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• W2098002295 abstract "Article15 October 1999free access Functional interaction between GCN5 and polyamines: a new role for core histone acetylation Kerri J. Pollard Kerri J. Pollard Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA, 01605 USA Search for more papers by this author Michael L. Samuels Michael L. Samuels Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA, 01605 USA Search for more papers by this author Kimberly A. Crowley Kimberly A. Crowley Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA, 01605 USA Search for more papers by this author Jeffrey C. Hansen Jeffrey C. Hansen Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78284-7760 USA Search for more papers by this author Craig L. Peterson Corresponding Author Craig L. Peterson Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA, 01605 USA Search for more papers by this author Kerri J. Pollard Kerri J. Pollard Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA, 01605 USA Search for more papers by this author Michael L. Samuels Michael L. Samuels Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA, 01605 USA Search for more papers by this author Kimberly A. Crowley Kimberly A. Crowley Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA, 01605 USA Search for more papers by this author Jeffrey C. Hansen Jeffrey C. Hansen Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78284-7760 USA Search for more papers by this author Craig L. Peterson Corresponding Author Craig L. Peterson Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA, 01605 USA Search for more papers by this author Author Information Kerri J. Pollard1, Michael L. Samuels1, Kimberly A. Crowley1, Jeffrey C. Hansen2 and Craig L. Peterson 1 1Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA, 01605 USA 2Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78284-7760 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:5622-5633https://doi.org/10.1093/emboj/18.20.5622 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Polyamines are organic polycations essential for a wide variety of cellular functions, including nuclear integrity and chromosome condensation. Here we present genetic evidence that depletion of cellular polyamines partially alleviates the defects in HO and SUC2 expression caused by inactivation of the GCN5 histone acetyltransferase. In addition, the combination of polyamine depletion and a sin− allele of the histone H4 gene leads to almost complete bypass of the transcriptional requirement for GCN5. In contrast, polyamine depletion does not alter the transcriptional requirements for the SWI/SNF chromatin remodeling complex nor does depletion lead to global defects in transcriptional regulation. In addition to these genetic studies, we show that polyamines facilitate oligomerization of nucleosomal arrays in vitro, and that polyamine-mediated condensation requires intact core histone N-terminal domains and is inhibited by histone hyperacetylation. Our studies suggest that polyamines are repressors of transcription in vivo, and that one role of histone hyperacetylation is to antagonize the ability of polyamines to stabilize highly condensed states of chromosomal fibers. Introduction Polyamines are small, ubiquitous organic polycations that have been implicated in a wide variety of physiological functions including protein translation, membrane stabilization and cell proliferation (reviewed in Tabor and Tabor, 1984). As one might expect, biosynthesis of polyamines is essential for viability of both prokaryotic and eukaryotic cells. The most common polyamines are putrescine, spermidine and spermine, which contain two, three or four charged amine groups, respectively. Eukaryotic organisms contain all three of these amines at abundant levels (high micromolar to millimolar). Much attention has focused on the roles of polyamines in various disease processes. For instance, polyamines have been implicated in autoimmune disorders such as systemic lupus erythematosus (Brooks, 1994). Polyamines also accumulate in cancer cells, and high levels of polyamines are found in the urine from cancer patients (reviewed in Russel and Duri, 1978; Chanda and Ganguly, 1988). Increased levels of ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, are associated with many types of cancer (Pegg, 1988), and overproduction of ODC can lead to acquisition of the transformed cell phenotype (Tabib and Bachrach, 1998). These observations have led to the development of inhibitors of polyamine biosynthesis, and several such drugs have been used successfully in the treatment of some cancers and protozoan diseases, particularly African trypanosomiasis (McCann et al., 1987; Fairlamb, 1990a, b). In terms of polyamine function in the nucleus, several studies have implicated polyamines in the formation of higher order chromosomal fibers in vitro and in vivo (Belmont et al., 1989; Belmont and Bruce, 1994), and spermidine and spermine have been shown to facilitate condensation of chromatin fragments in vitro (Colson and Houssier, 1989). Spermidine, spermine and related analogs have also been shown to interact specifically with nucleosome core particles and DNA in vitro (Morgan et al., 1989). Higher order folding of chromatin requires both cation-dependent charge neutralization and the flexible N-terminal domains of the core histones (reviewed in Fletcher and Hansen, 1996). These 25–40 amino acid N-terminal ‘tails’ are exposed at the surface of the nucleosome core particle (van Holde, 1988; Luger et al., 1997) and contain the sites for post-translational histone acetylation. Recent studies have used nucleosomal array model systems to define the roles of the core histone N-termini and histone acetylation in chromatin condensation (Garcia-Ramirez et al., 1992, 1995; Tse and Hansen, 1997; Tse et al., 1998b). The DNA template for reconstitution of model arrays is composed of 12 tandem repeats of a 208 bp 5S rRNA gene from Lytechinus variegatus (the 208-12 template). When these nucleosomal arrays are incubated in physiological mixtures of monovalent and divalent salt, they both fold extensively and oligomerize (Schwarz and Hansen, 1994). Importantly, although oligomerization has commonly been referred to throughout the literature as ‘precipitation’ or ‘aggregation’, and as such is often dismissed as biologically irrelevant, it has been shown recently that the oligomerization transition is a highly cooperative and fully reversible process (Schwarz et al., 1996) that shares many characteristics known to be involved in chromosomal fiber formation in vivo (reviewed extensively in Fletcher and Hansen, 1996; also see Sen and Crothers, 1986; Widom, 1986; Belmont and Bruce, 1994). Furthermore, both higher order folding and oligomerization are absolutely dependent on the presence of the N-terminal domains (Tse and Hansen, 1997) and are diminished by histone hyperacetylation (Tse et al., 1998b), consistent with a fundamental role for the core histone N-termini in all steps leading to chromosomal fiber condensation (reviewed in Fletcher and Hansen, 1996; Hansen, 1997; Hansen et al., 1998; Luger and Richmond, 1998). Gcn5p is the founding member of a growing family of histone acetyltransferases (Neuwald and Landsman, 1997), which includes several proteins previously identified as transcriptional coactivators. The GCN5 gene was identified initially as a positive regulator of amino acid biosynthetic genes (Georgakopoulos and Thireos, 1992), and subsequently as a putative transcriptional adaptor (Marcus et al., 1994). More recent studies have shown that Gcn5p has intrinsic histone acetyltransferase activity (Brownell et al., 1996), and that it is the catalytic subunit of several, distinct histone acetyltransferase complexes (Grant et al., 1997; Pollard and Peterson, 1997; Ruiz-Garcia et al., 1997; Saleh et al., 1997), and that Gcn5p preferentially acetylates condensed nucleosomal arrays when assayed under optimal conditions in vitro (Tse et al., 1998a). GCN5-dependent histone acetylation is recruited to the promoter region of a small number of genes in yeast, and has been shown to precede and be required for activated transcription (Kuo et al., 1998; Krebs et al., 1999). Gcn5p activity may disrupt a domain of condensed chromatin surrounding the target gene, or acetylation may promote or inhibit the binding of non-histone proteins to the promoter region. Here we describe genetic and biochemical studies that indicate a functional link between GCN5-dependent histone acetylation and polyamine function in vivo and in vitro. We have isolated the ARG3 gene as a multicopy suppressor of the transcriptional defects caused by a mutation in GCN5. Overexpression of Arg3p appears to partially restore transcriptional activity in a gcn5 mutant by limiting the availability of ornithine for polyamine biosynthesis. Consistent with this apparent link, a deletion of the SPE1 gene, which encodes the rate-limiting enzyme for polyamine biosynthesis (ODC), also partially alleviates the defects in HO and SUC2 expression of a gcn5 mutant. Furthermore, we find that the combination of a spe1 deletion and a sin− allele of the histone H4 gene leads to almost complete bypass of the transciptional requirement for GCN5. In complementary in vitro studies, we find both that polyamines facilitate the reversible oligomerization of nucleosomal arrays, and that polyamine-dependent oligomerization requires the core histone N-terminal tails and is diminished by histone acetylation. These studies support the idea that polyamines contribute to transcriptional repression in vivo by stabilizing condensed chromatin fibers, and that histone acetyltransferases, such as Gcn5p, promote transcriptional induction in part by counteracting polyamine-dependent chromatin condensation. Results Multicopy ARG3 alleviates transcriptional defects due to a gcn5 mutation We have used a genetic screen to identify genes that when present in high copy number rescue the transcriptional defects caused by a mutation in the yeast GCN5 gene, which encodes the catalytic subunit of at least three histone acetyltransferase complexes (Grant et al., 1997; Pollard and Peterson, 1997; Saleh et al., 1997). The yeast strain that we have used for this hunt harbors a null allele of GCN5 and an HO–LacZ fusion gene integrated at the chromosomal ho locus. Because HO–LacZ expression requires the GCN5 product (Pollard and Peterson, 1997; Perez-Martin and Johnson, 1998), this gcn5− strain is white in β-galactosidase filter assays. A high copy (100–200 copies/cell) yeast genomic library (Nasmyth and Reed, 1980) was introduced into this gcn5− strain. Of the initial 2500 transformants (approximately one genome equivalent), 60 candidates were isolated that were blue in β-galactosidase filter assays (expressing HO–LacZ). Of these 60 initial positives, 30 plasmids were able to re-confer suppression of the gcn5− phenotype following recovery from yeast and passage through bacteria. From this pool, PCR analysis identified one plasmid that contained the GCN5 gene; this isolate was eliminated from further characterization. After partial sequencing of the genomic inserts of five of the most potent suppressors, we found that two of the plasmids contained overlapping restriction fragments harboring the ARG3 locus as well as several additional genes. To confirm that ARG3 was responsible for high copy suppression, the ARG3 gene was subcloned into the high copy vector and introduced into the gcn5− strain. The isolated ARG3 gene was able to suppress the defect in HO–LacZ expression to a similar level to that of the original, larger clone (data not shown; see Figure 1). In addition, multicopy ARG3 also alleviated the defect in HO–LacZ expression due to a deletion of ADA2 or ADA3, two other components of ADA–GCN5 acetyltransferase complexes (data not shown) (Grant et al., 1997; Pollard and Peterson, 1997; Saleh et al., 1997). Figure 1.Multicopy ARG3 partially alleviates the defect in HO–LacZ expression due to mutations in GCN5. Expression of a chromosomal HO–LacZ fusion was measured by liquid β-galactosidase assays in wild-type (CY432), gcn5− (CY563) and the gcn5− (CY563) strain harboring a high copy plasmid containing the ARG3 ORF (pARG3). Assays were performed with cells grown in the presence or absence of additional ornithine in the growth media. Assays were performed in triplicate and the standard error was <20%. Download figure Download PowerPoint The ARG3 gene encodes ornithine transcarbamoylase, a mitochondrial enzyme responsible for the conversion of ornithine into citrulline, one step of the urea cycle. The only other use of ornithine pools in the cytoplasm is for production of intracellular polyamines (see Figure 2A). To explain our genetic results, we hypothesized that overexpression of ARG3 may deplete the ornithine pool, thereby limiting the production of polyamines, and that depletion of cellular polyamines may be responsible for the suppression of gcn5− transcriptional defects due to their effects on chromatin structure. To test this possibility, we supplemented the growth media with ornithine, then re-evaluated the ability of multicopy ARG3 to alleviate the HO–LacZ defect in the gcn5 mutant (Figure 1). The gcn5 mutant has 3% of the wild-type level of HO–LacZ expression. In the presence of multicopy ARG3, expression is enhanced >3-fold, to 10% of the wild-type level. This effect of multicopy ARG3 on HO–LacZ expression is similar in magnitude to the suppression observed previously for semi-dominant mutations in the genes encoding histone H3 or H4 (Pollard and Peterson, 1997; see also Figure 5). When additional ornithine is present in the growth media, the effect of multicopy ARG3 is decreased such that HO–LacZ expression is enhanced only 1.5-fold in the gcn5 mutant (Figure 1). In contrast, addition of ornithine increases expression of HO–LacZ 1.4-fold in the wild-type strain. These data are consistent with our hypothesis that multicopy ARG3 leads to a depletion of ornithine pools, and that this depletion is required to observe suppression of the gcn5 mutant phenotype. Figure 2.Polyamine depletion partially alleviates gcn5− transcriptional defects. (A) The biosynthetic pathway of polyamine production in Saccharomyces cerevisiae. (B) Depletion of intracellular polyamines. HO–LacZ expression in wild-type (CY773), spe1Δ (CY765), gcn5Δ (CY761) or a spe1Δ gcn5Δ (CY769) double mutant, grown in YPD rich media or polyamine-free media for 4 days. β-galactosidase assays were performed in triplicate and the standard error was <20%. (C) Polyamine add back. The indicated concentrations of spermidine were added to cultures of gcn5Δ (CY761) or spe1Δ gcn5Δ (CY769) double mutants after 4 days of polyamine depletion. HO–LacZ expression was measured as described in Figure 1. (D) SUC2 expression. Levels of invertase activity (SUC2 expression) in wild-type (CY773), spe1Δ (CY765), gcn5Δ (CY761) or spe1Δ gcn5Δ (CY769) double mutants were determined after 4 days growth in polyamine-free media. Invertase activities from three independent cultures were averaged and the standard error was <20%. HO–LacZ was measured in parallel and is shown for comparison. Download figure Download PowerPoint Figure 3.Polyamine depletion and histone sin mutations are additive for alleviation of gcn5 transcription defects. HO–LacZ expression in wild-type (CY773), spe1Δ (CY765), gcn5Δ (CY761) or spe1Δ gcn5Δ (CY769) double mutants grown in polyamine-free media. Strains harbor either a wild-type copy of histone H4 or a semi-dominant (sin−) allele of histone H4 on ARS/CEN plasmids (Kruger et al., 1995; Wechser et al., 1997). Download figure Download PowerPoint Polyamine depletion alleviates gcn5− transcriptional defects Because ornithine is the sole precursor for polyamine biosynthesis (Figure 2A), depletion of cytosolic ornithine would result in decreased levels of polyamines. To test directly whether polyamine depletion can alleviate transcriptional defects due to a deletion of GCN5, we generated a congenic set of strains that harbor deletions of both GCN5 and SPE1, SPE1 encodes ODC, which converts ornithine to putrescine (Figure 2A) (Schwartz et al., 1995). In spe1 mutants, polyamines can not be synthesized, and cellular levels of polyamines decline during growth in polyamine-free media (PFM) (Schwartz et al., 1995). Following 5–6 days of growth in PFM, spe1 cells arrest reversibly in the G1 phase of the cell cycle (Balasundaram et al., 1991). Wild-type, spe1, gcn5 and spe1 gcn5 mutants were grown in PFM for 4 days to deplete polyamine pools, then expression of HO–LacZ was analyzed (Figure 2B). Following 4 days of depletion, spe1 mutants grow slowly, but contain sufficient levels of polyamines to perform essential functions. In the presence of SPE1, growth in PFM had little effect on HO–LacZ expression in either a GCN5+ or gcn5− strain. In contrast, expression in the gcn5 spe1 double mutant was increased nearly 6-fold in comparison with a gcn5− strain grown in PFM (3.7 versus 21% of the wild-type level). Furthermore, the effect of the spe1 mutation was nearly eliminated by growth in rich media [Figure 2B, yeast extract/peptone/dextrose (YPD)] or by addition of spermidine to the PFM (see Figure 2C). Thus, depletion of cellular polyamines can partially alleviate the defect in HO–LacZ expression due to deletion of GCN5. The GCN5 gene product is also required for full expression of the SUC2 gene (Pollard and Peterson, 1997). To test whether polyamine depletion has a more general effect on GCN5-dependent transcription, we assayed expression of SUC2 after depletion of polyamines (Figure 2D). After 4 days of growth in PFM, the gcn5 spe1 double mutant had nearly a 3-fold higher level of SUC2 expression than the level observed in the gcn5 single mutant (8 versus 20% of wild-type levels). In contrast, SUC2 expression in the GCN5 spe1 single mutant was decreased to 82% of the wild-type level. Thus, depletion of polyamines partially alleviates both the HO–LacZ and SUC2 transcriptional defects caused by loss of GCN5 function. Recently we have found that growth of gcn5 mutants is extremely sensitive to low concentrations of t-butyl-hydroperoxide (tBOOH) which is a stable inducer of the oxidative stress response in yeast (Kuge and Jones, 1994). The inability of gcn5 mutants to grow on this medium suggests that GCN5 might be required for expression of one or more genes induced by oxidative stress. To test whether a deletion of SPE1 might alleviate this growth defect, serial dilutions of wild-type, spe1, gcn5 and gcn5 spe1 cells were spotted onto media that contained or lacked tBOOH (Figure 3). Whereas the gcn5 mutant shows a severe growth defect on plates containing tBOOH, the gcn5 spe1 double mutant grows nearly as well as the wild-type strain (upper panel). Thus, polyamine depletion alleviates many of the phenotypes of a gcn5 mutant. Figure 4.Polyamine depletion alleviates the sensitivity of gcn5 mutants to oxidative stress. Strains were grown to an OD600 of 1.0 and diluted to a concentration of 2 × 105 cells/ml in phosphate-buffered saline. A 5 μl aliquot of 4-fold dilutions was spotted onto plates in the absence or presence of 200 μM t-butyl-hydroperoxide (−/+ tBOOH). The spots were allowed to dry and the plates were incubated at 30°C for 3 days. Strains used were wild-type (CY773), spe1Δ (CY765), gcn5Δ (CY761), swi2Δ (CY778), spe1Δ gcn5Δ (CY769) and spe1Δ swi2Δ (CY777). Download figure Download PowerPoint Genetic relationship among polyamines, GCN5 and SWI/SNF complex GCN5 is required for expression of many of the same genes that require the SWI/SNF chromatin remodeling complex (Pollard and Peterson, 1997). In addition, GCN5 and SWI/SNF subunit genes show similar genetic interactions with chromatin components (Pollard and Peterson, 1997; Perez-Martin and Johnson, 1998). For instance, semi-dominant sin− alleles of the genes encoding histones H3 and H4 can partially alleviate the transcriptional defects of gcn5 or swi/snf mutations. Due to the similarities between these two groups of mutants, we tested whether polyamine depletion could alleviate the defects in growth and transcription due to a deletion of the SWI2/SNF2 gene, which encodes the catalytic subunit of the SWI/SNF complex. First, we tested whether a deletion of SPE1 could alleviate the slow growth of a swi2 mutant on media containing tBOOH (Figure 3, lower panel). Unlike the case for gcn5, deletion of SPE1 did not alleviate the slow growth of a swi2 mutant, in fact the swi2 spe1 double mutant was more sensitive to this inducer of oxidative stress. Next, we analyzed expression of the HO–LacZ fusion gene in an isogenic set of SWI+ SPE+, spe1, swi2 and swi2 spe1 strains (Figure 4A). A swi2 deletion decreases HO–LacZ expression to 0.7% of the wild-type level; this transcriptional defect is not suppressed by depletion of polyamines (0.9% of wild type; Figure 4A). Importantly, these results demonstrate that the ability of spe1 to suppress the transcriptional defects of a gcn5 mutant is not due to stabilization or enhanced translation of a low level of lacZ transcripts. These results also indicate that depletion of polyamines does not bypass requirements for all types of chromatin remodeling complexes. Figure 5.Polyamine depletion does not alleviate the requirement for the SWI/SNF chromatin remodeling complex. (A) HO–LacZ expression in wild-type (CY773), spe1Δ (CY765), gcn5Δ (CY761), swi2Δ (CY778), or spe1Δ gcn5Δ (CY769) and spe1Δ swi2Δ (CY777) double mutants grown in PFM for 4 days. (B) Growth rates of yeast strains in PFM. Spe1−, spe1− gcn5− and spe1− swi2− cells were grown in normal or polyamine-free media, with appropriate dilution to maintain an OD600 between 0.05 and 0.8. Doubling times were calculated after growth in culture at the times indicated. Download figure Download PowerPoint One possibility that we considered is that the inability of a spe1 mutation to alleviate the phenotypes of a swi2 mutant is due to the slow growth of this strain, which might delay the kinetics of polyamine depletion. To investigate this possibility, we determined the growth rates of spe1, gcn5 spe1 and swi2 spe1 cells grown in media that contain (SD) or lack (PFM) polyamines (Figure 4B). Similarly to previous studies, we found that the growth rate of spe1 cells began to slow dramatically after ∼3 days of growth in PFM, and cells stopped dividing after 5 days of growth (Figure 4B and data not shown). Likewise, the gcn5 spe1 double mutant showed similar kinetics of growth arrest in PFM. Importantly, the growth rate of the swi2 spe1 double mutants also began to decline after 3 days of culturing in PFM and growth arrest was achieved after 4–5 days (Figure 4B and data not shown). Thus, the slow growth of the swi2 mutant does not appear to slow the kinetics of polyamine depletion. Polyamines and an intact histone octamer contribute equally to transcriptional repression in the absence of GCN5 The genetic interactions between GCN5 and histone H4 (Pollard and Peterson, 1997; Perez-Martin and Johnson, 1998) indicate that one role for GCN5 is to antagonize transcriptional repression mediated by nucleosomes. To examine the genetic relationship between polyamines and histones, we introduced a low copy plasmid containing either a wild-type copy of HHF2 (histone H4) or a semi-dominant sin− allele (Kruger et al., 1995; Wechser et al., 1997) of HHF2 (hhf2-7) into the gcn5 and gcn5 spe1 mutant strains. The semi-dominant histone H4 allele leads to a 4.5-fold increase in HO–LacZ expression in the gcn5 mutant (Figure 5), which is similar to levels of suppression seen with polyamine depletion (Figure 2B). However, when the spe1 deletion is combined with a sin− HHF2 allele, HO–LacZ expression increases 14-fold to nearly 60% of the wild-type level (Figure 5). Thus, the combination of polyamine depletion and a sin− allele of the histone H4 gene almost completely alleviates the GCN5 dependence of HO–LacZ expression. Furthermore, these data suggest that polyamines and the SIN domain of histone H4 act independently to contribute to transcriptional repression. Depletion of polyamines does not lead to global transcriptional defects Our genetic studies indicate that polyamines behave formally as a repressor of transcription, and one role of GCN5 is to antagonize these repressive effects. One possibility is that polyamines control expression of a small subset of genes, perhaps only those genes regulated by GCN5 (e.g. HO and SUC2). Alternatively, the presence or absence of polyamines may exert a global influence on gene expression. To test these possibilities, we investigated the expression of four different genes after SPE1+ or spe1− cells were grown in PFM for 4 days. Cells were either harvested immediately for RNA isolation, or treated for 20 min with copper to induce expression of the CUP1 and SSA4 genes. Figure 6 shows the Northern blot analysis of SSA4, CUP1, DED1 and ADH1 expression after polyamine depletion. In the case of the constitutively expressed genes, DED1 and ADH1, polyamine depletion did not increase or decrease the steady-state level of RNA. Likewise, polyamine depletion did not increase the basal, uninduced level of SSA4 or CUP1 expression, nor did depletion alter the response of SSA4 or CUP1 to copper treatment. Thus, for the four genes tested, polyamine depletion did not influence either the basal or induced level of gene expression. These results are consistent with our analysis of HO and SUC2 expression, where no significant changes were observed unless GCN5 was inactivated (Figure 2). These results reinforce the specificity of the genetic interactions between GCN5 and polyamines, and are consistent with the view that polyamines may inhibit expression of only a small subset of genes. Figure 6.Polyamine depletion does not lead to global changes in gene expression. Spe1− or spe1− gcn5− cells were grown in minimal medium that contains (SD) or lacks polyamines (PFM) for 72 h, and then either not treated or treated with 1 mM copper sulfate for 20 min (−/+ Cu). A single nylon membrane containing mRNA from these cells was hybridized with radiolabeled probes as indicated. Download figure Download PowerPoint Polyamines facilitate reversible oligomerization of nucleosomal arrays How do polyamines act to inhibit transcription in vivo? One possibility is that polyamines stabilize or facilitate the formation of higher order condensed chromatin structures. In this view, GCN5-dependent acetylation would antagonize the repressive effects of polyamines by destabilizing condensed chromatin domains. This could occur either if GCN5 directly acetylates the polyamines themselves, or if GCN5-dependent acetylation of the core histone N-termini counteracts the ability of polyamines to cause chromatin condensation. Two different polyamine acetylases have been described, the cytoplasmic spermidine/spermine N1-acetyltransferase and the nuclear spermidine N8-acetyltransferase (reviewed in Morgan, 1998). Polyamines can also be acetylated by crude preparations of nuclear histone acetyltransferases (Wong et al., 1991). Polyamine acetylation initiates the catabolism of polyamines in vivo and it may also facilitate their transport across nuclear or plasma membranes. As is the case for histones, acetylation of polyamines neutralizes positive charge and is expected to disrupt the binding of polyamines to negatively charged binding sites (i.e. chromatin). We find, however, that native GCN5-containing histone acetyltransferase complexes (Pollard and Peterson, 1997) are unable to incorporate [3H]acetate into putrescine, spermidine or spermine (data not shown). Therefore, to investigate polyamine effects on chromatin structure, we have used defined model systems to determine directly whether polyamines can facilitate condensation of nucleosomal arrays in vitro. The DNA template for reconstitution of model arrays is composed of 11–12 head-to-tail repeats of a 208 bp 5S rRNA gene from Lytechinus variegatus (the 208-11 and 208-12 templates). Each repeat can rotationally and translationally position a nucleosome after in vitro reconstitution with purified histone octamers (Hansen and van Holde, 1991). When these model nucleosomal arrays are incubated in low salt TE buffer, they assume an extended structure that sediments at 28S. Addition of monovalent cations (Na+) induces folding of the arrays to an intermediate 40S level, whereas 1–2 mM Mg2+ results in formation of extensively folded structures that sediment at 55S and a" @default.
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• W2098002295 title "Functional interaction between GCN5 and polyamines: a new role for core histone acetylation" @default.
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