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Distal chromatin structure influences local nucleosome positions and gene expression.

Jansen A, van der Zande E, Meert W, Fink GR, Verstrepen KJ - Nucleic Acids Res. (2012)

Bottom Line: In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity.Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes.Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Systems Biology, VIB, Bio-Incubator, Gaston Geenslaan 1, B-3001, Leuven, Belgium.

ABSTRACT
The positions of nucleosomes across the genome influence several cellular processes, including gene transcription. However, our understanding of the factors dictating where nucleosomes are located and how this affects gene regulation is still limited. Here, we perform an extensive in vivo study to investigate the influence of the neighboring chromatin structure on local nucleosome positioning and gene expression. Using truncated versions of the Saccharomyces cerevisiae URA3 gene, we show that nucleosome positions in the URA3 promoter are at least partly determined by the local DNA sequence, with so-called 'anti-nucleosomal elements' like poly(dA:dT) tracts being key determinants of nucleosome positions. In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity. Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes. Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression.

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Progressive 5′–3′ deletion of the URA3 promoter results in a gradual decrease in URA3 expression. (A) The URA3 promoter has a poly(dA:dT)-rich region required for basal expression between positions −206 and −157, a UAS bound by the transcriptional activator Ppr1 between positions −154 and −139 (dashed underline) and two functionally distinct (constitutive and regulatory) TATA elements between positions −109 and −80. The promoter has eight different transcription initiation sites, consisting of sites inducible by Ppr1 at positions −41, −38, −33, −28 and −19, and constitutive sites at positions −60, −55 and −47. DNA-bound Ppr1 is not transcriptionally active and Ppr1-induced expression requires increased levels of dihydroorotic acid (DHO). Adapted from Ref. (27). (B) Truncation of the URA3 promoter leads to changes in growth on SC-ura and 5-FOA medium. Mutants are labeled by the number of remaining nucleotides in the URA3 promoter. Δpoly(dA:dT) indicates a mutant strain only deleted for the URA3 promoter poly(dA:dT) sequence. These mutants also show differences in doubling times (C), normalized mean Ura3-YFP protein abundance (D) and normalized URA3 mRNA expression (E) for growth in liquid YPD (black bars) and liquid SC-ura (gray bars) media. In YPD medium, URA3 protein and mRNA levels for strains 161–100 did not differ significantly from the negative control. In SC-ura medium, we were unable to obtain reproducible URA3 protein and mRNA levels for strains 161–100 because of the very poor growth of these strains in this medium. Error bars denote standard deviation.
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gkr1311-F1: Progressive 5′–3′ deletion of the URA3 promoter results in a gradual decrease in URA3 expression. (A) The URA3 promoter has a poly(dA:dT)-rich region required for basal expression between positions −206 and −157, a UAS bound by the transcriptional activator Ppr1 between positions −154 and −139 (dashed underline) and two functionally distinct (constitutive and regulatory) TATA elements between positions −109 and −80. The promoter has eight different transcription initiation sites, consisting of sites inducible by Ppr1 at positions −41, −38, −33, −28 and −19, and constitutive sites at positions −60, −55 and −47. DNA-bound Ppr1 is not transcriptionally active and Ppr1-induced expression requires increased levels of dihydroorotic acid (DHO). Adapted from Ref. (27). (B) Truncation of the URA3 promoter leads to changes in growth on SC-ura and 5-FOA medium. Mutants are labeled by the number of remaining nucleotides in the URA3 promoter. Δpoly(dA:dT) indicates a mutant strain only deleted for the URA3 promoter poly(dA:dT) sequence. These mutants also show differences in doubling times (C), normalized mean Ura3-YFP protein abundance (D) and normalized URA3 mRNA expression (E) for growth in liquid YPD (black bars) and liquid SC-ura (gray bars) media. In YPD medium, URA3 protein and mRNA levels for strains 161–100 did not differ significantly from the negative control. In SC-ura medium, we were unable to obtain reproducible URA3 protein and mRNA levels for strains 161–100 because of the very poor growth of these strains in this medium. Error bars denote standard deviation.

Mentions: To investigate the influence of chromatin context on local nucleosome positioning, we inserted the URA3 gene at various locations in the S. cerevisiae genome and explored the consequences for URA3 nucleosome distribution. URA3 was chosen because the nucleosome structure of this gene is well characterized and reflects the nucleosome pattern of a typical yeast gene, containing a 5′ NFR that is surrounded by two highly localized nucleosomes (Figure 1A, 2A) (27,32,43). Moreover, expression of URA3 can be easily estimated by growth on two different substrates: SC-ura, a growth medium lacking uracil and allowing growth only if URA3 is induced, and 5-FOA, allowing growth in the absence of basal URA3 transcription (44,45).Figure 1.


