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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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Schematic representation of centromeric chromatin with a CENP-A containing nucleosome. In this model, the internucleosomal linker DNA is alternatively occupied by histone H1 and CENP-B. The arrows indicate the protein associations detected in this study.
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Figure 5: Schematic representation of centromeric chromatin with a CENP-A containing nucleosome. In this model, the internucleosomal linker DNA is alternatively occupied by histone H1 and CENP-B. The arrows indicate the protein associations detected in this study.

Mentions: The impact of H1 on centromere structure and function is elusive. As CENP-B, also H1 might contribute to the positioning of successive nucleosomes and the pattern of nucleosome arrangement (49). CENP-B binds to the sequence-specific 17-bp boxes present on α-satellite DNA repeats and H1 is able to suppress the binding of other proteins to the same linker (71) suggesting that CENP-B and H1 might bind to neighbouring linkers (Figure 5). One could speculate that both linker DNA binding proteins, H1 and CENP-B, might cooperate leading to a more condensed and/or stable centromere-specific chromatin structure (113–115) which might contribute to function and identity of centromeric chromatin (113,115). In this case, the presence of CENP-B might influence H1 binding. This view, however, is not supported by our CENP-B knock down data. Within the experimental error range, H1 showed unchanged dynamics at centromeric chromatin in CENP-B down-regulated interphase cells. Furthermore, loss of CENP-B does not offer an additional H1 binding site since, due to its mode of interaction at the exit of both linker DNAs, only one H1 can be found per nucleosome. On the other hand, linker histone H1 might be able to partly take over CENP-B function in CENP-B-depleted centromeric chromatin. This would provide an explanation for the maintenance of an active kinetochore state despite the lack of CENP-B and/or CENP-B boxes. The molecular properties of CENP-B (18,116) are, however, distinct from those of H1: CENP-B, in contrast to H1, binds to a specific DNA binding site, the CENP-B box. Furthermore, CENP-B dimerizes tail-to-tail at its C-termini (29,30) also in vivo (S. Orthaus, unpublished results). We found no sequence homology and no subdomain similarities between CENP-B and H1. Thus, H1 is not expected to be able to fully replace CENP-B. If α-satellite chromatin forms a 30-nm fiber structure, CENP-B (and H1) might bind at the interior of the solenoid as discussed by Pluta et al. (28). This would be consistent with a tandem but not with a tail-to-tail dimerization of CENP-B (J. Sühnel and S. Diekmann, unpublished data). On the other hand, the negative charge of CENP-B is expected to destabilize this 30-nm structure (28) or might even induce another. Currently, the centromeric chromatin structure is unclear.Figure 5.


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)

Schematic representation of centromeric chromatin with a CENP-A containing nucleosome. In this model, the internucleosomal linker DNA is alternatively occupied by histone H1 and CENP-B. The arrows indicate the protein associations detected in this study.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Schematic representation of centromeric chromatin with a CENP-A containing nucleosome. In this model, the internucleosomal linker DNA is alternatively occupied by histone H1 and CENP-B. The arrows indicate the protein associations detected in this study.
Mentions: The impact of H1 on centromere structure and function is elusive. As CENP-B, also H1 might contribute to the positioning of successive nucleosomes and the pattern of nucleosome arrangement (49). CENP-B binds to the sequence-specific 17-bp boxes present on α-satellite DNA repeats and H1 is able to suppress the binding of other proteins to the same linker (71) suggesting that CENP-B and H1 might bind to neighbouring linkers (Figure 5). One could speculate that both linker DNA binding proteins, H1 and CENP-B, might cooperate leading to a more condensed and/or stable centromere-specific chromatin structure (113–115) which might contribute to function and identity of centromeric chromatin (113,115). In this case, the presence of CENP-B might influence H1 binding. This view, however, is not supported by our CENP-B knock down data. Within the experimental error range, H1 showed unchanged dynamics at centromeric chromatin in CENP-B down-regulated interphase cells. Furthermore, loss of CENP-B does not offer an additional H1 binding site since, due to its mode of interaction at the exit of both linker DNAs, only one H1 can be found per nucleosome. On the other hand, linker histone H1 might be able to partly take over CENP-B function in CENP-B-depleted centromeric chromatin. This would provide an explanation for the maintenance of an active kinetochore state despite the lack of CENP-B and/or CENP-B boxes. The molecular properties of CENP-B (18,116) are, however, distinct from those of H1: CENP-B, in contrast to H1, binds to a specific DNA binding site, the CENP-B box. Furthermore, CENP-B dimerizes tail-to-tail at its C-termini (29,30) also in vivo (S. Orthaus, unpublished results). We found no sequence homology and no subdomain similarities between CENP-B and H1. Thus, H1 is not expected to be able to fully replace CENP-B. If α-satellite chromatin forms a 30-nm fiber structure, CENP-B (and H1) might bind at the interior of the solenoid as discussed by Pluta et al. (28). This would be consistent with a tandem but not with a tail-to-tail dimerization of CENP-B (J. Sühnel and S. Diekmann, unpublished data). On the other hand, the negative charge of CENP-B is expected to destabilize this 30-nm structure (28) or might even induce another. Currently, the centromeric chromatin structure is unclear.Figure 5.

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