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

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

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I) NUCLEOSOME POSITIONING II) NUCLEOSOME REMODELING BCH 6415 SPRING 2006 Dr. YANG (Part I)
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Translational and rotational positioning of nucleosomes How does nucleosome positioning/phasing affect transcription factor binding and transcriptional regulation?
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Translational phasing/positioning of nucleosomes
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Translational Phasing Translational Phasing – The same DNA sequence is wrapped around the histone octamer in all the cells in the population. Translationally Unphased Tranlationally Phased
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ATG EXON 1 Hybridization Probe Bcl I Bcl I Bcl I MNase Cleavages Mapping MNase Cleavage Sites: Indirect end-labeling
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Translational Positioning of Nucleosomes at the Translational Positioning of Nucleosomes at the HPRT HPRT Promoter Promoter
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ACTIVE ACTIVE HPRT HPRT ALLELE ALLELE +490 -260 -140 +1 +280 -460 +90 -70 -660 -840 ATG EXON 1 ATG EXON 1 INACTIVE INACTIVE HPRT HPRT ALLELE ALLELE -260 -660 -330 -530 -800
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Rotational phasing/positioning of nucleosomes [ Also, potential effect of rotational positioning when transcription factors bend the DNA upon binding (e.g., TBP): can’t bend the DNA into or away from the octamer surface, only “tangential” to octamer surface (i.e., upward or downward from plane of screen/paper) (See: Imbalzano et al. Nature 370:481; 1994) ]
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Rotational Phasing Rotational Phasing The helical orientation of the DNA sequence relative the the surface of the histone octamer is the same in the cell population. Rotationally Phased Rotationally Unphased
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Accessibility of DNase I to DNA Wrapped Accessibility of DNase I to DNA Wrapped Around a Nucleosome Around a Nucleosome
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} PO 4 -5’ Primer Extension OH-3' Blunt end Ligation DS linker OH-5' 3'-O 4 P Denaturation and annealing of gene specific PCR primer OH-5' 20 cycles of PCR w/ OH-5' OH-3‘ 5'-HO extension primer PCR primer LMPCR product location of hybridization probe location of labeling primer + * { } { } Primer sequences for PCR amplification * * * * * * * Ligation-Mediated PCR Single-stranded fragment of cleaved genomic DNA in region of interest 3'-O 4 P 3'-O 4 P 3'-O 4 P OH-3' 3’-OH OH-5' 5’-OH OH-3' OH-5' DNase I In Vivo Footprinting Naked Enhancement Protections Naked DNA DNase DNA Sequencing Gel In Vivo DNase In Vivo Treatment Ligation-mediated PCR Visualizing Rotational Positioning of Nucleosomes In Vivo See: Hornstra and Yang, Anal. Biochem. 213:179; 1993
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ATG EXON 1 ACTIVE ALLELE +490 -260 -140 +1 +280 -460 +90 -70 -660 -840 INACTIVE ALLELE -260 -660 -330 -530 -800 ATG EXON 1
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NUCLEOSOME REMODELING
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Nucleosome phasing-remodeling.BCH6415.Spr2006.Print Part I...

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