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Multiple sequence-directed possibilities provide a pool of nucleosome position choices in different states of activity of a gene.

Vinayachandran V, Pusarla RH, Bhargava P - Epigenetics Chromatin (2009)

Bottom Line: While the DNA sequences may help decide their locations, the observed positions in vivo are end-results of chromatin remodeling, the state of gene activity and binding of the sequence-specific factors to the DNA, all of which influence nucleosome positions.Thus, the observed nucleosome locations in vivo do not reflect the true contribution of DNA sequence to the mapped position.On a gene locus, multiple nucleosome positions are directed by a gene sequence to provide a pool of possibilities, out of which the preferred ones are selected by the chromatin remodeler and transcription factor of the gene under different states of activity of the gene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Cellular and Molecular Biology, (Council of Scientific and Industrial Research), Uppal Road, Hyderabad-500007, India. vinesh@ccmb.res.in

ABSTRACT

Background: Genome-wide mappings of nucleosome occupancy in different species have shown presence of well-positioned nucleosomes. While the DNA sequences may help decide their locations, the observed positions in vivo are end-results of chromatin remodeling, the state of gene activity and binding of the sequence-specific factors to the DNA, all of which influence nucleosome positions. Thus, the observed nucleosome locations in vivo do not reflect the true contribution of DNA sequence to the mapped position. Moreover, the naturally occurring nucleosome-positioning sequences are known to guide multiple translational positionings.

Results: We show that yeast SNR6, a gene transcribed by RNA polymerase III, constitutes nucleosome-positioning sequence. In the absence of a chromatin remodeler or any factor binding, the gene sequence confers a unique rotational phase to nucleosomes in the gene region, and directs assembly of several translationally positioned nucleosomes on approximately 1.2 kb DNA from the gene locus, including the short approximately 250 bp gene region. Mapping of all these gene sequence-directed nucleosome positions revealed that the array of nucleosomes in the gene upstream region occupy the same positions as those observed in vivo but the nucleosomes on the gene region can be arranged in three distinct registers. Two of these arrangements differ from each other in the position of only one nucleosome, and match with the nucleosome positions on the gene in repressed and active states in vivo, where the gene-specific factor is known to occupy the gene in both the states. The two positions are interchanged by an ATP-dependent chromatin remodeler in vivo. The third register represents the positions which block the access of the factor to the gene promoter elements.

Conclusion: On a gene locus, multiple nucleosome positions are directed by a gene sequence to provide a pool of possibilities, out of which the preferred ones are selected by the chromatin remodeler and transcription factor of the gene under different states of activity of the gene.

No MeSH data available.


Related in: MedlinePlus

Nucleosomes on the SNR6 gene are rotationally positioned. (A) Schematic representation of the selected gene regions, PCR-amplified from p-539H6 with their names and sizes is given. (B) Gel mobility shift assay of chromatin assembled over end-labeled 601c, SNR6ab and SNR6us DNA fragments (lanes 2, 4 and 6, respectively). Lanes 1, 3 and 5 show free DNA controls. Gray oval on the left-hand side marks the position of the centrally positioned nucleosome. (C) to (F) Hydroxyl radical cleavage of the DNA. Numbers identify the cleavage peaks of chromatin in base pairs. (C) and (F) Digestion times are given in minutes; 0 min. represents undigested DNA. Cleavage pattern of the chromatin samples, C, are shown in the lanes 3 and 4 for the top strand gel and lanes 4 and 5 for bottom strand gels. Lanes 1 and 2 show digestion pattern of naked DNA (N), while a 10 bp DNA ladder (Invitrogen) was end-labeled and used as size marker in the lanes M. (D) Profile of the digestion pattern of the top strand of SNR6ab chromatin. (E) Profile of the digestion pattern of the top strand of SNR6us chromatin from the gel shown in panel (F).
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Figure 3: Nucleosomes on the SNR6 gene are rotationally positioned. (A) Schematic representation of the selected gene regions, PCR-amplified from p-539H6 with their names and sizes is given. (B) Gel mobility shift assay of chromatin assembled over end-labeled 601c, SNR6ab and SNR6us DNA fragments (lanes 2, 4 and 6, respectively). Lanes 1, 3 and 5 show free DNA controls. Gray oval on the left-hand side marks the position of the centrally positioned nucleosome. (C) to (F) Hydroxyl radical cleavage of the DNA. Numbers identify the cleavage peaks of chromatin in base pairs. (C) and (F) Digestion times are given in minutes; 0 min. represents undigested DNA. Cleavage pattern of the chromatin samples, C, are shown in the lanes 3 and 4 for the top strand gel and lanes 4 and 5 for bottom strand gels. Lanes 1 and 2 show digestion pattern of naked DNA (N), while a 10 bp DNA ladder (Invitrogen) was end-labeled and used as size marker in the lanes M. (D) Profile of the digestion pattern of the top strand of SNR6ab chromatin. (E) Profile of the digestion pattern of the top strand of SNR6us chromatin from the gel shown in panel (F).

