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Histone H1 subtypes differentially modulate chromatin condensation without preventing ATP-dependent remodeling by SWI/SNF or NURF.

Clausell J, Happel N, Hale TK, Doenecke D, Beato M - PLoS ONE (2009)

Bottom Line: To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling.In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF.We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.

View Article: PubMed Central - PubMed

Affiliation: Centre de Regulació Genòmica (CRG), Universitat Pompeu Fabra, Barcelona, Spain.

ABSTRACT
Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.

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Role of H1 domains in chromatin affinity and nucleosome spacing.(A) Centrally positioned mononucleosomes were assembled on a 220 bp DNA fragment corresponding to the nucleosome B in the MMTV promoter. After a glycerol gradient purification, mononucleosome particles were incubated with increasing amounts of the following H1.4 domain mutants: the globular domain (GH1.4), the N-terminal plus the globular domain (N-GH1.4), and the globular plus the C-terminal domain (GH1.4-C), and analyzed on a 0.7% (w/v) agarose gel. A lane with wild type H1.4 is shown as control. The different particles are numbered on the left margin: 1) Mononucleosome + H1.4; 2) Mononucleosome + GH1.4-C; 3) Mononucleosome + N-GH1.4; 4) Mononucleosome + GH1.4. The symbol ‘*’ indicates the appearance of aggregates. The amount of each protein is indicated at the base of the gel (lanes 2–17). Protein concentrations are also expressed in 1×10−1 µM units. (B) MNase digestion of minichromosomes assembled with the indicated linker histone. The amount of linker histone added (in nanograms per 200 ng of DNA) is indicated bellow each lane. Lettering is as in (A). Numbers on the left margin refer to the size of the markers (lane M) in base pairs. (C) MNase digestions of minichromosomes with increasing amounts of GH1.0. Also shown is the digestion in the absence of linker histone (−H1) and in the presence of H1.4 (+H1.4). Markers and the amount of protein added are indicated as in (B).
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pone-0007243-g004: Role of H1 domains in chromatin affinity and nucleosome spacing.(A) Centrally positioned mononucleosomes were assembled on a 220 bp DNA fragment corresponding to the nucleosome B in the MMTV promoter. After a glycerol gradient purification, mononucleosome particles were incubated with increasing amounts of the following H1.4 domain mutants: the globular domain (GH1.4), the N-terminal plus the globular domain (N-GH1.4), and the globular plus the C-terminal domain (GH1.4-C), and analyzed on a 0.7% (w/v) agarose gel. A lane with wild type H1.4 is shown as control. The different particles are numbered on the left margin: 1) Mononucleosome + H1.4; 2) Mononucleosome + GH1.4-C; 3) Mononucleosome + N-GH1.4; 4) Mononucleosome + GH1.4. The symbol ‘*’ indicates the appearance of aggregates. The amount of each protein is indicated at the base of the gel (lanes 2–17). Protein concentrations are also expressed in 1×10−1 µM units. (B) MNase digestion of minichromosomes assembled with the indicated linker histone. The amount of linker histone added (in nanograms per 200 ng of DNA) is indicated bellow each lane. Lettering is as in (A). Numbers on the left margin refer to the size of the markers (lane M) in base pairs. (C) MNase digestions of minichromosomes with increasing amounts of GH1.0. Also shown is the digestion in the absence of linker histone (−H1) and in the presence of H1.4 (+H1.4). Markers and the amount of protein added are indicated as in (B).

Mentions: To further investigate the properties of the H1 domains, we generated domain deletion mutants of the H1.4 subtype. GH1.4 is a mutant containing only the GD, N-GH1.4 contains the N-terminal and the GD, and GH1.4-C contains the GD plus the C-terminal domain of H1.4. We assessed the binding properties of the H1.4 domain mutants to mononucleosome particles assembled on a 220 bp DNA fragment using gel retardation assays (Figure 4A; for H1.4 titration see Figure S2). Titration of increasing amounts of each protein showed that wild-type H1.4 and the GH1.4-C mutant had similar affinities, as they promoted gel retardation at similar protein concentrations (lanes 2 and 4), while N-GH1.4 and GH1.4 exhibited a much lower affinity for these particles. A weak retardation is promoted by N-GH1.4, as seen in lanes 10–12. A more precise comparison based on µM concentrations is also shown in Figure 4A. The C-terminus also conferred on H1.4 its nucleosome aggregating properties, as shown in Figure 4A (lane 7). For the concentration at which H1.4 causes aggregation, no aggregation was generated by mutants lacking the C-terminal domain.


