Toggle involving cis-interfering noncoding RNAs controls variegated gene expression in yeast

Abstract

The identification of specific functional roles for the numerous long noncoding (nc)RNAs found in eukaryotic transcriptomes is currently a matter of intense study amid speculation that these ncRNAs have key regulatory roles. We have identified a pair of cis-interfering ncRNAs in yeast that contribute to the control of variegated gene expression at the FLO11 locus by implementing a regulatory circuit that toggles between two stable states. These capped, polyadenylated ncRNAs are transcribed across the large intergenic region upstream of the FLO11 ORF. As with mammalian long intervening (li)ncRNAs, these yeast ncRNAs (ICR1 and PWR1) are themselves regulated by transcription factors (Sfl1 and Flo8) and chromatin remodelers (Rpd3L) that are key elements in phenotypic transitions in yeast. The mechanism that we describe explains the unanticipated role of a histone deacetylase complex in activating gene expression, because Rpd3L mutants force the ncRNA circuit into a state that silences the expression of the adjacent variegating gene.

Fig. 1.

The HDAC Rpd3L is a net activator of FLO11 transcription. (A) FLO11 promoter activity was assayed in haploids containing the P_FLO11-URA3 reporter at the endogenous locus. Four-fold serial dilutions were spotted onto SC, -Ura, and 5-FOA (0.1%) media. Cells with active FLO11 promoters are Ura⁺ and 5-FOA^S, whereas silenced cells are Ura⁻ and 5-FOA^R. (B) Northern blot analysis with a probe for FLO11 (3502–4093 bp) shows that reporter assays reflect average steady-state FLO11 mRNA levels. (C) Pseudohyphal growth is lost in Rpd3L⁻ diploids, but is restored by P_TEF-FLO11 on a 2-μ plasmid. C1, cti6/cti6 + vector; C2, cti6/cti6 + P_TEF-FLO11; C3, WT + vector; C4, WT + P_TEF-FLO11. (D) Loss of Rpd3L function abolishes haploid adhesion. The same plate, before (Left) and after (Right) washing, is shown. P_TEF-FLO11 on a 2-μ plasmid restores adhesion. (part 1) WT; (part 2) flo11; (part 3) cti6 + P_TEF-FLO11; (part 4) cti6 + vector; (part 5) rxt2 + P_TEF-FLO11; (part 6) rxt2 + vector; (part 7) rpd3 + P_TEF-FLO11; (part 8) rpd3 + vector.

Fig. 2.

Rpd3L localization to the FLO11 promoter alters transcription factor binding and chromatin remodeling. (A) ChIP-chip experiments were performed using a functional Myc-tagged allele of Rpd3 in a WT haploid (Fig. S2B). The plot shows fold enrichment of Rpd3-Myc in chromatin immunoprecipitated (IP) with an anti-Myc antibody normalized to the whole-cell extract (WCE). (Inset) Quantitative PCR was performed on IP and WCE using primers specific for the FLO11 promoter (−1,400 bp), for positive binding control P_INO1, and for unbound regions APL2 and ARG2. Data were normalized to unbound region ARK1 and are expressed as fold enrichment ± SD. (B) Localization of Flo8 using a Myc-tagged allele in WT and mutant haploids was assayed by qPCR with primers specific for the FLO11 promoter on IP (anti-Myc) and WCE. Data were normalized to unbound region ACT1 and are expressed as fold enrichment ± SEM. (C) Localization of TBP was assayed by ChIP-chip in haploid WT, cti6, and sfl1 cells. The plot shows fold enrichment of TBP at the FLO11 promoter in IP (anti-TBP) normalized to WCE. (D) Localization of histone H4 was assayed by ChIP-chip in haploid WT, cti6, and sfl1 cells. The plot shows fold enrichment of H4 at the FLO11 promoter in IP (anti-H4) normalized to WCE.

Fig. 3.

