The Keogh Lab - AECOM
Latest updates (12.10.10): Publication citation updated
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Chromatin
Lab / People
| Research InterestsOur lab investigates the role of chromatin in various DNA transactions including transcription, DNA Double-Strand Break (DSB) repair and chromosome transmission. To achieve this we use a combination of high-throughput genetic, genomic and biochemical approaches in budding and fission yeasts (S.cerevisiae and Sz.pombe). Our most recent publications include:
Bandyopadhyay et al (2010) Science 330:1385 Although cellular behaviors are dynamic, the networks that govern these behaviors have been mapped primarily as static snapshots. Using an approach called differential epistasis mapping, we have discovered widespread changes in genetic interaction among yeast kinases, phosphatases and transcription factors as the cell responds to DNA damage. Differential interactions uncover many gene functions that go undetected in static conditions. They are very effective at identifying DNA repair pathways, highlighting new damage-dependent roles for the Slt2 kinase, Pph3 phosphatase, and histone variant Htz1. The data also reveal that protein complexes are generally stable in response to perturbation, but the functional relations between these complexes are substantially reorganized. Differential networks chart a new type of genetic landscape that is invaluable for mapping cellular responses to stimuli. (PDF) (Supplementary) (This work is the subject of a Science Perspectives article by Friedman & Schuldiner [Science 330:1327])
Mehta et al (2010) J Biol Chem 285:39855 The multi-functional histone variant Htz1 (S. cerevisiae H2A.Z) is acetylated on up to four N-terminal lysines: K3, K8, K10 and K14. It has thus been posited that specific acetylated forms of the histone could regulate distinct roles. Antibodies against Htz1-K8Ac, K10Ac, and K14Ac show that all three modifications are added by Esa1 acetyltransferase and removed by Hda1 deacetylase. Completely unacetylatable htz1 alleles exhibit widespread interactions in genome-scale genetic screening. However singly-mutated (e.g. htz1-K8R) or singly-acetylable (e.g. the triple mutant htz1-K3R / K10R / K14R) alleles show no significant defects in these analyses. This suggests that the N-terminal acetylations on Htz1 are internally redundant. Further supporting this proposal, each acetylation decays with similar kinetics when Htz1 transcription is repressed, and global proteomic screening did not find a single condition in which one Htz1Ac was differentially regulated. However while the individual acetylations on Htz1 may be redundant, they are not dispensable. Completely unacetylatable htz1 alleles display genetic interactions and phenotypes in common with and distinct from htz1Δ. In addition each Htz1 N-terminal lysine is deacetylated by Hda1 in response to benomyl and reacetylated when this agent is removed. Such active regulation suggests that acetylation plays a significant role in Htz1 function. (PDF) Mehta et al (2010) J Biol 9:3 How much functional specialization can one component histone confer on a single nucleosome? The histone variant H2A.Z seems to be an extreme example. Genome-wide distribution maps show non-random (and evolutionarily conserved) patterns, with localized enrichment or depletion giving a tantalizing suggestion of function. Multiple post-translational modifications on the protein suggest further regulation. An additional layer of complexity has now been uncovered: the vertebrate form is actually encoded by two non-allelic genes that differ by expression pattern and three amino acids. (Review: PDF) Kim et al (2009) Nat Struct Mol Biol 16:1286 Histone variant H2A.Z has a conserved role in chromosome stability, although it remains unclear how this is mediated. Here we demonstrate that the fission yeast Swr1 ATPase inserts H2A.Z (Pht1) into chromatin and Kat5 acetyltransferase (Mst1) acetylates it. Deletion or an unacetylatable mutation of Pht1 leads to genomic instability, primarily caused by chromosome entanglement and breakage at anaphase. This leads to the loss of telomere-proximal markers, though telomere protection and repeat-length are unaffected by the absence of Pht1. Strikingly, the chromosome entanglement in pht1∆ anaphase cells can be rescued by forcing chromosome condensation before anaphase onset. We show that the condensin complex, required for the maintenance of anaphase chromosome condensation, prematurely dissociates from chromatin in the absence of Pht1. This and other findings suggest an important role for H2A.Z in the architecture of anaphase chromosomes. (PDF) (Supplementary) Fiedler et al (2009) Cell 136:952 Reversible protein phosphorylation is a signaling mechanism involved in all cellular processes. To create a systems view of the signaling apparatus in budding yeast, we generated an E-MAP (epistatic miniarray profile) comprised of 100,000 pair-wise, quantitative genetic interactions, including virtually all protein kinases and phosphatases and key cellular regulators. Quantitative genetic interaction mapping reveals factors working in compensatory pathways (negative genetic interactions; e.g. synthetic lethality) or those operating in linear pathways (positive genetic interactions; e.g. suppression). Within kinases, phosphatases, and their substrates, we found an enrichment of positive genetic interactions. To develop a global view of the signaling apparatus, we isolated “triplet genetic motifs” and assembled these into a higher-order map. The resulting network view provides new insights into signaling pathway regulation, and revealed a link between the cell cycle kinase, Cak1, the Fus3 MAP kinase, and a pathway that regulates chromatin integrity during transcription by RNA polymerase II. (PDF) Ahn et al (2009) EMBO J 28:205 As RNA polymerase II (RNApII) transitions from initiation to elongation, Mediator and the basal transcription factors TFIID, TFIIA, TFIIH, and TFIIE remain at the promoter as part of a scaffold complex, whereas TFIIB and TFIIF dissociate. The yeast Ctk1 kinase associates with elongation complexes and phosphorylates serine 2 in the YSPTSPS repeats of the Rpb1 C-terminal domain, a modification that couples transcription to mRNA 3'-end processing. The higher eukaryotic kinase Cdk9 not only performs a similar function, but also functions at the 5'-end of genes in the transition from initiation to elongation. In strains lacking Ctk1, many basal transcription factors cross-link throughout transcribed regions, apparently remaining associated with RNApII until it terminates. Consistent with this observation, preinitiation complexes formed on immobilized templates with transcription extracts lacking Ctk1 leave lower levels of the scaffold complex behind after escape. Taken together, these results suggest that Ctk1 is necessary for the release of RNApII from basal transcription factors. Interestingly, this function of Ctk1 is independent of its kinase activity, suggesting a structural function of the protein. (PDF) _________________________________________________Contact DetailsDepartment of Cell Biology, Albert Einstein College of Medicine _________________________________________________ |
