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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.

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Acceptor bleaching FRET in vivo reveals the presence of linker histone H1° at human centromeres. The column ‘Intensity image’ shows confocal images that were acquired in the donor channel (upper panels) and the acceptor channel (lower panels) before (left panels) and after (right panels) bleaching of the acceptor. Intensities are encoded by colour ranging from blue (low intensty) to red (high intensity). The analysed centromere is shown in the inserts at a magnified scale. The column ‘Time course’ shows a plot of the time course of the fluorescence intensities that were recorded in the donor (cyan line) and the acceptor (green line) channel. Data were averaged over the indicated ROI and normalized to the maximum of the averaged intensities obtained in the respective channel. The dotted green and blue lines mark the bleaching period. The horizontal dashed red lines indicate the mean of the donor fluorescence intensities of the two measurements performed before photobleaching of the acceptor (IDA) and donor intensity recorded in the first image after acceptor photobleaching (ID). (A) Acceptor photobleaching of centromeres that contained Cerulean-CENP-A/EYFP–H1°, (B) CENP-B-Cerulean/EYFP-H1° or (C) Cerulean-CENP-C/EYFP-H1°. In all experiments, acceptor photobleaching resulted in an increase of the donor fluorescence intensity. This indicates that FRET occurs between the donor and acceptor fluorophores and that the respective proteins are in close vicinity to each other (<10 nm). Bars = 5 µm.
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Figure 3: Acceptor bleaching FRET in vivo reveals the presence of linker histone H1° at human centromeres. The column ‘Intensity image’ shows confocal images that were acquired in the donor channel (upper panels) and the acceptor channel (lower panels) before (left panels) and after (right panels) bleaching of the acceptor. Intensities are encoded by colour ranging from blue (low intensty) to red (high intensity). The analysed centromere is shown in the inserts at a magnified scale. The column ‘Time course’ shows a plot of the time course of the fluorescence intensities that were recorded in the donor (cyan line) and the acceptor (green line) channel. Data were averaged over the indicated ROI and normalized to the maximum of the averaged intensities obtained in the respective channel. The dotted green and blue lines mark the bleaching period. The horizontal dashed red lines indicate the mean of the donor fluorescence intensities of the two measurements performed before photobleaching of the acceptor (IDA) and donor intensity recorded in the first image after acceptor photobleaching (ID). (A) Acceptor photobleaching of centromeres that contained Cerulean-CENP-A/EYFP–H1°, (B) CENP-B-Cerulean/EYFP-H1° or (C) Cerulean-CENP-C/EYFP-H1°. In all experiments, acceptor photobleaching resulted in an increase of the donor fluorescence intensity. This indicates that FRET occurs between the donor and acceptor fluorophores and that the respective proteins are in close vicinity to each other (<10 nm). Bars = 5 µm.

Mentions: The flexible N-terminus of CENP-A sticks out of the nucleosome (47), expected to be close to linker binding proteins. We tested this hypothesis using AB-FRET and FLIM in interphase HEp-2 cells. EYFP-H1° showed a non-homogenous distribution within the cell nucleus which partially overlapped with Cerulean-CENP-A at centromeres (Figure 3A ‘Intensity image, pre-bleach’). After complete bleaching of the acceptor EYFP-H1° at centromeric regions, a fluorescence intensity increase of Cerulean-CENP-A was observed (Figure 3A ‘Intensity image, post-bleach’ and ‘Time course’). A FRET efficiency of 23 ± 3% was calculated indicating an association between both proteins (n = 11, single interphase kinetochores of 11 different cells). This result was confirmed by FLIM using the FRET pair EGFP-CENP-A and mCherry-H1°. The donor fluorescence lifetime at 241 centromeres in 10 different cells was measured and each single centromere was fitted by a mono-exponential decay. A large population of centromeres could be observed with a fluorescence decay time close to the control value (donor-only EGFP-CENP-A with τm = 2.17 ± 0.03 ns, see above). This indicates that most donors have no FRET acceptor in their close vicinity. However, a second population (23%) of centromeres was found with a donor fluorescence lifetime <2.09 ns (mean control value of 2.17 ns – 3 SD of 0.03 ns; Figure 4A).Figure 3.


