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Histone H1: Subtypes and phosphorylation in cell life and death
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 97 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1086
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:liu:diva-15925ISBN: 978-91-7393-757-3 (print)OAI: oai:DiVA.org:liu-15925DiVA: diva2:128386
Public defence
2009-01-16, Berzeliussalen, Hälsouniversitetet, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-01-20 Created: 2008-12-16 Last updated: 2009-06-24Bibliographically approved
List of papers
1. Characterization of sequence variations in human histone H1.2 and H1.4 subtypes
Open this publication in new window or tab >>Characterization of sequence variations in human histone H1.2 and H1.4 subtypes
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2005 (English)In: The FEBS Journal, ISSN 1742-464X, Vol. 272, no 14, 3673 -3683 p.Article in journal (Refereed) Published
Abstract [en]

In humans, eight types of histone H1 exist (H1.1–H1.5, H1°, H1t and H1oo), all consisting of a highly conserved globular domain and less conserved N- and C-terminal tails. Although the precise functions of these isoforms are not yet understood, and H1 subtypes have been found to be dispensable for mammalian development, it is now clear that specific functions may be assigned to certain individual H1 subtypes. Moreover, microsequence variations within the isoforms, such as polymorphisms or mutations, may have biological significance because of the high degree of sequence conservation of these proteins. This study used a hydrophilic interaction liquid chromatographic method to detect sequence variants within the subtypes. Two deviations from wild-type H1 sequences were found. In K562 erythroleukemic cells, alanine at position 17 in H1.2 was replaced by valine, and, in Raji B lymphoblastoid cells, lysine at position 173 in H1.4 was replaced by arginine. We confirmed these findings by DNA sequencing of the corresponding gene segments. In K562 cells, a homozygous GCC→GTC shift was found at codon 18, giving rise to H1.2 Ala17Val because the initial methionine is removed in H1 histones. Raji cells showed a heterozygous AAA→AGA codon change at position 174 in H1.4, corresponding to the Lys173Arg substitution. The allele frequency of these sequence variants in a normal Swedish population was found to be 6.8% for the H1.2 GCC→GTC shift, indicating that this is a relatively frequent polymorphism. The AAA→AGA codon change in H1.4 was detected only in Raji cells and was not present in a normal population or in six other cell lines derived from individuals suffering from Burkitt's lymphoma. The significance of these sequence variants is unclear, but increasing evidence indicates that minor sequence variations in linker histones may change their binding characteristics, influence chromatin remodeling, and specifically affect important cellular functions.

Place, publisher, year, edition, pages
Wiley InterScience, 2005
Keyword
HILIC, linker histones, sequence variants, SNP, tumor cell lines
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16381 (URN)10.1111/j.1742-4658.2005.04793.x (DOI)
Available from: 2009-01-20 Created: 2009-01-20 Last updated: 2009-06-03Bibliographically approved
2. Histone H1 Dephosphorylation Is Not a General Feature in Early Apoptosis
Open this publication in new window or tab >>Histone H1 Dephosphorylation Is Not a General Feature in Early Apoptosis
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2008 (English)In: Biochemistry, ISSN 0006-2960, Vol. 47, 7539-7547 p.Article in journal (Refereed) Published
Abstract [en]

Histone H1 is a family of nucleosomal proteins that exist in a number of subtypes. These subtypes can be modified after translation in various ways, above all by phosphorylation. Increasing levels of H1 phosphorylation has been correlated with cell cycle progression, while both phosphorylation and dephosphorylation of histone H1 have been linked to the apoptotic process. Such conflicting results may depend on which various apoptosis-inducing agents cause apoptosis via different apoptotic pathways and often interfere with cell proliferation. Therefore, we investigated the relation between apoptosis and H1 phosphorylation in Jurkat cells after apoptosis induction via both the extrinsic and intrinsic pathways and by taking cell cycle effects into account. After apoptosis induction by anti-Fas, no significant dephosphorylation, as measured by capillary electrophoresis, or cell cycle-specific effects were detected. In contrast, H1 subtypes were rapidly dephosphorylated when apoptosis was induced by camptothecin. We conclude that histone H1 dephosphorylation is not connected to apoptosis in general but may be coupled to apoptosis by the intrinsic pathway or to concomitant growth inhibitory signaling.

Place, publisher, year, edition, pages
ACS Publications, 2008
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16382 (URN)10.1021/bi702311x (DOI)
Available from: 2009-01-20 Created: 2009-01-20 Last updated: 2009-04-28Bibliographically approved
3. Translocation of Histone H1 Subtypes Between Chromatin and Cytoplasm During Mitosis in Normal Human Fibroblasts
Open this publication in new window or tab >>Translocation of Histone H1 Subtypes Between Chromatin and Cytoplasm During Mitosis in Normal Human Fibroblasts
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2010 (English)In: Cytometry Part A, ISSN 1552-4922, E-ISSN 1552-4930, Vol. 77A, no 5, 478-484 p.Article in journal (Refereed) Published
Abstract [en]

Histone H1 is an important constituent of chromatin which undergoes major structural rearrangements during mitosis. However, the role of H1, multiple H1 subtypes and H1 phosphorylation is still unclear. In normal human fibroblasts, phosphorylated H1 was found located in nuclei during prophase and in both cytoplasm and condensed chromosomes during metaphase, anaphase and telophase as detected by immunocytochemistry. Moreover, we detected remarkable differences in the distribution of the histone H1 subtypes H1.2, H1.3 and H1.5 during mitosis. H1.2 was found in chromatin during prophase, and almost solely in the cytoplasm of metaphase and early anaphase cells. In late anaphase it appeared in both chromatin and cytoplasm, and again in chromatin during telophase. H1.5 distribution pattern resembled that of H1.2, but some H1.5 remained situated in chromatin during metaphase and early anaphase. H1.3 was detected in chromatin in all cell cycle phases. We propose therefore, that H1 subtype translocation during mitosis is controlled by phosphorylation, in combination with H1 subtype inherent affinity. We conclude that H1 subtypes, or their phosphorylated variants, may be signalling molecules in mitosis or that they leave chromatin in a regulated way to give access for chromatin condensing factors or transcriptional regulators during mitosis.

Place, publisher, year, edition, pages
John Wiley & Sons, 2010
Keyword
Histone H1, Chromatin, Cell cycle, Mitosis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16383 (URN)10.1002/cyto.a.20851 (DOI)000277174000009 ()20104577 (PubMedID)
Available from: 2009-01-20 Created: 2009-01-20 Last updated: 2017-08-30Bibliographically approved
4. Histone H1 interphase phosphorylation pattern becomes largely established during G1/S transition in proliferating cells
Open this publication in new window or tab >>Histone H1 interphase phosphorylation pattern becomes largely established during G1/S transition in proliferating cells
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(English)Manuscript (Other academic)
Abstract [en]

Histone H1 is an important constituent of chromatin, and is believed to be involved in regulation of chromatin structure. During the cell cycle, chromatin becomes locally decondensed in S phase, highly condensed during metaphase and again decondensed before re-entry into G1. This has been connected to increasing phosphorylation of H1 histones during the cell cycle. However, many of these experiments have been performed in non-human and human cancer   cell lines, and by the use of cell synchronization techniques and cell cycle-arresting drugs. In this study, we have investigated the H1 subtype composition and phosphorylation pattern in the cell cycle. Exponentially growing normal human activated T cells and Jurkat lymphoblastoid cells were sorted by fluorescence activated cell sorting into G1, S and G2/M populations, without the use of cell cycle arresting drugs. We found that the H1.5 protein level increased after T-cell activation. Our data indicate that serine phosphorylation of H1 subtypes occurred to a large extent in late G1 phase or early S, while some additional serine phosphorylation took place during S, G2 and M phases. Furthermore, our data suggest that the newly synthesized H1 molecules during S phase also achieve a similar phosphorylation pattern as the previous ones. Jurkat cells showed more extended H1.5 phosphorylation in G1 compared with T cells, a difference that can be explained by faster cell growth and/or the presence of enhanced H1 kinase activity in G1 in Jurkat cells. In conclusion, our data is consistent with a model where a major part of interphase H1 serine phosphorylation takes place within a narrow time window during the G1/Stransition. This implies that H1 serine phosphorylation may be coupled to changes in chromatin structure necessary for DNA replication.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16384 (URN)
Available from: 2009-01-20 Created: 2009-01-20 Last updated: 2010-01-14Bibliographically approved

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