A regulatory circuit of two lncRNAs and a master regulator directs cell fate in yeast

Abstract

Transcription of long noncoding RNAs (lncRNAs) regulates local gene expression in eukaryotes. Many examples of how a single lncRNA controls the expression of an adjacent or nearby protein-coding gene have been described. Here we examine the regulation of a locus consisting of two contiguous lncRNAs and the master regulator for entry into yeast meiosis, IME1. We find that the cluster of two lncRNAs together with several transcription factors form a regulatory circuit by which IME1 controls its own promoter and thereby promotes its own expression. Inhibition or stimulation of this unusual feedback circuit affects timing and rate of IME1 accumulation, and hence the ability for cells to enter meiosis. Our data demonstrate that orchestrated transcription through two contiguous lncRNAs promotes local gene expression and determines a critical cell fate decision.

Fig. 1

Transcription of IRT2 promotes entry into meiosis. a Scheme of the IME1 locus consisting of: IRT1, IRT2/MUT1573, and the IME1 gene. b IRT2 expression in SK1 diploid cells (FW1511) during entry into meiosis. Cells were grown in rich medium till saturation, shifted and grown in pre-sporulation medium for another 16 h, and transferred to sporulation medium (SPO). Samples for northern blot were taken at the indicated time points. A probe directed to the upstream region in the IME1 promoter was used to detect IRT2. To control for loading, membranes were also probed for SCR1. c IRT2 expression in S288C diploid cells (FW631) during entry into meiosis. Cells were grown till saturation in rich medium, and subsequently shifted to SPO. Samples were taken at the indicated time points. IRT2 levels were quantified by reverse transcription and quantitative PCR. The signals were normalized to ACT1 levels. The means ± SEM of n = 2 experiments are shown. d Scheme of IME1 and RME1 alleles used in e and f. e Quantification of cells that completed meiotic divisions (MI + MII) in diploid S288C cells that were either wild type (FW631), expressed IRT2 from the inducible CUP1 promoter (pCUP-IRT2) (FW2668) or harbored a deletion of RME1 (rme1Δ, FW1497). Cells were grown as described in c, at 2 h in SPO, pCUP-IRT2 cells were either not treated (−Cu) or treated with copper sulfate (+Cu). Cells were fixed after 72 h in SPO, stained, and DAPI masses of n = 200 cells were counted. Cells with two or more masses were considered to have completed at least one meiotic division. Means ± SEM of n = 5 experiments are shown. ***p < 0.0005; ****p < 0.0001 (Student’s t test). f Quantification of cells that completed meiotic divisions (MI + MII) in SK1 diploid cells that were either wild type (FW1511), pCUP-IRT2 (FW5254), rme1Δ (FW2340), harbored deletions in the a1α2 repressor sites of the RME1 promoter (RME1-H, FW1196), harbored pCUP-IRT2 and rme1Δ (FW2476), or pCUP-IRT2 and RME1-H (FW2385). Cells grown and treated as described in e. Means ± SEM of at least n = 4 experiments are shown. *p < 0.05; ****p < 0.0001 (Student’s t test)

Fig. 2

IRT2 transcription interferes with Rme1 binding, and promotes IME1 expression a IRT2, IRT1, and IME1 expression in diploid cells harboring pCUP-IRT2 and RME1-H tagged with the V5 epitope tag (FW2060) by northern blot. Cells were pre-grown in rich medium, pre-sporulation medium, before shifted to SPO and were either not treated (−Cu) or treated with copper sulfate (+Cu) after 45 min in SPO. Samples were taken at the indicated time points. Northern blot membranes were hybridized with a probe that detects both IRT1 and IRT2, and a probe that detects IME1. As a loading control, the ribosomal RNA is displayed. b Similar as a, except that binding of Rme1 to the IME1 promoter was determined by chromatin immunoprecipitation using V5-antibodies coupled to agarose beads in cells that were either not treated (−Cu), or treated with copper sulfate (+Cu). Samples were taken at the indicated time point after treatment, formaldehyde crosslinked, and chromatin extracts were prepared. Rme1-DNA complexes were isolated by immunoprecipitation, and the recovered DNA fragments were quantified by qPCR using a primer pair directed against the Rme1-binding sites in the IME1 promoter. The signals were normalized to the silent mating locus (HMR), where Rme1 does not bind. The means ± SEM of n = 4 experiments are shown. c Similar as a, except that kinetics of meiotic divisions were determined. Means ± SEM of n = 3 experiments are shown. *Indicates the time of treatment with copper sulfate

