Biology subjects

Skibbens, R. V.

Publications and source records attributed to Skibbens, R. V..

6 recordsLinked to original sources

bioRxiv2025

Aberrant cohesin function in Saccharomyces cerevisiae activates Mcd1 degradation to promote cell lethality

The cohesin complex is composed of core ring proteins (Smc1, Smc3 and Mcd1) and associated factors (Pds5, Scc3, and Rad61) that bind via Mcd1. Extrusion (looping from within a single DNA molecule) and cohesion (the tethering together of two different DNA molecules) underlie the many roles that cohesins play in chromosome segregation, gene transcription, DNA repair, chromosome condensation, replication fork progression, and genome organization. While cohesin functions flank the activities of critical cell checkpoints (including spindle assembly and DNA damage checkpoints), the extent to which checkpoints directly target cohesins, in response to aberrant cohesin function, remains unknown. Based on prior evidence that cells mutated for cohesin contain reduced Mcd1 protein, we tested whether loss of Mcd1 is based simply on cohesin instability or integrity. The results show that Mcd1 loss persists even in rad61 cells, which contain elevated levels of stable chromosome-bound cohesins, and also in scc2-4, which do not affect cohesin complex integrity. In fact, re-elevating Mcd1 levels suppresses the temperature-sensitive growth defects of all cohesin alleles tested, revealing that Mcd1 loss is a fundamental mechanism through which cohesins are inactivated to promote cell lethality. Our findings further reveal that cells that exhibit aberrant cohesin function employ E3 ligases (such as San1) to target Mcd1 for degradation. This mechanism of degradation appears unique in that Mcd1 is reduced during S phase, when Mcd1 levels typically peak and despite a dramatic upregulation in MCD1 transcription. We infer from these latter findings that cells contain a negative feedback mechanism used to maintain Mcd1 homeostasis. AUTHOR SUMMARYCohesins are central to almost all aspects of DNA regulation (chromosome segregation, gene transcription, DNA repair, chromosome condensation, replication fork progression, and genomic organization). Cohesin also play key roles in cell checkpoints: cohesin mutations activate the spindle assembly checkpoint while double strand DNA breaks can elicit a new round of cohesin establishment. In the current study, we provide evidence for a novel cohesin surveillance system that employs E3 ligases that directly target Mcd1, a core component of the cohesin ring structure, for degradation. We further describe a feedback mechanism through which cells dramatically induce MCD1 transcription to maintain Mcd1 homeostasis. Finally, we provide evidence that requires the re-evaluation of phenotypes associated with other cohesin gene mutations.

cell biology↗

bioRxiv2024

Fdo1, Fkh1, Fkh2 and the Swi6-Mbp1 MBF complex regulate Mcd1 levels to impact eco1 rad61 cell growth in Saccharomyces cerevisiae

Cohesins promote proper chromosome segregation, gene transcription, genomic architecture, DNA condensation, and DNA damage repair. Mutations in either cohesin subunit or regulatory genes can give rise to severe developmental abnormalities (such as Robert Syndrome and Cornelia de Lange Syndrome) and also are highly correlated with cancer. Despite this, little is known about cohesin regulation. Eco1 (ESCO2/EFO2 in humans) and Rad61 (WAPL in humans) represent two such regulators but perform opposing roles. Eco1 acetylation of cohesin during S phase, for instance, stabilizes cohesin-DNA binding to promote sister chromatid cohesion. On the other hand, Rad61 promotes the dissociation of cohesin from DNA. While Eco1 is essential, ECO1 and RAD61 co-deletion results in yeast cell viability, but only within a limited temperature range. Here, we report that eco1 rad61 cell lethality is due to reduced levels of the cohesin subunit Mcd1. Results from a suppressor screen further reveals that FDO1 deletion rescues the temperature sensitive (ts) growth defects exhibited by eco1 rad61 double mutant cells by increasing Mcd1 levels. Regulation of MCD1 expression, however, appears more complex. Elevated expression of MBP1, which encodes a subunit of the MBF transcription complex, also rescues eco1 rad61 cell growth defects. Elevated expression of SWI6, however, which encodes the Mbp1-binding partner of MBF, exacerbates eco1 rad61 cell growth and also abrogates the Mpb1-dependent rescue. Finally, we identify two additional transcription factors, Fkh1 and Fkh2, that impact MCD1 expression. In combination, these findings provide new insights into the nuanced and multi-faceted transcriptional pathways that impact MCD1 expression.

cell biology↗

bioRxiv2022

G1-Cyclin2 (Cln2) promotes chromosome hypercondensation in eco1/ctf7 rad61 null cells during hyperthermic stress in Saccharomyces cerevisiae

