Biology subjects

Brown, J. A. R.

Publications and source records attributed to Brown, J. A. R..

4 recordsLinked to original sources

bioRxiv2026

Dual regulation of chemical stress-induced DDI2/3 expression by a transcription factor Fzf1 and nucleosome in Saccharomyces cerevisiae

DDI2 and DDI3 (DDI2/3) are duplicated genes in Saccharomyces cerevisiae that exhibit strong induction by a transcription factor Fzf1 in response to chemical treatments like cyanamide (CY) and methyl methanesulfonate (MMS). Although, like DDI2/3, SSU1, YHB1 and YNR064C also contain an Fzf1-binding consensus sequence CS2 and are coordinately regulated by Fzf1, these genes are only modestly induced by CY and MMS. To identify additional cis-acting elements in the DDI2/3 promoter, we made DDI2/3 promoter deletions in a reporter system and identified upstream repressing sequences (URS) spanning 480 nucleotides. To test a hypothesis that the chromatin structure constitutes the URS, we utilized a yeast strain capable of histone H3/H4 depletion by shifting carbon sources. Following histone depletion, DDI2/3 were strongly induced in an Fzf1 dependent manner, while YHB1 was repressed. Interestingly, under histone depletion conditions, CY or MMS treatment further increased expression of all Fzf1-regulated genes to comparable levels in an Fzf1 dependent manner. A genome-wide MNase-seq analysis showed that CY treatment reduced the nucleosome occupancy at the mapped DDI2/3 URS region in wild-type cells, but not in in fzf1{Delta} cells. These findings collectively indicate that Fzf1 plays dual roles in regulating the DDI2/3 response to CY. Firstly, it binds CS2 and serves as a transcription activator. Secondly, it is required for the chromatin remodeling at URS. This two-tier regulation at the DDI2/3 promoter helps to explain why DDI2/3 achieve much higher fold induction by CY and MMS than other Fzf1-regulated genes, suggesting Fzf1 to be a candidate pioneer transcription factor.

molecular biology↗

bioRxiv2024

Human MeCP2 binds to promoters and inhibits transcription in an unmethylated S. cerevisiae genome

MeCP2 is a DNA-binding transcriptional regulator that is present at near-histone levels in mammalian cortical neurons. Originally identified as a DNA methylation reader, MeCP2 has been proposed to repress transcription by recruiting corepressors to methylated DNA. While some genome-wide occupancy studies support a preference for methylated DNA, others suggest that MeCP2 binding is more influenced by DNA sequence and accessibility than methylation status. Moreover, multiple studies also suggest a role for MeCP2 in gene activation. To clarify MeCP2 function we expressed MeCP2 in Saccharomyces cerevisiae, which lacks DNA methylation and known MeCP2 corepressors. We find that MeCP2 is toxic to yeast and globally inhibits transcription, indicating that MeCP2 can have significant functional impacts without DNA methylation or mammalian corepressors. A subset of MeCP2 mutations that cause the neurodevelopmental disorder Rett syndrome, particularly those that map to the DNA binding domain, alleviate the toxicity of MeCP2 in yeast. Consistent with the importance of DNA binding for toxicity in yeast, we show that MeCP2 binds to the yeast genome, with increased occupancy at GC-rich, nucleosome-depleted sequences. These findings present yeast as a useful tool for analyzing MeCP2 and reveal MeCP2 properties that are not strictly dependent on DNA methylation or mammalian corepressors. SummaryMeCP2, a transcription regulator found in vertebrates, is proposed to repress transcription by recruiting corepressors to methylated DNA. In this work Brown et al. show that MeCP2 expressed in Saccharomyces cerevisiae, which lacks DNA methylation and mammalian co-repressors, binds promoters and inhibits transcription. A subset of MeCP2 mutations that cause the neurodevelopmental disorder, Rett syndrome, rescues MeCP2-induced phenotypes in yeast, supporting the relevance of these results to MeCP2 function in mammals.

molecular biology↗

bioRxiv2024

PAN deadenylase is required for mitosis in response to microtubule stress in yeast and humans

