Affinity of linker histones for chromatin in situ analyzed using DAPI as a cytochemical probe

Abstract

Background:It is generally assumed that linker histones contribute to condensation of chromatin and to the repression of genetic activity by binding to chromatin with increased affinity. The aim of this study is to investigate a possible correlation between linker histone binding to chromatin in situ and chromatin condensation using a cytochemical approach that largely preserves nuclear structure. Methods: Ionically bound H1 was extracted with increasing concentrations of sodium chloride. Thereafter, the cells were fixed in formaldehyde and stained with 50 nM 4Ј,6-diamidino-2-phenylindole (DAPI) and fluorescence intensity was measured by image cytofluorometry. Results:The association between linker histones and chromatin was stronger in cultured human fibroblasts and

Histone H5—chromatin interactions in situ are strongly modulated by H5 C‐terminal phosphorylation

“…Thereafter, they were reconstituted by incubation with either nonphosphorylated or in vitro phosphorylated H5 histones at physiological ionic strength. The salt extraction of linker histones leads to an increase in FI which is proportional to the sum of DAPI binding sites that become available when H1 is detached from chromatin (44–46). We have recently applied this method to study linker histone–chromatin interactions in normal human fibroblast nuclei after depletion of the endogenous linker histones and reconstitution with H1 subfractions (47).…”

“…The analysis of salt extraction curves was performed as described previously (44, 45) using a least‐squares curve fitting to a linker histone binding equation (33). The salt extraction curves were normalized using an average of the three highest FIs after extraction as 100%.…”

“…Linker histones are bound to chromatin mainly through ionic interactions and they can be selectively extracted using salt concentrations in the range of 0.3 to 0.7 M NaCl. This property of linker histones was used to develop a method to investigate H1‐chromatin interactions in situ using the DNA‐binding fluorochrome DAPI as an indirect probe (44–46). Furthermore, our method was extended to study the affinity of a particular H1 subfraction reconstituted into nuclei after depletion of the endogenous linker histones (47).…”

Possible role of H1 histone in replication timing

“…H1 histone binding to metaphase chromosomes is much weaker than to interphase chromatin (Wu et al 1986;Bordignon et al 2001;Loberg & Rundquist 2006;Green et al 2010). Most H1 histone is translocated from metaphase chromosomes to the cytoplasm before its relocation to chromatin at telophase and early G1 phase ( Fig.…”