Yox1 or Yhp1 is required to restrict transcription of ECB elements to the M/G1 interval of the cell cycle. (A) Transcription driven by the wild-type ECB element of MCM3 through the cell cycle (solid symbols), or the equivalent reporter (open symbols) lacking the YOX site. (B–D) Parallel analyses of the MCM3:lacZ transcription in yhp1, yox1, or yox1yhp1 cells (open symbols) compared to wild type (solid symbols). The primary S1 protection data for the MCM3:lacZ reporter analyzed in wild-type and yox1yhp1 cells are provided in E and F, respectively.

Transcription is derepressed during the cell cycle in yox1yhp1 cells. The profiles of seven cell-cycle-regulated transcripts taken from the microarray experiment (A–F) or as measured by S1 protection (G,H) are graphed, as indicated, from wild-type (solid symbols) and yox1yhp1 (open symbols) cells. (I) The primary data for panel H, which monitors SWI4 90mer: lacZ transcription through the cell cycle. CLN1 mRNA is monitored as a control for cell-cycle synchrony. SWI4 and lacZ levels normalized to ACT1, an invariant transcript, are graphed in G and H.

Mcm1, Yox1, and Yhp1 bind to ECB elements. (Lanes 1–4) Gel retardation assays were carried out using the MCM3 ECB element and cell extracts from wild type (WT), yox1, yhp1, or yox1yhp1, as indicated. (Lanes 5–14) Gel retardation assays with the wild-type MCM3 ECB or the same sequence carrying multiple mutations in either the YOX site (y−) or the Mcm1 site (m−) as a probe. Extracts were used from wild-type cells or cells carrying myc-tagged Yox1 or Yhp1 as indicated above. Antibodies to Mcm1 (lane 6) or to the myc tag (lanes 10,13) were also added. ** indicates the upper complex, containing Mcm1, Yox1, and/or Yhp1. * marks the complex of Mcm1 with DNA.

Constitutive overproduction of Yox1 is highly deleterious to cells. (A) FACS profiles of wild-type cells, and cells after 0, 2, and 4 h of induction of Yhp1 or Yox1 overproduction from the GAL promoter. (B) Colony formation on a YEP galactose plate of wild-type cells compared to GAL:YOX1 and GAL:YHP1 cells. (C) Wild-type and yox1yhp1 G1 daughter cells were collected by elutriation, and their DNA content was followed by FACS analysis. (D) Mean cell volume (in femtoliters) was measured with a Coulter Z2 size analyzer and plotted as a function of time. (E) The kinetics of budding were followed and plotted as a function of mean cell volume. Wild-type (open symbols), yox1yhp1 (closed symbols).

Regulation of ECB-driven transcription and its role in G1 progression. Activation of ECB elements occurs in M/G1 phase of the cell cycle, involving constitutively bound Mcm1 and possibly another activator. Examples of the three prominent classes of ECB-regulated genes are listed. Yox1, transcribed in late G1 by Swi4 and Swi6, rises to the level required to repress ECB function, thereby inhibiting its own synthesis. This negative feedback sustains ECB-activated transcription until Yox1 reaches the threshold required to affect repression. YOX1, CLN1, CLN2, CLB5, and CLB6 are coordinately transcribed. This coordination enables these cyclins to accumulate to the threshold required to drive cells into S phase, and then be turned off rapidly. Yhp1 is made in late S phase. Either Yox1 or Yhp1 can bind to ECBs late in the cycle and repress their activity. Elimination of Yox1 and Yhp1 is required to reactivate the ECB elements late in M phase.

(A) Alignment of homeodomains of human Phox1 and its two closest budding yeast relatives, Yox1 and Yhp1. Shaded residues are conserved in all three sequences. Asterisks indicate residues important for DNA binding (Mann 1995). Lowercase residues below represent those that are typically conserved across the domain in most homeodomain proteins (Galliot et al. 1999). (B) Yox1 and Yhp1 both coimmunoprecipitate with Mcm1. (Left panel) The left three lanes show crude lysates from cells carrying the wild-type (WT) or the myc-tagged allele of Yox1 or Yhp1 as indicated. The right three lanes show the same lysates after immunoprecipitation with polyclonal antibodies to the myc tag. Samples were resolved by SDS-PAGE and immunoblotted with antibodies to Mcm1. “IgG” indicates immunoglobulin (55kD). (Right panel) The same strains were immunoprecipitated with Mcm1 antibodies and immunoblotted with antibodies to the myc epitope.

Cell-cycle regulation of Yox1 expression results in the cell-cycle regulation of ECB activity. (A) α-factor-synchronized cells were analyzed at 10-min intervals for lacZ transcript driven by the MCM3 ECB element. (B) DNA binding to the MCM3 ECB probe. Asterisks denote ECB-specific complexes as in Figure 6. (C) Gel retardation assay as in B to which antibodies to the myc tag were added. (D) Immunoblot of the cell extracts used in B and C detecting Yox1–myc with anti-myc antibodies. (E) CHIP assays performed with anti-myc polyclonal antibodies on the MCM3 and CDC47 promoters with extracts from synchronized Yox1–myc cells. “NC” indicates the negative control. PCR was performed on chromatin fragments before (INPUT) and after IP. (F) Genomic MCM3 mRNA was monitored from α-factor synchronized wild-type cells (solid symbols) and compared to the same transcript from yox1yhp1 cells expressing Yox1 constitutively from the GAL^s:YOX1 construct (open symbols).

Twenty-eight genes negatively regulated by Yox1 and/or Yhp1 share a common promoter element. Genome-wide transcript analysis of wild-type (A) and yox1yhp1 (B) cells through the cell cycle. Cells were synchronized with α-factor and monitored at 10-min intervals through two cell cycles. The compiled data are represented in rows for each gene and columns for each timepoint. Red indicates increased abundance and green indicates decreased abundance of the time course cDNA with respect to the invariant control cDNA. Black indicates the ratio of time course cDNA to control of 1. The first row represents the value for each timepoint averaged over all 28 genes. (C) Transcripts elevated in yox1yhp1 cells are repressed two- to thirtyfold by overproduction of Yox1. Transcript levels of these 28 genes after 4 h of overproduction of Yox1 were compared by microarray analysis to those of wild-type cells grown under the same conditions. These values, for each gene listed, are expressed as a ratio of the level in the Yox1 overproducer over the wild type. (D) The position (in bases upstream from the translational start site) and the sequences of the YOX- and Mcm1-binding sites in each putative M/G1-specific ECB element are shown, with shading to indicate bases that conform to the consensus sequence below. “C” in the position column indicates reverse orientation to that listed.