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Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

Lopez R, Sarg B, Lindner H, Bartolomé S, Ponte I, Suau P, Roque A - Nucleic Acids Res. (2015)

Bottom Line: Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation.A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation.Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Biociencias, Universidad Autónoma de Barcelona, 08193, Bellaterra, Barcelona, Spain.

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DLS analysis of short chromatin fragments. Panel (A) corresponds to DLS size distributions of short chromatin fragments in 1.6-mM MgCl2. The left panel corresponds to unphosphorylated chromatin and the right panel corresponds to phosphorylated chromatin. Chromatin samples were at 0.2 mg/ml in Tris 10-mM, NaCl 35-mM plus 1.6-mM MgCl2, and incubated at 30°C for 1 h, 5 h or overnight (on). The DLS spectrum at the bottom of the left panel corresponds to the initial chromatin sample in 1.6-mM MgCl2. For each peak the hydrodynamic diameter (d), percentage of intensity (I) and percentage of the volume fraction (V) are shown. (B) Plot of hydrodynamic diameter values of the more aggregated peak in the DLS scan of each sample. Light gray, unphosphorylated chromatin. Dark gray, phosphorylated chromatin. Error bars correspond to the standard deviation.
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Figure 5: DLS analysis of short chromatin fragments. Panel (A) corresponds to DLS size distributions of short chromatin fragments in 1.6-mM MgCl2. The left panel corresponds to unphosphorylated chromatin and the right panel corresponds to phosphorylated chromatin. Chromatin samples were at 0.2 mg/ml in Tris 10-mM, NaCl 35-mM plus 1.6-mM MgCl2, and incubated at 30°C for 1 h, 5 h or overnight (on). The DLS spectrum at the bottom of the left panel corresponds to the initial chromatin sample in 1.6-mM MgCl2. For each peak the hydrodynamic diameter (d), percentage of intensity (I) and percentage of the volume fraction (V) are shown. (B) Plot of hydrodynamic diameter values of the more aggregated peak in the DLS scan of each sample. Light gray, unphosphorylated chromatin. Dark gray, phosphorylated chromatin. Error bars correspond to the standard deviation.

Mentions: In order to observe the effects of phosphorylation on chromatin aggregation, samples were incubated in the presence of CDK2 for 1 h, 5 h and overnight at 30°C. Incubation at 30°C induced, by itself, the aggregation of part of the sample in a time-dependent manner when chromatin was in the presence of 1.6-mM MgCl2 (Supplementary Figure S8C). However, no significant aggregation was observed in 1-mM MgCl2 upon incubation at 30°C (Supplementary Figure S8B). Chromatin aggregation was thus dependent at the same time on the presence of a critical concentration of Mg2+ ions and the temperature and time of incubation. The effects of phosphorylation on chromatin aggregation were, therefore, evaluated in comparison with the degree of aggregation of samples incubated during the same time periods at 30°C, but without kinase (Figures 5 and 6 and Supplementary Table S6).


Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

Lopez R, Sarg B, Lindner H, Bartolomé S, Ponte I, Suau P, Roque A - Nucleic Acids Res. (2015)

DLS analysis of short chromatin fragments. Panel (A) corresponds to DLS size distributions of short chromatin fragments in 1.6-mM MgCl2. The left panel corresponds to unphosphorylated chromatin and the right panel corresponds to phosphorylated chromatin. Chromatin samples were at 0.2 mg/ml in Tris 10-mM, NaCl 35-mM plus 1.6-mM MgCl2, and incubated at 30°C for 1 h, 5 h or overnight (on). The DLS spectrum at the bottom of the left panel corresponds to the initial chromatin sample in 1.6-mM MgCl2. For each peak the hydrodynamic diameter (d), percentage of intensity (I) and percentage of the volume fraction (V) are shown. (B) Plot of hydrodynamic diameter values of the more aggregated peak in the DLS scan of each sample. Light gray, unphosphorylated chromatin. Dark gray, phosphorylated chromatin. Error bars correspond to the standard deviation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482070&req=5

Figure 5: DLS analysis of short chromatin fragments. Panel (A) corresponds to DLS size distributions of short chromatin fragments in 1.6-mM MgCl2. The left panel corresponds to unphosphorylated chromatin and the right panel corresponds to phosphorylated chromatin. Chromatin samples were at 0.2 mg/ml in Tris 10-mM, NaCl 35-mM plus 1.6-mM MgCl2, and incubated at 30°C for 1 h, 5 h or overnight (on). The DLS spectrum at the bottom of the left panel corresponds to the initial chromatin sample in 1.6-mM MgCl2. For each peak the hydrodynamic diameter (d), percentage of intensity (I) and percentage of the volume fraction (V) are shown. (B) Plot of hydrodynamic diameter values of the more aggregated peak in the DLS scan of each sample. Light gray, unphosphorylated chromatin. Dark gray, phosphorylated chromatin. Error bars correspond to the standard deviation.
Mentions: In order to observe the effects of phosphorylation on chromatin aggregation, samples were incubated in the presence of CDK2 for 1 h, 5 h and overnight at 30°C. Incubation at 30°C induced, by itself, the aggregation of part of the sample in a time-dependent manner when chromatin was in the presence of 1.6-mM MgCl2 (Supplementary Figure S8C). However, no significant aggregation was observed in 1-mM MgCl2 upon incubation at 30°C (Supplementary Figure S8B). Chromatin aggregation was thus dependent at the same time on the presence of a critical concentration of Mg2+ ions and the temperature and time of incubation. The effects of phosphorylation on chromatin aggregation were, therefore, evaluated in comparison with the degree of aggregation of samples incubated during the same time periods at 30°C, but without kinase (Figures 5 and 6 and Supplementary Table S6).

Bottom Line: Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation.A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation.Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Biociencias, Universidad Autónoma de Barcelona, 08193, Bellaterra, Barcelona, Spain.

Show MeSH
Related in: MedlinePlus