Mentions: We used DNase I footprinting (Figure 2) and hydroxyl radical cleavage (Figure 3) to find the presence of a 10 bp ladder, characteristic of rotationally positioned nucleosomes, on the gene region. DNase I footprinting of both the strands of the chromatin assembled on the plasmid pCS6 (Figure 2, panels A and B) shows a frequency of cut with ~8 to 12 bp periodicity. DNase I cuts two strands of DNA with a 4 bp stagger and there may be an error of 1 to 2 bp in upper parts of the gel in identifying the cut positions. Nevertheless, combining the mapping on both the strands, a 10 bp periodicity of cuts in the whole region can be seen, suggesting that ~180 bp DNA sequence between the boxes A and B (from +33 bp to +213 bp) may have a rotational nucleosome positioning signal.


Multiple sequence-directed possibilities provide a pool of nucleosome position choices in different states of activity of a gene.

Vinayachandran V, Pusarla RH, Bhargava P - Epigenetics Chromatin (2009)

Nucleosomes on the SNR6 gene are rotationally positioned. (A) Schematic representation of the selected gene regions, PCR-amplified from p-539H6 with their names and sizes is given. (B) Gel mobility shift assay of chromatin assembled over end-labeled 601c, SNR6ab and SNR6us DNA fragments (lanes 2, 4 and 6, respectively). Lanes 1, 3 and 5 show free DNA controls. Gray oval on the left-hand side marks the position of the centrally positioned nucleosome. (C) to (F) Hydroxyl radical cleavage of the DNA. Numbers identify the cleavage peaks of chromatin in base pairs. (C) and (F) Digestion times are given in minutes; 0 min. represents undigested DNA. Cleavage pattern of the chromatin samples, C, are shown in the lanes 3 and 4 for the top strand gel and lanes 4 and 5 for bottom strand gels. Lanes 1 and 2 show digestion pattern of naked DNA (N), while a 10 bp DNA ladder (Invitrogen) was end-labeled and used as size marker in the lanes M. (D) Profile of the digestion pattern of the top strand of SNR6ab chromatin. (E) Profile of the digestion pattern of the top strand of SNR6us chromatin from the gel shown in panel (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667510&req=5

