Nucleosome phasing-remodeling.BCH6415.Spr2006.Print Part II

Nucleosome phasing-remodeling.BCH6415.Spr2006.Print Part II...

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I) NUCLEOSOME POSITIONING II) NUCLEOSOME REMODELING BCH 6415 SPRING 2006 Dr. YANG (Part II)
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Human SWI/SNF interconverts a nucleosome between its base state and a stable remodeled state G. Schnitzler, S. Sif, RE Kingston Cell 94:17; 1998 32 P-labelled reconstituted mononucleosomes incubated w/ SWI/SNF, then subjected to gel-shift analysis Conclusion: formation of a novel gel-shift band that is SWI/SNF, ATP, & salt dependent (added after addition of SWI/SNF) added before (1) or after SWI/SNF(2) Western blot of gel-shift gel using anti-BRG1 Ab Conclusion: Novel band does not contain SWI/SNF; unlikely it is nucleosome bound to SWI/SNF
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Human SWI/SNF interconverts a nucleosome between its base state and a stable remodeled state G. Schnitzler, S. Sif, RE Kingston Cell 94:17; 1998 1) Novel band isolated by preparative gel-shift 2) Eluted novel band subjected to DNase I cleavage Result: complex in novel band (lane 3) has altered DNase I cleavage pattern compared to standard nucleosome core (lanes 1 & 2) , and resembles cleavage pattern of SWI/SNF-modified nucleosome (ln 7). Conclusion: novel band does not resemble std. core, but is more similar to SWI/SNF-remodeled nucleosome. Silver-stained 2-D gels of nucleosome cores and SWI/SNF-treated cores. The novel band (panel D), as well as nucleosome and no other detectable proteins; (e.g., SWI/SNF proteins, TF’s) Preparative Gel-shift + = DNase I enhanced - = DNase I protected Silver-stained 2-D gels
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Conclusion: The SWI/SNF-remodeled structure in the novel band may be some form of a nucleosome dimer. Also, the DNA histone ratios are similar to normal nucleosome cores (data not shown). G. Schnitzler, S. Sif, RE Kingston Cell 94:17; 1998 Novel band (isolated from prep. gel-shift) sedimented in glycerol gradient: novel band sediments faster than mononucs. Novel band subjected to gel filtration column: elutes faster than mononucs. and at ~dinucs. Novel band seidments at S-value of approx. a dinucleosome B = bare DNA N = novel band C = mononucleos. cores - + - + - End-labeled gradient-isolated novel band and cores digested w/ restriction enzymes or MNase. PstI, HindIII, and MNase showed increased digesttion/accessibility of novel band vs. core. Conclusion: DNA in the novel band (i.e., nucleosome dimer) has altered accessibilty to both MNase and restriction enzymes compared to standard mononucleosome cores.
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Conclusion: SWI/SNF (+ATP) can convert novel/remodeled nucleosomal structure back to normal nucleosomal cores: SWI/SNF creates equilibrium between standard nucleosomes and novel nucleosomal structure Conclusion: Gal4 binds with higher affinity to the novel nucleosomal structure compared to standard nucleosomes -> SWI/SNF-mediated remodeling facilitates TF binding 3.2-fold Model of SWI/SNF action: 2 normal nucleosome cores (A) are bound by SWI/SNF and converted via the energy of ATP hydrolysis to SWI/SNF-bound novel species (C) before release (D). The reaction also works in reverse, establishing an equilibrium
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Nucleosome phasing-remodeling.BCH6415.Spr2006.Print Part II...

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