Histone H1 Subtypes and phosphorylation in cell life and death

Abstract: The genetic information of a human diploid cell is contained within approximately 2 metres of linear DNA. The DNA molecules are compacted and organized in various ways to fit inside the cell nucleus. Various kinds of histones are involved in this compaction. One of these histones, histone H1 is the topic of the present thesis. In addition to its structural role, H1 histones have been implicated in various processes, for example gene regulation and inhibition of chromatin replication.H1 histones, also termed linker histones, are relatively conserved proteins, and the various subtypes seem to have different and important functions even though redundancy between the subtypes has been demonstrated. Despite the sequence conservation of H1 subtypes, two sequence variations were detected within the H1.2 and H1.4 subtypes using hydrophilic interaction liquid chromatographic separation of H1 proteins from K562 and Raji cell lines in Paper I in the present thesis. The variations were confirmed by genetic analysis, and the H1.2 sequence variation was also found in genomic DNA of normal blood donors, in an allele frequency of 6.8%. The H1.4 sequence variation was concluded to be Raji specific. The significance of H1 microsequence variants is unclear, since the physiological function of H1 histones remains to be established.H1 histones can be phosphorylated at multiple sites. Changes in H1 phosphorylation has been detected in apoptosis, the cell cycle, gene regulation, mitotic chromatin condensation and malignant transformation. Contradictory data have been obtained on H1 phosphorylation in apoptosis, and many results indicate that H1 dephosphorylation occurs during apoptosis. We and others hypothesized that cell cycle effects by the apoptosis inducers may have affected previous studies. In Paper II, the H1 phosphorylation pattern was investigated in early apoptosis in Jurkat cells, taking cell cycle effects into account. In receptor-mediated apoptosis, apoptosis occurs with a mainly preserved phosphorylation pattern, while Camptothecin induced apoptosis results in rapid dephosphorylation of H1 subtypes, demonstrating that H1 dephosphorylation is not a general event in apoptosis, but may occur upon apoptosis induction via the mitochondrial pathway. The dephosphorylation may also be a result of early cell cycle effects or signalling.Therefore, the H1 phosphorylation pattern in the cell cycle of normal activated T cells was investigated in Paper IV in this thesis. Some studies, which have been made using cancer cell lines from various species and cell synchronization, have indicated a sequential addition of phosphate groupsacross the cell cycle. Normal T cells and cell sorting by flow cytometry were used to circumvent side-effects from cell synchronization. The data demonstrate that a pattern with phosphorylated serines is established in late G1/early S phase, with some additional phosphorylation occurring during S, and further up-phosphorylation seems to occur during mitosis. Malignant transformation may lead to an altered G1 H1 phosphorylation pattern, as was demonstrated using sorted Jurkat T lymphoblastoid cells.During mitosis, certain H1 subtypes may be relocated to the cytoplasm. In Paper III, the location of histones H1.2, H1.3 and H1.5 during mitosis was investigated. Histone H1.3 was detected in cell nuclei in all mitotic stages, while H1.2 was detected in the nucleus during prophase and telophase, and primarily in the cytoplasm during metaphase and early anaphase. H1.5 was located mostly to chromatin during prophase and telophase, and to both chromatin and cytoplasm during metaphase and anaphase. Phosphorylated H1 was located in chromatin in prophase, and in both chromatin and cytoplasm during metaphase, anaphase and telophase, indicating that the mechanism for a possible H1 subtype relocation to the cytoplasm is phosphorylation.In conclusion, data obtained during this thesis work suggest that H1 histones and their phosphorylation may participate in the regulation of events in the cell cycle, such as S-phase progression and mitosis, possibly through altered interactions with chromatin, and/or by partial or complete removal of subtypes or phosphorylated variants from chromatin.