Limits...
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.

Show MeSH
Chromatin restriction enzyme accessibility assay employing the library approach. (A) Overview of the positions of HaeIII restriction endonuclease sites in the niiA-niaD region. Restriction site positions are indicated by vertical blue arrows and numbers below the arrows indicate cutting site positions in the bidirectional promoter region. Asterisks above two sites (145 and 151) indicate cutting events obtained in the assay at positions which do not correspond to a HaeIII site and which probably represent sites of HaeIII star activity. Nucleosome positions, transcriptional start points and analytical PCR primer positions are as described in Figure 2. (B) Overlapping fragment size profiles in the niiA-niaD region obtained by fragment size analysis of libraries derived from HaeIII-treated chromatin. Primers and colour code are as in Figure 2. Two libraries are compared: (i) ‘induced’ indicates the profiles obtained from the library constructed from HaeIII chromatin digestion of cells treated with nitrate; and (ii) ‘repressed’ indicates the profiles obtained from the library constructed from HaeIII chromatin digestion of cells treated with ammonium. As in Figure 2, ‘P’ indicates signals originating from non-incorporated labelled primers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2665225&req=5

Figure 3: Chromatin restriction enzyme accessibility assay employing the library approach. (A) Overview of the positions of HaeIII restriction endonuclease sites in the niiA-niaD region. Restriction site positions are indicated by vertical blue arrows and numbers below the arrows indicate cutting site positions in the bidirectional promoter region. Asterisks above two sites (145 and 151) indicate cutting events obtained in the assay at positions which do not correspond to a HaeIII site and which probably represent sites of HaeIII star activity. Nucleosome positions, transcriptional start points and analytical PCR primer positions are as described in Figure 2. (B) Overlapping fragment size profiles in the niiA-niaD region obtained by fragment size analysis of libraries derived from HaeIII-treated chromatin. Primers and colour code are as in Figure 2. Two libraries are compared: (i) ‘induced’ indicates the profiles obtained from the library constructed from HaeIII chromatin digestion of cells treated with nitrate; and (ii) ‘repressed’ indicates the profiles obtained from the library constructed from HaeIII chromatin digestion of cells treated with ammonium. As in Figure 2, ‘P’ indicates signals originating from non-incorporated labelled primers.

Mentions: Nucleosome positioning in a region with known sequence can also be analysed by accessibility to specific restriction enzymes. Indirect end-labelling following electrophoretic separation (36) or quantitative PCR amplification after restriction enzyme digestion (27) has been used for this purpose. We have tested here if the library-based method can also be used for restriction enzyme accessibility tests and analysed the niiA-niaD Intergenic region (IGR) locus using the HaeIII restriction enzyme. The relative positions of all HaeIII sites in the IGR are shown in Figure 3A. The fragment analysis in Figure 3B shows that the majority of HaeIII sites are accessible under open chromatin, nitrate-induced conditions (chromatogram ‘induced’), but accessibility is restricted under ammonium-repressing conditions (chromatogram ‘repressed’).Figure 3.


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)

Chromatin restriction enzyme accessibility assay employing the library approach. (A) Overview of the positions of HaeIII restriction endonuclease sites in the niiA-niaD region. Restriction site positions are indicated by vertical blue arrows and numbers below the arrows indicate cutting site positions in the bidirectional promoter region. Asterisks above two sites (145 and 151) indicate cutting events obtained in the assay at positions which do not correspond to a HaeIII site and which probably represent sites of HaeIII star activity. Nucleosome positions, transcriptional start points and analytical PCR primer positions are as described in Figure 2. (B) Overlapping fragment size profiles in the niiA-niaD region obtained by fragment size analysis of libraries derived from HaeIII-treated chromatin. Primers and colour code are as in Figure 2. Two libraries are compared: (i) ‘induced’ indicates the profiles obtained from the library constructed from HaeIII chromatin digestion of cells treated with nitrate; and (ii) ‘repressed’ indicates the profiles obtained from the library constructed from HaeIII chromatin digestion of cells treated with ammonium. As in Figure 2, ‘P’ indicates signals originating from non-incorporated labelled primers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Chromatin restriction enzyme accessibility assay employing the library approach. (A) Overview of the positions of HaeIII restriction endonuclease sites in the niiA-niaD region. Restriction site positions are indicated by vertical blue arrows and numbers below the arrows indicate cutting site positions in the bidirectional promoter region. Asterisks above two sites (145 and 151) indicate cutting events obtained in the assay at positions which do not correspond to a HaeIII site and which probably represent sites of HaeIII star activity. Nucleosome positions, transcriptional start points and analytical PCR primer positions are as described in Figure 2. (B) Overlapping fragment size profiles in the niiA-niaD region obtained by fragment size analysis of libraries derived from HaeIII-treated chromatin. Primers and colour code are as in Figure 2. Two libraries are compared: (i) ‘induced’ indicates the profiles obtained from the library constructed from HaeIII chromatin digestion of cells treated with nitrate; and (ii) ‘repressed’ indicates the profiles obtained from the library constructed from HaeIII chromatin digestion of cells treated with ammonium. As in Figure 2, ‘P’ indicates signals originating from non-incorporated labelled primers.
Mentions: Nucleosome positioning in a region with known sequence can also be analysed by accessibility to specific restriction enzymes. Indirect end-labelling following electrophoretic separation (36) or quantitative PCR amplification after restriction enzyme digestion (27) has been used for this purpose. We have tested here if the library-based method can also be used for restriction enzyme accessibility tests and analysed the niiA-niaD Intergenic region (IGR) locus using the HaeIII restriction enzyme. The relative positions of all HaeIII sites in the IGR are shown in Figure 3A. The fragment analysis in Figure 3B shows that the majority of HaeIII sites are accessible under open chromatin, nitrate-induced conditions (chromatogram ‘induced’), but accessibility is restricted under ammonium-repressing conditions (chromatogram ‘repressed’).Figure 3.

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