Brian Fleharty

Aneuploidy Underlies Rapid Adaptive Evolution of Yeast Cells Deprived of a Conserved Cytokinesis Motor

The ability to evolve is a fundamental feature of biological systems, but the mechanisms underlying this capacity and the evolutionary dynamics of conserved core processes remain elusive. We show that yeast cells deleted of MYO1, encoding the only myosin II normally required for cytokinesis, rapidly evolved divergent pathways to restore growth and cytokinesis. The evolved cytokinesis phenotypes correlated with specific changes in the transcriptome. Polyploidy and aneuploidy were common genetic alterations in the best evolved strains, and aneuploidy could account for gene expression changes due directly to altered chromosome stoichiometry as well as to downstream effects. The phenotypic effect of aneuploidy could be recapitulated with increased copy numbers of specific regulatory genes in myo1Delta cells. These results demonstrate the evolvability of even a well-conserved process and suggest that changes in chromosome stoichiometry provide a source of heritable variation driving the emergence of adaptive phenotypes when the cell division machinery is strongly perturbed.

Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation

The SAGA (Spt–Ada–Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C‐terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face‐to‐face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

Histone H3 Lysine 36 Dimethylation (H3K36me2) Is Sufficient to Recruit the Rpd3s Histone Deacetylase Complex and to Repress Spurious Transcription

Histone methylation is associated with both transcription activation and repression. However, the functions of different states of methylation remain largely elusive. Here, using methyl-lysine analog technology, we demonstrate that the histone deacetylase complex, Rpd3S, can distinguish the nucleosomes methylated to different extents and that K36me2 is sufficient to target Rpd3S in vitro. Through a genome-wide survey, we identified a few mutants in which the level of K36me3 is significantly reduced, whereas the level of K36me2 is sustained. Transcription analysis and genome-wide histone modification studies on these mutants suggested that K36me2 is sufficient to target Rpd3S in vivo, thereby maintaining a functional Set2-Rpd3S pathway.

Delayed Correlation of mRNA and Protein Expression in Rapamycin-treated Cells and a Role for Ggc1 in Cellular Sensitivity to Rapamycin

To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Δggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression under TOR control.

Cohesinopathy mutations disrupt the subnuclear organization of chromatin

Nuclear morphology, chromosomal condensation, and transcriptional-mediated localization of genes to the nuclear periphery are disturbed by mutations in cohesin pathway genes.

Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation

BackgroundHistone acetyltransferase enzymes (HATs) are implicated in regulation of transcription. HATs from different families may overlap in target and substrate specificity.ResultsWe isolated the elp3+ gene encoding the histone acetyltransferase subunit of the Elongator complex in fission yeast and characterized the phenotype of an Δelp3 mutant. We examined genetic interactions between Δelp3 and two other HAT mutants, Δmst2 and Δgcn5 and used whole genome microarray analysis to analyze their effects on gene expression.ConclusionsComparison of phenotypes and expression profiles in single, double and triple mutants indicate that these HAT enzymes have overlapping functions. Consistent with this, overlapping specificity in histone H3 acetylation is observed. However, there is no evidence for overlap with another HAT enzyme, encoded by the essential mst1+ gene.

Analysis of dynamic changes in retinoid-induced transcription and epigenetic profiles of murine Hox clusters in ES cells

The clustered Hox genes, which are highly conserved across metazoans, encode homeodomain-containing transcription factors that provide a blueprint for segmental identity along the body axis. Recent studies have underscored that in addition to encoding Hox genes, the homeotic clusters contain key noncoding RNA genes that play a central role in development. In this study, we have taken advantage of genome-wide approaches to provide a detailed analysis of retinoic acid (RA)-induced transcriptional and epigenetic changes within the homeotic clusters of mouse embryonic stem cells. Although there is a general colinear response, our analyses suggest a lack of strict colinearity for several genes in the HoxA and HoxB clusters. We have identified transcribed novel noncoding RNAs (ncRNAs) and their cis-regulatory elements that function in response to RA and demonstrated that the expression of these ncRNAs from both strands represent some of the most rapidly induced transcripts in ES cells. Finally, we have provided dynamic analyses of chromatin modifications for the coding and noncoding genes expressed upon activation and suggest that active transcription can occur in the presence of chromatin modifications and machineries associated with repressed transcription state over the clusters. Overall, our data provide a resource for a better understanding of the dynamic nature of the coding and noncoding transcripts and their associated chromatin marks in the regulation of homeotic gene transcription during development.

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation

ABSTRACT In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1. Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes.