression of SER3 by directing a high level of nucleosomes over SRG1, which overlaps the SER3 promoter. Repression by SRG1 transcription is dependent on the Spt6 and Spt16 transcription elongation factors. Significantly, spt6 and spt16 mutations reduce nucleosome levels over the SER3 promoter without reducing intergenic SRG1 transcription, strongly suggesting that nucleosome levels, not transcription levels, cause SER3 repression. Finally, we show that spt6 and spt16 mutations allow transcription factor access to the SER3 promoter. Our results raise the possibility that transcription of ncDNA may contribute to nucleosome positioning on a genome-wide scale where, in some cases, it negatively impacts protein-DNA interactions.

Figure 1.

Nucleosome positions and relative occupancy at SER3 in the presence and absence of SRG1 transcription. (A) Schematic of SER3 locus, including the 3′ 161 bp of AIM9 (−1000 to −839 relative to SER3 ATG) and the 5′ 600 bp of the SER3 ORF. The arrows at −475 and −75 indicate the transcription start sites of SRG1 and SER3, respectively. Blocks of intergenic sequence identity between S. cerevisiae and four related yeast strains are marked, including the SRG1 and SER3 TATAs (black boxes), sequences required for SER3 activation (white boxes), and a Cha4-binding site (gray box). The scale represents the distance from the SER3 translation start (+1). The tiled black bars above the scale indicate the DNA fragments amplified by qPCR to quantify nucleosome position and relative occupancy (see Supplemental Table S2 for details). (B) Nucleosome scanning assay was performed on wild-type (FY4, FY2097, and FY1350) and srg1-1 (YJ582, FY2250, and YJ585) cells that were grown in YPD medium (SER3 repressed) at 30°C. Using qPCR, the relative MNase protection of each SER3 template was calculated as a ratio to the control GAL1 NB template found within a well-positioned nucleosome in the GAL1–10 promoter (see Supplemental Fig. S1). Each point on the graph shows the mean ± SEM from three independent experiments that are plotted at the midpoint of each PCR product. Results for amplicons SER3-5 to SER3-41 are shown. Below the graph, a diagram of the SER3 locus indicates the positions of nucleosomes (gray ovals) extrapolated from the MNase protection data. The block arrows indicate the transcription activity of SRG1 and SER3, respectively. srg1-1 strains have a mutated TATA sequence (marked by an X) that inhibits SRG1 transcription, causing SER3 derepression.

Figure 2.

Effect of serine on nucleosome positions and relative occupancy at SER3. (A) Nucleosome scanning assay was performed on wild-type cells (FY2097 and FY4) that were grown at 30°C in SC + serine media (+ serine) and then shifted to SC − serine media (− serine) for 25 min as described in Figure 1. Each point on the graph shows the mean relative MNase protection ± SEM from four independent experiments (two for each strain) plotted at the midpoint of each PCR product. Results for amplicons SER3-7 to SER3-41 are shown. (B) Northern analysis of SER3 and SRG1 was performed on a wild-type (FY2097) and two ser3-100 strains (YJ275 and FY2099) that have a mutated SER3 TATA. Cells were grown at 30°C in SC + serine media (+ serine) and then shifted to SC − serine media (− serine) for 25 min. SCR1 serves as a loading control. (C) Nucleosome scanning assay was performed on ser3-100 strains (YJ275 and FY2099) as described in A.

Figure 3.

Repression of SER3 is dependent on Spt6/Spn1(Iws1) and the FACT complex. (A) Northern analysis of SER3, SRG1, and SCR1 (loading control) was performed on wild-type (FY4), spt6-1004 (FY2425), spt6-140 (FY111), spt6-14 (FY1221), iws1-7 (GHY1199), and iws1-13 (GHY1200) strains. Cells were grown in YPD at 30°C to mid-log and then shifted for 60 min to 37°C. (B) Northern analysis of SER3, SRG1, and SCR1 (loading control) was performed on wild-type (FY4), spt16-197 (FY346), spt16-11 (TF8030-1), spt16-22 (YJ832), spt16-23 (YJ833), spt16-24 (TF7783-24), pob3-7 (TF8031-1), and nhp6aΔ∷URA3 nhp6bΔ∷URA3 (FY1411) strains that were grown in YPD. (C) ChIP analysis was performed on chromatin isolated from wild-type (YJ877, YJ878, YJ879, and YJ884), spt6-1004 (YJ886, YJ887, YJ888, and YJ892), and spt16-197 (YJ841, YJ842, and YJ843) strains expressing Rpb1-C13myc and untagged control strains (FY4, FY5, and YJ586). Rpb1-C13myc was immunoprecipitated with α-myc A14 antibody from chromatin prepared from cells that were grown in YPD at 30°C. The amount of immunoprecipitated DNA was determined by qPCR as a percentage of the input material and is expressed as the fold enrichment over a control region of chromosome V that lacks ORFs (Supplemental Table S2, No ORF). Each bar represents the mean ± SEM from at least three independent experiments. Below the graph is a schematic of SER3 with black bars corresponding to the regions amplified by qPCR (see Supplemental Table S2 for details).

Figure 4.

Nucleosome positions and relative occupancy at SER3 in spt6-1004 and spt16-197 mutants. (A) Nucleosome scanning assay was performed on wild-type (FY2134, YJ864, and YJ847), spt6-1004 (FY2180, YJ855, YJ862), and spt16-197 (FY346, YJ859, and YJ916) strains that were grown in YPD at 30°C as described in Figure 1. The light-gray ovals over the SRG1 transcription unit in the spt16-197 strain reflect that this region is slightly more protected from MNase digestion as compared with the spt6-1004 strain. (B) Histone H3 ChIP was performed on chromatin isolated from wild-type (FY4, FY5, and YJ586), spt6-1004 (YJ886, YJ887, and YJ888), and spt16-197 (YJ844, YJ845, and YJ846) cells that were grown in YPD. The amount of immunoprecipitated DNA was determined by qPCR as a percentage of the input material and is expressed as the fold enrichment over GAL1 NB (see Supplemental Fig. S1). Each bar represents the mean ± SEM of at least three independent experiments. Below the graph is a schematic of SER3 with black bars corresponding to the regions amplified by qPCR (see Supplemental Table S2 for details).

Figure 5.

spt6-1004 and spt16-197 mutants are defective for transcription interference at SER3. (A) Gal4 ChIP was performed on wild-type (YJ871, YJ872, and YJ873), spt6-1004 (YJ875, YJ876, and YJ850), spt16-197 (YJ867, YJ868, and YJ869), and positive control srg1-1 (FY2260) cells that all contain the ser3∷GAL7UAS allele. Chromatin was prepared from cells grown at 30°C in YPraf to 0.8 × 10⁷ cells per milliliter, and then for an additional 4 h at 30°C after the addition of 2% galactose. Gal4 ChIP signals were determined by qPCR at the three SER3 locations (left histogram), and at GAL1 as a positive control (right histogram). All values were normalized to a control region located near the telomere of chromosome VI (TELVI) (Supplemental Table S2) and represent the mean ± SEM. Below the graph is a diagram of the ser3∷GAL7UAS allele in which the putative SER3 UAS region was replaced with the GAL7 UAS region containing two Gal4-binding sites (white box). The black bars indicate the regions of SER3 amplified by qPCR. (B) TBP ChIP was performed on chromatin isolated from wild-type (FY4, FY5, YJ586, and KY719), spt6-1004 (YJ886, YJ887, YJ888, and YJ892), spt16-197 (YJ841, YJ842, YJ843, and YJ844), and positive control srg1-1 (FY2471, YJ582, YJ583, and YJ585) strains that were grown in YPD at 30°C as described in Figure 3C.

Figure 6.

Repression of SER3 does not require histone methyltransferases or the Rpd3S and Set3C histone deacetylase complexes. Northern analysis of SER3, SRG1, and SCR1 (loading control) was performed on wild-type (YJ586), srg1-1 (FY2471), set1Δ (KY938), set2Δ (KY912), dot1Δ (KY934), rco1Δ (KY1235), set1Δset2Δ (KY1822), and set3Δ (KY1806) strains that were grown in YPD at 30°C.

Figure 7.

A model for SER3 regulation by SRG1 intergenic transcription. When serine is available to the cells, DNA-bound Cha4 recruits SAGA and Swi/Snf to initiate SRG1 transcription, possibly by remodeling the two nucleosomes located at the 5′ end of SRG1 to expose the SRG1 transcription start site. RNA Pol II transcribes SRG1 and, through Spt6 and Spt16, disassembles nucleosomes in its path and then reassembles them in its wake. As a result, nucleosomes continuously occupy the SER3 UAS where they repress SER3 by occluding the SER3 promoter from transcription factor binding. In the absence of serine, SRG1 transcription is repressed, possibly due to the presence of two nucleosomes at its 5′ end that encompass its transcription start site. In the absence of SRG1 transcription, the SER3 UAS is depleted of nucleosomes, allowing an as yet unknown activator (Act) and/or TBP and RNA Pol II to bind and activate SER3 transcription.