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Promoter-proximal transcription factor binding is transcriptionally active when coupled with nucleosome repositioning in immediate vicinity.

Yadav VK, Thakur RK, Eckloff B, Baral A, Singh A, Halder R, Kumar A, Alam MP, Kundu TK, Pandita R, Pandita TK, Wieben ED, Chowdhury S - Nucleic Acids Res. (2014)

Bottom Line: These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown.For example, physiologic TF binding studies involve many TFs, consequently, it is difficult to assign nucleosome reorganization to the binding site occupancy of any particular TF.Therefore, several aspects remain unclear: does TF binding influence nucleosome (re)organizations locally or impact the chromatin landscape at a more global level; are all or only a fraction of TF binding a result of reorganization in nucleosome occupancy and do all TF binding and associated changes in nucleosome occupancy result in altered gene expression?

View Article: PubMed Central - PubMed

Affiliation: GNR Center for Genome Informatics, Institute of Genomics and Integrative Biology, Delhi, India.

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Validation by quantitative real-time PCR (Polymerase Chain Reaction) for NME2 binding and nucleosome occupancy. Nucleosome positions and NME2 binding sites are shown before and after NME2 induction at six different loci in the left panel (genes shown in UCSC browser representation); right panel shows validation at the corresponding loci by quantitative real-time PCR for NME2 binding and nucleosome occupancy in cells before and after inducing NME2. Relative fold change is shown on x-axis. Experiments were performed in triplicate; error bars are for standard deviation (* and ** represent P < 0.05 and P < 0.01, respectively).
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Figure 4: Validation by quantitative real-time PCR (Polymerase Chain Reaction) for NME2 binding and nucleosome occupancy. Nucleosome positions and NME2 binding sites are shown before and after NME2 induction at six different loci in the left panel (genes shown in UCSC browser representation); right panel shows validation at the corresponding loci by quantitative real-time PCR for NME2 binding and nucleosome occupancy in cells before and after inducing NME2. Relative fold change is shown on x-axis. Experiments were performed in triplicate; error bars are for standard deviation (* and ** represent P < 0.05 and P < 0.01, respectively).

Mentions: To analyze nucleosome repositioning with respect to the NME2 target site after versus before NME2 induction, we calculated the distance of nucleosome shift between the two conditions as ΔNdisplacement (Figure 3C, left panel) and found ∼64% nucleosomes shifted by more than 300 bp in the NME2-induced condition. In order to test the significance of the noted nucleosome shift, we also calculated this distribution in A549 cells where NME2 was depleted (see below) relative to control A549 cells. Observed distributions were significantly altered following induction of NME2; in contrast, on NME2 depletion we did not find any repositioning in most cases (Wilcoxon rank sum test; P = 0.00016; Figure 3C, right panel). Next we plotted the 870-nucleosome positions (found within 300 bp of an NME2 target site in the un-induced condition) before and after NME2 induction. This showed a loss in organized nucleosome occurrence around NME2 binding sites in NME2-induced cells relative to the un-induced condition (Figure 3D). We further noted that expression of all the 791 genes with repositioned nucleosomes was significantly altered in the NME2-induced condition (P < 0.05, student's t-test, Figure 3D). For validation, we compared nucleosome occupancy and NME2 binding using quantitative real-time PCR in six genes. In all cases, low nucleosome occupancy signal was detected along with high NME2 occupancy in cells after NME2 induction as compared to that observed in un-induced cells (Figure 4).


Promoter-proximal transcription factor binding is transcriptionally active when coupled with nucleosome repositioning in immediate vicinity.

Yadav VK, Thakur RK, Eckloff B, Baral A, Singh A, Halder R, Kumar A, Alam MP, Kundu TK, Pandita R, Pandita TK, Wieben ED, Chowdhury S - Nucleic Acids Res. (2014)

Validation by quantitative real-time PCR (Polymerase Chain Reaction) for NME2 binding and nucleosome occupancy. Nucleosome positions and NME2 binding sites are shown before and after NME2 induction at six different loci in the left panel (genes shown in UCSC browser representation); right panel shows validation at the corresponding loci by quantitative real-time PCR for NME2 binding and nucleosome occupancy in cells before and after inducing NME2. Relative fold change is shown on x-axis. Experiments were performed in triplicate; error bars are for standard deviation (* and ** represent P < 0.05 and P < 0.01, respectively).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150765&req=5

Figure 4: Validation by quantitative real-time PCR (Polymerase Chain Reaction) for NME2 binding and nucleosome occupancy. Nucleosome positions and NME2 binding sites are shown before and after NME2 induction at six different loci in the left panel (genes shown in UCSC browser representation); right panel shows validation at the corresponding loci by quantitative real-time PCR for NME2 binding and nucleosome occupancy in cells before and after inducing NME2. Relative fold change is shown on x-axis. Experiments were performed in triplicate; error bars are for standard deviation (* and ** represent P < 0.05 and P < 0.01, respectively).
Mentions: To analyze nucleosome repositioning with respect to the NME2 target site after versus before NME2 induction, we calculated the distance of nucleosome shift between the two conditions as ΔNdisplacement (Figure 3C, left panel) and found ∼64% nucleosomes shifted by more than 300 bp in the NME2-induced condition. In order to test the significance of the noted nucleosome shift, we also calculated this distribution in A549 cells where NME2 was depleted (see below) relative to control A549 cells. Observed distributions were significantly altered following induction of NME2; in contrast, on NME2 depletion we did not find any repositioning in most cases (Wilcoxon rank sum test; P = 0.00016; Figure 3C, right panel). Next we plotted the 870-nucleosome positions (found within 300 bp of an NME2 target site in the un-induced condition) before and after NME2 induction. This showed a loss in organized nucleosome occurrence around NME2 binding sites in NME2-induced cells relative to the un-induced condition (Figure 3D). We further noted that expression of all the 791 genes with repositioned nucleosomes was significantly altered in the NME2-induced condition (P < 0.05, student's t-test, Figure 3D). For validation, we compared nucleosome occupancy and NME2 binding using quantitative real-time PCR in six genes. In all cases, low nucleosome occupancy signal was detected along with high NME2 occupancy in cells after NME2 induction as compared to that observed in un-induced cells (Figure 4).

Bottom Line: These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown.For example, physiologic TF binding studies involve many TFs, consequently, it is difficult to assign nucleosome reorganization to the binding site occupancy of any particular TF.Therefore, several aspects remain unclear: does TF binding influence nucleosome (re)organizations locally or impact the chromatin landscape at a more global level; are all or only a fraction of TF binding a result of reorganization in nucleosome occupancy and do all TF binding and associated changes in nucleosome occupancy result in altered gene expression?

View Article: PubMed Central - PubMed

Affiliation: GNR Center for Genome Informatics, Institute of Genomics and Integrative Biology, Delhi, India.

Show MeSH