Distal chromatin structure influences local nucleosome positions and gene expression.

Jansen A, van der Zande E, Meert W, Fink GR, Verstrepen KJ - Nucleic Acids Res. (2012)

Progressive 5′–3′ deletion of the URA3 promoter results in a gradual decrease in URA3 expression. (A) The URA3 promoter has a poly(dA:dT)-rich region required for basal expression between positions −206 and −157, a UAS bound by the transcriptional activator Ppr1 between positions −154 and −139 (dashed underline) and two functionally distinct (constitutive and regulatory) TATA elements between positions −109 and −80. The promoter has eight different transcription initiation sites, consisting of sites inducible by Ppr1 at positions −41, −38, −33, −28 and −19, and constitutive sites at positions −60, −55 and −47. DNA-bound Ppr1 is not transcriptionally active and Ppr1-induced expression requires increased levels of dihydroorotic acid (DHO). Adapted from Ref. (27). (B) Truncation of the URA3 promoter leads to changes in growth on SC-ura and 5-FOA medium. Mutants are labeled by the number of remaining nucleotides in the URA3 promoter. Δpoly(dA:dT) indicates a mutant strain only deleted for the URA3 promoter poly(dA:dT) sequence. These mutants also show differences in doubling times (C), normalized mean Ura3-YFP protein abundance (D) and normalized URA3 mRNA expression (E) for growth in liquid YPD (black bars) and liquid SC-ura (gray bars) media. In YPD medium, URA3 protein and mRNA levels for strains 161–100 did not differ significantly from the negative control. In SC-ura medium, we were unable to obtain reproducible URA3 protein and mRNA levels for strains 161–100 because of the very poor growth of these strains in this medium. Error bars denote standard deviation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3351160&req=5

gkr1311-F1: Progressive 5′–3′ deletion of the URA3 promoter results in a gradual decrease in URA3 expression. (A) The URA3 promoter has a poly(dA:dT)-rich region required for basal expression between positions −206 and −157, a UAS bound by the transcriptional activator Ppr1 between positions −154 and −139 (dashed underline) and two functionally distinct (constitutive and regulatory) TATA elements between positions −109 and −80. The promoter has eight different transcription initiation sites, consisting of sites inducible by Ppr1 at positions −41, −38, −33, −28 and −19, and constitutive sites at positions −60, −55 and −47. DNA-bound Ppr1 is not transcriptionally active and Ppr1-induced expression requires increased levels of dihydroorotic acid (DHO). Adapted from Ref. (27). (B) Truncation of the URA3 promoter leads to changes in growth on SC-ura and 5-FOA medium. Mutants are labeled by the number of remaining nucleotides in the URA3 promoter. Δpoly(dA:dT) indicates a mutant strain only deleted for the URA3 promoter poly(dA:dT) sequence. These mutants also show differences in doubling times (C), normalized mean Ura3-YFP protein abundance (D) and normalized URA3 mRNA expression (E) for growth in liquid YPD (black bars) and liquid SC-ura (gray bars) media. In YPD medium, URA3 protein and mRNA levels for strains 161–100 did not differ significantly from the negative control. In SC-ura medium, we were unable to obtain reproducible URA3 protein and mRNA levels for strains 161–100 because of the very poor growth of these strains in this medium. Error bars denote standard deviation.
Mentions: To investigate the influence of chromatin context on local nucleosome positioning, we inserted the URA3 gene at various locations in the S. cerevisiae genome and explored the consequences for URA3 nucleosome distribution. URA3 was chosen because the nucleosome structure of this gene is well characterized and reflects the nucleosome pattern of a typical yeast gene, containing a 5′ NFR that is surrounded by two highly localized nucleosomes (Figure 1A, 2A) (27,32,43). Moreover, expression of URA3 can be easily estimated by growth on two different substrates: SC-ura, a growth medium lacking uracil and allowing growth only if URA3 is induced, and 5-FOA, allowing growth in the absence of basal URA3 transcription (44,45).Figure 1.

Bottom Line: In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity.Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes.Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Systems Biology, VIB, Bio-Incubator, Gaston Geenslaan 1, B-3001, Leuven, Belgium.

ABSTRACT
The positions of nucleosomes across the genome influence several cellular processes, including gene transcription. However, our understanding of the factors dictating where nucleosomes are located and how this affects gene regulation is still limited. Here, we perform an extensive in vivo study to investigate the influence of the neighboring chromatin structure on local nucleosome positioning and gene expression. Using truncated versions of the Saccharomyces cerevisiae URA3 gene, we show that nucleosome positions in the URA3 promoter are at least partly determined by the local DNA sequence, with so-called 'anti-nucleosomal elements' like poly(dA:dT) tracts being key determinants of nucleosome positions. In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity. Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes. Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression.

Show MeSH
Related in: MedlinePlus