Swi4 subunit of the SBF and SBF targets are downregulated during early meiosis

A. A schematic of SBF and MBF complexes and the general functional groups of the genes they regulate. B. Samples from strain UB35246 were collected between 0-6 hours (h) in sporulation medium (SPO) and immunoblots were performed using α-Swi4, α-Swi6, and α-Mbp1 respectively. Hxk2 was used a loading control. Representative blots from one of two biological replicates are shown. C. Quantification of the immunoblots in (B). The signal at each time point was first normalized to Hxk2 loading control and then to the max signal. D. Scatterplot of RNA-seq data (RPKM) from (Brar et al., 2012) comparing 2 h in SPO vs. mitotic growth of well characterized SBF targets (pink) and MBF targets (teal). E. Wild type (UB22199) and pATG8-SWI4 (UB22226) cells were collected to perform RT-qPCR for CLN1, CLN2, CDC21, and RNR1 transcripts. Transcript abundance was quantified using primer sets specific for each respective gene from three technical replicates for each biological replicate. Quantification was performed in reference to PFY1 and then normalized to wild-type control. FC = fold change. Experiments were performed twice using biological replicates, mean value plotted with range. Differences in wild type versus pATG8-SWI4 transcript levels at 2 h in SPO compared with a two-tailed t-test (*, p = 0.0351 [CLN1]; **, p = 0.0013 [CLN2]; ns, p = 0.8488 [CDC21]; ns, p = 0.0859 [RNR1]). F. Live-cell imaging of strains containing the fluorescently tagged histone Htb1-mCherry for wild type (UB32085) and pATG8-SWI4 (UB32089). Experiments were performed twice using biological replicates, mean value plotted with range. Differences in meiotic progression tested by Mann Whitney test, two-tailed (**, p = 0.0045).

Regulation of Swi4 abundance is required for timely meiotic entry

A-B. Fixed imaging of cells marked with GFP-Ime1 and Htb1-mCherry. Wild type (UB22199) and pATG8-SWI4 (UB22226) cells were collected between 0-4 h in SPO. A. Representative images with merge at 0 h and 2 h in SPO. Representative cells outlined. Scale bar: 3 μm. B. Quantification as percent cells with nuclear Ime1. Experiments were performed twice using biological replicates, mean value plotted with range. Total of 200 cells analyzed per strain. Differences in the fraction of cells with nuclear Ime1 was compared using a two-tailed t-test (**, p = 0.0099 [1 h in SPO]; *, p = 0.0169 [2 h in SPO]; *, p = 0.0315 [3 h in SPO]; ns, two-tailed p = 0.4595 [4 h in SPO]). C-E. Fixed imaging of cells marked with GFP-Ime1 and Swi4-mCherry. Wild type (UB31378) and pATG8-SWI4 (UB31381) cells were collected at 0 h and 4 h in SPO. C. Example images with merge at 4 h in SPO. Example cells outlined (*low nuclear GFP-Ime1 with high nuclear Swi4-mCherry, **high nuclear GFP-Ime1 with low nuclear Swi4-mCherry). Scale bar: 3 μm. D. Scatterplot of GFP-Ime1 mean nuclear intensity and Swi4-mCherry mean nuclear intensity for wild type and pATG8-SWI4 cells at 0 h in SPO. See Materials and Methods for further details about image quantification. Dashed line is linear regression plotted for each condition and strain. A total number of 269 cells were analyzed. E. Same as in (D) but for wild type and. pATG8-SWI4 cells at 4 h in SPO. A total number of 341 cells were analyzed. Differences in mean nuclear GFP-Ime1 or Swi4-mCherry intensity between wild type and pATG8-SWI4 compared using a Mann-Whitney test, two-tailed (****, p < 0.0001 [wild type vs. pATG8-SWI4 (GFP-Ime1)]; ****, p < 0.0001 [wild type vs. pATG8-SWI4 (Swi4-mCherry)]). F. Volcano plot of DE-Seq2 analysis for pATG8-SWI4 versus wild type. Dashed line indicates padj (p value) = 0.05. Analysis was performed using mRNA-seq from two biological replicates. Wild type (UB22199) and pATG8-SWI4 (UB22226) cells were collected at 2 h in SPO. SBF targets (pink) (Iyer et al., 2001) and early meiotic genes (blue) defined by (Brar et al., 2012). Darker pink or darker blue, labeled dots are well studied targets in either gene set list. G. GSEA analysis of mRNA-seq comparing wild type vs. pATG8-SWI4 collected at 2 h in SPO. Vertical black bars represent the early meiotic cluster from (Brar et al., 2012) or SBF cluster from (Iyer et al., 2001). The heatmap indicates genes that are more enriched in pATG8-SWI4 (red, left-side) or genes that are enriched in wild type (blue, right-side). NES = normalized enrichment score. Enrichment was determined by comparing pATG8-SWI4 versus wild type. H-I. Live-cell imaging of cells in meiosis marked by Rec8-GFP and nuclear marker Htb1-mCherry for wild type (UB32085) and pATG8-SWI4 (UB32089). H. Movie montage with example images throughout meiosis for Rec8-GFP and Htb1-mCherry. Scale bar: 3 μm I. Quantification as percent of cells that entered meiosis assayed by nuclear Rec8 appearance. Experiments were performed using two biological replicates, mean value plotted with range. A total number of 452 cells were analyzed. Differences in meiotic progression compared by Mann Whitney test, two-tailed (***, p = 0.0005 [wild type vs. pATG8-SWI4]).

