Display Settings:

Items per page

Results: 8

1.
Figure 8

Figure 8. Model for regulation by Whi5, Stb1, and histone deacetylase.. From: Recruitment of Cln3 Cyclin to Promoters Controls Cell Cycle Entry via Histone Deacetylase and Other Targets.

The SBF complex (green) recruits Whi5 (blue) and Stb1 (grey). The SBF complex also recruits the repressive Rpd3 histone deacetylase complex (black); this recruitment may be aided by Whi5 and/or Stb1. In a growth- and size-dependent way, the Cln3-Cdc28 kinase (orange-red) is recruited. This CDK phosphorylates Whi5 and Stb1 and possibly Swi6, resulting in the loss of the Rpd3 complex and the loss of Whi5. The transcriptional activation domain of Swi6 is revealed, possibly aided by an activating function of phospho-Stb1.

Hongyin Wang, et al. PLoS Biol. 2009 September;7(9):e1000189.
2.
Figure 2

Figure 2. Suppressors of cln3 bck2.. From: Recruitment of Cln3 Cyclin to Promoters Controls Cell Cycle Entry via Histone Deacetylase and Other Targets.

Various mutations were tested for ability to suppress lethality of a cln3 bck2 rme1 {MET-CLN2} strain on +methionine medium. The parental strain (N497) is cln3 bck2 rme1 {MET-CLN2}; other strains (top to bottom: N452, N453, N451, N499, and N454) have one additional mutation as indicated. Serial 4-fold dilutions were spread on −methionine (where MET-CLN2 is expressed) or +methionine (where MET-CLN2 is repressed). The HOS3 gene encodes a histone deacetylase, but this histone deacetylase was not identified in our suppressor screen and serves as a negative control.

Hongyin Wang, et al. PLoS Biol. 2009 September;7(9):e1000189.
3.
Figure 6

Figure 6. Cln3 associates with SBF and ChIPs to the CLN2 promoter.. From: Recruitment of Cln3 Cyclin to Promoters Controls Cell Cycle Entry via Histone Deacetylase and Other Targets.

(A) Cln3 co-immunoprecipitates with Swi6. Strains (HWL72, HWL112, HWL130) with various combinations of CLN3 or CLN3-FLAG (pCM273), SWI6 or SWI6-Myc, or SWI4 or swi4, were grown and extracts made. In the left half of (A, these extracts were tested for the presence of the FLAG-tagged Cln3 and the Myc-tagged Swi6 by Western blotting with Anti-FLAG or Anti-Myc antibody). In the right half, proteins were immunoprecipitated with Anti-Myc antibody (directed against Swi6-Myc), and then these immunoprecipitates were tested by Western blotting for the presence of Swi6-Myc and Cln3-FLAG. (B) As 6A, except that the immunoprecipitation is done with the anti-FLAG antibody, followed by Western analysis with the anti-Myc antibody. (C) A ChIP experiment as in Figure 4B, for association of Cln3-TAP (HWL49, HWL63) with the CLN2 promoter.

Hongyin Wang, et al. PLoS Biol. 2009 September;7(9):e1000189.
4.
Figure 7

Figure 7. Cln3 may be titrated by SBF binding sites.. From: Recruitment of Cln3 Cyclin to Promoters Controls Cell Cycle Entry via Histone Deacetylase and Other Targets.

Cells (BF305-15d, HWL148, HWL145, and HWL146) were transformed with an empty vector (pBA70, grey line) or the same vector carrying an insert with four tandem copies of an SBF binding site (“pSCB”) (pMT3579, black line). Cells were grown to early log phase, and fractionated by centrifugal elutriation. Fractions containing small unbudded cells with a mode volume of about 20 fl were chosen and re-inoculated. Cell size (x-axis, in fl) and budding (y-axis, percent) were followed with time. The median experiment of five total wild-type experiments is shown in the upper left. “Critical size” was defined to be the size (in fl) at which 50% of the cells became budded. This critical size is shown next to each curve.

Hongyin Wang, et al. PLoS Biol. 2009 September;7(9):e1000189.
5.
Figure 5

Figure 5. Dependency analysis.. From: Recruitment of Cln3 Cyclin to Promoters Controls Cell Cycle Entry via Histone Deacetylase and Other Targets.

(A) ChIP analysis of proteins at the CLN2 promoter in various mutants. Cells (left panel: S288c, HWL99, HWL110, OBS1, and HWL117; right panel S288c, HWL110, HWL119, and OBS5) were CLN3 BCK2 CDC34 in exponential growth. The TAP-tagged protein being assayed is indicated, as is any additional mutation in the strain. The arrowhead (>) indicates the band containing the SBF binding sites. Other bands are as drawn in Figure 4A. (B) ChIP analysis of proteins at the CLN2 promoter in various mutants. As in Figure 4A, but with a different selection of mutants. The arrowhead indicates the band containing the SBF binding sites. (C) ChIP analysis of Sin3-TAP at the CLN2 promoter in wild-type, swi4, swi6, and untagged (negative control) strains. Ten independent experiments were done for each of the four genotypes. Results for all 40 experiments were obtained and tested statistically after “blinding” the samples (Table 1). The two median-most experiments for each of the four genotypes are shown here. The ratio of the intensity of the SBF band (arrowhead) to the sum of the intensities of the upper two bands (“dyn” plus “up”) is shown at the bottom of each gel lane.

Hongyin Wang, et al. PLoS Biol. 2009 September;7(9):e1000189.
6.
Figure 3

Figure 3. Analysis of CLN1 and CLN2 expression in the cln3 bck2 stb1 and cln3 bck2 whi5 strains.. From: Recruitment of Cln3 Cyclin to Promoters Controls Cell Cycle Entry via Histone Deacetylase and Other Targets.

qPCR was used to measure the expression of CLN1 and CLN2 in two of the suppressor strains. The parental strain is cln3 bck2 rme1 {MET-CLN2} (N497), and the two suppressor strains have, in addition, stb1 (N451) or whi5 (N499), as indicated. (A) The left panel shows quantitation of CLN1 and CLN2 mRNAs by qPCR, in the absence (−met) or presence (+met) of methionine. (B) The right panel shows the PCR products from (A) separated by agarose gel electorphoresis. The plasmid-borne allele of CLN2 is CLN2-NLS-I; it carries a tag that increases the length of the mRNA and the PCR product, allowing expression of genomic CLN2 to be distinguished from expression of plasmid-borne MET-CLN2-NLS-I. This demonstrates that the increased CLN2 expression caused by stb1 and whi5 is specific for the genomic copy of CLN2. The qPCR measurement in (A) includes both forms of CLN2, suggesting that the increased expression of genomic CLN2 in the stb1 and whi5 mutants is about 3-fold. The primers for amplification of ACT1 flank the ACT1 intron, yet only the mRNA-specific band was obtained, showing that no DNA was present in the RNA preparation. Error bars show the standard error of the mean.

Hongyin Wang, et al. PLoS Biol. 2009 September;7(9):e1000189.
7.
Figure 1

Figure 1. Responsiveness to CLN3.. From: Recruitment of Cln3 Cyclin to Promoters Controls Cell Cycle Entry via Histone Deacetylase and Other Targets.

Various mutants containing bck2 and CLN3 under the control of the GAL promoter (GAL-CLN3) as the only allele of CLN3 were grown in YEP raffinose medium (CLN3 off, solid grey line), split into two aliquots, and galactose was added to one aliquot (CLN3 on, dotted black line). Cell volume distributions were measured with a Coulter Channelyzer (left column), and photomicrographs were taken (right two columns). The median cell size (fl) as measured by the Coulter Channelyzer is shown in the bottom left corner of each photograph, and the percentage budding is shown in the bottom right corner. A shift to smaller cell sizes and to higher percent budding in galactose shows responsiveness to CLN3. The changes in budding are statistically significant at the p<0.01 level for all strains except the whi5 stb1 strain (4th from top) and the whi5 sin3 strain (5th from top). The cell size distribution is not shown for GAL-CLN3 bck2 cells in raffinose (top panel) because these extremely large cells are off-scale. Strains used, from top to bottom, are: LC517, LC520, LC518, LC504, LC524, LC521, and LC523.

Hongyin Wang, et al. PLoS Biol. 2009 September;7(9):e1000189.
8.
Figure 4

Figure 4. Proteins at the CLN2 promoter.. From: Recruitment of Cln3 Cyclin to Promoters Controls Cell Cycle Entry via Histone Deacetylase and Other Targets.

(A) Map of the CLN2 promoter, showing the location of the up, sbf, and dwn probes in the BBP1-CLN2 intergenic region. The sbf probe overlaps the consensus SBF binding sites. The dyn probe is in the middle of the DYN1 open reading frame, several kilobases from the nearest promoter. On the right is shown the relative order of these probes on gels; i.e., the dyn probe has the lowest mobility, and the dwn probe the highest mobility. (B) Proteins at the CLN2 promoter as a function of CLN3 induction. GAL-CLN3 expression was induced (left) or not (right) by the addition of galactose. Samples were taken from 0 to 30 min. Proteins were cross-linked to DNA and processed as described, and TAP-tagged proteins (from strains HWL61, HWL59, HWL51, HWL62, HWL77, and HWL128) were immunoprecipitated. Coprecipitated DNA fragments were identified by PCR using probes amplifying fragments described above in (A). Whole cell extract (WCE) was also amplified, as a positive control. Samples (HWL49) from cells lacking any TAP-tagged protein were processed as a negative control (No tag). The parental strain was HWL49, with partial genotype GAL-CLN3 bck2 cdc34-2. (C) As (B), but TAP-tagged Sin3 is being assayed by ChIP at the YOX1 promoter, instead of at the CLN2 promoter. (D) Kinetics of induction of the CLN2 mRNA, by Northern analysis. 5S RNA serves as a loading control. (E) GAL-CDC20 cdc20 SIN3-TAP cells were arrested in M-phase in glucose, then GAL-CDC20 was induced at 0 time with galactose medium. The amount of Sin3-TAP associated with the CLN2 promoter was assayed during a timecourse. Budding initiates at about 50 min.

Hongyin Wang, et al. PLoS Biol. 2009 September;7(9):e1000189.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk