Histone H5-chromatin interactions in situ are strongly modulated by H5 C-terminal phosphorylation.
We used linker histone-depleted normal human fibroblast nuclei as templates to study how phosphorylation affects histone H5 binding to chromatin in situ. Permeabilized cells were treated with 0.7 M NaCl to extract the native linker histones. Histone H5 was purified from chicken erythrocytes and phosphorylated in vitro by recombinant cdk5/p35 kinase. High performance capillary electrophoresis (HPCE) showed that the phosphorylated protein contained a mixture of multiply phosphorylated forms. Control experiments, using mass spectrometry, revealed that up to five SPXK motifs in the C terminus were phosphorylated, but also that about 10% of the protein contained one phosphoserine in the N-terminus. Reconstitution of H1-depleted fibroblast nuclei with nonphosphorylated or phosphorylated H5 was performed at physiological ionic strength. The bound H5 was then extracted using NaCl concentrations in the range of 0.15 to 0.7 M. The release of the H5 molecules was monitored by DAPI staining and image cytofluorometry. Our results show that H5 phosphorylation substantially reduced its affinity for chromatin in situ, which support previous observations indicating that C-terminal phosphorylation may be essential for the biological functions of linker histones.
Institute of Molecular Biology, Bulgarian Academy of Sciences, BG-1113 Sofia, Bulgaria.
Nora N
NN
Kostova
Ljuba
L
Srebreva
Dimiter V
DV
Markov
Bettina
B
Sarg
Herbert H
HH
Lindner
Ingemar
I
Rundquist
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens / Q000097:blood; D002843:Chromatin / Q000378:metabolism*; D051360:Cyclin-Dependent Kinase 5 / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005347:Fibroblasts; D005434:Flow Cytometry; D006657:Histones / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding
D048868:Adaptor Proteins, Signal Transducing; C500695:CDCA5 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D006657:Histones; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human