Removal of the SBF targets Cln1 or Cln2 partially rescues the meiotic entry delay in the pATG8-SWI4 mutant

A. Immunoblotting was performed on samples collected for wild type (UB29326) and pATG8-SWI4 (UB29328) between 0-6 h in SPO using α-V5 antibody to track Cln1-3V5. Hxk2 was used a loading control. Representative blots from one of two biological replicates are shown. B. Quantification of (A). C. Same as in (A) but for wild type (UB29330) and pATG8-SWI4 (UB29332) cells using α-V5 antibody to track Cln2-3V5. Hxk2 was used a loading control. Representative blots from one of two biological replicates are shown. D. Quantification of (C). E. Live-cell imaging of meiotic cells marked by Rec8-GFP and nuclear marker Htb1-mCherry, with the following genotypes: wild type (UB32085), pATG8-SWI4 (UB32089), and pATG8-SWI4; cln1Δ (UB34536). Quantification of cells that entered meiosis assayed by the initial timing of nuclear Rec8 appearance. Experiments were performed using two biological replicates, mean value plotted with range. A total number of 883 cells were analyzed. Differences in meiotic progression compared by Mann-Whitney test, two-tailed (*, p = 0.0111 [pATG8-SWI4 vs. pATG8-SWI4; cln1Δ]). cln1Δ alone (not shown) has similar meiotic progression kinetics relative to wild type. F. Same as (E) but with the following genotypes: wild type (UB32085), pATG8-SWI4 (UB32089), and pATG8-SWI4; cln2Δ (UB34165). A total number of 610 cells were analyzed. Differences in meiotic progression compared by Mann-Whitney test, two-tailed (*, p = 0.0478 [pATG8-SWI4 vs. pATG8-SWI4; cln2Δ]). cln2Δ alone (not shown) has similar meiotic progression kinetics relative to wild type.

Tethering of Ime1 to Ume6 is sufficient to overcome the meiotic block exerted by G1 cyclin overexpression

A. Sporulation efficiency of cells at 24 h in SPO media wild type (UB22199), pATG8-CLN1 (UB32820), pATG8-CLN2 (UB25959), pCUP-GFP-IME1 (UB34641), pCUP1-GFP-IME1; pATG8-CLN2 (UB35057), PUS1-⍺GFP (UB35593), PUS1-⍺GFP; pATG8-CLN2 (UB35982), UME6-⍺GFP (UB35300), and UME6-⍺GFP; pATG8-CLN2 (UB35177). Experiments shown in this figure were performed using two biological replicates, mean value plotted with range. Total of 200 cells counted per strain. See Supplementary Table 2 for statistics. B-C. Fixed imaging of cells marked with GFP-Ime1 and Htb1-mCherry. Wild type (UB22199) and pATG8-CLN2 (UB25959) cells were collected between 0-3 h in SPO. B. Representative images with merge at 2 h in SPO. Representative cells outlined. Scale bar: 3 μm. C. Quantification of cells with nuclear Ime1. Experiments were performed using two biological replicates, mean value plotted with range. Total of 200 cells analyzed per strain. Differences in percent of cells with nuclear Ime1 was compared by two-tailed t-test (**, p = 0.00917 [1 h in SPO]; **, p = 0.0044 [2 h in SPO]; *, p = 0.0122 [3 h in SPO]). D. Schematic depicting use of pCUP1 promoter (pCUP1-GFP-IME1) to rescue Ime1 transcript and protein levels. E-F Fixed imaging of cells marked with GFP-Ime1 and Htb1-mCherry. Cells with the following genotypes were collected at 2 h in SPO: wild type (UB22199), pATG8-CLN2 (UB35106), pCUP1-GFP-IME1 (UB34641), and pCUP1-GFP-IME1; pATG8-CLN2 (UB35057). E. Representative images with merge and representative cells outlined. Scale bar: 3 μm. F. GFP-Ime1 mean nuclear intensity measured for a single z-slice. A total number of 433 cells were analyzed. Differences in mean nuclear intensity compared by Mann-Whitney test, two tailed, (****, p < 0.0001 [pCUP1-IME1 vs. pCUP1-IME1; pATG8-CLN2]). G. Schematic of nanobody trap strategy with PUS1-⍺GFP and GFP-Ime1 to rescue Ime1 nuclear localization in meiosis. H-I. Fixed imaging of cells marked with GFP-Ime1 and Htb1-mCherry. Cells with the following genotypes were collected at 2 h in SPO: wild type (UB22199), pATG8-CLN2 (UB35106), PUS1-⍺GFP (UB35593), and PUS1-⍺GFP; pATG8-CLN2 (UB35982). H. Representative images with merge and example cells outlined. Scale bar: 3 μm. I. GFP-Ime1 mean nuclear intensity measured for a single z-slice. A total number of 934 cells were analyzed. Differences in mean nuclear intensity compared by Mann-Whitney test, two-tailed, (****, p < 0.0001 [wild type vs. pATG8-CLN2]; not significant (ns), p = 0.6563 [PUS1-⍺GFP vs. pATG8-CLN2; PUS1-⍺GFP]; not significant (ns), p = 0.8881 [wildtype vs. pATG8-CLN2; PUS1-⍺GFP]). J. Schematic of nanobody trap strategy with UME6-⍺GFP and GFP-Ime1 to rescue Ime1-Ume6 interaction in meiosis. K-L Fixed imaging of cells marked with GFP-Ime1 and Htb1-mCherry. Cells with the following genotypes were collected at 2 h in SPO: wild type (UB22199), pATG8-SWI4 (UB35106), UME6-⍺GFP (UB35300), and UME6-⍺GFP; pATG8-CLN2 (UB35177). K. Representative images with merge and representative cells outlined. Scale bar: 3 μm. L. GFP-Ime1 mean nuclear intensity measured for a single z-slice. A total number of 1220 cells were analyzed. Differences in mean nuclear intensity compared by Mann-Whitney test, two-tailed (****, p < 0.0001 [wild type vs. pATG8-CLN2]; ****, p < 0.0001 [UME6-⍺GFP vs. pATG8-CLN2; UME6-⍺GFP]; *, p = 0.0354 [wildtype vs. pATG8-CLN2; UME6-⍺GFP]).

Ime1-dependent expression of a LUTI from the SWI4 locus leads to a reduction in Swi4 protein levels during meiotic entry

