1. Volume 23, Issue 4, Pages 483-496 (August 2006)
Constraining G1-Specific Transcription to Late G1 Phase: The MBF-Associated Corepressor Nrm1 Acts via Negative Feedback 
Robertus A.M. de Bruin, Tatyana I. Kalashnikova, Charly Chahwan, W. Hayes McDonald, James Wohlschlegel, John Yates, Paul Russell, Curt Wittenberg 
Molecular Cell 
Volume 23, Issue 4, Pages (August 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Nrm1 Is a Component of MBF
(A and B) Nrm1 associates specifically with Swi6 and Mbp1. The number of spectra derived from peptides having a mass consistent with peptides predicted for Swi6, Swi4, Mbp1, or Nrm1 (Unique/No., unique peptides/total number of peptides) as identified by MudPIT analysis of TAP-immunopurified protein Swi4, Swi6, or Mbp1 (A) or Nrm1 (B) complexes. “% coverage” represents the percentage of coverage of the full-length protein by the unique peptides identified by MudPIT.
(C) Nrm1 interacts with Mbp1, but not with Swi4. Extracts were prepared from wild-type strains carrying NRM1-13xmyc alone or along with MBP1-TAP or SWI4-TAP. Whole-cell extract (WCE) from asynchronous cultures was probed with anti-myc to detect Nrm1-13xmyc. IgG immune complexes (IgG-IP) were probed with anti-myc to detect Nrm1-13xmyc or peroxidase-conjugated anti-peroxidase IgG (PAP) to detect Mbp1-TAP or Swi4-TAP.
(D) Nrm1 interacts with MBF. Extracts were prepared from wild-type or swi6Δ strains carrying NRM1-13xmyc and MBP1-TAP. WCE from asynchronous cultures was probed with PAP to detect Mbp1-TAP or anti-myc to detect Nrm1-myc. Anti-myc (anti-myc-IP) and anti-IgG immune complexes (IgG-IP) were probed with PAP to detect Mbp1-TAP or anti-myc to detect Nrm1-13xmyc.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2
Nrm1 Binds Transcriptionally Repressed G1-Specific Genes via DNA Bound MBF
(A) Nrm1 accumulates and binds to the RNR1 promoter late in G1 phase. Small unbudded cells were isolated by centrifugal elutriation, inoculated into fresh medium, and allowed to progress synchronously through the cell cycle. Samples were taken at 15 min intervals, budding index was determined, and mRNA was analyzed by real-time RT-PCR (RT-PCR). The transcript levels are expressed as a percentage of highest level (100%) after normalization of all values to the ACT1 mRNA. (Middle) Nrm1 protein levels were determined in WCE probed with anti-myc to detect Nrm1-myc in cells from the same time course and with anti-PSTAIRE antibody to detect Cdc28 protein level. (Bottom) Chromatin immunoprecipitation (ChIP) of RNR1 (MBF-dependent gene) and HXT3 (MBF-independent gene) promoter DNA by Nrm1-myc in cells from the same time course. ChIPs of untagged genes (no tag) and WCE are negative and positive controls.
(B) Nrm1 binds the RNR1 promoter via MBF. ChIP of RNR1 (MBF-dependent gene), PCL1 (SBF-dependent gene), and HXT3 (SBF/MBF-independent gene) promoter DNA from wild-type cells by Swi4-myc, Whi5-myc and Mbp1-myc and ChIP of the same promoters from wild-type (wt), swi4Δ, and mbp1Δ strains by Nrm1-myc immunoprecipitation.
(C) Swi6 remains associated to the MBF-regulated promoter during the cell cycle. Budding index and indicated mRNA levels in cells arrested by α factor (0 min) or released from the arrest for the indicated interval (10–130 min). Transcript levels represent the percentage relative to the highest level (100%) observed after normalization to the ACT1 mRNA level in the same sample. Quantitative PCR (qPCR) of chromatin-immunoprecipitated indicated promoter DNA by Swi6-myc from cells during the same time course, and from an untagged strain (no tag) at 0 and 130 min, are represented as a percentage relative to the highest level (100%). Efficiency of immunoprecipitation was determined by calculating (immunoprecipitated DNA/input DNA) / (ACT1 immunoprecipitated DNA/ACT1 input DNA).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3
NRM1 Is Required for Repression of MBF Targets and Is Itself an MBF Target
(A) Nrm1 is essential for repression of MBF targets. Wild-type (wt) and nrm1Δ cells carrying either ura3::YIp::URA3 (left) or ura3::YIpGAL1-SIC1ΔP::URA3 (right) were grown in raffinose media and synchronized by α factor and subsequently released into galactose media. CLN2 (top; SBF-dependent gene) and RNR1 (middle; MBF-dependent gene) mRNA levels and budding index (bottom) in cells arrested by α factor (0 min) or released from the arrest for the indicated interval (20–140 min).
