Papers by Michael-Christopher Keogh
Nature, Jan 26, 2006
One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation ... more One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create gammaH2AX-containing nucleosomes. Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB. This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins, chromatin remodellers and cohesins. Multiple mechanisms for eliminating gammaH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of gammaH2AX in vivo and efficiently d...
Molecular and cellular biology, 2008
Acetylation of Saccharomyces cerevisiae histone H3 on K56 by the histone acetyltransferase (HAT) ... more Acetylation of Saccharomyces cerevisiae histone H3 on K56 by the histone acetyltransferase (HAT) Rtt109 is important for repairing replication-associated lesions. Rtt109 purifies from yeast in complex with the histone chaperone Vps75, which stabilizes the HAT in vivo. A whole-genome screen to identify genes whose deletions have synthetic genetic interactions with rtt109Delta suggests Rtt109 has functions in addition to DNA repair. We show that in addition to its known H3-K56 acetylation activity, Rtt109 is also an H3-K9 HAT, and we show that Rtt109 and Gcn5 are the only H3-K9 HATs in vivo. Rtt109's H3-K9 acetylation activity in vitro is enhanced strongly by Vps75. Another histone chaperone, Asf1, and Vps75 are both required for acetylation of lysine 9 on H3 (H3-K9ac) in vivo by Rtt109, whereas H3-K56ac in vivo requires only Asf1. Asf1 also physically interacts with the nuclear Hat1/Hat2/Hif1 complex that acetylates H4-K5 and H4-K12. We suggest Asf1 is capable of assembling into ...
Genome Medicine, 2014
Background: The evolutionarily conserved DNA mismatch repair (MMR) system corrects base-substitut... more Background: The evolutionarily conserved DNA mismatch repair (MMR) system corrects base-substitution and insertion-deletion mutations generated during erroneous replication. The mutation or inactivation of many MMR factors strongly predisposes to cancer, where the resulting tumors often display resistance to standard chemotherapeutics. A new direction to develop targeted therapies is the harnessing of synthetic genetic interactions, where the simultaneous loss of two otherwise non-essential factors leads to reduced cell fitness or death. High-throughput screening in human cells to directly identify such interactors for disease-relevant genes is now widespread, but often requires extensive case-by-case optimization. Here we asked if conserved genetic interactors (CGIs) with MMR genes from two evolutionary distant yeast species (Saccharomyces cerevisiae and Schizosaccharomyzes pombe) can predict orthologous genetic relationships in higher eukaryotes. Methods: High-throughput screening was used to identify genetic interaction profiles for the MutSα and MutSβ heterodimer subunits (msh2Δ, msh3Δ, msh6Δ) of fission yeast. Selected negative interactors with MutSβ (msh2Δ/msh3Δ) were directly analyzed in budding yeast, and the CGI with SUMO-protease Ulp2 further examined after RNA interference/drug treatment in MSH2-deficient and -proficient human cells.
The EMBO Journal, 2009
As RNA polymerase II (RNApII) transitions from initiation to elongation, Mediator and the basal t... more 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 0 -end processing. The higher eukaryotic kinase Cdk9 not only performs a similar function, but also functions at the 5 0 -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.
mRNA Processing and Metabolism, 2004
The chromatin immunoprecipitation (ChIP) technique has been used to determine where and under wha... more The chromatin immunoprecipitation (ChIP) technique has been used to determine where and under what conditions DNA binding proteins associate with specific DNA sequences. Proteins are crosslinked in vivo with formaldehyde, and chromatin is then isolated and sheared. The protein of interest is then immunoprecipitated and the associated DNA sequences identified via PCR. Although this technique was originally designed to assay DNA binding proteins, it can also be used to monitor mRNA processing factors associated with transcription complexes.
Proceedings of the National Academy of Sciences, 2004
NuA4, the only essential histone acetyltransferase complex in Saccharomyces cerevisiae, acetylate... more NuA4, the only essential histone acetyltransferase complex in Saccharomyces cerevisiae, acetylates the N-terminal tails of histones H4 and H2A. Affinity purification of NuA4 revealed the presence of three previously undescribed subunits, Vid21/Eaf1/ Ydr359c, Swc4/Eaf2/Ygr002c, and Eaf7/Ynl136w. Experimental analyses revealed at least two functionally distinct sets of polypeptides in NuA4: (i) Vid21 and Yng2, and (ii) Eaf5 and Eaf7. Vid21 and Yng2 are required for bulk histone H4 acetylation and are functionally linked to the histone H2A variant Htz1 and the Swr1 ATPase complex (SWR-C) that assembles Htz1 into chromatin, whereas Eaf5 and Eaf7 have a different, as yet undefined, role. Mutations in Htz1, the SWR-C, and NuA4 cause defects in chromosome segregation that are consistent with genetic interactions we have observed between the genes encoding these proteins and genes encoding kinetochore components. Because SWR-C-dependent recruitment of Htz1 occurs in both transcribed and centromeric regions, a NuA4/SWR-C/Htz1 pathway may regulate both transcription and centromere function in S. cerevisiae.
Nature Structural & Molecular Biology, 2009
Histone variant H2A.Z has a conserved role in genome stability, although it remains unclear how t... more Histone variant H2A.Z has a conserved role in genome 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 genome 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.
Nature, 2006
One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation ... more One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create gH2AXcontaining nucleosomes 1 . Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB 3 . This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins 4,5 , chromatin remodellers 6 and cohesins 7,8 . Multiple mechanisms for eliminating gH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of LETTERS
Molecular Cell, 2004
The 26S proteasome, which consists of a 19S regula-1 Kings College Circle Toronto, Ontario M5S 1A... more The 26S proteasome, which consists of a 19S regula-1 Kings College Circle Toronto, Ontario M5S 1A8 tory cap and 20S catalytic core, degrades polyubiquitinated proteins in eukaryotic cells (Voges et al., 1999). Canada 3 Department of Biological Chemistry In addition to directing ubiquitin-dependent proteolysis, the proteasome has been shown to have nonproteolytic and Molecular Pharmacology Harvard Medical School roles in recent studies (Voges et al., 1999). Although no direct link between the proteasome and the repair of Boston, Massachusetts 02115 DNA DSBs has been reported, recent work has suggested a role for the 19S proteasome in nucleotide excision repair (NER) mediated by the repair protein Rad23 Summary (Russell et al., 1999a; Schauber et al., 1998). Russell et al. (1999a) further demonstrated that the 19S protea-Affinity purification of the yeast 19S proteasome resome, but not the 20S core, functions in NER and that vealed the presence of Sem1 as a subunit. Its human this process is independent of proteolysis. Furthermore, homolog, DSS1, was found likewise to copurify with the 19S proteasome has been shown to have not only the human 19S proteasome. DSS1 is known to associa stimulatory role in NER, but a negative role as well ate with the tumor suppressor protein BRCA2 involved which is mediated by Rad23 (Gillette et al.
Molecular Cell, 2005
Phosphorylated histone H2AX (g-H2AX) forms foci over large chromatin domains surrounding doublest... more Phosphorylated histone H2AX (g-H2AX) forms foci over large chromatin domains surrounding doublestranded DNA breaks (DSB). These foci recruit DSB repair proteins and dissolve during or after repair is completed. How g-H2AX is removed from chromatin remains unknown. Here, we show that protein phosphatase 2A (PP2A) is involved in removing g-H2AX foci. The PP2A catalytic subunit [PP2A(C)] and g-H2AX coimmunoprecipitate and colocalize in DNA damage foci and PP2A dephosphorylates g-H2AX in vitro. The recruitment of PP2A(C) to DNA damage foci is H2AX dependent. When PP2A(C) is inhibited or silenced by RNA interference, g-H2AX foci persist, DNA repair is inefficient, and cells are hypersensitive to DNA damage. The effect of PP2A on g-H2AX levels is independent of ATM, ATR, or DNA-PK activity.
Molecular Cell, 2003
of modifications that can facilitate or inhibit processes such as transcription, DNA replication,... more of modifications that can facilitate or inhibit processes such as transcription, DNA replication, DNA repair, and 112 College Street Toronto, Ontario chromosome segregation. These posttranslational chemical modifications include acetylation, phosphorylation, Canada M5G 1L6 2 Department of Medical Genetics and Microbiology ADP-ribosylation, ubiquitination, and methylation (Fischle et al., 2003). Variation in nucleosome structure also re-University of Toronto
Molecular and Cellular Biology, 2003
The Saccharomyces cerevisiae cyclin-dependent kinase (CDK) Bur1 (Sgv1) may be homologous to mamma... more The Saccharomyces cerevisiae cyclin-dependent kinase (CDK) Bur1 (Sgv1) may be homologous to mammalian Cdk9, which functions in transcriptional elongation. Although Bur1 can phosphorylate the Rpb1 carboxyterminal domain (CTD) kinase in vitro, it has no strong specificity within the consensus heptapeptide YSPTSPS for Ser2 or Ser5. BUR1 mutants are sensitive to the drugs 6-azauracil and mycophenolic acid and interact genetically with the elongation factors Ctk1 and Spt5. Chromatin immunoprecipitation experiments show that Bur1 and its cyclin partner Bur2 are recruited to transcription elongation complexes, cross-linking to actively transcribing genes. Interestingly, Bur1 shows reduced cross-linking to transcribed regions downstream of polyadenylation sites. In addition, bur1 mutant strains have a reduced cross-linking ratio of RNA polymerase II at the 3 end of genes relative to promoter regions. Phosphorylation of CTD serines 2 and 5 appears normal in mutant cells, suggesting that Bur1 is not a significant source of cotranscriptional Rpb1 phosphorylation. These results show that Bur1 functions in transcription elongation but may phosphorylate a substrate other than the CTD. Downloaded from 7014 KEOGH ET AL. MOL. CELL. BIOL. on January 28, 2016 by guest http://mcb.asm.org/ Downloaded from FIG. 8. Bur1 is required for transcription elongation but not cotranscriptional CTD phosphorylation. (A)
Molecular and Cellular Biology, 2002
Basal transcription factor TFIIH phosphorylates the RNA polymerase II (RNApII) carboxy-terminal d... more Basal transcription factor TFIIH phosphorylates the RNA polymerase II (RNApII) carboxy-terminal domain (CTD) within the transcription initiation complex. The catalytic kinase subunit of TFIIH is a member of the cyclin-dependent kinase (Cdk) family, designated Kin28 in Saccharomyces cerevisiae and Cdk7 in higher eukaryotes. Together with TFIIH subunits cyclin H and Mat1, Cdk7 kinase is also found in a trimer complex known as Cdk activating kinase (CAK). A yeast trimer complex has not previously been identified, although a Kin28-Ccl1 dimer called TFIIK has been isolated as a breakdown product of TFIIH. Here we show that a trimeric complex of Kin28-Ccl1-Tfb3 exists in yeast extracts. Several Kin28 point mutants that are defective in CTD phosphorylation were created. Consistent with earlier studies, these mutants have no transcriptional defect in vitro. Like other Cdks, Kin28 is activated by phosphorylation on T162 of the T loop. Kin28 T162 mutants have no growth defects alone but do demonstrate synthetic phenotypes when combined with mutant versions of the cyclin partner, Ccl1. Surprisingly, these phosphorylation site mutants appear to destabilize the association of the cyclin subunit within the context of TFIIH but not within the trimer complex.
Journal of Biological Chemistry, 2005
The breast-and ovarian-specific tumor suppressor BRCA1, when associated with BARD1, is an ubiquit... more The breast-and ovarian-specific tumor suppressor BRCA1, when associated with BARD1, is an ubiquitin ligase. We have shown here that this heterodimer ubiquitinates a hyperphosphorylated form of Rpb1, the largest subunit of RNA polymerase II. Two major phosphorylation sites have been identified in the Rpb1 carboxyl terminal domain, serine 2 (Ser-2) or serine 5 (Ser-5) of the YSPTSPS heptapeptide repeat. Only the Ser-5 hyperphosphorylated form is ubiquitinated by BRCA1/ BARD1. Overexpression of BRCA1 in cells stimulated the DNA damage-induced ubiquitination of Rpb1. Similar to the in vitro reaction, the stimulation of Rpb1 ubiquitination by BRCA1 in cells occurred only on those molecules hyperphosphorylated on Ser-5 of the heptapeptide repeat. In vitro, the carboxyl terminus of BRCA1 (amino acids 501-1863) was dispensable for the ubiquitination of hyperphosphorylated Rpb1. In cells, however, efficient Rpb1 ubiquitination required the carboxyl terminus of BRCA1, suggesting that interactions mediated by this region were essential in the complex milieu of the nucleus. These results link the BRCA1-dependent ubiquitination of the polymerase with DNA damage.
Genes & Development, 2006
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Papers by Michael-Christopher Keogh