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Found 1 result for PubMed for id: 200148068
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. 2020 Jun 1;10(6):2057-2068.
doi: 10.1534/g3.120.401131.

Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A in Saccharomyces cerevisiae

Jessica R Eisenstatt  1 , Lars Boeckmann  1 , Wei-Chun Au  1 , Valerie Garcia  1 , Levi Bursch  1 , Josefina Ocampo  2 , Michael Costanzo  3   4 , Michael Weinreich  5 , Robert A Sclafani  6 , Anastasia Baryshnikova  7 , Chad L Myers  8 , Charles Boone  3   4 , David J Clark  2 , Richard Baker  9 , Munira A Basrai  10
Affiliations

Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A in Saccharomyces cerevisiae

Jessica R Eisenstatt et al. G3 (Bethesda). .

Abstract

The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4) by E3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and psh1Δ strains display synthetic dosage lethality (SDL) with GALCSE4 We previously performed a genome-wide screen and identified five alleles of CDC7 and DBF4 that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with GALCSE4 We determined that cdc7-7 strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4 Mutation of MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a cdc7-7 strain. We determined that mcm5-bob1 does not rescue the SDL and defects in proteolysis of GALCSE4 in a cdc7-7 strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a cdc7-7psh1Δ strain were similar to that of cdc7-7 and psh1Δ strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1 Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.

Keywords: CENP-A; Cdc7; Centromere; Cse4; DDK; Psh1.

