dnase i  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: H2A.Z facilitates Sox2-nucleosome interaction by promoting DNA and histone H3 tail mobility

doi: 10.1093/nar/gkag371

Figure Lengend Snippet: H2A.Z modulates an altered Lin28B NCP conformation in the presence of Sox2. ( A ) Lin28B sequence depicting potential binding motifs (TTGT) for Sox2 HMG (blue) and Oct4 POU S and POU HD domains (orange). ( B ) EMSA gels of nucleosomes with increasing Sox2 (left) or Oct4 (right) concentrations and quantified fraction bound (specific complex or total, mean ± SD) showing similar binding with both H2A and H2A.Z. The laddering effect with H2A.Z could be an artifact of some free DNA present. ( C ) DNaseI digestion profiles of H2A and H2A.Z Lin28B nucleosomes (100 nM) with or without Sox2 (10×), showing multiple footprints (decreased signal) and hypersensitive sites (increased signal) with Sox2. The locations of Sox2 footprints (gray, open) and TTGT motifs (blue) are indicated. The nucleosome dyad region, showing higher DNaseI and Sox2 sensitivity with H2A.Z, is highlighted in red. NCP cartoons (right) depicting approximate locations of Sox2 motifs and DNaseI footprints, and the position of H2A/H2A.Z (light blue).

Article Snippet: Binding reactions were set up with or without Sox2 HMG (0.1 μM or 1 μM) and Oct4 CtoS (0.3 μM or 3 μM), and incubated at 25°C for 45 min. DNaseI (NEB) was added at 0.5 U (or 0.125 U) for NCP and 0.1 U (or 0.025 U) for DNA and incubated for 5 min at 25°C, inactivated by 40 μl quench buffer [10 mM Tris–HCl pH 7.5, 50 mM EDTA, 2% SDS, 300 ng/μl glycogen (Invitrogen)], and heating at 75°C for 30 min, as previously described [ ].

Techniques: Sequencing, Binding Assay