(B) Mbp1 binds to the NRM1 promoter. ChIP of NRM1 promoter DNA by Mbp1-myc and Swi4-myc. Immunoprecipitation from a strain containing untagged genes (no tag) is presented as a negative control. WCE is presented as a positive control.
(C) NRM1 is a preferred MBF target. Comparison of NRM1 mRNA levels in wild-type (MBP1 SWI4) and mbp1Δ, swi4Δ, and mbp1Δswi4Δ mutants. NRM1 mRNA levels were determined in cells arrested by α factor (0 min) or released from the arrest for the indicated interval (20–90 min) by real-time RT-PCR. The transcript levels are expressed as a percentage of highest level (100%) observed in wild-type samples after normalization of all values to the ACT1 mRNA level. Budding indexes of wild-type, swi4Δ, and mbp1Δ cells, and SIC1 mRNA levels for swi4Δ mbp1Δ cells, as an indicator of synchrony of the release from α-factor arrest, are presented along with the analysis of additional genes in Figure 4D.
(D) NRM1 is an MBF target. qPCR of chromatin-immunoprecipitated (ChIP) NRM1 promoter DNA in an untagged strain (no tag) and Mbp1-myc and Swi4-myc expressed in wild-type (Mbp1-myc and Swi4-myc) and swi4Δ or mbp1Δ cells (Mbp1-myc swi4Δ and Swi4-myc mbp1Δ), respectively. Analysis of additional genes in the same experiment is presented in Figure S4. Bars represent the specific signal derived from NRM1 promoter DNA detected by qPCR in the relevant ChIP analysis. Background signal for each target DNA in each qPCR was determined by the internal ACT1 background signal (SBF/MBF independent) multiplied by the ratio of (target DNA) / (ACT1 signal detected) in the untagged stain. The average value from three independent experiments, each run in triplicate, is presented as the standard error of the mean.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4 MBF Is Essential for Repression of MBF Targets
(A) SBF and MBF binding to G1-specific promoters as determined by the genome-wide ChIP analysis of Iyer et al. (2001) and Simon et al. (2001) The number of SCB and MCB motifs present in the promoter sequence of the G1-specific genes is indicated.
(B) MBF binds to SBF target promoters. ChIP of CDC21, DUN1, and RNR1 (MBF-dependent) and SVS1 promoter DNA (SBF-dependent) by Swi4-myc and Mbp1-myc in asynchronous wild-type (wt) and mbp1Δ or swi4Δ cells, respectively. No epitope tag (no tag) and WCE are presented as controls.
(C) Redundant binding of SBF and MBF to G1-specific targets. Bars represent specific SVS1, POL1, CDC21, and CDC28 promoter DNA signal detected by qPCR in the relevant ChIP analysis. Background signal for each target DNA in each qPCR was determined by the internal ACT1 background signal (SBF/MBF independent) multiplied by the ratio of (target DNA) / (ACT1 signal detected) in the untagged stain. The average value from three independent experiments, each run in triplicate, is presented as the standard error of the mean.
(D) Mbp1 represses G1-specific transcription outside of G1 phase. mRNA levels (qRT-PCR; expressed as percentage of highest level after normalization to the ACT1 mRNA) in α factor-synchronized wild-type, swi4Δ, mbp1Δ, and swi4Δ mbp1Δ cells. (Right) Budding index of wild-type, swi4Δ, and mbp1Δ cells and SIC1 mRNA levels for swi4Δ mbp1Δ cells is presented as an indicator of synchrony. Viability of swi4Δ mbp1Δ cells was maintained by constitutive expression of GAL1-CLN2.
(E) Mbp1 is necessary for repression of RNR1 in late G1 phase. A wild-type (MBP1) and mbp1Δ strain carrying YIpGAL1-SIC1ΔP was synchronized with α factor (0 min) in raffinose media and released into galactose media. CLN2 (SBF-dependent) and RNR1 (MBF-dependent) mRNA levels and budding index (right) in cells arrested by α factor (0 min) or released from the arrest for the interval indicated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5
nrm1Δ Results in HU Resistance and rad53Δ Lethality Bypass, whereas Stabilizing Nrm1 Results in HU Hypersensitivity
(A) nrm1Δ leads to HU resistance. Serial 4-fold dilutions of wild-type (NRM1) and nrm1Δ cells were spotted on YEPD plates (two upper lanes) and YEPD plates containing 300 mM concentration of HU (two lower lanes).