Figure 3: Nucleosomes on the SNR6 gene are rotationally positioned. (A) Schematic representation of the selected gene regions, PCR-amplified from p-539H6 with their names and sizes is given. (B) Gel mobility shift assay of chromatin assembled over end-labeled 601c, SNR6ab and SNR6us DNA fragments (lanes 2, 4 and 6, respectively). Lanes 1, 3 and 5 show free DNA controls. Gray oval on the left-hand side marks the position of the centrally positioned nucleosome. (C) to (F) Hydroxyl radical cleavage of the DNA. Numbers identify the cleavage peaks of chromatin in base pairs. (C) and (F) Digestion times are given in minutes; 0 min. represents undigested DNA. Cleavage pattern of the chromatin samples, C, are shown in the lanes 3 and 4 for the top strand gel and lanes 4 and 5 for bottom strand gels. Lanes 1 and 2 show digestion pattern of naked DNA (N), while a 10 bp DNA ladder (Invitrogen) was end-labeled and used as size marker in the lanes M. (D) Profile of the digestion pattern of the top strand of SNR6ab chromatin. (E) Profile of the digestion pattern of the top strand of SNR6us chromatin from the gel shown in panel (F).
Mentions: We used DNase I footprinting (Figure 2) and hydroxyl radical cleavage (Figure 3) to find the presence of a 10 bp ladder, characteristic of rotationally positioned nucleosomes, on the gene region. DNase I footprinting of both the strands of the chromatin assembled on the plasmid pCS6 (Figure 2, panels A and B) shows a frequency of cut with ~8 to 12 bp periodicity. DNase I cuts two strands of DNA with a 4 bp stagger and there may be an error of 1 to 2 bp in upper parts of the gel in identifying the cut positions. Nevertheless, combining the mapping on both the strands, a 10 bp periodicity of cuts in the whole region can be seen, suggesting that ~180 bp DNA sequence between the boxes A and B (from +33 bp to +213 bp) may have a rotational nucleosome positioning signal.

Bottom Line: While the DNA sequences may help decide their locations, the observed positions in vivo are end-results of chromatin remodeling, the state of gene activity and binding of the sequence-specific factors to the DNA, all of which influence nucleosome positions.Thus, the observed nucleosome locations in vivo do not reflect the true contribution of DNA sequence to the mapped position.On a gene locus, multiple nucleosome positions are directed by a gene sequence to provide a pool of possibilities, out of which the preferred ones are selected by the chromatin remodeler and transcription factor of the gene under different states of activity of the gene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Cellular and Molecular Biology, (Council of Scientific and Industrial Research), Uppal Road, Hyderabad-500007, India. vinesh@ccmb.res.in

ABSTRACT

Background: Genome-wide mappings of nucleosome occupancy in different species have shown presence of well-positioned nucleosomes. While the DNA sequences may help decide their locations, the observed positions in vivo are end-results of chromatin remodeling, the state of gene activity and binding of the sequence-specific factors to the DNA, all of which influence nucleosome positions. Thus, the observed nucleosome locations in vivo do not reflect the true contribution of DNA sequence to the mapped position. Moreover, the naturally occurring nucleosome-positioning sequences are known to guide multiple translational positionings.

Results: We show that yeast SNR6, a gene transcribed by RNA polymerase III, constitutes nucleosome-positioning sequence. In the absence of a chromatin remodeler or any factor binding, the gene sequence confers a unique rotational phase to nucleosomes in the gene region, and directs assembly of several translationally positioned nucleosomes on approximately 1.2 kb DNA from the gene locus, including the short approximately 250 bp gene region. Mapping of all these gene sequence-directed nucleosome positions revealed that the array of nucleosomes in the gene upstream region occupy the same positions as those observed in vivo but the nucleosomes on the gene region can be arranged in three distinct registers. Two of these arrangements differ from each other in the position of only one nucleosome, and match with the nucleosome positions on the gene in repressed and active states in vivo, where the gene-specific factor is known to occupy the gene in both the states. The two positions are interchanged by an ATP-dependent chromatin remodeler in vivo. The third register represents the positions which block the access of the factor to the gene promoter elements.

Conclusion: On a gene locus, multiple nucleosome positions are directed by a gene sequence to provide a pool of possibilities, out of which the preferred ones are selected by the chromatin remodeler and transcription factor of the gene under different states of activity of the gene.

No MeSH data available.


Related in: MedlinePlus