Eco1/Ctf7 is a highly conserved acetyltransferase that activates cohesin complexes and is critical for sister chromatid cohesion, chromosome condensation, DNA damage repair, nucleolar integrity, and gene transcription. Mutations in the human homolog of ECO1 (ESCO2/EFO2), or in genes that encode cohesin subunits, result in severe developmental abnormalities and intellectual disabilities referred to as Roberts Syndrome (RBS) and Cornelia de Lange Syndrome (CdLS), respectively. In yeast, deletion of ECO1 results in cell inviability. Co-deletion of RAD61 (WAPL in humans), however, produces viable yeast cells. These eco1 rad61 double mutants, however, exhibit a severe temperature-sensitive growth defect, suggesting that Eco1 or cohesins respond to hyperthermic stress through a mechanism that occurs independent of Rad61. Here, we report that deletion of the G1 cyclin CLN2 rescues the temperature sensitive lethality otherwise exhibited by eco1 rad61 mutant cells, such that the triple mutant cells exhibit robust growth over a broad range of temperatures. While Cln1, Cln2 and Cln3 are functionally redundant G1 cyclins, neither CLN1 nor CLN3 deletions rescue the temperature-sensitive growth defects otherwise exhibited by eco1 rad61 double mutants. We further provide evidence that CLN2 deletion rescues hyperthermic growth defects independent of START and impacts the state of chromosome condensation. These findings reveal novel roles for Cln2 that are unique among the G1 cyclin family and appear critical for cohesin regulation during hyperthermic stress.

cell biology↗

bioRxiv2020

Esco2 and Cohesin Regulate CRL4 Ubiquitin Ligase ddb1 Expression and Thalidomide Teratogenicity

Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) are severe developmental maladies that arise from mutation of cohesin (including SMC3, CdLS) and ESCO2 (RBS). Though ESCO2 activate cohesin, CdLS and RBS etiologies are currently considered non-synonymous and for which pharmacological treatments are unavailable. Here, we identify a unifying mechanism that integrates these genetic maladies to pharmacologically-induced teratogenicity via thalidomide. Our results reveal that Esco2 and cohesin co-regulate a component of CRL4 ubiquitin ligase through which thalidomide exerts teratogenic effects. These findings are the first to link RBS and CdLS to thalidomide teratogenicity and offers new insights into treatments.

developmental biology↗

bioRxiv2020

PCNA Antagonizes Cohesin-dependent Roles in Genomic Stability

PCNA sliding clamp binds factors through which histone deposition, chromatin remodeling, and DNA repair are coupled to DNA replication. PCNA also directly binds Eco1/Ctf7 acetyltransferase, which in turn activates cohesins and establishes cohesion between nascent sister chromatids. While increased recruitment thus explains the mechanism through which elevated levels of chromatin-bound PCNA rescue eco1 mutant cell growth, the mechanism through which PCNA instead worsens cohesin mutant cell growth remains unknown. Possibilities include that elevated levels of long-lived chromatin-bound PCNA reduce either cohesin deposition onto DNA or cohesin acetylation. Instead, our results reveal that PCNA increases the levels of both chromatin-bound cohesin and cohesin acetylation. Beyond sister chromatid cohesion, PCNA also plays a critical role genomic stability such that high levels of chromatin-bound PCNA destabilize the DNA replication fork. At a relatively modest increase of chromatin-bound PCNA, however, fork stability and progression appear normal in wildtype cells. Our results reveal that even this moderate increase of PCNA sensitizes cohesin mutant cells to DNA damaging agents and in a process that involves the DNA damage response kinase Mec1(ATR), but not Tel1(ATM). These and other findings document that PCNA mis-regulation results in genome instabilities that normally are resolved by cohesin and suggest that elevating levels of chromatin-bound PCNA may target cohesinopathic cells linked to cancer.

genetics↗

bioRxiv2019

Promotion of hyperthermic-induced rDNA hypercondensation in Saccharomyces cerevisiae

Ribosome biogenesis is tightly regulated through stress-sensing pathways that impact genome stability, aging and senescence. In Saccharomyces cerevisiae, ribosomal RNAs are transcribed from rDNA located on the right arm of chromosome XII. Numerous studies reveal that rDNA decondenses into a puff-like structure during interphase and condenses into a tight loop-like structure during mitosis. Intriguingly, a novel and additional mechanism of increased mitotic rDNA compaction (termed hypercondensation) was recently discovered that occurs in response to temperature stress (hyperthermic-induced) and is rapidly reversible. Here, we report that neither changes in condensin nor cohesin binding dynamics appear to play a critical role in hyperthermic-induced rDNA hypercondensation - differentiating this architectural state from normal mitotic condensation (requiring cohesins and condensins) and the premature condensation (requiring condensins) that occurs during interphase in response to nutrient starvation. A candidate genetic approach revealed that deletion of either Hsp82 or Hsc82 (Hsp90 heat shock paralogs) result in significantly reduced hyperthermic-induced rDNA hypercondensation. Intriguingly, Hsp inhibitors do not impact rDNA hypercondensation. In combination, these findings suggest that Hsp90 either stabilizes client proteins, which are sensitive to very transient thermic challenges, or directly promotes rDNA hypercondensation during preanaphase. Our findings further reveal that the high mobility group protein Hmo1 is a negative regulator of mitotic rDNA condensation, distinct from its role in promoting premature-condensation of rDNA during interphase upon nutrient starvation.

cell biology↗