Histone H2A C-Terminus Regulates Chromatin Dynamics, Remodeling, and Histone H1 Binding
Figure 4
The H2A C-terminal tail is important for nucleosome stability and chromatin remodeling in vitro.
(A) Differential positioning and thermal shift of mononucleosomes containing either full-length H2A, H2A1-122 or H2A1-114. Nucleosomes were reconstituted on a linear MMTV NucA DNA fragment and incubated at 45°C for increasing periods of time. Nucleosome positions were analyzed on a native 5% polyacrylamide gel and visualized by ethidium bromide staining (left panel). Quantification of the relative repositioning is shown in the right panel. (B) C-terminal deletions affect chromatin remodeling. In vitro chromatin remodeling assay with mononucleosomes containing H2A1-129, H2A1-122 or H2A1-114 that were assembled on linear DNA fragment (Drosophila Hsp70 promoter) and incubated with the indicated chromatin remodeling complex with or without ATP. Upper panel: ISWI. Middle panel: SNF2H. Lower panel: ACF. Lanes 1, 5, 9: reconstituted mononucleosomes alone; lanes 2, 6, 10: + remodeling factor, no ATP; lanes 3, 7, 11 and 4, 8, 12: + remodeling factor, + ATP. Nucleosome positions were analyzed by native gel electrophoresis and staining with ethidium bromide. Black arrowheads indicate positions to which nucleosomes were moved, white arrowheads indicate positions from which nucleosomes were removed. (C) Kinetics of nucleosome remodeling assayed with a 601 remodeling template. Nucleosomes containing the wildtype H2A and the indicated C-terminal truncations (300 ng), as indicated on the left, were incubated without (lane 1), or with 100 ng of the indicated remodeling enzyme and ATP (lanes 2 to 6). The remodeling reaction was incubated from 1 up to 40 min and analyzed as described above. Nucleosome positions are indicated. Black triangles indicate new nucleosome positions observed with H2A1-122 and H2A1-114.