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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

Exo III mapping of the nucleosomes on the 5' Half SNR6 DNA fragments. Chromatin was assembled on the 248 bp DNA fragments labeled at the 5' end of either the top strand (panel A) or the bottom strand (panel B). A 10 bp DNA ladder (Invitrogen) was end-labeled and used as size marker in the lanes M. Sizes of the marker bands are given in the left-hand side of both the panels while arrows on the right-hand side give the sizes of the DNA fragments due to the pauses of Exo III. Lanes 1 and 5 in both the panels show the uncut DNA. Other lanes show naked DNA or chromatin digested for different times by Exo III. (C) Schematic representation of mapping results from the panels A and B. Insert represents the cloned genomic DNA from +62 to +256 bp positions in the SNR6 gene while the flanking 48 and 34 bp are vector-derived DNA. The numbers on right-hand side are the Exo III pauses seen in the gels and represent the nucleosome boundaries.
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Figure 5: Exo III mapping of the nucleosomes on the 5' Half SNR6 DNA fragments. Chromatin was assembled on the 248 bp DNA fragments labeled at the 5' end of either the top strand (panel A) or the bottom strand (panel B). A 10 bp DNA ladder (Invitrogen) was end-labeled and used as size marker in the lanes M. Sizes of the marker bands are given in the left-hand side of both the panels while arrows on the right-hand side give the sizes of the DNA fragments due to the pauses of Exo III. Lanes 1 and 5 in both the panels show the uncut DNA. Other lanes show naked DNA or chromatin digested for different times by Exo III. (C) Schematic representation of mapping results from the panels A and B. Insert represents the cloned genomic DNA from +62 to +256 bp positions in the SNR6 gene while the flanking 48 and 34 bp are vector-derived DNA. The numbers on right-hand side are the Exo III pauses seen in the gels and represent the nucleosome boundaries.

Mentions: To dissect the gene sequence further with respect to its nucleosome positioning capability, we reconstituted mononucleosomes over DNA fragments of ~240 to 270 bp sizes (Figure 4A) carrying the two halves of the gene and mapped the locations of nucleosomes on them by using exonuclease III (Exo III) digestion assay. As compared with the IEL method, which does not differentiate between different rotational positions, Exo III mapping of nucleosomes positioned on the 5'-half DNA (Figure 5) shows the presence of several translational positions with a unique rotational phase. Mapping of the Exo III-resistant positions on both the strands, which are not well resolved in the top of the gel (lanes 6–8, panel A), shows the presence of multiple positions with 10 bp differences (lanes 6–8, panels A-D). Exo III shows a prominent pause at the +1 (marked by asterisk, 122 bp band) and -110 (232 bp band) base pair positions on the bottom strand of SNR6 (panels C and D). Out of several possibilities, mapping of both the boundaries of at least four major positions (Table 1, position nos. 7 to 10), reveals the nucleosome boundaries are 155 bp apart. Taking 156 bp as the nucleosomal size, a sequence-based nucleosome position from -116 to +40 bp, similar to position number 7 (Table 1) has been predicted on the SNR6 DNA ([26], ). Application of 145 bp nucleosome size to the most interior Exo III pauses at -20 bp on the bottom strand and +25 bp on the top strand (a comparatively weaker pause at 152 bp size DNA in lanes 6–8, panel A) would place the ends of the two nucleosomes at +125 and -120 bp, respectively, (positions 11 and 6, Table 1) at the extreme ends of this DNA (panel E). Thus, -20 to +25 bp of SNR6 represents a DNA stretch, central and common in all possible nucleosome positions on the 5' half of SNR6 (panel E), which brings the +2 bp of SNR6 to the center of all the possible positions. Exo III shows a pause at the +1 bp position (panels D and E, the asterisk), which shows alignment of two nucleosomes on its two sides in the 5'-half DNA (Figure 4B), suggesting this may be a central, reference position for organization of nucleosomes on this helically phased DNA. These results suggest that depending on the context, nucleosomes either exclude or assemble over the +1 site. The possibility of multiple positions at uniform intervals on the 5'-half DNA suggests that the protection seen upstream of +110 bp position in the plasmid p-539H6 (Figure 1B) may be due to the translationally positioned nucleosomes with unique rotational settings and not due to a unique translational position. Thus, a similar positioning may be observed in vivo due to this sequence, present as part of the full-length gene.


