Transcription of two long noncoding RNAs mediates mating-type control of gametogenesis in budding yeast

Abstract

The cell-fate decision leading to gametogenesis is essential for sexual reproduction. In S. cerevisiae, only diploid MATa/α but not haploid MATa or MATα cells undergo gametogenesis, known as sporulation. We find that transcription of two long noncoding RNAs (lncRNAs) mediates mating-type control of sporulation. In MATa or MATα haploids, expression of IME1, the central inducer of gametogenesis, is inhibited in cis by transcription of the lncRNA IRT1, located in the IME1 promoter. IRT1 transcription recruits the Set2 histone methyltransferase and the Set3 histone deacetylase complex to establish repressive chromatin at the IME1 promoter. Inhibiting expression of IRT1 and an antisense transcript that antagonizes the expression of the meiotic regulator IME4 allows cells expressing the haploid mating type to sporulate with kinetics that are indistinguishable from that of MATa/α diploids. Conversely, expression of the two lncRNAs abolishes sporulation in MATa/α diploids. Thus, transcription of two lncRNAs governs mating-type control of gametogenesis in yeast.

Figure 1. The non-coding RNA IRT1 is transcribed through the IME1 promoter

(A) Mating type control of IME1 expression. See text for details. (B) Overview of the IME1 locus. The location of IME1, the non-coding RNA IRT1 (formerly SUT643) and MUT1573 is shown. The arrows show direction of transcription. (C) MATa/α (A4962) and MATa/a (A28374) cells were grown to OD_600nm=1.8 in YPD (Y) for 24 hours followed by growth in BYTA medium overnight. Cells were then transferred into sporulation medium (SPO) to induce sporulation. Samples were taken at the indicated times to examine IME1 and IRT1 RNA levels. The cartoon above the blot indicates the location of the probes used to detect IME1 and IRT1. (D) Haploid MATa (A4841), MATa/α (A4962) and MATa/a (A28374) diploid cells were induced to sporulate as described in (C) and IRT1 and IME1 RNA level were analyzed at the indicated time points by RT-PCR. RNA levels were normalized to ACT1 expression. The data are represented as mean +/− SEM from multiple experiments. See also Figure S1. (E, F) MATa (A4841) cells were induced to sporulate. After 6 hours thiolutin (3µg/ml) was added and IRT1 and ACT1 RNA levels were determined at the indicated times.

Figure 2. IRT1 and IME1 RNA levels are mutually exclusive

(A) MATa/α diploids (A24333) and MATa haploids (A10931) were induced to sporulate. Samples were taken at the indicated time points to examine IME1 and IRT1 RNA in single cells. Merged images of IRT1 (red) and IME1 (green) transcripts are shown. DNA is shown in blue. (B, C) Quantification of the percentage of cells with no transcripts (open triangles), two or more transcript of IRT1 (open circles), IME1 (closed circles), or of both (closed triangles) is shown. At least 450 cells were analyzed per time-point (see Table S3). See also Figures S2 and S3.

Figure 3. Rme1-dependent IRT1 transcription inhibits IME1 expression

(A) Analysis of IRT1 and IME1 expression in MATa/a irt1-T (A30070; truncated IRT1) and MATa/a rme1Δ (A30195) cells progressing through sporulation in a synchronous manner. RNA samples were taken from cells grown in YPD (Y), or SPO medium for 0, 4, 8, and 12 hours. rRNA is shown as a loading control. (*) marks the truncated version of the IRT1 transcript. (**) marks the MUT1573 transcript, which accumulates during late stages of sporulation. The MATa/α and MATa/a controls for this experiment are shown in Figure 1C as these experiments were performed at the same time. (B) MATa/α (open circles; A4962), MATa/a rme1Δ (closed circles; A30195), MATa/a irt1-T (open triangles; A30070) diploid cells and MATa irt1-T (closed triangles; A30067) haploid cells were induced to sporulate. The number of cells that had undergone either one or both meiotic divisions was determined at the indicated times (n=100). (C) MATa (A4841), MATa rme1Δ (A30075) and MATa irt1-T (A30067) cells were induced to sporulate. Cells were harvested either before transfer into SPO medium or after a 14 day incubation in SPO medium. 5-fold serial dilutions were spotted onto YPD plates. (D) Sequence conservation and position of the two RME1 binding sites with respect to the IME1 translation start-site across different Saccharomyces species are shown. (E) Analysis of Rme1 occupancy at the RME1 bindings sites upstream of the IRT1 transcription start site (primer pair one), where Rme1 is known to bind and at the transcription start site of IME1 (primer pair two), where Rme1 is not known to bind. Rme1 binding was determined at the indicated times in MATa RME1-3xV5 (A30108) cells grown in sporulation-inducing conditions. ChIP signals were normalized to the HMR locus, which does not bind Rme1. The data are represented as mean +/− SEM from multiple experiments. See also Figure S4.

Figure 4. IRT1 represses IME1 in cis

(A) Structure of the duplicated IRT1 locus. The plasmid backbone harboring URA3 and lacZ is located between the two IRT1 genes. (B) MATa IRT1 (A4841), MATa irt1-T (A30067), MATa IRT1 IRT1 (A30197) and MATa IRT1 irt1-T (A30199) cells were induced to sporulate. Samples were taken at the indicated times to determine the amount of IME1 and IRT1 RNA. (C) Strains described in (B) were induced to sporulate. Cells were harvested either before transfer into SPO medium or after a14 day incubation. 5-fold serial dilutions were spotted onto YPD plates. (D) MATa rme1Δ (A30075) and MATa rme1Δ pGPD1-IRT1 (A30134) cells were induced to sporulate. Samples were taken at the indicated times to determine the amount of IRT1 and IME1 RNA. See also Figure S5.

