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On the Mechanism of Gene Silencing in Saccharomyces cerevisiae.

Steakley DL, Rine J - G3 (Bethesda) (2015)

Bottom Line: Multiple mechanisms have been proposed for gene silencing in Saccharomyces cerevisiae, ranging from steric occlusion of DNA binding proteins from their recognition sequences in silenced chromatin to a specific block in the formation of the preinitiation complex to a block in transcriptional elongation.Moreover, unlike previous suggestions, we found no evidence for stalled RNA polymerase II within silenced chromatin.The effectiveness of the Sir protein-based silencing mechanism to block transcription activated by Gal4 at promoters in the domain of silenced chromatin was marginal, yet it improved when tested against mutant forms of the Gal4 protein, highlighting a role for specific activators in their sensitivity to gene silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, California Institute of Quantitative Biosciences, Stanley Hall, University of California Berkeley, Berkeley, California 94720.

No MeSH data available.


Related in: MedlinePlus

Analysis of HMR Gal1pro:a1 transcription by wild-type Gal4 vs. gal4L331P. a1 mRNA expression from GAL1pro::a1 as determined by qRT-PCR of mRNA from strains bearing Gal4 or the gal4L331P mutant in SIR4 and sir4∆ in galactose medium. All a1 expression values were normalized as in Figure 5B. All cultures were seeded from saturated overnight growth. Each bar represents the average and SE of three biological replicates.
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fig9: Analysis of HMR Gal1pro:a1 transcription by wild-type Gal4 vs. gal4L331P. a1 mRNA expression from GAL1pro::a1 as determined by qRT-PCR of mRNA from strains bearing Gal4 or the gal4L331P mutant in SIR4 and sir4∆ in galactose medium. All a1 expression values were normalized as in Figure 5B. All cultures were seeded from saturated overnight growth. Each bar represents the average and SE of three biological replicates.

Mentions: Abf1 is the transcription factor responsible for activating transcription from MATa (McBroom and Sadowski 1995) and therefore is susceptible to silencing at HMR, whose regulatory region sequence is identical to MATa. To explore why Gal4 was largely immune from silencing at HMR whereas Abf1 is sensitive, we tested whether mutant forms of Gal4 that compromise some aspect of its function would become sensitive to silencing. A point mutant in the central domain of Gal4 (gal4L331P) compromises its ability to activate transcription but not its ability to bind DNA (Johnston and Dover 1988; Mylin et al. 1990). As expected, relative to wild-type Gal4, the gal4L331P mutant protein showed a 25-fold reduced ability to activate transcription of HMR GAL1pro::a1 in sir4∆ strains (Figure 9, note the y-axis scale). In SIR4 cells a1 transcript levels were even further reduced (Figure 9, right panel), with 12-fold instead of the 2.5-fold reduction of wild-type Gal4. Thus, in this example, a weaker activator exhibited a greater degree of sensitivity to silencing than a stronger version of that same activator. This relationship between sensitivity to silencing and activator strength of Gal4 was observed for some other alleles of GAL4, but exceptions such as gal4-763∆851 indicate that silencing sensitivity is influenced by more than simply activator strength (Table 4).


On the Mechanism of Gene Silencing in Saccharomyces cerevisiae.

Steakley DL, Rine J - G3 (Bethesda) (2015)

Analysis of HMR Gal1pro:a1 transcription by wild-type Gal4 vs. gal4L331P. a1 mRNA expression from GAL1pro::a1 as determined by qRT-PCR of mRNA from strains bearing Gal4 or the gal4L331P mutant in SIR4 and sir4∆ in galactose medium. All a1 expression values were normalized as in Figure 5B. All cultures were seeded from saturated overnight growth. Each bar represents the average and SE of three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4528331&req=5

fig9: Analysis of HMR Gal1pro:a1 transcription by wild-type Gal4 vs. gal4L331P. a1 mRNA expression from GAL1pro::a1 as determined by qRT-PCR of mRNA from strains bearing Gal4 or the gal4L331P mutant in SIR4 and sir4∆ in galactose medium. All a1 expression values were normalized as in Figure 5B. All cultures were seeded from saturated overnight growth. Each bar represents the average and SE of three biological replicates.
Mentions: Abf1 is the transcription factor responsible for activating transcription from MATa (McBroom and Sadowski 1995) and therefore is susceptible to silencing at HMR, whose regulatory region sequence is identical to MATa. To explore why Gal4 was largely immune from silencing at HMR whereas Abf1 is sensitive, we tested whether mutant forms of Gal4 that compromise some aspect of its function would become sensitive to silencing. A point mutant in the central domain of Gal4 (gal4L331P) compromises its ability to activate transcription but not its ability to bind DNA (Johnston and Dover 1988; Mylin et al. 1990). As expected, relative to wild-type Gal4, the gal4L331P mutant protein showed a 25-fold reduced ability to activate transcription of HMR GAL1pro::a1 in sir4∆ strains (Figure 9, note the y-axis scale). In SIR4 cells a1 transcript levels were even further reduced (Figure 9, right panel), with 12-fold instead of the 2.5-fold reduction of wild-type Gal4. Thus, in this example, a weaker activator exhibited a greater degree of sensitivity to silencing than a stronger version of that same activator. This relationship between sensitivity to silencing and activator strength of Gal4 was observed for some other alleles of GAL4, but exceptions such as gal4-763∆851 indicate that silencing sensitivity is influenced by more than simply activator strength (Table 4).

Bottom Line: Multiple mechanisms have been proposed for gene silencing in Saccharomyces cerevisiae, ranging from steric occlusion of DNA binding proteins from their recognition sequences in silenced chromatin to a specific block in the formation of the preinitiation complex to a block in transcriptional elongation.Moreover, unlike previous suggestions, we found no evidence for stalled RNA polymerase II within silenced chromatin.The effectiveness of the Sir protein-based silencing mechanism to block transcription activated by Gal4 at promoters in the domain of silenced chromatin was marginal, yet it improved when tested against mutant forms of the Gal4 protein, highlighting a role for specific activators in their sensitivity to gene silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, California Institute of Quantitative Biosciences, Stanley Hall, University of California Berkeley, Berkeley, California 94720.

No MeSH data available.


Related in: MedlinePlus