Histone H1 subtypes differentially modulate chromatin condensation without preventing ATP-dependent remodeling by SWI/SNF or NURF.

Clausell J, Happel N, Hale TK, Doenecke D, Beato M - PLoS ONE (2009)

Role of H1 domains in chromatin affinity and nucleosome spacing.(A) Centrally positioned mononucleosomes were assembled on a 220 bp DNA fragment corresponding to the nucleosome B in the MMTV promoter. After a glycerol gradient purification, mononucleosome particles were incubated with increasing amounts of the following H1.4 domain mutants: the globular domain (GH1.4), the N-terminal plus the globular domain (N-GH1.4), and the globular plus the C-terminal domain (GH1.4-C), and analyzed on a 0.7% (w/v) agarose gel. A lane with wild type H1.4 is shown as control. The different particles are numbered on the left margin: 1) Mononucleosome + H1.4; 2) Mononucleosome + GH1.4-C; 3) Mononucleosome + N-GH1.4; 4) Mononucleosome + GH1.4. The symbol ‘*’ indicates the appearance of aggregates. The amount of each protein is indicated at the base of the gel (lanes 2–17). Protein concentrations are also expressed in 1×10−1 µM units. (B) MNase digestion of minichromosomes assembled with the indicated linker histone. The amount of linker histone added (in nanograms per 200 ng of DNA) is indicated bellow each lane. Lettering is as in (A). Numbers on the left margin refer to the size of the markers (lane M) in base pairs. (C) MNase digestions of minichromosomes with increasing amounts of GH1.0. Also shown is the digestion in the absence of linker histone (−H1) and in the presence of H1.4 (+H1.4). Markers and the amount of protein added are indicated as in (B).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2748705&req=5

pone-0007243-g004: Role of H1 domains in chromatin affinity and nucleosome spacing.(A) Centrally positioned mononucleosomes were assembled on a 220 bp DNA fragment corresponding to the nucleosome B in the MMTV promoter. After a glycerol gradient purification, mononucleosome particles were incubated with increasing amounts of the following H1.4 domain mutants: the globular domain (GH1.4), the N-terminal plus the globular domain (N-GH1.4), and the globular plus the C-terminal domain (GH1.4-C), and analyzed on a 0.7% (w/v) agarose gel. A lane with wild type H1.4 is shown as control. The different particles are numbered on the left margin: 1) Mononucleosome + H1.4; 2) Mononucleosome + GH1.4-C; 3) Mononucleosome + N-GH1.4; 4) Mononucleosome + GH1.4. The symbol ‘*’ indicates the appearance of aggregates. The amount of each protein is indicated at the base of the gel (lanes 2–17). Protein concentrations are also expressed in 1×10−1 µM units. (B) MNase digestion of minichromosomes assembled with the indicated linker histone. The amount of linker histone added (in nanograms per 200 ng of DNA) is indicated bellow each lane. Lettering is as in (A). Numbers on the left margin refer to the size of the markers (lane M) in base pairs. (C) MNase digestions of minichromosomes with increasing amounts of GH1.0. Also shown is the digestion in the absence of linker histone (−H1) and in the presence of H1.4 (+H1.4). Markers and the amount of protein added are indicated as in (B).
Mentions: To further investigate the properties of the H1 domains, we generated domain deletion mutants of the H1.4 subtype. GH1.4 is a mutant containing only the GD, N-GH1.4 contains the N-terminal and the GD, and GH1.4-C contains the GD plus the C-terminal domain of H1.4. We assessed the binding properties of the H1.4 domain mutants to mononucleosome particles assembled on a 220 bp DNA fragment using gel retardation assays (Figure 4A; for H1.4 titration see Figure S2). Titration of increasing amounts of each protein showed that wild-type H1.4 and the GH1.4-C mutant had similar affinities, as they promoted gel retardation at similar protein concentrations (lanes 2 and 4), while N-GH1.4 and GH1.4 exhibited a much lower affinity for these particles. A weak retardation is promoted by N-GH1.4, as seen in lanes 10–12. A more precise comparison based on µM concentrations is also shown in Figure 4A. The C-terminus also conferred on H1.4 its nucleosome aggregating properties, as shown in Figure 4A (lane 7). For the concentration at which H1.4 causes aggregation, no aggregation was generated by mutants lacking the C-terminal domain.

Bottom Line: To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling.In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF.We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.

View Article: PubMed Central - PubMed

Affiliation: Centre de Regulació Genòmica (CRG), Universitat Pompeu Fabra, Barcelona, Spain.

ABSTRACT
Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.

Show MeSH
Related in: MedlinePlus