Fig. 3

Ime1 and Ume6 control IRT2 expression. a Schematic overview of the IME1 locus indicating the position of the Ume6-binding site (left). Nucleotide coordinates of the Ume6 site relative to IME1 AUG in different Saccharomyces species (right). b Binding of Ume6 to the IME1 promoter measured by chromatin immunoprecipitation using V5 tagged Ume6 (FW2978). A wild-type (FW1509) and an Ume6-binding deletion mutant (pIME1-Δu6, FW2000) strains were also included in the analysis. The means ± SEM of n = 3 experiments are shown. c Northern blot of IRT2 expression in cells that were pre-grown in rich medium and pre-sporulation medium, before shifted to SPO. Wild-type (FW1511), single, and double mutants harboring pIME1-Δu6 and/or a 3′ end mutation in IME1 (ime1-t) (FW2449, FW2370, and FW2571) strains were used for the analysis. To control for loading, the membrane was also probed for SCR1. d IRT1, IRT2, and IME1 expression detected by northern blot in cells expressing IME1 from the copper-inducible promoter (pCUP-IME1, FW3006). pIME1-Δu6 (FW2842) cells were also included in the analysis. Cells were pre-grown in rich and pre-sporulation medium before shifted to SPO and were either not treated (−Cu) or treated with copper sulfate (+Cu) at 1 h in SPO. Northern blot membranes were hybridized with a probe that detects both IRT1 and IRT2, and a probe that detects IME1. As a loading control, the ribosomal RNA is displayed

Fig. 4

Ime1 changes local chromatin via IRT2 and promotes its own expression. a Diploid cells harboring pCUP-IME1 (FW3006) were grown in rich and pre-sporulation medium before shifted to SPO, and were either not treated (−Cu) or treated with copper sulfate (+Cu) after 1 h in SPO. Samples were taken at 0 and 6 h in SPO. Chromatin extracts were treated with micrococcal nuclease (MNase). Mononucleosome DNA fragments were isolated and quantified using 14 primers pairs. The signals were normalized to a no MNase input. Means ± SEM of n = 3. b Diploid cells with pCUP1-IME1 and RME1-H-V5 (FW1366) were induced to enter meiosis and were either not treated (−Cu) or treated (+Cu) after 1 h in SPO. Samples for chromatin immunoprecipitation were taken at 0 or 2 h after induction, and quantified by qPCR. The signals were normalized to the silent mating locus (HMR). Means ± SEM of n = 3. c IRT1, IRT2, and IME1 expression in cells harboring RME1-H and pCUP-IME1 (FW2270). Cells were grown as described in a. Northern blot membranes were hybridized with probes that detects IRT1, IRT2, and IME1. As a loading control, the ribosomal RNA is shown. d IRT1, IRT2, and IME1 expression in diploid cells harboring RME1-H (FW1196) or RME1-H together with the set2Δset3Δ mutant (FW1312) during entry in meiosis. Cells were grown and samples were taken as described in c. Northern blot membrane was probed for IRT1, IRT2, and IME1. SCR1 expression was used as loading control. e Cells containing one copy of pCUP-IME1, one copy of pIME1-GFP-IME1 (pIME1) (FW5291), or pIME1-Δu6-GFP-IME1 (pIME1-Δu6) (FW5295) were induced to enter meiosis in the absence (−Cu) or presence (+Cu) of IME1 expression. Signals were quantified (n = 100 cells) per time point. Means ± error bars represent the 95% confidence interval. f Cells containing RME1-H and one copy of pCUP-IME1, one copy of pIME1-LacZ (FW5337), or a mutant pIME1-EIt-LacZ plasmid lacking part of the IRT2 sequence including the Ume6 motif (FW5341) were induced to enter meiosis. The graph displays the ratio of β galactosidase activity signals from IME1 induced samples versus not-induced samples. Means ± SEM of n = 3. *Treatment with copper sulfate

