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A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions.

Basheer A, Berger H, Reyes-Dominguez Y, Gorfer M, Strauss J - Nucleic Acids Res. (2009)

Bottom Line: To close this gap between the traditional and the high-throughput procedures we have developed a method in which a condition-specific, genome-wide chromatin fragment library is produced and then used for locus-specific DNA fragment analysis.To validate the method, we used, as a test locus, the well-studied promoter of the divergently transcribed niiA and niaD genes coding for nitrate assimilation enzymes in Aspergillus.Additionally, we have used the condition-specific libraries to study nucleosomal positioning at two different loci, the promoters of the general nitrogen regulator areA and the regulator of secondary metabolism, aflR.

View Article: PubMed Central - PubMed

Affiliation: Austrian Research Centers, Department of Applied Genetics and Cell Biology, BOKU University Vienna, Vienna, Austria.

ABSTRACT
Traditional chromatin analysis methods only test one locus at the time or use different templates for each locus, making a standardized analysis of large genomic regions or many co-regulated genes at different loci a difficult task. On the other hand, genome-wide high-resolution mapping of chromatin accessibility employing massive parallel sequencing platforms generates an extensive data set laborious to analyse and is a cost-intensive method, only applicable to the analysis of a limited set of biological samples. To close this gap between the traditional and the high-throughput procedures we have developed a method in which a condition-specific, genome-wide chromatin fragment library is produced and then used for locus-specific DNA fragment analysis. To validate the method, we used, as a test locus, the well-studied promoter of the divergently transcribed niiA and niaD genes coding for nitrate assimilation enzymes in Aspergillus. Additionally, we have used the condition-specific libraries to study nucleosomal positioning at two different loci, the promoters of the general nitrogen regulator areA and the regulator of secondary metabolism, aflR.

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Either locus-specific or adaptor-binding primers can be labelled for analytical PCRs and fragment size analysis. The fragment size profiles of analytical PCRs using either the labelled locus-specific primer (green line profiles) or the labelled adaptor-binding primer (blue line profiles) are compared. Two independent A-B adaptor fragment libraries have been tested for this comparison and both profiles represent the MNase accessibility of the locus AN7802.3 at different time points 1 and 2. ‘P’ indicates non-incorporated primer signals.
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Figure 5: Either locus-specific or adaptor-binding primers can be labelled for analytical PCRs and fragment size analysis. The fragment size profiles of analytical PCRs using either the labelled locus-specific primer (green line profiles) or the labelled adaptor-binding primer (blue line profiles) are compared. Two independent A-B adaptor fragment libraries have been tested for this comparison and both profiles represent the MNase accessibility of the locus AN7802.3 at different time points 1 and 2. ‘P’ indicates non-incorporated primer signals.

Mentions: The approach used throughout this study requires one labelled primer for each locus amplification step. When multiple loci need to be analysed in one condition-specific chromatin fragment library, this strategy becomes costly due to the increased costs of primer labelling. We therefore have tested if also the opposite primer binding to the adaptor (primer Amplifyer-Ain) can be labelled and non-labelled locus-specific primers can be used for amplification. This strategy would considerably reduce consumables costs for multi-locus analysis. To this end, we generated two additional A-B adaptor fragment libraries and compared chromatin accessibility profiles in both libraries between the different primer labelling strategies. Figure 5 shows the results of both libraries comparing profiles obtained with gene-specific primer labelling (blue line profiles) with profiles obtained with adaptor primer labelling (green line profiles). Labelled and non-labelled gene-specific primers are amplifying an A. nidulans locus involved in secondary metabolism regulation (aflR promoter, gene number AN7802.3). Both, the green line (adaptor-specific labelled primer) and the blue line (gene-specific labelled primer) profiles match very well in both libraries indicating that labelling the adaptor-binding primer for fragment amplification is a suitable strategy to economize multi-locus chromatin accessibility analyses with the library-based method. The difference in peak intensities reflects the differences in PCR efficiencies in the individual reactions. Both reactions start with the same amount of FAM-labelled primers, in the case of the adaptor-specific primer the higher peak intensities of the PCR products correlate with the lower intensity of the primer peak.Figure 5.


A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions.