A. Genome browser views of RNA-seq data (Brar et al., 2012) of the SWI4 locus. SWI4LUTItranscription start site is ∼1045bp upstream of SWI4 ORF translation start site. B. A schematic of LUTI-based gene regulation. Top: Mitotic growth, SWI4LUTI is repressed due to Ume6-Rpd3-Sin3 complex and SWI4canonis induced by one or more transcription factors including Ace2, Mbp1 and Swi5, leading to Swi4 protein production. Bottom: Meiosis-specific expression of SWI4LUTI by Ime1-Ume6 leads to downregulation of Swi4 protein production due to combined effect of transcriptional and translation interference. SWI4LUTI5′ leader contains 7 AUG uORFs but only one is shown in the model for simplicity. Schematic is adapted from (Tresenrider et al., 2021). C. Representative smFISH images collected from premeiotic and meiotic cells for detecting SWI4canon and SWI4LUTI. Cells with pCUP1-IME1/pCUP1-IME4 meiotic synchronization system were induced to enter meiosis with 50 µM CuSO4 after 2 h in SPO. Premeiotic cells were collected before IME1/4 induction and meiotic cells were collected 2 h post IME1/4 induction from strain UB14273. Q 670 probes (green) hybridize to shared region within SWI4 CDS. CF590 probes hybridize to the unique 5′ leader region of SWI4LUTI (depicted on the schematic shown above the images). DNA was stained with DAPI. Scale bar: 3 μm. D. Quantification of smFISH shown in (C), plotted as relative frequency histograms of cells with SWI4canonand SWI4LUTItranscripts per cell. Data pooled from two independent biological replicates. Dashed line indicates median number of transcripts per cell. Each histogram is normalized with maximum bin height being the same across all histograms. A total number of 44 cells counted for premeiotic and 102 cells counted in meiotic prophase. Differences in premeiotic versus meiotic were compared by Mann-Whitney test, two-tailed (***, p = 0.0007 [SWI4canon]; ****, p < 0.0001 [SWI4LUTI]). E. RNA blot performed on cells collected between 0-6 h in SPO. All strains carry a SWI4-3V5 tagged allele. Probe was specific for 3V5. Methylene blue staining of rRNA bands was used as a loading control. Matched immunoblotting was performed against Swi4-3V5 using α-V5 and normalized to Hxk2 loading control for each sample. Cells collected are wild type (UB22199) and ΔLUTI (UB23012). Representative blots from one of two biological replicates are shown. F. Quantification of immunoblot in (E). G. Performed as described in (E) for wild type (UB21386) and ΔuORF (UB23636) strains. Representative blots from one of two biological replicates are shown. H. Quantification of immunoblot in (G). I. Immunoblot using α-V5 performed on cells collected between 0-7 h in SPO from a strain carrying pCUP1-IME1 and SWI4-3V5 alleles (UB34641). Swi4-3V5 abundance was normalized to Hxk2 loading control. Cells were induced to enter meiosis with 50 µM CuSO4 after 2 h preincubation in SPO. Representative blots from one of two biological replicates are shown. J. Quantification of immunoblot in (I). K. Genome browser view of Ume6-ChIP at the SWI4 locus (adapted from Tresenrider et al., 2021).

SWI4LUTIis integrated into a larger regulatory network to regulate SBF activity during meiotic entry

A. Volcano plot of DESeq2 analysis for ΔLUTI versus wild type. Dashed line indicates padj (p-value) = 0.05. Analysis was performed with mRNA-seq in duplicate. Wild type (UB27083) and ΔLUTI (UB26874) collected at 2 h in SPO. SBF targets (pink) and early meiotic genes (blue) defined by (Iyer et al., 2001 and; Brar et al., 2012). Darker pink or darker blue, labeled dots are well studied targets in either gene set list. B. Top: Schematic of the anchor-away system using WHI5-mCherry-FRB (WHI5-AA) and RPL13a-FKBP12 alleles. Bottom: Fixed imaging of cells marked with WHI5-mCherry-FRB (WHI5-AA) with DNA stained with DAPI. 1 µM rapamycin added at 0 h in SPO to induce nuclear exclusion of Whi5 (UB25431) strain collected at 0.5 h in SPO. Scale bar: 3 μm. Cells are rapamycin resistant due to mutated TOR1 (tor1-1) and frp1Δ (yeast FKBP12 homolog) to reduce competition between for binding of Frb and Fkbp12. C. Same as in (A) but for wild type (UB27083) and WHI5-AA (UB25431) collected at 2 h in SPO. D. Same as in (A) but for wild type (UB27083) and ΔLUTI; WHI5-AA (UB25428) collected at 2 h in SPO. E. Live-cell imaging of cells in meiosis marked by Rec8-GFP and nuclear marker Htb1-mCherry. The following genotypes were imaged: wild type (UB35987), ΔLUTI (UB35989), WHI5-AA (UB35991), ΔLUTI; WHI5-AA (UB35989). Quantification as percent of cells that entered meiosis assayed by nuclear Rec8 appearance. Experiments were performed using two biological replicates, mean value plotted with range. Differences in meiotic progression compared by Mann-Whitney test, two-tailed (*, p = 0.0112 [wild type vs. ΔLUTI; WHI5-AA]). F. Model of SBF regulation during meiotic entry. Ime1 downregulates Swi4 protein expression via induction of SWI4LUTI while Whi5 represses SBF activity in parallel to LUTI-based mechanism to prevent expression of SBF targets, including G1 cyclins, which perturb meiotic entry via blocking interaction between Ime1 and its cofactor Ume6.