Figure 5. IRT1 transcription inhibits Pog1 recruitment and increases nucleosome occupancy at the IME1 promoter

(A, B) Wild-type (A4962) and pog1Δ (A30194) MATa/α diploid cells were induced to sporulate. IME1 RNA levels (A) and the percentage of cells that have undergone at least one meiotic division (B) were determined at the indicated times. (C) Graphical overview of the IRT1/IME1 locus. The positions of the nine primer pairs used to determine Pog1 and histone H3 occupancy are shown. (D) MATa/α diploid cells carrying a POG1-3xV5 fusion (A30236) were induced to sporulate. Pog1 binding throughout the IME1 promoter was determined after 0 or 3 hours in SPO medium. ChIP signals were normalized to the HMR locus, where Pog1 is not known to bind. The data are represented as mean +/− SEM from multiple experiments. (E) Pog1 binding to the IME1 promoter was determined in MATa/α diploid and MATa haploid cells (A30235) at the indicated times. Primer pair 4 was used for this analysis. (F) Wild-type (A30235) and irt1-T MATa cells (A30246) were induced to sporulate and Pog1 binding was determined at the indicated times. (G-J) Relative histone H3 occupancy across the IRT1/IME1 locus in MATa haploid (A4841) and MATa/α diploid (A4962) cells. Cells were grown either in YPD (exponential phase or to saturation) or induced to sporulate for 0 or 3 hours.

Figure 6. SET2 and SET3 are required for IRT1 transcription mediated repression of the IME1 promoter

(A) The positions of the primer pairs used in ChIP experiments for panel B, C, and H are shown. (B, C) Relative occupancy of histone H3 lysine 4 dimethylation (B) and lysine 36 methylation (using an antibody directed against histone H3 lysine 36 trimethylation) (C) across the IRT1/IME1 locus in MATa haploid (A4841) cells. The data are represented as mean +/− SEM from multiple experiments. (D) Wild-type (A4841), irt1-T (A30067), set2Δ (A31995), set3Δ (A31999), set2Δ set3Δ (A32040) MATa cells were induced to sporulate. Samples were taken at the indicated times to determine the amount of IME1 and IRT1 RNA. (E, F) Wild-type (A10931), set2Δ (A31992), set3Δ (A31998), and set2Δ set3Δ (A32051) MATa haploid cells were induced to sporulate to examine IME1 and IRT1 RNAs in single cells. (E) shows set2Δ set3Δ cells that harbor IRT1 (red) and IME1 (green) transcripts. DNA is shown in blue. Quantification of the percentage of single cells that harbor no transcripts, two or more transcripts of IRT1, IME1, or IRT1 and IME1 (n=3; SEM) (F). At least 450 cells were analyzed per strain. (G) Wild-type (A30235), set2Δ (A32036), set3Δ (A32033) and set2Δ set3Δ (A32049) MATa haploid cells carrying a POG1-3xV5 fusion were induced to sporulate and Pog1 occupancy in the IME1 promoter was determined. (H) Relative histone H3 occupancy across the IRT1/IME1 locus after 6 hours in SPO medium. (I) MATa/α (closed circles; A4962), MATa/α set2Δ set3Δ (open circles; A32041) and MATa/a set2Δ set3Δ (closed triangles; A32059) cells were induced to sporulate. Samples were taken at the indicated times to determine the number of cells that had undergone either one or both meiotic division. (J) Model for IRT1 mediated repression of IME1 involving Set2 and Set3. See text for details. See also Figure S6.

Figure 7. Transcription of two lncRNAs conveys mating type control of sporulation

(A) MATa/α (A4962; closed circles), MATa/a irt1-T (A30070; open circles), MATa/a pTEF-IME4 (A30133; closed triangles), and MATa/a pTEF-IME4 irt1-T (A30100; open triangles) cells were induced to sporulate. The percentage of cells that had completed at least one meiotic division was determined at the indicated times (n=100). (B) MATa/α cells (closed circles), MATa/a cells in which the a1–α2 binding sites in the IME4-AS promoter were replaced by tetO sequences (MATa/a pIME4-3’-tetO; open circles; A30217), MATa/a cells in which the a1–α2 binding sites in the RME1 promoter were replaced by tetO sequences (MATa/a pRME1-tetO; closed triangles; A30231) and MATa/a cells expressing both fusions (open triangles; A30219) all carrying a TetR-Tup1 fusion were induced to sporulate. The percentage of cells having completed at least one meiotic division was determined at the indicated times (n=100). (C) MATa/α strains carrying various combinations of a1–α2 binding site mutations are listed (#1 (A32019), #2 (A32020), #3 (A32021), #4 (A32022), #5 (A32023), #6 (A32024), #7 (A32025), and #8 (A32026)). (D) The percentage of sporulated cells of strains in (C) was determined after 48 hours in SPO medium. (E) Wild-type (A4962), set2Δ (A31996), set3Δ (A32001), and set2Δ set3Δ (A32041) MATa/α cells were induced to sporulate. The percentage of cells that had completed at least one meiotic division was determined at the indicated times (n=100). (F) MATa/α diploid cells carrying deletions in the two a1–α2 binding sites of the RME1 promoter (A32022, A32035, A32034 and A32057) were analyzed as in (E). See also Figure S7.