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Rpd3


EVOLUTIONARY HOMOLOGS part 2/3

Yeast histone deacetylases target specific genes

The histone deacetylase RPD3 can be targeted to certain genes through its interaction with DNA-binding regulatory proteins. RPD3 can then repress gene transcription. In the yeast Saccharomyces cerevisiae, association of RPD3 with the transcriptional repressors SIN3 and UME6 results in repression of reporter genes containing the UME6-binding site. RPD3 can deacetylate all histone H4 acetylation sites in cell extracts. However, it is unknown how H4 proteins located at genes near UME6-binding sites are affected, nor whether the effect of RPD3 is localized to the promoter regions. The mechanism by which RPD3 represses gene activity has been studed by examining the acetylation state of histone proteins at UME6-regulated genes. Antibodies specific for individual acetylation sites in H4 have been used to immunoprecipitate chromatin fragments. A deletion of RPD3 or SIN3, but not of the related histone-deacetylase gene HDA1, results in increased acetylation of the lysine 5 residue of H4 in the promoters of the UME6-regulated INO1, IME2 and SPO13 genes. As increased acetylation of this residue is not merely a consequence of gene transcription, acetylation of this site may be essential for regulating gene activity (Rundlett, 1998).

Full activation of the HO promoter in vivo requires the Gcn5 histone acetyltransferase protein. Defects in this protein can be suppressed by deletion of the RPD3 gene, which encodes a histone deacetylase. These results suggest an interplay between acetylation and deacetylation of histones in the regulation of the HO gene. Mutations in either the H4 or the H3 histone gene, as well as mutations in the SIN1 gene, which encodes an HMG1-like protein, strongly suppress the defects produced by the gcn5 mutant. These results suggest a hierarchy of action in the process of chromatin remodeling (Perez-Martin, 1998).

In a screen for extragenic suppressors of a silencing defective rap 1s hmr delta A strain, recessive mutations in 21 different genes were found that restored repression to HMR. Three of these SDS (suppressors of defective silencing) genes have been characterized. SDS16 and SDS6 are known transcriptional modifiers[SIN3(RPD1/UME4/SDI1/GAM2) and RPD3(SDI2), respectively], while the third is a novel gene, SDS3. SDS3 shares the meiotic functions of SIN3 and RPD3 in that it represses IME2 in haploid cells and is necessary for sporulation in diploid cells. However, sds3 mutations differ from sin3 and rpd3 mutations in that they do not derepress TRK2. These sds mutations suppress a variety of cis- and trans-defects that impair the establishment of silencing at HMR. Any one of the sds mutations slightly increases telomere position effect while a striking synergistic increase in repression is observed in a rap 1s background. Epistasis studies suggest that SDS3 works in a different pathway from RPD3 and SIN3 to affect silencing at HMR. Together these results show that defects in certain general transcriptional modifiers can have a pronounced influence on position-effect gene silencing in yeast (Vannier, 1996).

In Saccharomyces cerevisiae, TRK1 and TRK2 encode the high- and low-affinity K+ transporters, respectively. In cells containing a deletion of TRK1, transcription levels of TRK2 are extremely low and are limiting for growth in media containing low levels of K+ (Trk- phenotype). Recessive mutations in RPD1 and RPD3 suppress the TRK2, conferring an approximately fourfold increase in transcription. rpd3 mutations confer pleiotropic phenotypes, including (1) mating defects, (2) hypersensitivity to cycloheximide, (3) inability to sporulate as homozygous diploids, and (4) constitutive derepression of acid phosphatase. RPD3 was cloned and is predicted to encode a 48-kDa protein with no extensive similarity to proteins contained in current data bases. Deletion of RPD3 is not lethal but confers phenotypes identical to those caused by spontaneous mutations. RPD3 is required for both full repression and full activation of transcription of target genes including PHO5, STE6, and TY2. RPD3 is the second gene required for this function, since RPD1 is also required. The effects of mutations in RPD1 and RPD3 are not additive, suggesting that these genes are involved in the same transcriptional regulatory function or pathway (Vidal, 1991).

The Sin3-Rpd3 histone deacetylase complex, conserved between human and yeast, represses transcription when targeted by promoter-specific transcription factors. SIN3 and RPD3 also affect transcriptional silencing at the HM mating loci and at telomeres in yeast. Interestingly, however, deletion of the SIN3 and RPD3 genes enhances silencing, implying that the Sin3-Rpd3 complex functions to counteract, rather than to establish or maintain, silencing. Sin3, Rpd3, and Sap30, a novel component of the Sin3-Rpd3 complex, affect silencing not only at the HMR and telomeric loci, but also at the rDNA locus. The effects on silencing at all three loci are dependent upon the histone deacetylase activity of Rpd3. Enhanced silencing associated with sin3 mutation, rpd3 mutation, and sap30 mutation is differentially dependent upon Sir2 and Sir4 at the telomeric and rDNA loci and is also dependent upon the ubiquitin-conjugating enzyme Rad6 (Ubc2). SAP30 is a component of the Sin3 corepressor complex involved in N-CoR-corepressor-mediated repression by specific transcription factors. The Cac3 subunit of the CAF-I chromatin assembly factor and Sin3-Rpd3 exert antagonistic effects on silencing. Strikingly, deletion of GCN5, which encodes a histone acetyltransferase, enhances silencing in a manner similar to deletion of RPD3 (Sun, 1999).

The following model is proposed to explain the role of Rpd3 in silencing. Accordingly, Rpd3 would play a direct role in silencing by affecting the relative levels of the 'inactive' (heterochromatin) and 'active' (euchromatin) forms of histone H4. This ratio would affect the efficiency of formation of silent chromatin in much the same way that components of silent chromatin and a transcriptional activator compete to establish either the silent or active state of gene expression at telomeres following the disassembly of silent chromatin during DNA replication. This effect was proposed to account for the random nature of phenotypic switching in variegated gene expression. This model is consistent with the recent identification of multiple genes associated with DNA replication and chromatin modification in a genetic screen for rDNA silencing defects. In the mutant sin3, rpd3, or sap30 strains, the relative levels of H4 acetylated at K5 and K12 would increase due to loss of histone deacetylase activity. H4 acetylated at K12 is the inactive form, thereby accounting for enhanced silencing associated with loss of Sin3-Rpd3 function. This scenario is dependent upon substrate specificity of Rpd3 for H4 K12. Indeed, rpd3 mutation enhances acetylation of H4 residues K5 and K12 (Sun, 1999 and references).

This model would also account for the enhanced silencing associated with gcn5 mutants. Accordingly, the Gcn5 histone acetyltransferase would directly affect silencing by catalyzing acetylation of H4 residues K8 and K16. Consistent with this premise, an H4 K16Q replacement, which simulates acetylated K16, disrupts the interaction between H4 and Sir3. This result led to the proposal that K16 hypoacetylation might be important for H4 interaction with Sir3 in heterochromatin. In the gcn5 mutant strain, the levels of H4 acetylated at K8 and K16 would decrease, thereby increasing the relative levels of the inactive form of H4 acetylated at K5 and K12. Again, this proposal is consistent with the specificity of Gcn5 for H4 residues K8 and K16. This model is also applicable for histone H3, because H3 can be a substrate for Rpd3, Gcn5, and CAF-I, and is a structural component of silent chromatin. However, the specific acetylation pattern of H3 in silent chromatin has yet to be defined (Sun, 1999 and references).

To facilitate inheritance of silencing, CAF-I would ensure that only appropriately acetylated inactive histones (both newly synthesized and recycled from the previous cell cycle) are assembled into silent chromatin. CAF-I might also exclude histones with the active acetylation pattern from being recycled into silent chromatin. In the case of a derepressed silent locus from the previous cell cycle, this function would be especially relevant. In the cac mutants, new nucleosomes must be assembled by an alternative pathway. Cac1 protein, a subunit of yeast chromatin assembly factor I, is required for the stable inheritance of transcriptionally repressed chromatin at telomeres. If the alternative assembly complex lacks the substrate specificity of CAF-I, then the increased level of inactive histones associated with the absence of either Rpd3 or Gcn5 would facilitate silent chromatin assembly. This would account for the offsetting effects of cac3 mutants and either rpd3 mutation, sin3 mutation, or sap30 mutation (Sun, 1999 and references).

A second function of CAF-I would be to ensure that local Sir2, Sir3, and Sir4 protein concentrations are sufficiently elevated to permit assembly of a strong silencer. This conclusion is based on improved silencing associated with elevated levels of Sir2, Sir3, or Sir4 in cac1 mutants, and on disruption of silencing associated with limiting amounts of Sir2 or Sir3 in an otherwise wild-type background. Therefore, the decreased local SIR protein concentrations associated with cac3 mutation would partially weaken the enhanced silencing caused by sin3 mutation, rpd3 mutation, and sap30 mutation. This is consistent with the observation that loss of the Sin3-Rpd3 complex does not bypass the SIR protein requirement for maintaining silencing (Sun, 1999 and references).

A key feature of this model is that the acetylation state of histones affects the efficiency of assembly of silent chromatin. The model does not propose that the acetylation pattern at silent loci would necessarily change upon deletion of RPD3 or GCN5. Indeed, chromatin immunoprecipitation experiments, demonstrating that rpd3 mutation and sin3 mutation alter the acetylation pattern of histone H4 at Ume6-regulated promoters, show that the H4 acetylation pattern at a telomeric locus is unchanged by rpd3 mutation and sin3 mutation, despite the dramatic effects of these mutations on telomeric silencing (Sun, 1999 and references).

Transcriptional silencing in Saccharomyces cerevisiae occurs at several genetic loci, including the ribosomal DNA (rDNA). Silencing at telomeres (telomere position effect [TPE]) and the cryptic mating-type loci (HML and HMR) depends on the silent information regulator genes, SIR1, SIR2, SIR3, and SIR4. However, silencing of polymerase II-transcribed reporter genes integrated within the rDNA locus (rDNA silencing) requires only SIR2. The mechanism of rDNA silencing is therefore distinct from TPE and HM silencing. Few genes other than SIR2 have so far been linked to the rDNA silencing process. To identify additional non-Sir factors that affect rDNA silencing, a genetic screen designed to isolate mutations which alter the expression of reporter genes integrated within the rDNA was performed. Two classes of mutants were isolated: those with a loss of rDNA silencing (lrs) phenotype and those with an increased rDNA silencing (irs) phenotype. Using transposon mutagenesis, lrs mutants were found in 11 different genes, and irs mutants were found in 22 different genes. Surprisingly, no genes involved in rRNA transcription were isolated. Instead, multiple genes associated with DNA replication and modulation of chromatin structure were isolated. Two gene classes, and two previously uncharacterized genes, LRS4 and IRS4 are described. Further characterization of the lrs and irs mutants reveals that many have alterations in rDNA chromatin structure. Several lrs mutants, including those in the cdc17 and rfc1 genes, cause lengthened telomeres, consistent with the hypothesis that telomere length modulates rDNA silencing. Mutations in the HDB (RPD3) histone deacetylase complex paradoxically increase rDNA silencing by a SIR2-dependent, SIR3-independent mechanism. Mutations in rpd3 also restore mating competence selectively to sir3Delta MATalpha strains, suggesting restoration of silencing at HMR in a sir3 mutant background (Smith, 1999).

Diploid yeast undergo meiosis under certain conditions of nutrient limitation, which triggers a transcriptional cascade involving two key regulatory genes. IME1 is a positive activator of IME2, which activates downstream genes. Gcn5, a histone H3 acetylase, plays a central role in initiation of meiosis via effects on IME2 expression. An allele, gcn5-21, was isolated as a mutant defective in spore formation. gcn5-21 fails to carry out meiotic DNA replication, recombination, or meiotic divisions. This mutant also fails to induce IME2 transcription; IME1 transcription, however, is essentially normal. Further investigation shows that during wild-type meiosis the IME2 promoter undergoes an increase in the level of bound acetylated histone H3. This increase is contemporaneous with meiotic induction of IME2 transcription and is absent in gcn5-21. In contrast, the RPD3 gene, which encodes a histone H4 deacetylase and is known to be required for repression of basal IME2 transcription in growing yeast cells, is not involved in induction of IME2 transcription or IME2 histone acetlyation during meiosis. These and other results suggest that Gcn5 and Rpd3 play distinct roles, modulating transcription initiation in opposite directions under two different cellular conditions. These roles are implemented via opposing effects of the two gene products upon acetylation of two different histones. gcn5 and rpd3 single mutants are not defective in meiosis if acetate is absent and respiration is promoted by a metabolically unrelated carbon source. Perhaps intracellular acetate levels regulate meiosis by controlling histone acetylation patterns (Burgess, 1999).