(B) Inactivation of Nrm1 bypasses the lethality caused by rad53Δ or mec1Δ. Growth of wild-type (left; NRM1) and nrm1Δ (right), rad53Δ (top), or mec1Δ (bottom) strains carrying YCpRAD52::URA3 or YCpMEC1::URA3, respectively, on 5-FOA to select against the YCpRAD52::URA3 or YCpMEC1::URA3 plasmids.
(C) Stabilization of the Nrm1ΔN protein. Protein levels of wild-type Nrm1 (top, α-myc) and Nrm1ΔN (bottom, anti-myc) expressed from the GAL1 promoter after α factor release in galactose for 15 min followed by addition of glucose. Same blots were probed with anti-PSTAIRE antibody as a loading control.
(D) Constitutive expression of Nrm1ΔN represses MBF-regulated transcripts during late-G1 phase. A wild-type (NRM1) and GAL-NRM1ΔN strain was synchronized by α factor (0 min) and then released in galactose media. CLN2 (SBF-dependent gene) and RNR1 (MBF-dependent gene) mRNA levels, expressed as a percentage of highest level (100%) observed in wild-type cells, and budding index (right) in cells arrested by α factor (0 min) or released from the arrest for the indicated interval are shown.
(E) Constitutive expression of NRM1ΔN leads to hypersensitivity to HU. Serial 4-fold dilutions of wild-type (NRM1), nrm1Δ, GAL-NRM1, and GAL-NRM1ΔN cells were spotted on YEP Galactose (YEPG) plates (top) and YEPG plates containing 200 mM concentration of HU (bottom).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

7. Figure 6 Functionally Analogous Role for the SpNrm1 Homolog
(A) Nrm1 is conserved among yeasts. Aligned amino acid sequence of Saccharomyces cerevisiae (Sc), Kluyveromyces lactis (Kl), and Schizosaccharomyces pombe (Sp). Identical amino acids are indicated in black; similar residues are indicated in gray.
(B) Functional parallels of proteins involved in G1-specific transcriptional regulation in Saccharomyces cerevisiae and Schizosaccharomyces pombe.
(C) SpNrm1 interacts with Res2. Extracts from wild-type strains carrying res2+-13xmyc alone or with nrm1+-TAP (left) or nrm1+-HA alone or with res2+-TAP (right). Whole WCE and IgG immune complexes (IgG;IP) were probed with anti-myc, anti-HA, or PAP to detect SpNrm1-TAP or Res2-TAP.
(D) nrm1+ is a G1-specific transcript. Temperature-sensitive cdc25-22 cells were arrested in G2/M by incubation at 35.5°C (0 min) and subsequently released into the cell cycle at 25°C. Samples were taken at 20 min intervals and septation and mRNA (qRT-PCR, expressed as a percentage of highest level after normalization to the act1+ mRNA) were analyzed.
(E) Res2 and SpNrm1 bind to the G1-specific nrm1+ promoter. ChIP of cdc22+, cdc18+, nrm1+, and act1+ promoter DNA from wild-type (wt) strain by Res2-3xHA and SpNrm1-3xHA. No epitope tag (no tag) and WCE are presented as controls.
(F) SpNrm1 is essential for repression of MBF targets. Small unseptated wild-type and nrm1Δ cells were isolated by centrifugal elutriation and allowed to progress synchronously through the cell cycle. cdc22+mRNA levels ([left]; determined by qRT-PCR, expressed as percentage of highest level after normalization to the act1+ mRNA) are shown for the indicated interval.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

8. Figure 7 Parallel Pathways for G1/S Control in Eukaryotes
(A) Model depicting the mechanism of MBF-regulated transcription in budding and fission yeasts. In early G1 phase, transcriptional repression is maintained by a complex containing MBF bound to target promoters. Transcription is activated by inactivation of the repressive activity of MBF or by loss of a corepressor. Transcription is then inactivated, during the G1/S transition, by binding of the MBF-associated corepessor Nrm1.
(B) Model depicting the known pathways of G1-specific transcriptional regulation in the budding yeast. Whi5 represses SBF-dependent transcription during early G1 phase. Whi5 is inactivated via phosphorylation by Cln3/CDK, relieving transcriptional repression and activating SBF-specific transcription. Upon exit from G1 phase, transcriptional repression of SBF targets results from disassociation of the transcription factor from promoters promoted by Clb/CDK. MBF-regulated transcription is repressed by a complex involving MBF. Transcription is activated via an unknown mechanism involving Cln3/CDK and culminates in the repression of that same set of genes by NRM1, a preferred target of MBF (SBF can also contribute to NRM1 expression). Nrm1 accumulates and binds to MBF at its target promoters repressing transcription. Consequently, MBF target genes are controlled by an autoregulatory mechanism as cells exit G1 phase.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions