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Silencing of the mating-type locus HMR in Saccharomyces cerevisiae requires DNA elements called silencers. To establish HMR silencing, the origin recognition complex binds the HMR-E silencer and recruits the silent information regulator (Sir)1 protein. Sir1 in turn helps establish silencing by stabilizing binding of the other Sir proteins, Sir2-4. However, silencing is semistable even in sir1Delta cells, indicating that SIR1-independent establishment mechanisms exist. Furthermore, the requirement for SIR1 in silencing a sensitized version of HMR can be bypassed by high-copy expression of FKH1 (FKH1(hc)), a conserved forkhead transcription factor, or by deletion of the S phase cyclin CLB5 (clb5Delta). FKH1(hc) caused only a modest increase in Fkh1 levels but effectively reestablished Sir2-4 chromatin at HMR as determined by Sir3-directed chromatin immunoprecipitation. In addition, FKH1(hc) prolonged the cell cycle in a manner distinct from deletion of its close paralogue FKH2, and it created a cell cycle phenotype more reminiscent to that caused by a clb5Delta. Unexpectedly, and in contrast to SIR1, both FKH1(hc) and clb5Delta established silencing at HMR using the replication origins, ARS1 or ARSH4, as complete substitutes for HMR-E (HMRDeltaE::ARS). HMRDeltaE::ARS1 was a robust origin in CLB5 cells. However, initiation by HMRDeltaE::ARS1 was reduced by clb5Delta or FKH1(hc), whereas ARS1 at its native locus was unaffected. The CLB5-sensitivity of HMRDeltaE::ARS1 did not result from formation of Sir2-4 chromatin because sir2Delta did not rescue origin firing in clb5Delta cells. These and other data supported a model in which FKH1 and CLB5 modulated Sir2-4 chromatin and late-origin firing through opposing regulation of a common pathway.
Mol Biol Cell 2008 Feb
PMID:Conversion of a replication origin to a silencer through a pathway shared by a Forkhead transcription factor and an S phase cyclin. 1804 95

In budding yeast, telomeres, the ribosomal DNA array, and HM loci are transcriptionally silenced by chromatin complexes containing Sir proteins. Hek2, a protein containing three evolutionary conserved RNA-binding K-homology domains, was identified as a suppressor of telomeric silencing [telomeric position effect (TPE)]. To explore the mechanisms of Hek2p action in gene silencing, we examined its relationship with Sir proteins. This search revealed an epistatic interaction between HEK2 and SIR1 at telomeres. Both single mutations, sir1Delta and hek2Delta, enhanced TPE, whereas the effect of double mutation, sir1Delta hek2Delta, did not exceed that of the single mutations. The results of chromatin immunoprecipitation analysis demonstrate that the TPE enhancement observed in these mutants is associated with increased binding of Sir2 protein to telomeres. At the HMR locus, hek2Delta rescues the silencing defect caused by sir1Delta mutation and reverses the loss of Sir2p and Sir3p. These data suggest that the epistatic interaction of HEK2 and SIR1 reflects competition between telomeres and HMR for Sir2/3 factors where HEK2 acts to suppress silencing. Because chromatin immunoprecipitation analysis reveals the presence of Hek2p at a subtelomeric region and HMR, its silencing effects at these loci are likely direct. These observations suggest that HEK2 regulates the composition of Sir complexes at HMR and telomeres.
J Mol Biol 2008 Jan 25
PMID:Epistatic interaction between the K-homology domain protein HEK2 and SIR1 at HMR and telomeres in yeast. 1806 21

Gene regulation involves long-range communication between silencers, enhancers, and promoters. In Saccharomyces cerevisiae, silencers flank transcriptionally repressed genes to mediate regional silencing. Silencers recruit the Sir proteins, which then spread along chromatin to encompass the entire silenced domain. In this report we have employed a boundary trap assay, an enhancer activity assay, chromatin immunoprecipitations, and chromosome conformation capture analyses to demonstrate that the two HMR silencer elements are in close proximity and functionally communicate with one another in vivo. We further show that silencing is necessary for these long-range interactions, and we present models for Sir-mediated silencing based upon these results.
Mol Cell Biol 2008 Mar
PMID:Long-range communication between the silencers of HMR. 1819 43

Saccharomyces cerevisiae chromosome III encodes 11 autonomously replicating sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species.
Mol Cell Biol 2008 Aug
PMID:Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site. 1857 88

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also Xu et al., Mol. Cell 2006.
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PMID:A rapid technique for the visualization of live immobilized yeast cells. 1870 80