Swi4 and Ime1 protein abundance, Related to Figure 1

A. Wild type (UB22199) and pATG8-SWI4 (UB22226) cells collected between 0-4 h in SPO. Immunoblot performed using a-V5 to quantify Swi4-3V5 abundance. Normalized to Hxk2 loading control. B. Quantification of the immunoblot in (A). C. Immunoblot using aGFP to quantify GFP-Ime1 abundance. Normalized to Hxk2 loading control. D. Quantification of the immunoblot in (C).

Gene ontology analysis for mRNA-seq comparing pATG8-SWI4 to wild type, Related to Figure 2

A. Gene ontology analysis for genes that have significant increased expression by DESeq2 (see Materials and Methods) in pATG8-SWI4 (UB2226) and wild type (UB22199). Term size is the number of genes within a defined term. Enrichment score calculated for each term and plotted. B. same as in (A) but for genes that have significant decreased expression.

Ime1 protein levels when CLN2 is overexpressed and rescue of IME1 transcript levels and protein abundance with pCUP1-IME1, Related to Figure 4

A. Immunoblot was performed on wild type (UB22199) and pATG8-CLN2 (UB35106) cells collected between 0-6 h in SPO. Immunoblotted using α-GFP to quantify GFP-Ime1 abundance. Normalized to Hxk2 loading control. Representative blots from one of two biological replicates are shown. All blots in this figure were performed the same way. B. Quantification of the immunoblot in (A). C. RT-qPCR was performed on IME1 transcript for samples collected in (A). Quantification was performed in reference to levels of meiotic housekeeping gene PFY1 and then normalized to wild type. FC = fold change. Experiments were performed in duplicate, mean value plotted with range. D. Same as in (C) but for pCUP1-GFP-IME1 (UB34641) and pCUP1-GFP-IME1; pATG8-CLN2 (UB35057). E. Immunoblot was performed on samples from wild type (UB22199), pATG8-CLN2 (UB35106), pCUP1-GFP-IME1 (UB34641), and pCUP1-GFP-IME1; pATG8-CLN2 (UB35057) strains collected between 0-4 h in SPO. 50 μM CuSO4 after 2 h in SPO was added to all cells. Representative blots from one of two biological replicates are shown. F. Quantification of the immunoblot in (E).

Expression of SWI4LUTI in Ume6(T99N) mutant, Related to Figure 5

Fold change expression in wild type or Ume6(T99N) mutant between pre meiotic conditions and meiotic prophase (Tresenrider et al., 2021)

Whi5 localization in early meiosis, Swi4 protein levels matching Figure 6A, 6C, 6D, and GSEA data for ΔLUTI; WHI5-AA, Related to Figure 6

A. Fixed imaging of cells marked with GFP-Ime1 and Whi5-mCherry with DNA stained with DAPI. Representative images with merge at 0 h and 2 h in SPO. Representative cells outlined. B. Left: Cells with the following genotypes were collected between 0-6 h in SPO: wild type (UB27083), ΔLUTI (UB26874), WHI5-AA (UB25431), ΔLUTI; WHI5-AA (UB25428). Immunoblotted using α-V5 to quantify Swi4-3V5 abundance. Normalized to Hxk2 loading control. Representative blots from one of two biological replicates are shown. Right: Quantification C. GSEA analysis of mRNA-seq comparing wildtype vs. ΔLUTI; WHI5-AA (UB25428) cells collected at 2 h in SPO. Vertical black bars represent early meiotic gene cluster from (Brar et al., 2012) or SBF cluster from (Iyer et al., 2001). The heatmap indicates genes that are more enriched in ΔLUTI; WHI5-AA (red, left-side) or genes that are enriched with wild type (blue, right-side). NES = normalized enrichment score. Enrichment was determined comparing wild type to ΔLUTI; WHI5-AA.