Limits...
Linker histone H1 is present in centromeric chromatin of living human cells next to inner kinetochore proteins.

Orthaus S, Klement K, Happel N, Hoischen C, Diekmann S - Nucleic Acids Res. (2009)

Bottom Line: The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome.These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap.By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1 degrees and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins.

View Article: PubMed Central - PubMed

Affiliation: Leibniz-Institute for Age Research - Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany.

ABSTRACT
The vertebrate kinetochore complex assembles at the centromere on alpha-satellite DNA. In humans, alpha-satellite DNA has a repeat length of 171 bp slightly longer than the DNA in the chromatosome containing the linker histone H1. The centromere-binding protein CENP-B binds specifically to alpha-satellite DNA with properties of a centromeric-linker histone. Here, we analysed if linker histone H1 is present at or excluded from centromeric chromatin by CENP-B. By immunostaining we detected the presence, but no enrichment or depletion of five different H1 subtypes at centromeric chromatin. The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome. These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap. By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1 degrees and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins.

Show MeSH
Linker histone H1 binds to the centromere in direct vicinity to CENP-A, -B and -C. Displayed are the histograms of the donor fluorescence lifetimes evaluated at all centromeres by FLIM. HEp-2 cells were co-transfected with (A) EGFP-CENP–A, mCherry-H1°, (B) EGFP-CENP–A, H1°-mCherry, (C) EGFP-CENP–A, mCherry-H1.2, (D) CENP–B-EGFP, mCherry-H1°, (E) EGFP-CENP–B, mCherry-H1°, (F) EGFP-CENP–B, mCherry-H1.2, (G) EGFP-CENP–C, mCherry-H1°, (H) EGFP-CENP–C, mCherry-H1.2 and (I) EGFP-CENP–C, mCherry-H1° in CENP-B depleted cells. In these living cells, the donor fluorescence lifetimes were measured by TCSPC. The histograms display the fitted fluorescence lifetime values of all single centromeres (black bars). In addition, lifetime distributions of the corresponding donor-only control experiments are depicted as grey bars. The heights of the bars represent the numbers of centromeres (y-axis, frequency) whose lifetimes fall within the indicated 0.3 ns range (x-axis, time).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2691837&req=5

Figure 4: Linker histone H1 binds to the centromere in direct vicinity to CENP-A, -B and -C. Displayed are the histograms of the donor fluorescence lifetimes evaluated at all centromeres by FLIM. HEp-2 cells were co-transfected with (A) EGFP-CENP–A, mCherry-H1°, (B) EGFP-CENP–A, H1°-mCherry, (C) EGFP-CENP–A, mCherry-H1.2, (D) CENP–B-EGFP, mCherry-H1°, (E) EGFP-CENP–B, mCherry-H1°, (F) EGFP-CENP–B, mCherry-H1.2, (G) EGFP-CENP–C, mCherry-H1°, (H) EGFP-CENP–C, mCherry-H1.2 and (I) EGFP-CENP–C, mCherry-H1° in CENP-B depleted cells. In these living cells, the donor fluorescence lifetimes were measured by TCSPC. The histograms display the fitted fluorescence lifetime values of all single centromeres (black bars). In addition, lifetime distributions of the corresponding donor-only control experiments are depicted as grey bars. The heights of the bars represent the numbers of centromeres (y-axis, frequency) whose lifetimes fall within the indicated 0.3 ns range (x-axis, time).

Mentions: AB-FRET measurements have the disadvantage that the measured fluorescence intensities depend on the fluorophore concentrations. Therefore, energy transfer between the tagged proteins was further analysed by FLIM. Since EGFP has a fluorescence decay dominated by a single exponential (103), EGFP as donor with mCherry as acceptor fluorophore was used for the fluorescence lifetime measurements (104). The detection of FRET between EGFP and mCherry requires the fluorophores to be close to one another within a distance of 10 nm. FRET results in a shortening of the donor fluorescence lifetime. Unfused EGFP and mCherry, co-transfected in living human cells at similar expression levels, showed no FRET, allowing us to exclude that the FRET detected for the protein fusions might be caused by an incidental association of the fluorescent proteins (data not shown). Furthermore, the fluorescence lifetime of the donor EGFP was determined in living HEp-2 cells expressing the fluorophore alone or fused to either CENP-A, CENP-B or CENP-C. In cells expressing EGFP, EGFP-CENP-A, CENP-B-EGFP, EGFP-CENP-B or EGFP-CENP-C, the average fluorescence lifetimes were τm = 2.32 ± 0.04 ns (mean ± SD, n = 8 cells), τm = 2.17 ± 0.03 ns (n = 175 centromeres of six cells), τm = 2.23 ± 0.03 ns (n = 175 centromeres of five cells), τm = 2.28 ± 0.04 ns (n = 162 centromeres of seven cells) and τm = 2.23 ± 0.04 ns (n = 203 centromeres of nine cells), respectively. The donor-only lifetime distributions (Figure 4) also represent potential environmental influences on the donor fluorescence.


Linker histone H1 is present in centromeric chromatin of living human cells next to inner kinetochore proteins.

Orthaus S, Klement K, Happel N, Hoischen C, Diekmann S - Nucleic Acids Res. (2009)

Linker histone H1 binds to the centromere in direct vicinity to CENP-A, -B and -C. Displayed are the histograms of the donor fluorescence lifetimes evaluated at all centromeres by FLIM. HEp-2 cells were co-transfected with (A) EGFP-CENP–A, mCherry-H1°, (B) EGFP-CENP–A, H1°-mCherry, (C) EGFP-CENP–A, mCherry-H1.2, (D) CENP–B-EGFP, mCherry-H1°, (E) EGFP-CENP–B, mCherry-H1°, (F) EGFP-CENP–B, mCherry-H1.2, (G) EGFP-CENP–C, mCherry-H1°, (H) EGFP-CENP–C, mCherry-H1.2 and (I) EGFP-CENP–C, mCherry-H1° in CENP-B depleted cells. In these living cells, the donor fluorescence lifetimes were measured by TCSPC. The histograms display the fitted fluorescence lifetime values of all single centromeres (black bars). In addition, lifetime distributions of the corresponding donor-only control experiments are depicted as grey bars. The heights of the bars represent the numbers of centromeres (y-axis, frequency) whose lifetimes fall within the indicated 0.3 ns range (x-axis, time).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691837&req=5

Figure 4: Linker histone H1 binds to the centromere in direct vicinity to CENP-A, -B and -C. Displayed are the histograms of the donor fluorescence lifetimes evaluated at all centromeres by FLIM. HEp-2 cells were co-transfected with (A) EGFP-CENP–A, mCherry-H1°, (B) EGFP-CENP–A, H1°-mCherry, (C) EGFP-CENP–A, mCherry-H1.2, (D) CENP–B-EGFP, mCherry-H1°, (E) EGFP-CENP–B, mCherry-H1°, (F) EGFP-CENP–B, mCherry-H1.2, (G) EGFP-CENP–C, mCherry-H1°, (H) EGFP-CENP–C, mCherry-H1.2 and (I) EGFP-CENP–C, mCherry-H1° in CENP-B depleted cells. In these living cells, the donor fluorescence lifetimes were measured by TCSPC. The histograms display the fitted fluorescence lifetime values of all single centromeres (black bars). In addition, lifetime distributions of the corresponding donor-only control experiments are depicted as grey bars. The heights of the bars represent the numbers of centromeres (y-axis, frequency) whose lifetimes fall within the indicated 0.3 ns range (x-axis, time).
Mentions: AB-FRET measurements have the disadvantage that the measured fluorescence intensities depend on the fluorophore concentrations. Therefore, energy transfer between the tagged proteins was further analysed by FLIM. Since EGFP has a fluorescence decay dominated by a single exponential (103), EGFP as donor with mCherry as acceptor fluorophore was used for the fluorescence lifetime measurements (104). The detection of FRET between EGFP and mCherry requires the fluorophores to be close to one another within a distance of 10 nm. FRET results in a shortening of the donor fluorescence lifetime. Unfused EGFP and mCherry, co-transfected in living human cells at similar expression levels, showed no FRET, allowing us to exclude that the FRET detected for the protein fusions might be caused by an incidental association of the fluorescent proteins (data not shown). Furthermore, the fluorescence lifetime of the donor EGFP was determined in living HEp-2 cells expressing the fluorophore alone or fused to either CENP-A, CENP-B or CENP-C. In cells expressing EGFP, EGFP-CENP-A, CENP-B-EGFP, EGFP-CENP-B or EGFP-CENP-C, the average fluorescence lifetimes were τm = 2.32 ± 0.04 ns (mean ± SD, n = 8 cells), τm = 2.17 ± 0.03 ns (n = 175 centromeres of six cells), τm = 2.23 ± 0.03 ns (n = 175 centromeres of five cells), τm = 2.28 ± 0.04 ns (n = 162 centromeres of seven cells) and τm = 2.23 ± 0.04 ns (n = 203 centromeres of nine cells), respectively. The donor-only lifetime distributions (Figure 4) also represent potential environmental influences on the donor fluorescence.

Bottom Line: The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome.These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap.By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1 degrees and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins.

View Article: PubMed Central - PubMed

Affiliation: Leibniz-Institute for Age Research - Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany.

ABSTRACT
The vertebrate kinetochore complex assembles at the centromere on alpha-satellite DNA. In humans, alpha-satellite DNA has a repeat length of 171 bp slightly longer than the DNA in the chromatosome containing the linker histone H1. The centromere-binding protein CENP-B binds specifically to alpha-satellite DNA with properties of a centromeric-linker histone. Here, we analysed if linker histone H1 is present at or excluded from centromeric chromatin by CENP-B. By immunostaining we detected the presence, but no enrichment or depletion of five different H1 subtypes at centromeric chromatin. The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome. These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap. By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1 degrees and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins.

Show MeSH