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)

Acceptor bleaching FRET in vivo reveals the presence of linker histone H1° at human centromeres. The column ‘Intensity image’ shows confocal images that were acquired in the donor channel (upper panels) and the acceptor channel (lower panels) before (left panels) and after (right panels) bleaching of the acceptor. Intensities are encoded by colour ranging from blue (low intensty) to red (high intensity). The analysed centromere is shown in the inserts at a magnified scale. The column ‘Time course’ shows a plot of the time course of the fluorescence intensities that were recorded in the donor (cyan line) and the acceptor (green line) channel. Data were averaged over the indicated ROI and normalized to the maximum of the averaged intensities obtained in the respective channel. The dotted green and blue lines mark the bleaching period. The horizontal dashed red lines indicate the mean of the donor fluorescence intensities of the two measurements performed before photobleaching of the acceptor (IDA) and donor intensity recorded in the first image after acceptor photobleaching (ID). (A) Acceptor photobleaching of centromeres that contained Cerulean-CENP-A/EYFP–H1°, (B) CENP-B-Cerulean/EYFP-H1° or (C) Cerulean-CENP-C/EYFP-H1°. In all experiments, acceptor photobleaching resulted in an increase of the donor fluorescence intensity. This indicates that FRET occurs between the donor and acceptor fluorophores and that the respective proteins are in close vicinity to each other (<10 nm). Bars = 5 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Acceptor bleaching FRET in vivo reveals the presence of linker histone H1° at human centromeres. The column ‘Intensity image’ shows confocal images that were acquired in the donor channel (upper panels) and the acceptor channel (lower panels) before (left panels) and after (right panels) bleaching of the acceptor. Intensities are encoded by colour ranging from blue (low intensty) to red (high intensity). The analysed centromere is shown in the inserts at a magnified scale. The column ‘Time course’ shows a plot of the time course of the fluorescence intensities that were recorded in the donor (cyan line) and the acceptor (green line) channel. Data were averaged over the indicated ROI and normalized to the maximum of the averaged intensities obtained in the respective channel. The dotted green and blue lines mark the bleaching period. The horizontal dashed red lines indicate the mean of the donor fluorescence intensities of the two measurements performed before photobleaching of the acceptor (IDA) and donor intensity recorded in the first image after acceptor photobleaching (ID). (A) Acceptor photobleaching of centromeres that contained Cerulean-CENP-A/EYFP–H1°, (B) CENP-B-Cerulean/EYFP-H1° or (C) Cerulean-CENP-C/EYFP-H1°. In all experiments, acceptor photobleaching resulted in an increase of the donor fluorescence intensity. This indicates that FRET occurs between the donor and acceptor fluorophores and that the respective proteins are in close vicinity to each other (<10 nm). Bars = 5 µm.
Mentions: The flexible N-terminus of CENP-A sticks out of the nucleosome (47), expected to be close to linker binding proteins. We tested this hypothesis using AB-FRET and FLIM in interphase HEp-2 cells. EYFP-H1° showed a non-homogenous distribution within the cell nucleus which partially overlapped with Cerulean-CENP-A at centromeres (Figure 3A ‘Intensity image, pre-bleach’). After complete bleaching of the acceptor EYFP-H1° at centromeric regions, a fluorescence intensity increase of Cerulean-CENP-A was observed (Figure 3A ‘Intensity image, post-bleach’ and ‘Time course’). A FRET efficiency of 23 ± 3% was calculated indicating an association between both proteins (n = 11, single interphase kinetochores of 11 different cells). This result was confirmed by FLIM using the FRET pair EGFP-CENP-A and mCherry-H1°. The donor fluorescence lifetime at 241 centromeres in 10 different cells was measured and each single centromere was fitted by a mono-exponential decay. A large population of centromeres could be observed with a fluorescence decay time close to the control value (donor-only EGFP-CENP-A with τm = 2.17 ± 0.03 ns, see above). This indicates that most donors have no FRET acceptor in their close vicinity. However, a second population (23%) of centromeres was found with a donor fluorescence lifetime <2.09 ns (mean control value of 2.17 ns – 3 SD of 0.03 ns; Figure 4A).Figure 3.

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