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Figures

Figure 1
Figure 1
DDK mutants exhibit synthetic dosage lethality (SDL) to GALCSE4. A. Validation of GALCSE4 SDL in cdc7 and dbf4 strains. Growth assays were done with wild type [BY4741 (for cdc7-4, dbf4-1, and dbf4-2) and RSY299 (for cdc7-7)], cdc7-4 (tsa131), dbf4-1 (tsa161), dbf4-2 (tsa162), and cdc7-7 (RSY302) strains transformed with vector (pMB433, vector) or GAL-HA-CSE4 (SB878, GALCSE4). Cells were spotted in fivefold serial dilutions on medium selective for the plasmid containing either glucose (2%, Cse4 expression off) or raffinose/galactose (2% each, Cse4 expression is on) and incubated at 23° for 3-5 days. Two independent transformants of dbf4-1, dbf4-2, and cdc7-4 strains and three independent transformants of cdc7-7 strains were assayed and a representative image is shown. B. The GALCSE4 SDL phenotype of a cdc7-7 strain is linked to the cdc7 mutant allele. Growth assays were done with cdc7-7 strains (RSY302 with pMB433 and RSY302 with pMB1597) transformed with empty vector (pRS425) or plasmid-born CDC7 (pMB1898). Cells were spotted in fivefold serial dilutions on medium selective for the plasmids with glucose (2%) or raffinose/galactose (2% each). Plates were incubated at the indicated temperature for 5-7 days. Three independent transformants for each strain were assayed and a representative image is shown.
Figure 2
Figure 2
Cdc7 regulates ubiquitin-mediated proteolysis of Cse4. A. Cse4 is stabilized in a cdc7 strain and B. Cse4 is stabilized in a dbf4 strain. Western blot analysis of protein extracts prepared from wild type (BY4741 for dbf4-1 and RSY299 for cdc7-7), (A) cdc7-7 (RSY302), and (B) dbf4-1 (TSA161) strains transformed with GAL-HA-CSE4 (pMB1597). Strains were grown to logarithmic phase of growth in raffinose-containing media (2%), and expression of GAL-HA-CSE4 was induced with galactose (2%) for four hours. Cells were then treated with cycloheximide (CHX, 10 µg/ml) and glucose (2%). Aliquots were taken at the indicated timepoints. Protein extracts were analyzed using Western blot analysis and blots were probed with anti-HA (Cse4) and anti-Tub2 (loading control). Quantification of the levels of HA-Cse4 remaining after treatment with CHX relative to Tub2 from two independent experiments is shown in the graphs. Error bars represent SEM. C. Ubiquitination of Cse4 is decreased in a cdc7 strain. Ub-pull down assays were performed using protein extracts from wild type and cdc7-7 strains as described above and lysates were incubated with Tandem Ubiquitin Binding Entity beads (LifeSensors). Input and ubiquitin-enriched (Pull down: Ub+) samples were analyzed via Western blot against HA (left). Arrow indicates the unmodified Cse4 band. Quantification of levels of poly-ubiquitinated Cse4 (Ubn-Cse4) normalized to the levels in the input from three independent experiments is shown in the graph, p-value < 0.05.
Figure 3
Figure 3
Cdc7 regulates stability of Cse4 independently of its role in initiation of DNA replication. A. A cdc7-7 mcm5-bob1 strain shows SDL with GALCSE4. Growth assays with wild type (RSY299), mcm5-bob1 (RSY867), cdc7-7 (RSY302), or cdc7-7 mcm5-bob1 (RSY847) strains transformed with vector (pMB433, vector) or GAL-HA-CSE4 (SB878, GALCSE4). Cells were spotted in fivefold serial dilutions on media selective for the plasmid containing either glucose (2%) or raffinose/galactose (2% each) and incubated at 23° for 3-5 days. Three independent transformants for each strain were assayed and the representative image is shown. B. A cdc7-7 mcm5-bob1 strain exhibits defects in Cse4 proteolysis. Western blot analysis of protein extracts from wild type (RSY299), mcm5-bob1 (RSY867), cdc7-7 (RSY302), or cdc7-7 mcm5-bob1 (RSY847) strains transformed with GAL-HA-CSE4 (pMB1597). Strains were grown to logarithmic phase of growth in raffinose-containing media (2%) and expression of GAL-HA-CSE4 was induced with galactose (2%) for four hours. Cells were then treated with cycloheximide (CHX, 10 µg/ml) and glucose (2%). Aliquots were taken at the indicated timepoints. Protein extracts were analyzed using Western blot analysis and blots were probed with anti-HA (Cse4) and anti-Tub2. (loading control). The graph shows the quantification of levels of HA-Cse4 remaining after treatment with CHX relative to Tub2 from two independent experiments. Error bars represent SEM.
Figure 4
Figure 4
Cdc7 prevents mislocalization of Cse4 to non-centromeric regions. A. Cse4 is mislocalized in a cdc7 strain. Localization of Cse4 was examined using chromosome spreads prepared from nocodazole arrested wild type (RSY299) and cdc7-7 (RSY302) strains transformed with GAL-HA-CSE4 (pMB1597). HA-Cse4 was labeled with Cy3 (red) and DNA with DAPI (blue). Representative images of cells showing normal localization counted as nuclei with one or two Cse4 foci (WT) and mislocalization counted as nuclei with more than two foci or a diffuse signal in the nucleus (cdc7-7). Arrow indicates HA-Cse4 foci. B. Quantification of Cse4 localization from A. The graph displays the quantification of Cse4 localization as a percentage over total cell count. The SEM of two independent experiments is shown, WT 1 or 2 foci vs. cdc7-7 1 or 2 Foci p-value = 0.0028; WT 3+ foci vs. cdc7-7 3+ Foci p-value = 0.0028. C. The kinetochore protein Mtw1 is not mislocalized in a cdc7-7 strain. Wild type (YMB9337) and cdc7-7 (YMB9338) cells were transformed with Mtw1-GFP on a plasmid (pMB1058), grown to logarithmic phase of growth, and analyzed for Mtw1-GFP (green) foci with live cell imaging. Representative images of cells showing single Mtw1-GFP foci are shown. Arrow indicates Mtw1-GFP foci. D. Quantification of Mtw1-GFP localization from C. The graph displays the quantification of cells with one or two GFP foci (normal) or with greater than three foci (mislocalized) with the SEM of two independent experiments; WT 1 or 2 foci vs. cdc7-7 1 or 2 Foci p-value = 0.1683; WT 3+ foci vs. cdc7-7 3+ Foci p-value = 0.1683.
Figure 5
Figure 5
Cse4 is mislocalized to non-centromeric regions in a cdc7-7 strain. ChIP-seq was performed using chromatin lysates from wild type (YMB10044) and cdc7-7 (YMB10041) strains. A. Flag-Cse4 is mislocalized in a cdc7-7 strain. Genome browser of input and ChIP samples for Chromosome I and Chromosome V in wild type (top) and cdc7-7 (bottom) strains overexpressing Flag-Cse4. Regions of CEN1 and CEN5 are shown. B. Flag-Cse4 is enriched at promoters in a cdc7-7 strain. The annotatePeaks tool of HOMER v5.10 (http://homer.ucsd.edu/homer/) was used to define genomic locations of Flag-Cse4 enrichment in the cdc7-7 strain. The genomic feature, peak number, percent of total peaks, region size, fold-enrichment (relative to sequence content), and LogP enrichment are indicated. C. FLAG-Cse4 is preferentially enriched at promoters in cdc7-7 and psh1Δ strains. Overlap between Flag-Cse4 enrichment in cdc7-7 and psh1Δ strains and at promoters.
Figure 6
Figure 6
Cdc7 regulates Psh1-mediated proteolysis of Cse4. A. Overexpression of UBI4 suppresses the SDL of a cdc7-7 GALCSE4 strain. Growth assays of wild type (RSY299) and cdc7-7 (RSY302) cells transformed with empty vector (pMB433, GALCSE4 -) or GAL-HA-CSE4 (pMB1597, GALCSE4+) and subsequently transformed with empty (pRS425, 2µ UBI4-) or UBI4 (pMB1604, UBI4+). Cells were spotted in fivefold serial dilutions on media selective for the plasmids containing either glucose (2%) or raffinose/galactose (2% each) and incubated at 23° or 37° as indicated for 3-5 days. Three independent transformants for each strain were assayed and the representative image is shown. B. The GALCSE4 SDL phenotype of the cdc7-7 psh1∆ strain is similar to that observed for psh1∆ and cdc7-7 strains. Growth assays of wild type, psh1∆, cdc7-7, and cdc7-7 psh1∆ strains with endogenously expressed Flag-Cse4 (vector; YMB10043, YMB10126, YMB10040, and YMB10124, respectively) or Flag-Cse4 expressed from a galactose-inducible promoter integrated into the genome (GALCSE4; YMB10044, YMB10127, YMB10041, and YMB10125, respectively) spotted in fivefold serial dilutions on to rich media containing either glucose (2%) or raffinose/galactose (2% each) and incubated at 23°C for 5 days. Three independent transformants for each strain were assayed and the representative image is shown. C. The Cse4 proteolysis defect in a cdc7-7 psh1∆ double mutant is similar to that observed for a psh1∆ strain. Western blot analysis of protein extracts from wild type (YMB10044), cdc7-7 (YMB10041), psh1∆ (YMB10127), and cdc7-7 psh1∆ (YMB10125) strains grown to logarithmic phase of growth in raffinose-containing media (2%). Expression of GAL-FLAG-CSE4 was induced with galactose (2%) for 1.75 hr. Cells were then treated with cycloheximide (CHX, 10 µg/ml) and glucose (2%). Aliquots were taken at the indicated timepoints. Protein extracts were analyzed using Western blot analysis and blots were probed with anti-FLAG (Cse4) and anti-Tub2 (loading control). The graph shows the quantification of the levels of FLAG-Cse4 remaining after treatment with CHX relative to Tub2 from two independent experiments. Error bars represent SEM. D. Mislocalization of Cse4 is not further enhanced in the cdc7-7 psh1∆ strain. Localization of Cse4 was examined using chromosome spreads prepared from nocodazole arrested wild type (YMB10044), cdc7-7 (YMB10041), psh1∆ (YMB10127), and cdc7-7 psh1∆ (YMB10125) strains. FLAG-Cse4 was labeled with Cy3 and DNA with DAPI. The graph displays quantification of Cse4 localization as a percentage over total cell count. The graph displays the SEM of two independent experiments, psh1Δ 3+ foci vs. cdc7-7 3+ Foci, cdc7-7 3+ foci vs. cdc7-7 psh1Δ 3+ Foci, and psh1Δ 3+ foci vs. cdc7-7 psh1Δ 3+ Foci p-value > 0.999.
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