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At Linkoping University, medical education focused on problem-based learning was introduced, with an unchanged budget, but a new method of resource management. The point system for teaching and administrative tasks gives better control of resources and increases departments' motivation to participate. (SK)

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 G1. 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 G1, S and G2/M populations. Results 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 G1 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 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. Conclusion Our data are consistent with a model in which a major part of interphase H1 phosphorylation takes place in G1 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 G1 may be affecting the G1/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.

Psoriasis is an inflammatory skin disorder characterized by the hyperproliferation of basal epidermal cells. It is regarded as T-cell mediated, but the role of keratinocytes (KCs) in the disease pathogenesis has reemerged, with genetic studies identifying KC-associated genes. We applied flow cytometry on KCs from lesional and nonlesional epidermis to characterize the phenotype in the germinative compartment in psoriasis, and we observed an overall increase in the stemness markers CD29 (2.4-fold), CD44 (2.9-fold), CD49f (2.8-fold), and p63 (1.4-fold). We found a reduced percentage of cells positive for the early differentiation marker cytokeratin 10 and a greater fraction of CD29 + and involucrin + cells in the psoriasis KCs than in nonlesional KCs. The up-regulation of stemness markers was more pronounced in the K10 + cells. Furthermore, the psoriasis cells were smaller, indicating increased proliferation. Treatment with IL-17 and IL-22 induced a similar expression pattern of an up-regulation of p63, CD44, and CD29 in normal KCs and increased the colony-forming efficiency and long-term proliferative capacity, reflecting increased stem cell-like characteristics in the KC population. These data suggest that IL-17 and IL-22 link the inflammatory response to the immature differentiation and epithelial regeneration by acting directly on KCs to promote cell stemness., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 International Society for Advancement of Cytometry.)

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 H1.5 was partitioned between chromatin and cytoplasm 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 theirphosphorylated forms, may leave chromatin in a regulated way to give access for chromatin condensing factors or transcriptional regulators during mitosis.

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.

Recent studies, using cytometric techniques based on fluorescence microscopy, have provided new information on how linker histones interact with chromatin in vivo or in situ. In particular, the use of green fluorescent proteins (GFPs) has enabled detailed studies of how individual H1 subtypes, and specific motifs in them, interact with chromatin in vivo. Furthermore, the development of cytochemical methods to study the interaction between linker histones and chromatin using DNA-binding fluorochromes as indirect probes for linker histone affinity in situ, in combination with highly sensitive and specific analytical methods, has provided additional information on the interactions between linker histones and chromatin in several cell systems. Such results verified that linker histones have a substantially higher affinity for chromatin in mature chicken erythrocytes than in frog erythrocytes, and they also indicated that the affinity decreased during differentiation of the frog erythrocytes. Furthermore, in cultured human fibroblasts, the linker histones showed a relatively high affinity for chromatin in interphase, whereas it showed a significantly lower affinity in highly condensed metaphase chromosomes. This method also enables the analysis of linker histone affinity for chromatin in H1-depleted fibroblasts reconstituted with purified linker histones. No consistent correlation between linker histone affinity and chromatin condensation has so far been detected.

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.

In humans, eight types of histone H1 exist (H1.1-H1.5, H1 degrees , 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.

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., (Copyright 2004 Wiley-Liss, Inc.)

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.

Methylation of the N-terminal region of histones was first described more than 35 years ago, but its biological significance has remained unclear. Proposed functions range from transcriptional regulation to the higher order packing of chromatin in progress of mitotic condensation. Primarily because of the recent discovery of the SET domain-depending H3-specific histone methyltransferases SUV39H1 and Suv39h1, which selectively methylate lysine 9 of the H3 N terminus, this posttranslational modification has regained scientific interest. In the past, investigations concerning the biological significance of histone methylation were largely limited because of a lack of simple and sensitive analytical procedures for detecting this modification. The present work investigated the methylation pattern of histone H4 both in different mammalian organs of various ages and in cell lines by applying mass spectrometric analysis and a newly developed hydrophilic-interaction liquid chromatographic method enabling the simultaneous separation of methylated and acetylated forms, which obviates the need to work with radioactive materials. In rat kidney and liver the dimethylated lysine 20 was found to be the main methylation product, whereas the monomethyl derivative was present in much smaller amounts. In addition, for the first time a trimethylated form of lysine 20 of H4 was found in mammalian tissue. A significant increase in this trimethylated histone H4 was detected in organs of animals older than 30 days, whereas the amounts of mono- and dimethylated forms did not essentially change in organs from young (10 days old) or old animals (30 and 450 days old). Trimethylated H4 was also detected in transformed cells; although it was present in only trace amounts in logarithmically growing cells, we found an increase in trimethylated lysine 20 in cells in the stationary phase.

