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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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Nucleosome positioning analysis at the areA gene promoter. (A) Overview of the 700 bp promoter region of areA. The FAM-labelled gene-specific primers used for analytical PCR and subsequent fragment analysis are shown as horizontal arrows and named according to their relative position within the promoter sequence. The colours of the primers represent the colour code of the fragment size profiles. Blue arrowheads indicate the position of predicted AreA binding GATA sites in the promoter of this positively autoregulated gene. The start of the areA coding region (ORF) is indicated by the bent arrow. A summary of MNase hypersensitive sites obtained from fragment size analysis of the in vitro control DNA is presented (indicated by vertical blue arrows). Numbers below the blue arrows indicate the exact nucleotide position of the MNase cut in the areA promoter region. Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the in vitro MNase digest library using the primers indicted above are shown below the locus overview. As in the other figures, ‘P’ indicates signals originating from non-incorporated labelled primers. (B) Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the same MNase digest libraries as used for the analysis of the niiA-niaD region (Figure 2). Analytical PCRs were carried out using labelled primers indicated in panel A. The areA promoter profiles of the two libraries (induced, repressed) are shown here. The nucleosome positioned within the reading frame of areA is depicted as orf 1 and nucleosomes in the promoter region are designated −1 and −2. Under conditions in which AreA is active on its own promoter (induced) additional MNase cutting sites are revealed in nucleosome −2 at position ∼650 bp, at the end of nucleosome −1 at position ∼930 bp as well as in the orf1 nucleosome. ‘P’ indicates non-incorporated primer signals.
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Figure 4: Nucleosome positioning analysis at the areA gene promoter. (A) Overview of the 700 bp promoter region of areA. The FAM-labelled gene-specific primers used for analytical PCR and subsequent fragment analysis are shown as horizontal arrows and named according to their relative position within the promoter sequence. The colours of the primers represent the colour code of the fragment size profiles. Blue arrowheads indicate the position of predicted AreA binding GATA sites in the promoter of this positively autoregulated gene. The start of the areA coding region (ORF) is indicated by the bent arrow. A summary of MNase hypersensitive sites obtained from fragment size analysis of the in vitro control DNA is presented (indicated by vertical blue arrows). Numbers below the blue arrows indicate the exact nucleotide position of the MNase cut in the areA promoter region. Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the in vitro MNase digest library using the primers indicted above are shown below the locus overview. As in the other figures, ‘P’ indicates signals originating from non-incorporated labelled primers. (B) Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the same MNase digest libraries as used for the analysis of the niiA-niaD region (Figure 2). Analytical PCRs were carried out using labelled primers indicated in panel A. The areA promoter profiles of the two libraries (induced, repressed) are shown here. The nucleosome positioned within the reading frame of areA is depicted as orf 1 and nucleosomes in the promoter region are designated −1 and −2. Under conditions in which AreA is active on its own promoter (induced) additional MNase cutting sites are revealed in nucleosome −2 at position ∼650 bp, at the end of nucleosome −1 at position ∼930 bp as well as in the orf1 nucleosome. ‘P’ indicates non-incorporated primer signals.

Mentions: We used this method to investigate the currently non-resolved nucleosomal pattern of the promoter region of areA (Figure 4). We used the same MNase-derived A-B adaptor fragment libraries (repressed and induced) as in previous experiments (Figures 2 and 3) and amplified fragments with three different FAM-labelled primers binding to the areA promoter region. Fragment size analysis of the amplification products allowed us to resolve three positioned nucleosomes under repressed conditions (Figure 4B, chromatogram ‘repressed’). Interestingly, positioned nucleosome −1 covers a region which contains three putative AreA-binding GATA sites. Platt and co-workers (23) have shown that AreA is positively autoregulated. These GATA sites might therefore be excluded by the nucleosome for binding AreA under repressive conditions, leading to disruption of the positive autoregulation loop under nitrogen metabolite repressing conditions. In contrast, nitrate induction leads to chromatin remodelling as seen from the elevated MNase accessibility in the promoter region and in the first nucleosome of the areA ORF. Comparison of the induced MNase patterns obtained by the traditional end-labelling method and the fragment chromatograms again showed a good correlation of digestion profiles (Supplementary Figure S1).Figure 4.


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)

Nucleosome positioning analysis at the areA gene promoter. (A) Overview of the 700 bp promoter region of areA. The FAM-labelled gene-specific primers used for analytical PCR and subsequent fragment analysis are shown as horizontal arrows and named according to their relative position within the promoter sequence. The colours of the primers represent the colour code of the fragment size profiles. Blue arrowheads indicate the position of predicted AreA binding GATA sites in the promoter of this positively autoregulated gene. The start of the areA coding region (ORF) is indicated by the bent arrow. A summary of MNase hypersensitive sites obtained from fragment size analysis of the in vitro control DNA is presented (indicated by vertical blue arrows). Numbers below the blue arrows indicate the exact nucleotide position of the MNase cut in the areA promoter region. Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the in vitro MNase digest library using the primers indicted above are shown below the locus overview. As in the other figures, ‘P’ indicates signals originating from non-incorporated labelled primers. (B) Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the same MNase digest libraries as used for the analysis of the niiA-niaD region (Figure 2). Analytical PCRs were carried out using labelled primers indicated in panel A. The areA promoter profiles of the two libraries (induced, repressed) are shown here. The nucleosome positioned within the reading frame of areA is depicted as orf 1 and nucleosomes in the promoter region are designated −1 and −2. Under conditions in which AreA is active on its own promoter (induced) additional MNase cutting sites are revealed in nucleosome −2 at position ∼650 bp, at the end of nucleosome −1 at position ∼930 bp as well as in the orf1 nucleosome. ‘P’ indicates non-incorporated primer signals.
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Figure 4: Nucleosome positioning analysis at the areA gene promoter. (A) Overview of the 700 bp promoter region of areA. The FAM-labelled gene-specific primers used for analytical PCR and subsequent fragment analysis are shown as horizontal arrows and named according to their relative position within the promoter sequence. The colours of the primers represent the colour code of the fragment size profiles. Blue arrowheads indicate the position of predicted AreA binding GATA sites in the promoter of this positively autoregulated gene. The start of the areA coding region (ORF) is indicated by the bent arrow. A summary of MNase hypersensitive sites obtained from fragment size analysis of the in vitro control DNA is presented (indicated by vertical blue arrows). Numbers below the blue arrows indicate the exact nucleotide position of the MNase cut in the areA promoter region. Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the in vitro MNase digest library using the primers indicted above are shown below the locus overview. As in the other figures, ‘P’ indicates signals originating from non-incorporated labelled primers. (B) Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the same MNase digest libraries as used for the analysis of the niiA-niaD region (Figure 2). Analytical PCRs were carried out using labelled primers indicated in panel A. The areA promoter profiles of the two libraries (induced, repressed) are shown here. The nucleosome positioned within the reading frame of areA is depicted as orf 1 and nucleosomes in the promoter region are designated −1 and −2. Under conditions in which AreA is active on its own promoter (induced) additional MNase cutting sites are revealed in nucleosome −2 at position ∼650 bp, at the end of nucleosome −1 at position ∼930 bp as well as in the orf1 nucleosome. ‘P’ indicates non-incorporated primer signals.
Mentions: We used this method to investigate the currently non-resolved nucleosomal pattern of the promoter region of areA (Figure 4). We used the same MNase-derived A-B adaptor fragment libraries (repressed and induced) as in previous experiments (Figures 2 and 3) and amplified fragments with three different FAM-labelled primers binding to the areA promoter region. Fragment size analysis of the amplification products allowed us to resolve three positioned nucleosomes under repressed conditions (Figure 4B, chromatogram ‘repressed’). Interestingly, positioned nucleosome −1 covers a region which contains three putative AreA-binding GATA sites. Platt and co-workers (23) have shown that AreA is positively autoregulated. These GATA sites might therefore be excluded by the nucleosome for binding AreA under repressive conditions, leading to disruption of the positive autoregulation loop under nitrogen metabolite repressing conditions. In contrast, nitrate induction leads to chromatin remodelling as seen from the elevated MNase accessibility in the promoter region and in the first nucleosome of the areA ORF. Comparison of the induced MNase patterns obtained by the traditional end-labelling method and the fragment chromatograms again showed a good correlation of digestion profiles (Supplementary Figure S1).Figure 4.

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