The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain
Section snippets
Materials and methods
Plasmids. Construction of plasmids used in this study and oligonucleotide primers used for cDNA amplification or mutagenesis are contained in supplementary material available online.
GST protein-binding assays. The GST and polyhistidine fusion proteins were purified from bacteria under native conditions and the recombinant polyhistidine CSD proteins were subjected to size exclusion chromatography on a Superdex 200 column (GE-Healthcare-Amersham) as described previously [14]. The polyhistidine
Identification of the CSD-binding motif in Sp100, LBR, ATRX, NIPBL, and HP1-BP74
In a previous study, we identified a short sequence in the KAP-1 corepressor protein required for binding directly to the CSD region of HP1 proteins [14], which was very similar to the HP1-binding sequence in CAF-1 p150 [15]. These sequences appear to minimally carry a PxVxL motif, similar to pentameric sequences found through phage display analysis with the D. melanogaster HP1 CSD [24]. Several other proteins with HP1-binding activity have been identified, however they have yet to be
Discussion
The work presented in this study has expanded the definition of the consensus sequence for CSD-binding by defining variants of the PxVxL sequence that are contained in physiologically relevant partners of HP1 proteins. We can draw the following conclusions from this work: (1) the binding of both variant and canonical PxVxL sequences is direct, (2) mutations in the CSD that abolish dimerization also abolish interaction with partners, (3) the surface of the CSD bound by each type of sequence is
Acknowledgments
We thank the members of the Rauscher laboratory for helpful discussions in support of this work. We are grateful to Dr. D. Picketts for the ATRX plasmid and Dr. J. Frey for the Sp100 antisera. We also thank the Wistar Institute Hybridoma Facility for production of monoclonal antibodies, the Wistar Institute Peptide Facility for synthesis of peptides, and the Wistar Institute Protein Microchemistry/Mass Spectrometry Laboratory. M.S.L. was supported by basic cancer research training Grant CA
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Present address: Department of Pharmacology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA.