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A novel method using DNA binding fluorochromes as indirect probes to study linker histone-chromatin interactions in situ
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-25682Local ID: 10058ISBN: 91-7219-582-7 (print)OAI: oai:DiVA.org:liu-25682DiVA: diva2:246230
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
List of papers
1. High Affinity Binding of 7-Aminoactinomycin D and 4' ,6-Diamidino-2-Phenylindole to Human Neutrophilic Granulocytes and Lymphocytes
Open this publication in new window or tab >>High Affinity Binding of 7-Aminoactinomycin D and 4' ,6-Diamidino-2-Phenylindole to Human Neutrophilic Granulocytes and Lymphocytes
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1995 (English)In: Cytometry, ISSN 0196-4763, E-ISSN 1097-0320, Vol. 20, no 4, 296-306 p.Article in journal (Refereed) Published
Abstract [en]

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 methods 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.

Keyword
DNA, chromatin structure, dye binding, human leukocytes
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79455 (URN)10.1002/cyto.990200405 (DOI)
Available from: 2012-08-01 Created: 2012-08-01 Last updated: 2017-12-07Bibliographically approved
2. DNA Binding Fluorochromes as Probes for Histone HI-Chromatin Interactions In Situ
Open this publication in new window or tab >>DNA Binding Fluorochromes as Probes for Histone HI-Chromatin Interactions In Situ
1997 (English)In: Cytometry, ISSN 0196-4763, E-ISSN 1097-0320, Vol. 28, no 3, 212-219 p.Article in journal (Refereed) Published
Abstract [en]

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 μM 7-AAMD or 50 nM DAPI. Lymphocytes stained with 7-AAMD showed a gradual increase from 11% to 36% of HCl 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.

Keyword
DNA, chromatin structure, linker histones, dye binding
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79456 (URN)10.1002/(SICI)1097-0320(19970701)28:3<212::AID-CYTO5>3.0.CO;2-F (DOI)
Available from: 2012-08-01 Created: 2012-08-01 Last updated: 2017-12-07Bibliographically approved
3. Affinity of linker histones for chromatin in situ analyzed using DAPI as a cytochemical probe
Open this publication in new window or tab >>Affinity of linker histones for chromatin in situ analyzed using DAPI as a cytochemical probe
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.

Keyword
linker histones, chromatin condensation, affinity, image cytofluorometry, in situ
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25257 (URN)10.1002/(SICI)1097-0320(20000501)40:1<1::AID-CYTO1>3.0.CO;2-G (DOI)9697 (Local ID)9697 (Archive number)9697 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
4. Linker histones are strongly bound to chromatin in mature avian erythrocytes and loosely bound to chromatin in mature amphibian erythrocytes
Open this publication in new window or tab >>Linker histones are strongly bound to chromatin in mature avian erythrocytes and loosely bound to chromatin in mature amphibian erythrocytes
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Linker histones are known to participate in the formation and maintenance of higher order chromatin strnctnres and they are also involved in transcription control. Mature nucleated erythrocytes provide an experimental model to study linker histone binding in cells with highly condensed chromatin and extremely low transcriptional activity. The aim of this study is to investigate linker histone binding in avian and amphibian erythrocytes !mown to contain different subsets of linlcer histones. We used DAPI as an indirect cytochemical probe for the analysis of linker histone affinity for chromatin in situ. We found that linlcer histones were strongly associated to chromatin iu matnre hen erythrocytes which are known to accumulate considerable amounts of H5. Linlcer histones in matnre frog erythrocytes, known to accumulate HIo, showed significantly lower affmity for chromatin. We conclude that linlcer histone affinity is not a key factor for chromatin condensation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79457 (URN)
Available from: 2012-08-01 Created: 2012-08-01 Last updated: 2012-08-01Bibliographically approved

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