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)

Exo III mapping of the nucleosomes on the 5' Half SNR6 DNA fragments. Chromatin was assembled on the 248 bp DNA fragments labeled at the 5' end of either the top strand (panel A) or the bottom strand (panel B). A 10 bp DNA ladder (Invitrogen) was end-labeled and used as size marker in the lanes M. Sizes of the marker bands are given in the left-hand side of both the panels while arrows on the right-hand side give the sizes of the DNA fragments due to the pauses of Exo III. Lanes 1 and 5 in both the panels show the uncut DNA. Other lanes show naked DNA or chromatin digested for different times by Exo III. (C) Schematic representation of mapping results from the panels A and B. Insert represents the cloned genomic DNA from +62 to +256 bp positions in the SNR6 gene while the flanking 48 and 34 bp are vector-derived DNA. The numbers on right-hand side are the Exo III pauses seen in the gels and represent the nucleosome boundaries.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Exo III mapping of the nucleosomes on the 5' Half SNR6 DNA fragments. Chromatin was assembled on the 248 bp DNA fragments labeled at the 5' end of either the top strand (panel A) or the bottom strand (panel B). A 10 bp DNA ladder (Invitrogen) was end-labeled and used as size marker in the lanes M. Sizes of the marker bands are given in the left-hand side of both the panels while arrows on the right-hand side give the sizes of the DNA fragments due to the pauses of Exo III. Lanes 1 and 5 in both the panels show the uncut DNA. Other lanes show naked DNA or chromatin digested for different times by Exo III. (C) Schematic representation of mapping results from the panels A and B. Insert represents the cloned genomic DNA from +62 to +256 bp positions in the SNR6 gene while the flanking 48 and 34 bp are vector-derived DNA. The numbers on right-hand side are the Exo III pauses seen in the gels and represent the nucleosome boundaries.
Mentions: To dissect the gene sequence further with respect to its nucleosome positioning capability, we reconstituted mononucleosomes over DNA fragments of ~240 to 270 bp sizes (Figure 4A) carrying the two halves of the gene and mapped the locations of nucleosomes on them by using exonuclease III (Exo III) digestion assay. As compared with the IEL method, which does not differentiate between different rotational positions, Exo III mapping of nucleosomes positioned on the 5'-half DNA (Figure 5) shows the presence of several translational positions with a unique rotational phase. Mapping of the Exo III-resistant positions on both the strands, which are not well resolved in the top of the gel (lanes 6–8, panel A), shows the presence of multiple positions with 10 bp differences (lanes 6–8, panels A-D). Exo III shows a prominent pause at the +1 (marked by asterisk, 122 bp band) and -110 (232 bp band) base pair positions on the bottom strand of SNR6 (panels C and D). Out of several possibilities, mapping of both the boundaries of at least four major positions (Table 1, position nos. 7 to 10), reveals the nucleosome boundaries are 155 bp apart. Taking 156 bp as the nucleosomal size, a sequence-based nucleosome position from -116 to +40 bp, similar to position number 7 (Table 1) has been predicted on the SNR6 DNA ([26], ). Application of 145 bp nucleosome size to the most interior Exo III pauses at -20 bp on the bottom strand and +25 bp on the top strand (a comparatively weaker pause at 152 bp size DNA in lanes 6–8, panel A) would place the ends of the two nucleosomes at +125 and -120 bp, respectively, (positions 11 and 6, Table 1) at the extreme ends of this DNA (panel E). Thus, -20 to +25 bp of SNR6 represents a DNA stretch, central and common in all possible nucleosome positions on the 5' half of SNR6 (panel E), which brings the +2 bp of SNR6 to the center of all the possible positions. Exo III shows a pause at the +1 bp position (panels D and E, the asterisk), which shows alignment of two nucleosomes on its two sides in the 5'-half DNA (Figure 4B), suggesting this may be a central, reference position for organization of nucleosomes on this helically phased DNA. These results suggest that depending on the context, nucleosomes either exclude or assemble over the +1 site. The possibility of multiple positions at uniform intervals on the 5'-half DNA suggests that the protection seen upstream of +110 bp position in the plasmid p-539H6 (Figure 1B) may be due to the translationally positioned nucleosomes with unique rotational settings and not due to a unique translational position. Thus, a similar positioning may be observed in vivo due to this sequence, present as part of the full-length gene.

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