Barrier-to-autointegration factor: major roles in chromatin decondensation and nuclear assembly

Barrier-to-autointegration factor (BAF) is a DNA-bridging protein, highly conserved in metazoans. BAF binds directly to LEM (LAP2, emerin, MAN1) domain nuclear membrane proteins, including LAP2 and emerin. We used site-directed mutagenesis and biochemical analysis to map functionally important residues in human BAF, including those required for direct binding to DNA or emerin. We also tested wild-type BAF and 25 point mutants for their effects on nuclear assembly in Xenopus egg extracts, which contain approximately 12 microM endogenous BAF dimers. Exogenous BAF caused two distinct effects: at low added concentrations, wild-type BAF enhanced chromatin decondensation and nuclear growth; at higher added concentrations, wild-type BAF completely blocked chromatin decondensation and nuclear growth. Mutants fell into four classes, including one that defines a novel functional surface on the BAF dimer. Our results suggest that BAF, unregulated, potently compresses chromatin structure, and that BAF interactions with both DNA and LEM proteins are critical for membrane recruitment and chromatin decondensation during nuclear assembly.     

Review: lamina-associated polypeptide 2 isoforms and related proteins in cell cycle-dependent nuclear structure dynamics

The lamina-associated polypeptide (LAP) 2 family comprises up to six alternatively spliced proteins in mammalian cells and three isoforms in Xenopus. LAP2beta is a type II integral protein of the inner nuclear membrane, which binds to lamin B and the chromosomal protein BAF, and may link the nuclear membrane to the underlying lamina and provide docking sites for chromatin. LAP2alpha shares only the N-terminus with the other isoforms and contains a unique C-terminus. It is a nonmembrane protein associated with the nucleoskeleton and may help to organize higher order chromatin structure by interacting with A-lamins and chromosomes. Recent studies using mutant proteins have just begun to unravel functions of LAP2 isoforms during postmitotic nuclear reassembly. LAP2alpha associates with chromosomes via an alpha-specific domain at early stages of assembly, possibly providing a structural framework for chromosome reorganization. The subsequent interaction of both LAP2alpha and LAP2beta with the chromosomal BAF may stabilize chromatin structure and target membranes to the chromosomes. At later stages LAP2 may regulate the assembly of lamins. LAP2 isoforms have been found to share a homologous approximately 40 amino acid long region, the LEM domain, with nuclear membrane proteins MAN1 and emerin, which has been implicated in Emery-Dreifuss muscular dystrophy.     

Structural characterization of the LEM motif common to three human inner nuclear membrane proteins.

BACKGROUND: Integral membrane proteins of the inner nuclear membrane are involved in chromatin organization and postmitotic reassembly of the nucleus. The discovery that mutations in the gene encoding emerin causes X-linked Emery-Dreifuss muscular dystrophy has enhanced interest in such proteins. A common structural domain of 50 residues, called the LEM domain, has been identified in emerin MAN1, and lamina-associated polypeptide (LAP) 2. In particular, all LAP2 isoforms share an N-terminal segment composed of such a LEM domain that is connected to a highly divergent LEM-like domain by a linker that is probably unstructured. RESULTS: We have determined the three-dimensional structures of the LEM and LEM-like domains of LAP2 using nuclear magnetic resonance and molecular modeling. Both domains adopt the same fold, mainly composed of two large parallel alpha helices. CONCLUSIONS: The structural LEM motif is found in human inner nuclear membrane proteins and in protein-protein interaction domains from bacterial multienzyme complexes. This suggests that LEM and LEM-like domains are protein-protein interaction domains. A region conserved in all LEM domains, at the surface of helix 2, could mediate interaction between LEM domains and a common protein partner.     