Basheer A, Berger H, Reyes-Dominguez Y, Gorfer M, Strauss J - Nucleic Acids Res. (2009)

Either locus-specific or adaptor-binding primers can be labelled for analytical PCRs and fragment size analysis. The fragment size profiles of analytical PCRs using either the labelled locus-specific primer (green line profiles) or the labelled adaptor-binding primer (blue line profiles) are compared. Two independent A-B adaptor fragment libraries have been tested for this comparison and both profiles represent the MNase accessibility of the locus AN7802.3 at different time points 1 and 2. ‘P’ indicates non-incorporated primer signals.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665225&req=5

Figure 5: Either locus-specific or adaptor-binding primers can be labelled for analytical PCRs and fragment size analysis. The fragment size profiles of analytical PCRs using either the labelled locus-specific primer (green line profiles) or the labelled adaptor-binding primer (blue line profiles) are compared. Two independent A-B adaptor fragment libraries have been tested for this comparison and both profiles represent the MNase accessibility of the locus AN7802.3 at different time points 1 and 2. ‘P’ indicates non-incorporated primer signals.
Mentions: The approach used throughout this study requires one labelled primer for each locus amplification step. When multiple loci need to be analysed in one condition-specific chromatin fragment library, this strategy becomes costly due to the increased costs of primer labelling. We therefore have tested if also the opposite primer binding to the adaptor (primer Amplifyer-Ain) can be labelled and non-labelled locus-specific primers can be used for amplification. This strategy would considerably reduce consumables costs for multi-locus analysis. To this end, we generated two additional A-B adaptor fragment libraries and compared chromatin accessibility profiles in both libraries between the different primer labelling strategies. Figure 5 shows the results of both libraries comparing profiles obtained with gene-specific primer labelling (blue line profiles) with profiles obtained with adaptor primer labelling (green line profiles). Labelled and non-labelled gene-specific primers are amplifying an A. nidulans locus involved in secondary metabolism regulation (aflR promoter, gene number AN7802.3). Both, the green line (adaptor-specific labelled primer) and the blue line (gene-specific labelled primer) profiles match very well in both libraries indicating that labelling the adaptor-binding primer for fragment amplification is a suitable strategy to economize multi-locus chromatin accessibility analyses with the library-based method. The difference in peak intensities reflects the differences in PCR efficiencies in the individual reactions. Both reactions start with the same amount of FAM-labelled primers, in the case of the adaptor-specific primer the higher peak intensities of the PCR products correlate with the lower intensity of the primer peak.Figure 5.

Bottom Line: To close this gap between the traditional and the high-throughput procedures we have developed a method in which a condition-specific, genome-wide chromatin fragment library is produced and then used for locus-specific DNA fragment analysis.To validate the method, we used, as a test locus, the well-studied promoter of the divergently transcribed niiA and niaD genes coding for nitrate assimilation enzymes in Aspergillus.Additionally, we have used the condition-specific libraries to study nucleosomal positioning at two different loci, the promoters of the general nitrogen regulator areA and the regulator of secondary metabolism, aflR.

View Article: PubMed Central - PubMed

Affiliation: Austrian Research Centers, Department of Applied Genetics and Cell Biology, BOKU University Vienna, Vienna, Austria.

ABSTRACT
Traditional chromatin analysis methods only test one locus at the time or use different templates for each locus, making a standardized analysis of large genomic regions or many co-regulated genes at different loci a difficult task. On the other hand, genome-wide high-resolution mapping of chromatin accessibility employing massive parallel sequencing platforms generates an extensive data set laborious to analyse and is a cost-intensive method, only applicable to the analysis of a limited set of biological samples. To close this gap between the traditional and the high-throughput procedures we have developed a method in which a condition-specific, genome-wide chromatin fragment library is produced and then used for locus-specific DNA fragment analysis. To validate the method, we used, as a test locus, the well-studied promoter of the divergently transcribed niiA and niaD genes coding for nitrate assimilation enzymes in Aspergillus. Additionally, we have used the condition-specific libraries to study nucleosomal positioning at two different loci, the promoters of the general nitrogen regulator areA and the regulator of secondary metabolism, aflR.

Show MeSH