Rpd3L, Sfl1, and Flo8 control a pair of ncRNAs transcribed upstream of FLO11. (A) Genome-wide transcription of polyadenylated [poly(A)] RNAs was profiled in haploid WT, Rpd3L⁻ (cti6), and sfl1 strains with strand-specific microarrays. Transcription detected near the FLO11 locus is shown. In the plots, each circle represents a probe with log signal intensity indicated on the y axis. Circles positioned above each x axis indicate Watson-strand transcription. Circles positioned below each x axis indicate Crick-strand transcription. Results from two arrays are shown. (Upper) Transcription in sfl1 (red circles) vs. cti6 (blue circles); (Lower) Transcription in WT (red circles) vs. cti6 (blue circles). Faded circles represent probes that were not called as part of a transcript in the analysis. A larger version of these plots is provided in Fig. S3. (B) Map of ncRNAs detected upstream of FLO11 and probes used in Northern blot analysis. Probes a, b, and c hybridize to regions located 284–819, 1653–2255, and 2631–3226 bases upstream of FLO11, respectively. Vertical lines at the 5′ ends of ICR1 and PWR1 ncRNAs show the range of start sites identified by RACE (Tables S2 and S3). Arrowheads at the 3′ ends of the ncRNAs indicate the range of stop sites identified by RACE (Tables S2 and S3). (C) Northern blot analysis was performed on poly(A) RNA from haploid WT (lane 1), cti6 (lane 2), sfl1 (lane 3), and ΔP_FLO11 (lane 4) where the entire intergenic region upstream of FLO11 is deleted. FLO11 is, by convention, encoded on the Crick strand; other transcripts encoded on this strand are designated “Crick-strand,” and those encoded on the complementary strand are designated “Watson-strand.” Crick-strand specific probes 1–3 detect the ∼3.2-kb ICR1 ncRNA. Watson-strand specific probes 2 and 3 detect a diffuse band with upper size of ∼1.2 kb representing the ncRNA PWR1. Load control (LC) = SCR1. (D) Northern blot analysis was performed on poly(A) RNA from haploid WT (lane 1), cti6 (lane 2), sfl1 (lane 3), flo8 (lane 4), cti6 flo8 (lane 5), sfl1 flo8 (lane 6), cti6 sfl1 (lane 7), cti6 sfl1 flo8 (lane 8), and ΔP_FLO11 (lane 9). LC = rRNA. (E) Quantitative PCR assay of transcription using primers tiled from +120 bp within the FLO11 ORF to 2280 bp upstream was performed for cti6, sfl1, and ΔP_FLO11 haploids. Detected transcription normalized to SCR1 levels is presented ±SD.

Fig. 4.

ICR1 represses FLO11 transcription in cis. (A) Schematic representation of transcriptional terminator constructs T1, T2, and T3 and control construct C inserted −3,041 bp upstream of FLO11. T1, Kluyveromyces lactis URA3 expressed under its own promoter and followed by its terminator (47); T2, S. cerevisiae HIS3 gene with its terminator (+1 to +817) (23); T3, HIS3 gene and its terminator followed by KanMX and the TEF terminator (48); C, HIS3 ORF (+1 to +663, no terminator) (23). (B) Quantitative PCR assay of FLO11 transcript levels was performed in haploid WT and Rpd3L⁻ (cti6) strains in which T1, T2, T3, or C was inserted (with no loss of endogenous sequence) 3,041 bp upstream of FLO11. FLO11 levels normalized to ACT1 are presented ± SD. (C) ICR1 and PWR1 levels were assayed by Northern blot analysis using strand-specific probes a and c, respectively. Strains: WT (lane 1); cti6 (lane 2); cti6 + T1 (lane 3); cti6 + T2 (lane 4); cti6 + T3 (lane 5); and cti6 + C (lane 6). Termination of ICR1 by T1, T2, or T3 increases PWR1 transcription. The larger PWR1 band in lane 3 is the size predicted due to insertion of T1 (1.4 kb) if the K. lactis URA3 terminator is unidirectional. The shorter PWR1 transcripts in lanes 4 and 5 suggest that the HIS3 terminator in the T2 and T3 constructs is bidirectional. (D) Haploid adhesion to YPD agar was assayed. The same plate, before (Upper) and after (Lower) washing, is shown. (E) In MATa/MATa cti6/cti6 diploids with one allele of FLO11 intact and the other precisely replaced by the URA3 reporter gene, insertion of T3 3,041 bp upstream restores expression of the downstream ORF only in cis. MATa/MATa diploids were used because FLO11 expression is dramatically reduced in MATa/MATα diploids compared with haploids (51).

Fig. 5.

Model for transcriptional variegation at the FLO11 locus involving a toggle between the ncRNAs ICR1 and PWR1. Competitive binding of Sfl1 or Flo8 at their respective binding domains [indicated by a blue line (for Flo8) or red lines (for Sfl1) on the DNA] (33) initiates events that contribute to either (i) a switch to the silenced FLO11 state (Sfl1-binding) or (ii) a switch to the competent state (Flo8 binding). Competition between Sfl1 and Flo8 determines which of two mutually exclusive ncRNA transcription programs occurs. ICR1 represses FLO11 transcription, whereas PWR1 promotes it. Localized chromatin condensation by Rpd3L at an upstream site (∼1,250 bp; Fig. 2A) could hinder the access of Sfl1 to its binding site, but promote Flo8 binding, toggling the FLO11 promoter toward a state competent for transcription of the protein-coding ORF.