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 4',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 rat smooth muscle cells than in both human T lymphocytes and granulocytes, and in particular, frog erythrocytes, which exhibited highly condensed chromatin. Our data indicate a lower affinity of linker histones for metaphase chromosomes than for interphase chromatin, and we observed a reduction in linker histone affinity during terminal differentiation of frog erythrocytes in vivo., Conclusions: Taken together, our findings imply that the affinity of linker histones for chromatin in situ is unrelated or inversely related to chromatin condensation., (Copyright 2000 Wiley-Liss, Inc.)

We have investigated using the DNA binding fluorochromes 7-aminoactinomycin (7-AAMD) and 4',6-diamidino-2-phenylindole (DAPI) as cytochemical probes for linker histone (H1)-chromatin interactions in situ. Human lymphocytes, permeabilized with digitonin, were exposed to increasing concentrations of sodium chloride to remove ionically bound H1 from the nuclei. The cells were stained to equilibrium with 1 microM 7-AAMD or 50 nM DAPI. Lymphocytes stained with 7-AAMD showed a gradual increase from 11% to 36% of HC1 treated cell fluorescence intensity when the salt concentration was increased from 0.15 to 0.7 M. The corresponding increase for DAPI was 53-68%. The 7-AAMD obviously showed higher sensitivity for H1-chromatin interactions that DAPI but had disadvantages such as high background fluorescence and an affinity that was dependent on the preparation procedure. DAPI had negligible background fluorescence, and its fluorescence intensity resembles the number of available high-affinity dye-binding sites when used at 50 nM. We conclude that both fluorochromes can be used as probes for H1-chromatin interactions in situ and that our method has a potential to provide new information on such interactions.

DNA-protein interactions, mainly DNA-histone interactions, are thought to play an essential role in the packing mechanisms of chromatin as well as in transcriptional control. In this context it is important to develop new methods to study DNA-protein interactions and structural changes associated with them. Paraformaldehyde (PFA) has been shown to crosslink proteins, mainly histones, to DNA; and in this short study we have used a system with the DNA-binding dye 7-aminoactinomycin D (7-AAMD) as an indirect probe for PFA fixation. The aim was to investigate the dynamics of fixation on the binding of this dye to intact human lymphocytes. The results show a decrease in accessibility of 7-AAMD,initially affecting both the nonspecific binding of 7-AAMD and the high affinity binding sites, and thereafter mainly the high affinity binding sites. We conclude that fixation with PFA is a long-term reaction that requires a fixation time of several hours at a sufficient concentration to be completed. Our findings suggest that staining with a low concentration of 7-AAMD after extensive PFA fixation may be used to obtain information on DNA-protein interactions in intact cells.

The binding behavior of the DNA binding dyes 7-aminoactinomycin D (7-AAMD) and 4',6-diamidino-2-phenylindole (DAPI) to human neutrophilic granulocytes and lymphocytes was studied by image cytofluorometry. Peripheral blood leukocytes were prefixed in paraformaldehyde (PFA) and attached to cover glasses. Different fixation, permeabilization, and acid extraction method were applied before the cells were stained to equilibrium using varying concentrations of 7-AAMD or DAPI. The apparent association constant and number of high affinity dye binding sites were estimated for the different cell types, dyes, and treatments. Acid-extracted cells, supposedly containing nucleosome-free DNA, were chosen to represent maximal dye binding. Only about 10% of the 7-AAMD binding sites remained in the unextracted PFA-fixed cells, and the apparent dye affinity was also reduced. We found no major difference in high affinity binding between the cell types, but granulocytes showed more fluorescence from less specifically bound 7-AAMD compared to lymphocytes. DAPI had a much higher affinity than 7-AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high affinity binding sites at a dye concentration of about 50 nM. We conclude that both dyes may be useful as probes for chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye affinities and numbers of high affinity binding sites in situ.

The binding of 4',6-diamidino-2-phenylindole (DAPI) and 7-aminoactinomycin D (7-AAMD) to chromatin of intact cultured fibroblast nuclei was studied by cytofluorometry. Staining was performed at equilibrium at varying dye concentrations. By using large volumes of dye solutions, the free dye concentration became approximately equal to total dye concentration, and the estimation of affinity and number of binding sites was performed by Scatchard analysis. Since all Scatchard plots showed a definite curvature, indicating more than one class of binding sites, a two component regression was performed, although only the highest affinity binding sites were determined. The observed affinity constants did not change substantially between different preparational procedures for any of the dyes. The number of DAPI binding sites increased about 30% after detergent extraction. No such increase was observed for 7-AAMD. Fixation in formaldehyde, instead of ethanol, after detergent extraction had no effect on DAPI binding, but decreased the number of 7-AAMD binding sites with about 50%. Extraction of basic proteins with HCl resulted in about 100% increase of the number of binding sites for both dyes. It should be feasible to use this technique as an assay for the identification of proteins or other nuclear components important for the maintenance of chromatin structure.