The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain

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Abstract

The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter–enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization.

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.

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