Interaction of Xenopus lamins A and LII with chromatin in vitro mediated by a sequence element in the carboxyterminal domain.
Morphological data suggest an interaction of the nuclear lamina with chromatin which markedly changes during the cell cycle. To study the molecular basis of this interaction we developed a novel lamin/chromatin binding assay that quantitated the binding of soluble, radiolabeled lamins to minichromosomes assembled in Xenopus laevis oocyte nuclear extracts. Lamins were derived from couple in vitro transcription and translation of the corresponding cDNAs. Chromatin binding was detected by monitoring the cofractionation with assembled minichromosomes in gel filtration and sucrose gradient centrifugation. Binding of lamins to chromatin increased with chromatin concentration and was accompanied by lamin polymerization. Lamins of the A-(Xenopus LA and human LC) as well as the B-type (Xenopus LI and LII) showed strikingly different chromatin binding capacities. Lamins A and LII bound efficiently of lamins LI and LC was detected. Using site-directed mutagenesis, we were able to define carboxy-terminal sequence elements of LA and LII required for the observed lamin/chromatin interaction that are rich in serine, threonine, and glycine residues. Competition experiments with a synthetic peptide containing the chromatin binding motif of lamin A corroborate the importance of these sequence elements in the lamin/chromatin interaction.
Höger
T H
TH
Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg.
Krohne
G
G
Kleinschmidt
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism*; D002875:Chromosomes / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D034904:Lamin Type A; D034882:Lamins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D002843:Chromatin; D034904:Lamin Type A; D034882:Lamins; D009687:Nuclear Proteins; D010455:Peptides