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- Gréen, Anna, et al.
(författare)
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Histone H1 interphase phosphorylation becomes largely established in G(1) or early S phase and differs in G(1) between T-lymphoblastoid cells and normal T cells
- 2011
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Ingår i: Epigenetics & Chromatin. - : BioMed Central. - 1756-8935. ; 4:15
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Tidskriftsartikel (refereegranskat)abstract
- Background: Histone H1 is an important constituent of chromatin, and is involved in regulation of its structure. During the cell cycle, chromatin becomes locally decondensed in S phase, highly condensed during metaphase, and again decondensed before re-entry into G(1). This has been connected to increasing phosphorylation of H1 histones through the cell cycle. However, many of these experiments have been performed using cell-synchronization techniques and cell cycle-arresting drugs. In this study, we investigated the H1 subtype composition and phosphorylation pattern in the cell cycle of normal human activated T cells and Jurkat T-lymphoblastoid cells by capillary electrophoresis after sorting of exponentially growing cells into G(1), S and G(2)/M populations. less thanbrgreater than less thanbrgreater thanResults: We found that the relative amount of H1.5 protein increased significantly after T-cell activation. Serine phosphorylation of H1 subtypes occurred to a large extent in late G(1) or early S phase in both activated T cells and Jurkat cells. Furthermore, our data confirm that the H1 molecules newly synthesized during S phase achieve a similar phosphorylation pattern to the previous ones. Jurkat cells had more extended H1.5 phosphorylation in G(1) compared with T cells, a difference that can be explained by faster cell growth and/or the presence of enhanced H1 kinase activity in G(1) in Jurkat cells. less thanbrgreater than less thanbrgreater thanConclusion: Our data are consistent with a model in which a major part of interphase H1 phosphorylation takes place in G(1) or early S phase. This implies that H1 serine phosphorylation may be coupled to changes in chromatin structure necessary for DNA replication. In addition, the increased H1 phosphorylation of malignant cells in G(1) may be affecting the G(1)/S transition control and enabling facilitated S-phase entry as a result of relaxed chromatin condensation. Furthermore, increased H1.5 expression may be coupled to the proliferative capacity of growth-stimulated T cells.
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| 2. |
- Kostova-Koleva, Nora N., et al.
(författare)
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Histone H5chromatin interactions in situ are strongly modulated by H5 C-terminal phosphorylation
- 2013
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Ingår i: Cytometry Part A. - : Wiley-Blackwell. - 1552-4922 .- 1552-4930. ; 83A:3, s. 273-279
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Tidskriftsartikel (refereegranskat)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.
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