Histone H2A C-Terminus Regulates Chromatin Dynamics, Remodeling, and Histone H1 Binding
Figure 5
The H2A C-terminal tail binds linker histone H1.
(A) Schematic representation of the construct used to identify H2A C-terminus interacting proteins. Proteins interacting with the HA Flag 8x C-terminus were identified by Mass Spectrometry after Flag affinity purification. Histone H1 peptides that bind to the H2A C-terminal tail as identified by mass spectrometry. DeltaCN and XCorr values are shown. (B) H1 co-immunoprecipitates with the 8x repeat of the H2A C-terminal tail. The HA-Flag 8x H2A C-terminus was purified and the eluate from the Flag Sepharose probed by immunoblotting with an H1 specific antibody. 10% of the input was loaded (lanes 1 and 2). As control, the empty vector without the 8x H2A C-terminus was used. Purified H1 was used as an immunoblot control (lane 5). (C) Deletion of the H2A C-terminus reduces H1 binding. Binding of H1 to in vitro reconstituted mononucleosomes containing either full-length or C-terminally truncated H2A. Mononucleosomes containing wild type H2A (lanes 1–6), H2A 1-122 (lanes 7–12) or H2A 1-114 (lanes 13–14) were reconstituted on a DNA fragment containing the 601 positioning sequence. The nucleosomes were incubated with increasing amounts of H1 (H1 to nucleosome ratios 1∶1 to 6∶1) and then analyzed on native polyacrylamide gels. Nucleoprotein complexes were visualized by ethidium bromide staining. (D) Partial MNase digestion of H1-nucleosome complexes shows a chromatosome stop. Nucleosomes and H1-nucleosome complexes reconstituted on a 208 bp 601 DNA fragment at a molar ratio of 6∶1 were incubated with increasing MNase concentrations and the resulting DNA cleavage products were analyzed by polyacrylamide gel electrophoresis and ethidium bromide staining next to a DNA standard (M). The position of the undigested DNA fragment (208 bp), the protected nucleosomal DNA (147 bp) and the chromatosome stop (arrow) are indicated.