The C-terminal domain is the primary determinant of histone H1 binding to chromatin in vivo.
We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1 in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 binding in vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.
Cross Cancer Institute and Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada.
Michael J
MJ
Hendzel
Melody A
MA
Lever
Ellen
E
Crawford
John P H
JP
Th'ng
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D017353:Gene Deletion; D018698:Glutamic Acid / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D002843:Chromatin; D006657:Histones; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D013912:Threonine; D018698:Glutamic Acid; D012694:Serine; D008239:Lysine; D000409:Alanine