HIGHLIGHTSO_LIPAN2-PAN3 catalytic activity is required for mitotic robustness under MT stress C_LIO_LICatalytic-dead Pan2-D1020A phenocopies pan2{Delta}: spindle defects and G2/M arrest C_LIO_LILoss of Pan2 catalytic activity amplifies nocodazole-induced changes in CLB1 and CLN3 mRNA C_LIO_LIPAN2 depletion in human cells produces multipolar spindles under MT stress C_LI IN BRIEFVerma et al. show that PAN2-PAN3 deadenylase catalytic activity is selectively required for mitotic fidelity under microtubule stress. Yeast pan2{Delta} cells exhibit spindle defects, G2/M arrest, and altered CLB1 and CLN3 mRNA levels under nocodazole. PAN2 knockdown in human cells produces multipolar spindles, demonstrating evolutionary conservation. Cytoplasmic deadenylation by PAN2-PAN3 and CCR4-NOT regulates mRNA stability and translation, but the contribution of individual deadenylases to mitotic progression is poorly defined. Here we show that PAN2-PAN3 catalytic activity is selectively required for mitotic robustness under microtubule stress. In budding yeast, the catalytically inactive Pan2-D1020A mutant fails to complement pan2{Delta} nocodazole sensitivity, leading to G2/M arrest, spindle defects, and increased cell death. pan2{Delta} exhibits synthetic genetic interactions with tubulin and tubulin-folding genes, and RNA-seq reveals dysregulation of cell-cycle transcripts including the cyclins CLB1 and CLN3. The phenotype is stress-conditional: pan2{Delta} cells proliferate normally without drug. In mammalian cells, PAN2 depletion sensitizes HEK293A cells to nocodazole and colchicine and produces prolonged metaphase with multipolar spindles. Together, these findings define a conserved, stress-conditional role for PAN2-PAN3 deadenylase activity in safeguarding mitotic fidelity when microtubules are compromised. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/591760v3_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@19e6662org.highwire.dtl.DTLVardef@53ba18org.highwire.dtl.DTLVardef@de9c99org.highwire.dtl.DTLVardef@fb0f9d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO See accompanying graphical abstract file. C_FIG

cell biology↗

bioRxiv2024

Rtt107 cooperates with Rad55 or Slx4 to maintain genome stability in Saccharomyces cerevisiae

A range of genome maintenance factors respond to endogenous and exogenous DNA damage to prevent mutations and cell death. The scaffold protein, Rtt107, is important for the growth of cells exposed to DNA-damaging agents in the budding yeast Saccharomyces cerevisiae. Rtt107 binds to a diverse array of partner proteins, such as Slx4, and responds to DNA damage by localizing to phosphorylated histone H2A. Rad55-Rad57, a heterodimer involved in DNA repair, also binds to Rtt107, but the function of the Rtt107-Rad55-Rad57 complex remains unclear. In addition to their sensitivity to DNA-damaging agents, rtt107{Delta} mutants exhibit spontaneous genome instability phenotypes, including spontaneous loss of heterozygosity (LOH) caused by crossovers and other genetic events. However, the binding partners with which Rtt107 interacts to prevent spontaneous genome instability have yet to be elucidated. Here, we showed that Rtt107 acts in the same pathway as Rad55 to limit LOH, specifically by preventing crossover events. A rad55-S404A phosphorylation site mutation largely disrupted the interaction between Rtt107 and Rad55-Rad57, resulting in increased LOH and crossover rates, consistent with the contribution of Rtt107-Rad55-Rad57 interaction to genome stability. Strikingly, an rtt107-K887M mutation that reduces Rtt107 recruitment to H2A did not result in an LOH phenotype, suggesting that the role of Rtt107 in preventing LOH is distinct from its function as an H2A-binding scaffold. Rtt107 did not function primarily in the same pathway as Rad55 to limit recombination at the sensitive ribosomal DNA (rDNA) locus, but instead acted with Slx4 to maintain rDNA stability, suggesting that interactions of Rtt107 with different partners prevented distinct types of instability. Taken together, our observations suggested that Rtt107 limits spontaneous LOH and crossover events in part by binding to Rad55 in a manner dependent on Rad55-S404. Author SummaryNumerous proteins are involved in the repair of damaged DNA and prevention of genome instability in cells, which would otherwise result in persistent changes to DNA. Genome maintenance pathways are evolutionarily conserved, and the budding yeast, Saccharomyces cerevisiae, is a powerful model organism for investigating the maintenance of genome integrity. Rtt107 is a scaffold protein expressed in yeast, containing conserved protein domains that are important for the function of genome maintenance proteins. Although the functions of Rtt107 in cells treated with DNA-damaging agents have been characterized in some detail, it remains unclear how Rtt107 prevents spontaneous genome instability in cells growing under normal conditions. Here, we found that Rtt107 prevents specific types of spontaneous genome instability and acts in the same pathway as the DNA repair protein, Rad55, to which it binds. Mutation of a possible Rtt107 binding site on Rad55 showed that Rtt107 indeed limited genome instability in part by binding to Rad55. Strikingly, Rtt107 also showed Rad55-independent roles in preventing ribosomal DNA (rDNA) instability, and in this context Rtt107 cooperated in part with its binding partner Slx4. Taken together, our results revealed the pathways by which the evolutionarily conserved protein Rtt107 limits spontaneous genome instability.

genetics↗