In Saccharomyces cerevisiae, silenced chromatin occurs at telomeres and the silent mating-type loci HMR and HML. At these sites, the Sir proteins are recruited to a silencer and then associate with adjacent chromatin. We used chromatin immunoprecipitation to compare the rates of Sir protein assembly at different genomic locations and discovered that establishment of silenced chromatin was much more rapid at HMR than at the telomere VI-R. Silenced chromatin also assembled more quickly on one side of HMR-E than on the other. Despite differences in spreading, the Sir proteins were recruited to HMR-E and telomeric silencers at equivalent rates. Additionally, insertion of HMR-E adjacent to the telomere VI-R increased the rate of Sir2p association with the telomere. These data suggest that HMR-E functions to both recruit Sir proteins and promote their assembly across several kilobases. Observations that association of Sir2p occurs simultaneously throughout HMR and that silencing at HMR is insensitive to coexpression of catalytically inactive Sir2p suggest that HMR-E acts by enabling assembly to occur in a nonlinear fashion. The ability of silencers to promote assembly of silenced chromatin over several kilobases is likely an important mechanism for maintaining what would otherwise be unstable chromatin at the correct genomic locations.
Mol Cell Biol 2009 Jan
PMID:A silencer promotes the assembly of silenced chromatin independently of recruitment. 1895 2

We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLalpha or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and alpha cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLalpha, unlike HMRa and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLalpha donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLalpha creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus.
Mol Cell Biol 2009 Feb
PMID:Regulation of nuclear positioning and dynamics of the silent mating type loci by the yeast Ku70/Ku80 complex. 1904 66

In Saccharomyces cerevisiae, transcriptional silencing occurs at the cryptic mating-type loci (HML and HMR), telomeres, and ribosomal DNA (rDNA; RDN1). Silencing in the rDNA is unusual in that polymerase II (Pol II) promoters within RDN1 are repressed by Sir2 but not Sir3 or Sir4. rDNA silencing unidirectionally spreads leftward, but the mechanism of limiting its spreading is unclear. We searched for silencing barriers flanking the left end of RDN1 by using an established assay for detecting barriers to HMR silencing. Unexpectedly, the unique sequence immediately adjacent to RDN1, which overlaps a prominent cohesin binding site (CARL2), did not have appreciable barrier activity. Instead, a fragment located 2.4 kb to the left, containing a tRNA(Gln) gene and the Ty1 long terminal repeat, had robust barrier activity. The barrier activity was dependent on Pol III transcription of tRNA(Gln), the cohesin protein Smc1, and the SAS1 and Gcn5 histone acetyltransferases. The location of the barrier correlates with the detectable limit of rDNA silencing when SIR2 is overexpressed, where it blocks the spreading of rDNA heterochromatin. We propose a model in which normal Sir2 activity results in termination of silencing near the physical rDNA boundary, while tRNA(Gln) blocks silencing from spreading too far when nucleolar Sir2 pools become elevated.
Mol Cell Biol 2009 May
PMID:Limiting the extent of the RDN1 heterochromatin domain by a silencing barrier and Sir2 protein levels in Saccharomyces cerevisiae. 1928 3

Saccharomyces cerevisiae Yta7 is a barrier active protein that modulates transcriptional states at the silent mating locus, HMR. Additionally, Yta7 regulates histone gene transcription and has overlapping functions with known histone chaperones. This study focused on deciphering the functional role of the noncanonical Yta7 bromodomain. By use of genetic and epistasis analyses, the Yta7 bromodomain was shown to be necessary for barrier activity at HMR and to have overlapping functions with histone regulators (Asf1 and Spt16). Canonical bromodomains can bind to acetylated lysines on histones; however, the Yta7 bromodomain showed an association with histones that was independent of posttranslational modification. Further investigation showed that regions of Yta7 other than the bromodomain conferred histone association. Chromatin immunoprecipitation-chip analyses revealed that the Yta7 bromodomain was not solely responsible for histone association but was also necessary for proper chromosomal positioning of Yta7. This work demonstrates that the Yta7 bromodomain engages histones for certain cellular functions like barrier chromatin maintenance and particular Spt16/Asf1 cellular pathways of chromatin regulation.
Mol Cell Biol 2009 Sep
PMID:A noncanonical bromodomain in the AAA ATPase protein Yta7 directs chromosomal positioning and barrier chromatin activity. 1958 Dec 91

The silenced chromatin at the cryptic mating-type loci (HML and HMR) of Saccharomyces cerevisiae requires a cell cycle event between early S phase and G(2)/M phase to achieve repression. Although DNA replication per se is not essential for silencing, mutations in many of the proteins involved in DNA replication affect silencing. Each of the four silencers, which flank the silenced loci, includes an origin recognition complex (ORC) binding site (ACS). ORC directly interacted with Sir1 and recruits Sir1 to the silencers. This study describes additional roles for ORC in the architecture of silenced chromatin. Using chromatin immunoprecipitation (ChIP) analysis, we found that ORC physically interacts throughout the internal regions of HMR as well as with silencers. This interaction depended on the presence of Sir proteins and, in part, on the HMR-I silencer. ORC remained associated with the internal regions of HMR even when these regions were recombinationally separated from the silencers. Moreover, ORC could be recruited to the silencers lacking an ACS through its Sir1 interaction.
Mol Cell Biol 2010 Feb
PMID:Expanded roles of the origin recognition complex in the architecture and function of silenced chromatin in Saccharomyces cerevisiae. 1994 82


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