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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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Effect of histone H1 subtypes on ATP-dependent chromatin remodeling.MMTV minichromosomes were reconstituted in the presence of the histone H1 subtypes, as indicated, purified, resuspended in a 60 mM KCl and 5 mM MgCl2 containing buffer, and incubated with Fok I that cleaves in nucleosome A and B of the MMTV promoter. Increasing nM concentrations of SWI/SNF (A) and NURF (B) complexes were added and incubated for 30 minutes at 30°C. The reaction was stopped and a linear extension PCR with a radiolabeled oligonucleotide was performed. The cleavage products were visualised in a 10% polyacrylamide denaturing gel and quantified using Image Quant software (Amersham). Minichromosomes of two independent purifications were tested and the results represented with the corresponding S.E.M in the graphs bellow. Abbreviations: Nuc A, nucleosome A; Nuc B, nucleosome B; U, uncut material; C, cut material.
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pone-0007243-g005: Effect of histone H1 subtypes on ATP-dependent chromatin remodeling.MMTV minichromosomes were reconstituted in the presence of the histone H1 subtypes, as indicated, purified, resuspended in a 60 mM KCl and 5 mM MgCl2 containing buffer, and incubated with Fok I that cleaves in nucleosome A and B of the MMTV promoter. Increasing nM concentrations of SWI/SNF (A) and NURF (B) complexes were added and incubated for 30 minutes at 30°C. The reaction was stopped and a linear extension PCR with a radiolabeled oligonucleotide was performed. The cleavage products were visualised in a 10% polyacrylamide denaturing gel and quantified using Image Quant software (Amersham). Minichromosomes of two independent purifications were tested and the results represented with the corresponding S.E.M in the graphs bellow. Abbreviations: Nuc A, nucleosome A; Nuc B, nucleosome B; U, uncut material; C, cut material.

Mentions: We next studied the influence of the H1 subtypes on ATP-dependent chromatin remodeling. Two different ATP-dependent chromatin remodeling complexes were used, yeast SWI/SNF and Drosophila NURF. Yeast SWI/SNF was purified from yeast strains expressing a tagged SNF2 subunit as previously described [53] and recombinant Drosophila NURF was purified from baculovirus infected S9 cells by affinity chromatography via the tagged SNF2H subunit [54]. We used Fok I restriction enzyme (RE) cleavage at 30°C as a measure of the increase in chromatin accessibility generated by these chromatin remodelers. Fok I cleaves MMTV minichromosomal DNA at two sites located in separated nucleosomes over the MMTV promoter (Nuc A and Nuc B, Figure 5 and Figure S5) and therefore it gives information of the effect in two different contexts.


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)

Effect of histone H1 subtypes on ATP-dependent chromatin remodeling.MMTV minichromosomes were reconstituted in the presence of the histone H1 subtypes, as indicated, purified, resuspended in a 60 mM KCl and 5 mM MgCl2 containing buffer, and incubated with Fok I that cleaves in nucleosome A and B of the MMTV promoter. Increasing nM concentrations of SWI/SNF (A) and NURF (B) complexes were added and incubated for 30 minutes at 30°C. The reaction was stopped and a linear extension PCR with a radiolabeled oligonucleotide was performed. The cleavage products were visualised in a 10% polyacrylamide denaturing gel and quantified using Image Quant software (Amersham). Minichromosomes of two independent purifications were tested and the results represented with the corresponding S.E.M in the graphs bellow. Abbreviations: Nuc A, nucleosome A; Nuc B, nucleosome B; U, uncut material; C, cut material.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2748705&req=5

pone-0007243-g005: Effect of histone H1 subtypes on ATP-dependent chromatin remodeling.MMTV minichromosomes were reconstituted in the presence of the histone H1 subtypes, as indicated, purified, resuspended in a 60 mM KCl and 5 mM MgCl2 containing buffer, and incubated with Fok I that cleaves in nucleosome A and B of the MMTV promoter. Increasing nM concentrations of SWI/SNF (A) and NURF (B) complexes were added and incubated for 30 minutes at 30°C. The reaction was stopped and a linear extension PCR with a radiolabeled oligonucleotide was performed. The cleavage products were visualised in a 10% polyacrylamide denaturing gel and quantified using Image Quant software (Amersham). Minichromosomes of two independent purifications were tested and the results represented with the corresponding S.E.M in the graphs bellow. Abbreviations: Nuc A, nucleosome A; Nuc B, nucleosome B; U, uncut material; C, cut material.
Mentions: We next studied the influence of the H1 subtypes on ATP-dependent chromatin remodeling. Two different ATP-dependent chromatin remodeling complexes were used, yeast SWI/SNF and Drosophila NURF. Yeast SWI/SNF was purified from yeast strains expressing a tagged SNF2 subunit as previously described [53] and recombinant Drosophila NURF was purified from baculovirus infected S9 cells by affinity chromatography via the tagged SNF2H subunit [54]. We used Fok I restriction enzyme (RE) cleavage at 30°C as a measure of the increase in chromatin accessibility generated by these chromatin remodelers. Fok I cleaves MMTV minichromosomal DNA at two sites located in separated nucleosomes over the MMTV promoter (Nuc A and Nuc B, Figure 5 and Figure S5) and therefore it gives information of the effect in two different contexts.

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