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A computational model of quantitative chromatin immunoprecipitation (ChIP) analysis.

Xie J, Crooke PS, McKinney BA, Soltman J, Brandt SJ - Cancer Inform (2008)

Bottom Line: We developed a computational model of quantitative ChIP analysis to elucidate the factors contributing to the method's resolution.The most important variables identified by the model were, in order of importance, the spacing of the PCR primers, the mean length of the chromatin fragments, and, unexpectedly, the type of fragment width distribution, with very small DNA fragments and smaller amplicons providing the best resolution of TF binding.One of the major predictions of the model was also validated experimentally.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.

ABSTRACT
Chromatin immunoprecipitation (ChIP) analysis is widely used to identify the locations in genomes occupied by transcription factors (TFs). The approach involves chemical cross-linking of DNA with associated proteins, fragmentation of chromatin by sonication or enzymatic digestion, immunoprecipitation of the fragments containing the protein of interest, and then PCR or hybridization analysis to characterize and quantify the genomic sequences enriched. We developed a computational model of quantitative ChIP analysis to elucidate the factors contributing to the method's resolution. The most important variables identified by the model were, in order of importance, the spacing of the PCR primers, the mean length of the chromatin fragments, and, unexpectedly, the type of fragment width distribution, with very small DNA fragments and smaller amplicons providing the best resolution of TF binding. One of the major predictions of the model was also validated experimentally.

No MeSH data available.


Related in: MedlinePlus

Results of quantitative ChIP analysis of upstream region of the mouse Band 3 gene(A) Results of quantitative ChIP analysis of a region upstream of the Band 3 gene, with the numbers denoting the relative position in this region. Data are expressed for each primer pair as a percentage of input DNA. Specific binding was determined by subtracting binding with rabbit IgG from that with affinity-purified rabbit Tal1 antibody. (B) Results of PCR analysis of the same collection of DNA fragments using primer pairs directing the amplification of longer DNA products. Data are expressed as a percentage of input DNA. The same scale is used for the ordinate in A and B so that the amount of TF binding could be compared directly.
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f3-cin-6-0137: Results of quantitative ChIP analysis of upstream region of the mouse Band 3 gene(A) Results of quantitative ChIP analysis of a region upstream of the Band 3 gene, with the numbers denoting the relative position in this region. Data are expressed for each primer pair as a percentage of input DNA. Specific binding was determined by subtracting binding with rabbit IgG from that with affinity-purified rabbit Tal1 antibody. (B) Results of PCR analysis of the same collection of DNA fragments using primer pairs directing the amplification of longer DNA products. Data are expressed as a percentage of input DNA. The same scale is used for the ordinate in A and B so that the amount of TF binding could be compared directly.

Mentions: To test the predictions of the computer model, quantitative ChIP analysis was carried out for a region upstream of the gene for the erythrocyte membrane protein Band 3. A series of primer pairs of different spacing was applied to a common set of DNA fragments, with some of the amplicons necessarily overlapping because of constraints in primer design. In preliminary studies, the helix-loop-helix TF Tal1 was found to occupy this region in murine erythroleukemia cells and was shown to be recruited by another TF, Runx1, to one or more of its five binding sites in the 106 bp interval defined by primer pair #4. Importantly, these primers also gave the greatest signal for Tal1 occupancy in quantitative ChIP analysis (Fig. 3A). Consistent with the computational model, shorter amplicons produced a binding isotherm more closely centered over the determined site(s) of Tal1 association (Fig. 3A), and signal strength diminished and the binding curve flattened with wider spacing between primers (compare Fig. 3B to Fig. 3A). Detailed analysis of the sizes of ethidium bromide-stained DNA fragments from this experiment showed that neither an exponential nor the normal distribution precisely fit the experimental data. Nevertheless, these studies bear out the major prediction of the model, that for a given mean DNA fragment size, the use of smaller amplicons would, in general, provide greater resolution than longer amplicons.


A computational model of quantitative chromatin immunoprecipitation (ChIP) analysis.

Xie J, Crooke PS, McKinney BA, Soltman J, Brandt SJ - Cancer Inform (2008)

Results of quantitative ChIP analysis of upstream region of the mouse Band 3 gene(A) Results of quantitative ChIP analysis of a region upstream of the Band 3 gene, with the numbers denoting the relative position in this region. Data are expressed for each primer pair as a percentage of input DNA. Specific binding was determined by subtracting binding with rabbit IgG from that with affinity-purified rabbit Tal1 antibody. (B) Results of PCR analysis of the same collection of DNA fragments using primer pairs directing the amplification of longer DNA products. Data are expressed as a percentage of input DNA. The same scale is used for the ordinate in A and B so that the amount of TF binding could be compared directly.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2367313&req=5

f3-cin-6-0137: Results of quantitative ChIP analysis of upstream region of the mouse Band 3 gene(A) Results of quantitative ChIP analysis of a region upstream of the Band 3 gene, with the numbers denoting the relative position in this region. Data are expressed for each primer pair as a percentage of input DNA. Specific binding was determined by subtracting binding with rabbit IgG from that with affinity-purified rabbit Tal1 antibody. (B) Results of PCR analysis of the same collection of DNA fragments using primer pairs directing the amplification of longer DNA products. Data are expressed as a percentage of input DNA. The same scale is used for the ordinate in A and B so that the amount of TF binding could be compared directly.
Mentions: To test the predictions of the computer model, quantitative ChIP analysis was carried out for a region upstream of the gene for the erythrocyte membrane protein Band 3. A series of primer pairs of different spacing was applied to a common set of DNA fragments, with some of the amplicons necessarily overlapping because of constraints in primer design. In preliminary studies, the helix-loop-helix TF Tal1 was found to occupy this region in murine erythroleukemia cells and was shown to be recruited by another TF, Runx1, to one or more of its five binding sites in the 106 bp interval defined by primer pair #4. Importantly, these primers also gave the greatest signal for Tal1 occupancy in quantitative ChIP analysis (Fig. 3A). Consistent with the computational model, shorter amplicons produced a binding isotherm more closely centered over the determined site(s) of Tal1 association (Fig. 3A), and signal strength diminished and the binding curve flattened with wider spacing between primers (compare Fig. 3B to Fig. 3A). Detailed analysis of the sizes of ethidium bromide-stained DNA fragments from this experiment showed that neither an exponential nor the normal distribution precisely fit the experimental data. Nevertheless, these studies bear out the major prediction of the model, that for a given mean DNA fragment size, the use of smaller amplicons would, in general, provide greater resolution than longer amplicons.

Bottom Line: We developed a computational model of quantitative ChIP analysis to elucidate the factors contributing to the method's resolution.The most important variables identified by the model were, in order of importance, the spacing of the PCR primers, the mean length of the chromatin fragments, and, unexpectedly, the type of fragment width distribution, with very small DNA fragments and smaller amplicons providing the best resolution of TF binding.One of the major predictions of the model was also validated experimentally.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.

ABSTRACT
Chromatin immunoprecipitation (ChIP) analysis is widely used to identify the locations in genomes occupied by transcription factors (TFs). The approach involves chemical cross-linking of DNA with associated proteins, fragmentation of chromatin by sonication or enzymatic digestion, immunoprecipitation of the fragments containing the protein of interest, and then PCR or hybridization analysis to characterize and quantify the genomic sequences enriched. We developed a computational model of quantitative ChIP analysis to elucidate the factors contributing to the method's resolution. The most important variables identified by the model were, in order of importance, the spacing of the PCR primers, the mean length of the chromatin fragments, and, unexpectedly, the type of fragment width distribution, with very small DNA fragments and smaller amplicons providing the best resolution of TF binding. One of the major predictions of the model was also validated experimentally.

No MeSH data available.


Related in: MedlinePlus