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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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Different H1 subtypes are present at centromeric chromatin, however, do not show any specific enrichment or reduction at human centromeres. HeLa cells were stained with (A) anti-H1°, (B) anti-H1.2/H1.5, (C) anti-H1.3, (D) anti-H1.5 and (E) anti-H1x antibodies and visualized by a species-specific secondary antibody fused to Alexa-Fluor 488 (green). Centromeres were stained with ACA (CY3, red). One confocal plane was examined per cell. Bars = 10 μm. The profiles (right) display the red and green fluorescence intensity along the indicated arrow in ‘Merge’.
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Figure 1: Different H1 subtypes are present at centromeric chromatin, however, do not show any specific enrichment or reduction at human centromeres. HeLa cells were stained with (A) anti-H1°, (B) anti-H1.2/H1.5, (C) anti-H1.3, (D) anti-H1.5 and (E) anti-H1x antibodies and visualized by a species-specific secondary antibody fused to Alexa-Fluor 488 (green). Centromeres were stained with ACA (CY3, red). One confocal plane was examined per cell. Bars = 10 μm. The profiles (right) display the red and green fluorescence intensity along the indicated arrow in ‘Merge’.

Mentions: First, we asked if one of the H1 subtypes might be centromere-specific and if any enrichment or depletion of particular histone H1 subtypes can be detected at human centromeres. Therefore, human HEp-2 cells were stained with specific antibodies against the H1 subtypes H1°, H1.2, H1.3, H1.5 and H1x. Co-staining of centromeres with ACA (anti-centromere antibody) serum (containing antibodies against human CENP-A, -B and -C) revealed the presence of these H1 subtypes at interphase centromeres at levels similar to those at other locations in the nucleus. Furthermore, no specific association or exclusion of the analysed H1 subtypes at centromeres was found as indicated by the absence of any correlation in the fluorescence intensity overlay between the stained H1 subtypes and the centromeres. Images together with intensity profiles along one centromere are displayed in Figure 1 and a statistical analysis including a higher number of centromeres is presented in Supplementary Table 1. We conclude that these H1 proteins are neither enriched nor depleted at interphase centromeres. More detailed studies were carried out for H1° and H1.2 fusion proteins that have been ectopically expressed in human HEp-2 cells. The correct full length expression of each fusion protein was controlled by western blot analysis (Supplementary Figure 1). Microscopical analysis of the transfected interphase cells, co-stained with an H1° antibody, ensured that the fusion proteins showed a similar distribution as the endogeneous linker histones (Supplementary Figure 2). For life cell experiments, we selected these two subtypes for the following reasons: H1° is expressed during the complete cell cycle, mainly in terminally differentiated cells with an intermediate chromatin binding affinity, and H1.2 distributes throughout the nucleus, it is one of the most predominant subtypes in most human cells (96). H1.2 distribution correlates with DNA concentration (97), it seems to be a basic subtype being responsible for a ground level of chromatin compaction.Figure 1.


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)

Different H1 subtypes are present at centromeric chromatin, however, do not show any specific enrichment or reduction at human centromeres. HeLa cells were stained with (A) anti-H1°, (B) anti-H1.2/H1.5, (C) anti-H1.3, (D) anti-H1.5 and (E) anti-H1x antibodies and visualized by a species-specific secondary antibody fused to Alexa-Fluor 488 (green). Centromeres were stained with ACA (CY3, red). One confocal plane was examined per cell. Bars = 10 μm. The profiles (right) display the red and green fluorescence intensity along the indicated arrow in ‘Merge’.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Different H1 subtypes are present at centromeric chromatin, however, do not show any specific enrichment or reduction at human centromeres. HeLa cells were stained with (A) anti-H1°, (B) anti-H1.2/H1.5, (C) anti-H1.3, (D) anti-H1.5 and (E) anti-H1x antibodies and visualized by a species-specific secondary antibody fused to Alexa-Fluor 488 (green). Centromeres were stained with ACA (CY3, red). One confocal plane was examined per cell. Bars = 10 μm. The profiles (right) display the red and green fluorescence intensity along the indicated arrow in ‘Merge’.
Mentions: First, we asked if one of the H1 subtypes might be centromere-specific and if any enrichment or depletion of particular histone H1 subtypes can be detected at human centromeres. Therefore, human HEp-2 cells were stained with specific antibodies against the H1 subtypes H1°, H1.2, H1.3, H1.5 and H1x. Co-staining of centromeres with ACA (anti-centromere antibody) serum (containing antibodies against human CENP-A, -B and -C) revealed the presence of these H1 subtypes at interphase centromeres at levels similar to those at other locations in the nucleus. Furthermore, no specific association or exclusion of the analysed H1 subtypes at centromeres was found as indicated by the absence of any correlation in the fluorescence intensity overlay between the stained H1 subtypes and the centromeres. Images together with intensity profiles along one centromere are displayed in Figure 1 and a statistical analysis including a higher number of centromeres is presented in Supplementary Table 1. We conclude that these H1 proteins are neither enriched nor depleted at interphase centromeres. More detailed studies were carried out for H1° and H1.2 fusion proteins that have been ectopically expressed in human HEp-2 cells. The correct full length expression of each fusion protein was controlled by western blot analysis (Supplementary Figure 1). Microscopical analysis of the transfected interphase cells, co-stained with an H1° antibody, ensured that the fusion proteins showed a similar distribution as the endogeneous linker histones (Supplementary Figure 2). For life cell experiments, we selected these two subtypes for the following reasons: H1° is expressed during the complete cell cycle, mainly in terminally differentiated cells with an intermediate chromatin binding affinity, and H1.2 distributes throughout the nucleus, it is one of the most predominant subtypes in most human cells (96). H1.2 distribution correlates with DNA concentration (97), it seems to be a basic subtype being responsible for a ground level of chromatin compaction.Figure 1.

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