Prior research has identified the recessive rec3-1ts mutation in Saccharomyces cerevisiae which, in homozygous diploid cells, confers a conditional phenotype resulting in reduced levels of spontaneous mitotic recombination and loss of sporulation at the restrictive temperature of 36 degrees C. A 3.4-kb genomic fragment that complements the rec3-1ts/rec3-1ts mutation and which maps to chromosome XIV, is identical to RPD3, a gene encoding a histone deacetylase. Sporulation is reduced in homozygous diploid strains containing the rec3-1ts allele at 24 degrees C, suggesting that this allele of RPD3 encodes a gene product with a reduced function. Sporulation is abolished in diploid strains homozygous for the rpd3Delta or rec3-1ts alleles, as well as in rpd3Delta/rec3-1ts heteroallelic diploids, at the non-permissive temperature. Acid-phosphatase expression has been shown to be RPD3 dependent. Acid-phosphatase activity is greater in diploid strains homozygous for the temperature-sensitive rec3-1ts allele than in RPD3/RPD3 strains and increases further when mutant strains are grown at 36 degrees C. The rpd3Delta/rpd3Delta strains were tested for their effects on spontaneous mitotic recombination. By assaying a variety of intra- and inter-genic recombination events distributed over three chromosomes, it was found that in the majority of cases spontaneous mitotic recombination is reduced in diploid rpd3Delta/rpd3Delta cells (relative to a RPD3/RPD3 control). Finally, although 90% of mitotic recombinant events are initiated in the G1 phase of the growth cycle (i.e., before DNA synthesis), RPD3 is not regulated in a cell-cycle-dependent manner. These data suggest that mitotic recombination, in addition to gene expression, is affected by changes in chromatin architecture mediated by RPD3 (Dora, 1999).

Transcriptional activation of eukaryotic genes often requires the function of histone acetyltransferases (HATs), which is expected to result in the hyperacetylation of histones within promoter nucleosomes. In Saccharomyces cerevisiae, the steady-state levels of Gcn5-dependent histone acetylation within a number of transcriptionally active promoters are inversely related to the rate of transcription. High acetylation levels were measured only when transcription was attenuated either by TATA element mutations or in a strain carrying a temperature-sensitive protein component of RNA polymerase II. In addition, it is shown that in one case the low levels of histone acetylation depends on the function of the Rpd3 histone deacetylase. These results point to the existence of an unexpected interplay of two opposing histone-modifying activities which operate on promoter nucleosomes following the initiation of RNA synthesis. Such interplay could ensure rapid turnover of chromatin acetylation states in continuously reprogrammed transcriptional systems (Topalidou, 2003).

The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. Rapamycin and nutrient starvation, conditions inhibiting TOR, are found to lead to significant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. Evidence is presented that TOR regulates association of Rpd3-Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site-specific deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3Delta and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, these results suggest a chromatin-mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability (Tsang, 2003).

Histone acetylation regulates the time of replication origin firing in yeast

The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. The state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p (see Drosophila CDC45) to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing (Vogelauer, 2002).

DNA synthesis in yeast follows a temporal pattern with origins of replication firing at different times during S phase. While an origin may itself contain some of the information for its timing, e.g., ARS301, a number of findings argue that the time of origin activation is influenced by its chromosomal position. For example, it has been demonstrated that proximity to the telomere can confer late activation on an origin. Late origins that are not close to telomeres may also be regulated by chromosomal context since they maintain their late replication on plasmids only if sizeable regions (~15 kb) of the surrounding chromosomal sequences are included on the plasmids. Elements that promote early replication may also exist; it was recently shown that origins near centromeres (within ~30 kb) replicate earlier than the genome average (Vogelauer, 2002).

The position effect on the time of origin activation is a dynamic process that is established in early G1 of every cell cycle. During G1, a prereplicative complex (pre-RC) that contains Orc1-6p, Cdc6p, and six minichromosome maintenance proteins (Mcm2-7p) is assembled at origins. The firing of a specific origin occurs only after the activation of S phase-specific cyclin dependent kinases (S-Cdkp) and the Cdc7p/Dbf4p kinase. In turn, activation of these kinases promotes the association of Cdc45p with the pre-RC, a step that is required for recruitment of DNA polymerase alpha. Since Cdc45p binding to late origins is delayed relative to binding at early origins, its association with origins may reflect the temporal order of their firing. Two kinases, Clb5p and Rad53p, seem to be implicated in the proper execution of the temporal program (Vogelauer, 2002 and references therein).

