ABSTRACT
We used linker histone-depleted normal human fibroblast nuclei as templates to study how phosphorylation affects histone H5 binding to chromatin in situ. Permeabilized cells were treated with 0.7 M NaCl to extract the native linker histones. Histone H5 was purified from chicken erythrocytes and phosphorylated in vitro by recombinant cdk5/p35 kinase. High performance capillary electrophoresis (HPCE) showed that the phosphorylated protein contained a mixture of multiply phosphorylated forms. Control experiments, using mass spectrometry, revealed that up to five SPXK motifs in the C terminus were phosphorylated, but also that about 10% of the protein contained one phosphoserine in the N-terminus. Reconstitution of H1-depleted fibroblast nuclei with nonphosphorylated or phosphorylated H5 was performed at physiological ionic strength. The bound H5 was then extracted using NaCl concentrations in the range of 0.15 to 0.7 M. The release of the H5 molecules was monitored by DAPI staining and image cytofluorometry. Our results show that H5 phosphorylation substantially reduced its affinity for chromatin in situ, which support previous observations indicating that C-terminal phosphorylation may be essential for the biological functions of linker histones.
Subject(s)
Chromatin/metabolism , Histones/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Motifs , Animals , Binding Sites , Cell Cycle Proteins/metabolism , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cells, Cultured , Chickens/blood , Cyclin-Dependent Kinase 5/metabolism , Erythrocytes/metabolism , Fibroblasts , Flow Cytometry , Humans , Phosphorylation , Protein BindingABSTRACT
Histone H1(0) is a linker histone subvariant present in tissues of low proliferation rate. It is supposed to participate in the expression and maintenance of the terminal differentiation phenotype. The aim of this work was to study histone H1(0) distribution in human breast carcinoma and its relationship with the processes of proliferation and differentiation. Most of the cells in carcinomas of moderate and high level of differentiation expressed histone H1(0) including cells invading connective and adipose tissues. In low differentiated tumours, the number of H1(0) expressing cells was considerably lower. Staining of myoepithelial cells, when seen, and of stromal fibroblasts was variable. The metastatic malignant cells in the lymph nodes also accumulated H1(0) but lymphocytes were always negative. All immunopositive malignant cells exhibited signs of polymorphism. Double H1(0)/Ki-67 staining showed that the growth fraction in more differentiated tumours belonged to the H1(0)-positive cells, while in poorly differentiated carcinomas it also included a cell subpopulation not expressing H1(0). If expressed, p27Kip1 was always found in H1(0)-positive cells. These findings are inconsistent with the widespread view that histone H1(0) is expressed only in terminally differentiated cells. Rather, they suggest that the protein is expressed in cells in a prolonged intermitotic period irrespective of their level of differentiation. Double H1(0)/Ki-67 immunostaining could be a useful tool in studying the growth fraction in tumours.
Subject(s)
Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Lobular/metabolism , Histones/metabolism , Adenoma/metabolism , Adenoma/pathology , Biomarkers, Tumor/metabolism , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/pathology , Carcinoma, Lobular/pathology , Female , Fibroadenoma/metabolism , Fibroadenoma/pathology , Fibrocystic Breast Disease/metabolism , Fibrocystic Breast Disease/pathology , Fluorescent Antibody Technique, Indirect , HumansABSTRACT
BACKGROUND: Linker histones constitute a family of lysine-rich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact. METHODS: A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy. RESULTS: Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin interactions after reconstitution with purified linker histone subfractions. Our results showed that exogenous linker histones bind to chromatin with lower affinity than the native ones. We detected no significant differences between the main H1 and H1 degrees histone fractions with respect to their affinity for chromatin or in their ability to condense chromatin. CONCLUSIONS: Linker histone interactions with chromatin are controlled also by mechanisms independent of linker histone subtype composition.