Fig. 5

Single-cell analysis of the IME1, IRT2, and IRT1 feedback cascade. a Schematic overview of IME1 locus used for time-lapse microscopy (top). Diploid cells were heterozygous for the IME1 locus: one copy of GFP fused to N-terminus of IME1 (pIME1-GFP-IME1) and one copy expressing the IME1 promoter fused to mCherry lacking the IRT2 sequence (pIME1-Δirt2-mCherry). Cells were grown to log phase in synthetic complete media loaded into a microfluidic device, induced to enter meiosis and imaged for up till 50 h (see supplemental information for details). Example images of phase, GFP, and mCherry of a single cell taken at start and end of a time-lapse experiment (bottom). The scale bar in right bottom corner represents 3.2 μm. b Example of traces of a single cell. GFP and mCherry fluorescence signals were monitored and overall signals were scaled (see supplemental information for details). The graph also displays the time of initiation of expression (onset time), the rate of accumulation, and maximum levels (max intensity) of pIME1-GFP-IME1 and pIME1-Δirt2-mCherry. c Summary of data obtained from time-lapse microscopy experiments. All strains harbored one copy of pIME1-GFP-IME1 and one copy of pIME1-Δirt2-mCherry, combined with either wild-type RME1 (FW4843), pRME1-Δaa1 (FW4844), or pRME1-Δaa1/pCUP-IRT2 (FW5051). Cells were imaged and GFP and mCherry fluorescence signals were quantified. Mean onset times, rate of accumulation, and max intensity were determined. The means ± SEM of n = 58 (FW4843), n = 88 (FW4844), and n = 64 (FW5051) cells are shown. All pairwise differences, except between FW4843 and FW4844 (left panel only), are statistically significant; p < 0.001 (Kolmogorov–Smirnov test)

Fig. 6

Distribution of IME1 expression among single S288C cells in the presence or absence of IRT2 transcription. a Distribution of IME1 transcripts levels among single cells during entry into meiosis in S288C as detected by single-molecule RNA fluorescence in situ hybridization. Wild-type (WT) (FW631), pCUP-IRT2 (FW2668), and pIME1-Δu6 (FW1390) cells were grown in rich medium till saturation and subsequently shifted to SPO. Samples were taken at the indicated time points, fixed, and hybridized with probes directed against IME1 and ACT1 mRNAs (see supplemental information for details). The black line indicates the median number of transcripts per cell for each time point. At least n = 120 cells were used for the analysis. b Same as a, but cells were binned according IME1 expression levels. c Quantification of cells that completed meiotic divisions in S288C cells described in a. Cells were fixed after 72 h in SPO, stained, and DAPI masses were counted. The means ± SEM of at least n = 5 experiments are shown. ***p < 0.0005 (Student’s t test)

Fig. 7

Model for the regulatory circuit consisting of IME1 and the lncRNAs, IRT2, and IRT1. a Model for Ime1 regulation by IRT1, IRT2, and IME1 (see Supplemental information for details). All the variables and connections between variables are displayed. b Simulation of IME1 transcription rate (It) and Ime1 protein accumulation (Ip) prior (s = 0, before 0 h) and during starvation (s = 1) for the wild-type IME1 promoter, in the absence of IRT2 (no IRT2), or in the absence of IRT1 and IRT2 (no IRT2 + IRT1). The y axis displays the level of s, It, or Ip in arbitrary units (AU) scaled between 0 and 1. c Similar as b except that Ime1 protein accumulation (Ip) was simulated during starvation periods of 1 h and 5 h, respectively. d Model of feedback cascade involving IME1, IRT2, and IRT1. In the presence of the regulatory circuit consisting of IME1, IRT2, and IRT1, Ime1 is able to stimulate its own expression. This increases the propensity for cells to undergo meiosis. Cells lacking the feedback signals from Ime1 to IRT2 and IRT1 show reduced IME1 expression and have a lower ability to enter meiosis