Emerin belongs to the "LEM domain" family of nuclear proteins, which contain a characteristic ϳ40-residue LEM motif. The LEM domain mediates direct binding to barrier to autointegration factor (BAF), a conserved 10-kDa chromatin protein essential for embryogenesis in Caenorhabditis elegans. In mammalian cells, BAF recruits emerin to chromatin during nuclear assembly. BAF also mediates chromatin decondensation during nuclear assembly. The LEM domain and central region of emerin are essential for binding to BAF and lamin A, respectively. However, two other conserved regions of emerin lacked ascribed functions, suggesting that emerin could have additional partners. We discovered that these "unascribed" domains of emerin mediate direct binding to a transcriptional repressor, germ cellless (GCL). GCL co-immunoprecipitates with emerin from HeLa cells. We determined the binding affinities of emerin for GCL, BAF, and lamin A and analyzed their oligomeric interactions. We showed that emerin forms stable complexes with either lamin A plus GCL or lamin A plus BAF. Importantly, BAF competed with GCL for binding to emerin in vitro, predicting that emerin can form at least two distinct types of complexes in vivo. Loss of emerin causes Emery-Dreifuss muscular dystrophy, a tissue-specific inherited disease that affects skeletal muscles, major tendons, and the cardiac conduction system. Although GCL alone cannot explain the disease mechanism, our results strongly support gene expression models for Emery-Dreifuss muscular dystrophy by showing that emerin binds directly to a transcriptional repressor, GCL, and by suggesting that emerin-repressor complexes might be regulated by BAF. Biochemical roles for emerin in gene expression are discussed.

Nesprin-1K K is a spectrin repeat (SR)-containing, transmembrane protein of the inner nuclear membrane, and is highly expressed in muscle cells. A yeast two-hybrid screen for nesprin-1K K-interacting proteins showed that nesprin-1K K interacted with itself. Blot overlay experiments revealed that nesprin-1K K's third SR binds the ¢fth SR. The carboxy-terminal half of nesprin-1K K directly bound lamin A, a nuclear intermediate ¢lament protein. Biochemical analysis demonstrated that nesprin-1K K dimers bind directly to the nucleoplasmic domain of emerin, an inner nuclear membrane protein, with an a⁄nity of 4 nM. Binding was optimal for full nucleoplasmic dimers of nesprin-1K K, since nesprin fragments SR1-5 and SR5-7 bound emerin as monomers with a⁄nities of 53 nM and 250 mM, respectively. We propose that membrane-anchored nesprin-1K K antiparallel dimers interact with both emerin and lamin A to provide sca¡olding at the inner nuclear membrane. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Using recombinant bead-conjugated emerin we affinity-purified seven proteins from HeLa cell nuclear lysates that bind emerin either directly or indirectly. These proteins were identified by mass spectrometry as nuclear αII-spectrin, non-muscle myosin heavy chain alpha, Lmo7 (a predicted transcription regulator; reported separately), nuclear myosin I, β-actin (reported separately), calponin 3 and SIKE. We now report emerin binds nuclear myosin I (NMI, a molecular motor) directly in vitro. Furthermore, bead-conjugated emerin bound nuclear αII-spectrin and NMI equally well with or without ATP (which stimulates motor activity), whereas ATP decreased actin binding by 65%. Thus αII-spectrin and NMI interact stably with emerin. To investigate the physiological relevance of these interactions, we used antibodies against emerin to affinity-purify emerin-associated protein complexes from HeLa cells, then further purified by ion exchange chromatography to resolve by net charge, and size exclusion chromatography yielding six distinct emerin-containing fractions (0.5 to 1.6 MDa). Western blotting suggested each complex had distinct components involved in nuclear architecture (e.g., NMI, αII-spectrin, lamins) or gene or chromatin regulation (BAF, transcription regulators, HDACs). Additional constituents were identified by mass spectrometry. One putative gene-regulatory complex (complex 32) included core components of the Nuclear Co-Repressor (NCoR) Complex, which mediates gene regulation by thyroid hormone and other nuclear receptors. 155/170; BAT, HLA-B-associated transcript 1; BCCIP, BRCA2 and CDKN1A-interacting protein; BSA, Bovine serum albumin; Btf, Bcl-2-associated transcription factor; Dnmt1, DNA methyltransferase 1; DRIP, Vitamin D receptor-interacting protein; EDMD, EmeryDreifuss muscular dystrophy; ERBA1, Thyroid hormone receptor alpha; FUBP1, far upstream element binding protein 1; G3BP1, RasGTPase-activating protein-binding protein; GCL, Germ-cell less; H1, histone 1; H3, histone 3; H3K9me, H3-lysine-9-methyl; HDAC, Histone deacetylase; Gps2, G protein pathway suppressor 2; HDGF, hepatoma-derived growth factor; hnRNP, heterogeneous nuclear ribonucleoprotein; HP1, heterochromatin protein 1; IQGAP1, IQ motif-containing GTPase-activating protein 1; KAP1, KRAB-associated protein 1; KCIP-1, Protein kinase C inhibitor protein 1; LBR, lamin B receptor; Lmo7, Lim domain only 7; Lsm, Sm-like; MCM, minichromosome maintenance; Mst-3, mammalian sterile 20 kinase-like 3; NCoR, Nuclear Co-repressor; NFAR, nuclear factor associated with double-stranded RNA; NF-Y, nuclear transcription factor Y; NMI, nuclear myosin I; NMMHCA, non-muscle myosin heavy chain A; NuMA, nuclear mitotic apparatus protein; NURD, nucleosome remodeling and histone deacetylation; PDCD4, programmed cell death 4; Rb, Retinoblastoma protein; RBD, repressor-binding domain; Rpd3, reduced potassium dependency 3; SAP18, Sin3-associated polypeptide 18 kD; SAP130, spliceosome-associated polypeptide 130 kD; SETDB1, SET-domain protein bifurcated 1; SF2, Splici...