The accessibility of origin DNA to the proteins that initiate replication is likely to be influenced by chromatin structure. The degree of chromatin compaction is thought to be modulated by the reversible acetylation of the amino terminal tails of histones. A direct role for reversible histone acetylation in transcription regulation has been demonstrated. Histone acetylation mediates gene activation, while deacetylation allows repression. In yeast, histone acetyltransferases and deacetylases function through their recruitment by transcriptional activators or repressors to specific upstream regulatory sequences. However, they do so in a background of global histone acetylation and deacetylation that affects not only promoters but also adjacent coding and noncoding regions (Vogelauer, 2002).

The global action of acetyltransferases (Gcn5p and Esa1p) and deacetylases (Rpd3p and Hda1p) suggests a possible role of histone acetylation in processes other than transcription, such as replication. This idea is supported by a study that suggests a relationship between general trichostatin A-induced acetylation and replication timing. Moreover, it has been found that a deletion of a long-distance upstream regulator of the human ß-globin locus can affect both acetylation and replication timing of this locus. In light of these experiments, it is also significant that Hbo1p, a putative human histone acetyltransferase coimmunoprecipitates with two components of the replication machinery, Orc1p and Mcm2p. At the telomere, delayed origin activation can be achieved through the presence of repressive proteins such as Sir3p that help form a condensed chromatin environment. While these data are consistent with the possibility that chromatin structure surrounding origins can regulate the time of origin firing, none of them demonstrate a cause and effect relationship between histone acetylation and replication in vivo (Vogelauer, 2002).

In this paper the possibility that histone acetylation and deacetylation can influence the timing of origin firing has been tested in vivo. It is demonstrated not only that histone deacetylation by Rpd3p delays origin firing but also that targeted acetylation of a late origin by Gcn5p causes it to fire earlier. These data show that histone acetylation is a regulator of the timing of origin firing (Vogelauer, 2002).

rRNA transcription in Saccharomyces cerevisiae is performed by RNA polymerase I and regulated by changes in growth conditions. During log phase, approximately 50% of the ribosomal DNA (rDNA) genes in each cell are transcribed and maintained in an open, psoralen-accessible conformation. During stationary phase, the percentage of open rDNA genes is greatly reduced. In this study it has been found that the Rpd3 histone deacetylase is required to inactivate (close) individual rDNA genes as cells entered stationary phase. Even though approximately 50% of the rDNA genes remain open during stationary phase in rpd3Delta mutants, overall rRNA synthesis is still reduced. Using electron microscopy of Miller chromatin spreads, it was found that the number of RNA polymerases transcribing each open gene in the rpd3Delta mutant is significantly reduced when cells grow past log phase. Bulk levels of histone H3 and H4 acetylation are reduced during stationary phase in an RPD3-dependent manner. However, histone H3 and H4 acetylation is not significantly altered at the rDNA locus in an rpd3Delta mutant. Rpd3 therefore regulates the number of open rDNA repeats (Sandmeier, 2002).

Yeast histone deacetylases and mitotic checkpoints

Expression of the human forkhead/winged helix transcription factor, CHES1 (checkpoint suppressor 1; FOXN3), suppresses sensitivity to DNA damage and restores damage-induced G(2)/M arrest in checkpoint-deficient strains of Saccharomyces cerevisiae. A functional glutathione S-transferase-Ches1 fusion protein binds in vivo to Sin3, a component of the S. cerevisiae Sin3/Rpd3 histone deacetylase complex. Checkpoint mutant strains with SIN3 deleted show increased resistance to UV irradiation, which is not further enhanced by CHES1 expression. Conversely, overexpression of SIN3 blocks the Ches1-mediated G(2)/M delay in response to DNA damage, which is consistent with Ches1 acting by inhibiting the Sin3/Rpd3 complex. Deletion of either SIN3 or RPD3 in rad9 or mec1 checkpoint mutant strains suppresses sensitivity to replication blocks and DNA damage resulting from Cdc9 ligase deficiency and UV irradiation. SIN3 or RPD3 deletions also restored G(2)/M arrest after DNA damage without concomitant Rad53 phosphorylation in mec1 mutant strains. This DNA damage response is absent in mad1 spindle checkpoint mutants. These data suggest that modulation of chromatin structure may regulate checkpoint responses in S. cerevisiae. Inhibition of histone deacetylation results in a DNA damage checkpoint response mediated by the spindle checkpoint pathway that compensates for loss of the primary DNA damage checkpoint pathway (Scott, 2003).

Histone deacetylases of other invertebrate species

continued: Rpd3 Evolutionary homologs part 3/3 | back to part 1/3 |


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