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Affinity of linker histones for chromatin in situ analyzed using DAPI as a cytochemical probe
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
2000 (English)In: Cytometry, ISSN 0196-4763, E-ISSN 1097-0320, Vol. 40, no 1, 1-9 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2000. Vol. 40, no 1, 1-9 p.
Keyword [en]
linker histones, chromatin condensation, affinity, image cytofluorometry, in situ
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25257DOI: 10.1002/(SICI)1097-0320(20000501)40:1<1::AID-CYTO1>3.0.CO;2-GLocal ID: 9697OAI: oai:DiVA.org:liu-25257DiVA: diva2:245585
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
In thesis
1. A novel method using DNA binding fluorochromes as indirect probes to study linker histone-chromatin interactions in situ
Open this publication in new window or tab >>A novel method using DNA binding fluorochromes as indirect probes to study linker histone-chromatin interactions in situ
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Over the years, a variety of methods have been used to study chromatin structure. These techniques have provided valuable information about nuclear framework, chromatin organisation, and the proteins that are involved in the formation and maintenance of chromatin. However, neither ultrastructural nor biochemical methods are suitable for investigating the dynamics of chromatin structure in structurally preserved nuclei. DNA binding fluorochromes can be used as indirect probes of chromatin structure, because their accessibility to DNA is detennined by the chromatin structure, which enables cytochemical studies of relatively undamaged cells. The aim of the work presented in this thesis was to develop a cytochemical method using the fluorocbromes 7-aminoactinomycin D (7-AAMD) and 4', 6-diamidino-2- phenylindole (DAPI) to study protein-DNA interactions in essentially intact cell nuclei.

Both fluorochromes proved to be useful as probes of chromatin structure, particularly to study Iinker histone-chromatin interactions. Suitable protocols for permeabilisation, salt extraction of linker histones, fixation, and staining were established. Permeabilisation with digitonin followed by linker histone extraction at various NaCl concentrations and subsequent fixation in paraformaldehyde was found to enable analysis of linker histone affinity for chromatin in situ. Although the cells became fragile during the salt extraction, this protocol provided individual cells that could be measured by image cytofluorometry.

In such analyses, DAPI had several advantages over 7-AAMD. At a concentration of 50 nM DAPI, all of the high-affinity binding sites were occupied, with only minor contributions from binding sites with lower affinity. This approach was used to study linker histone affinity in different types of cells, in cells in different stages of the cell cycle, and during cell differentiation in vivo. Our results indicate differences between the investigated cell types in regard to average Iinker histone affinity. It is generally assumed that increased linker histone affinity is associated with chromatin condensation, but that is not supported by our findings. Human fibroblasts showed substantially lower linker histone affinity for highly condensed metaphase chromatin than for interphase chromatin. Moreover, we found a tendency toward decreasing linker histone affinities in amphibian erytbroblasts that were developing into tenninally differentiated erythrocytes. However, as expected, mature avian erythrocytes showed strongly bound linker histones, probably due to their high arginine content. In conclusion, our results suggest that linker histone affinity is not directly involved in chromatin condensation.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2000. 42 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 625
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25682 (URN)10058 (Local ID)91-7219-582-7 (ISBN)10058 (Archive number)10058 (OAI)
Public defence
2000-05-11, Berzeliussalen, Hälsouniversitetet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-11-09Bibliographically approved

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Loborg, HelenaRundquist, Ingemar

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