Roles of LAP2 proteins in nuclear assembly and DNA replication: truncated LAP2beta proteins alter lamina assembly, envelope formation, nuclear size, and DNA replication efficiency in Xenopus laevis extracts

Humans express three major splicing isoforms of LAP2, a lamin- and chromatin-binding nuclear protein. LAP2beta and gamma are integral membrane proteins, whereas alpha is intranuclear. When truncated recombinant human LAP2beta proteins were added to cell-free Xenopus laevis nuclear assembly reactions at high concentrations, a domain common to all LAP2 isoforms (residues 1-187) inhibited membrane binding to chromatin, whereas the chromatin- and lamin-binding region (residues 1-408) inhibited chromatin expansion. At lower concentrations of the common domain, membranes attached to chromatin with a unique scalloped morphology, but these nuclei neither accumulated lamins nor replicated. At lower concentrations of the chromatin- and lamin-binding region, nuclear envelopes and lamins assembled, but nuclei failed to enlarge and replicated on average 2. 5-fold better than controls. This enhancement was not due to rereplication, as shown by density substitution experiments, suggesting the hypothesis that LAP2beta is a downstream effector of lamina assembly in promoting replication competence. Overall, our findings suggest that LAP2 proteins mediate membrane-chromatin attachment and lamina assembly, and may promote replication by influencing chromatin structure.     

LEM4/ANKLE-2 deficiency impairs post-mitotic re-localization of BAF,LAP2α and LaminA to the nucleus,causes nuclear envelope instability in telophase and leads to hyperploidy in HeLa cells

The human LEM-domain protein family is involved in fundamental aspects of nuclear biology. The LEM-domain interacts with the barrier-to-autointegration factor (BAF), which itself binds DNA. LEM-domain proteins LAP2, emerin and MAN1 are proteins of the inner nuclear membrane; they have important functions: maintaining the integrity of the nuclear lamina and regulating gene expression at the nuclear periphery.LEM4/ANKLE-2 has been proposed to participate in nuclear envelope reassembly after mitosis and to mediate dephosphorylation of BAF through binding to phosphatase PP2A. Here, we used CRISPR/Cas9 to create several cell lines deficient in LEM4/ANKLE-2. By using time-lapse video microscopy, we show that absence of this protein severely compromises the post mitotic re-association of the nuclear proteins BAF, LAP2α and LaminA to chromosomes. These defects give rise to a strong mechanical instability of the nuclear envelope in telophase and to a chromosomal instability leading to increased number of hyperploid cells. Reintroducing LEM4/ANKLE-2 in the cells by transfection could efficiently restore the telophase association of BAF and LAP2α to the chromosomes. This rescue phenotype was abolished for N- or C-terminally truncated mutants that had lost the capacity to bind PP2A. We demonstrate also that, in addition to binding to PP2A, LEM4/ANKLE-2 binds BAF through its LEM-domain, providing further evidence for a generic function of this domain as a principal interactor of BAF.     

TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts

Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.     

Distinct functions of the unique C terminus of LAP2alpha in cell proliferation and nuclear assembly

The non-membrane-bound lamina-associated polypeptide 2 isoform, LAP2alpha, forms nucleoskeletal structures with A-type lamins and interacts with chromosomes in a cell cycle-dependent manner. LAP2alpha contains a LEM (LAP2, emerin, and MAN1) domain in the constant N terminus that binds to chromosomal barrier-to-autointegration factor, and a C-terminal unique region that is essential for chromosome binding. Here we show that C-terminal LAP2alpha fragment efficiently bound to mitotic chromosomes and inhibited assembly of endogenous LAP2alpha, nuclear membranes, and lamins A/C in in vitro nuclear assembly assays. Full-length recombinant LAP2alpha, which bound to chromosomes, and N-terminal fragment, which did not bind, had no effect on assembly. This suggested an essential role for the LAP2alpha C terminus in chromosome association and for the N-terminal LEM domain in subsequent assembly stages. In vivo analysis upon transient expression of GFP-tagged LAP2alpha fragments confirmed that, unlike the N-terminal fragment, the C-terminal fragment was able to bind to chromosomes during mitosis, if expressed weakly. At higher expression levels, C-terminal LAP2alpha fragment and full-length protein led to cell cycle arrest in interphase and apoptosis, as shown by fluorescence-activated cell sorter analysis, time lapse microscopy, and BrdUrd incorporation assays. These data indicated distinct functions of LAP2alpha in cell cycle progression during interphase and in nuclear reassembly during mitosis.     

Solution NMR structure of the barrier-to-autointegration factor-Emerin complex

The barrier-to-autointegration factor BAF binds to the LEM domain (Em(LEM)) of the nuclear envelope protein emerin and plays an essential role in the nuclear architecture of metazoan cells. In addition, the BAF(2) dimer bridges and compacts double-stranded DNA nonspecifically via two symmetry-related DNA binding sites. In this article we present biophysical and structural studies on a complex of BAF(2) and Em(LEM). Light scattering, analytical ultracentrifugation, and NMR indicate a stoichiometry of one molecule of Em(LEM) bound per BAF(2) dimer. The equilibrium dissociation constant (K(d)) for the interaction of the BAF(2) dimer and Em(LEM), determined by isothermal titration calorimetry, is 0.59 +/- 0.03 microm. Z-exchange spectroscopy between corresponding cross-peaks of the magnetically non-equivalent subunits of the BAF(2) dimer in the complex yields a dissociation rate constant of 78 +/- 2s(-1). The solution NMR structure of the BAF(2)-Em(LEM) complex reveals that the LEM and DNA binding sites on BAF(2) are non-overlapping and that both subunits of the BAF(2) dimer contribute approximately equally to the Em(LEM) binding site. The relevance of the implications of the structural and biophysical data on the complex in the context of the interaction between the BAF(2) dimer and Em(LEM) at the nuclear envelope is discussed.     

Lamina-associated polypeptide 2beta (LAP2beta) is contained in a protein complex together with A- and B-type lamins

Lamina-associated polypeptide 2beta (LAP2beta) of vertebrates is an integral membrane protein of the inner nuclear membrane that is generated by alternative splicing from the LAP2 gene. In the majority of Xenopus somatic cells including cultured kidney epithelial cells (A6 cells) there is only one major LAP2 isoform expressed that has the highest similarities with the mammalian LAP2beta whereas isoforms corresponding in size to the mammalian LAP2gamma and alpha are not detectable. We selected A6 cells and A6 cells stably expressing GFP fusion proteins of Xenopus LAP2beta (XLAP2Pbeta) as a model system to study interactions between LAP2beta and lamins. In vitro binding experiments with GST-XLAP2beta fusion proteins and immunoprecipitations with antibodies to GFP revealed that XLAP2beta is part of a complex that contains A- and B-type lamins. For the targeting to the nuclear envelope and the in vivo formation of this complex, GFP fusion proteins were sufficient comprising only the carboxyterminal 135 amino acids of XLAP2beta or the comparable region of zebrafish LAP2beta. A highly conserved 36 amino acids long sequence is located in this region of LAP2beta that is part of the lamina-binding domain previously identified in rat LAP2beta. GFP-LAP2beta fusion proteins of Xenopus, zebrafish, and rat that contained this sequence do compete with endogenous LAP2 in transfected cells for the same binding sites in the lamina. Our data indicate that the lamina-binding site of LAP2beta has been highly conserved during vertebrate evolution and suggests that this region of LAP2beta mediates the interactions between polymers of A- and B-type lamins.     

Functional diversity of LAP2alpha and LAP2beta in postmitotic chromosome association is caused by an alpha-specific nuclear targeting domain

Lamina-associated polypeptide 2alpha (LAP2alpha) is a non-membrane-bound isoform of the LAP2 family implicated in nuclear structure organization. We show that during postmitotic nuclear assembly LAP2alpha associates with chromosomes prior to accumulation of the membrane-bound isoform LAP2beta, although both proteins contain the same putative chromatin interaction domains located in their common N-terminal regions. By transient and stable expression of various N- and C-terminal LAP2alpha deletion mutants in HeLa cells, we identified an approximately 350-amino-acid-long region in the C-terminal alpha-specific domain of the protein that is required for retention of LAP2alpha in interphase nuclei and for association with mitotic chromosomes, while the N-terminal domain seemed to be dispensable for these interactions. In vitro chromosome binding studies using recombinant LAP2alpha mutants revealed that this LAP2alpha-specific 'nuclear targeting domain' was essential and sufficient for association with chromosomes. These data suggested a functional diversity of chromosome binding properties of LAP2 isoforms.     

Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro

Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly.     

Two novel LEM-domain proteins are splice products of the annotated Drosophila melanogaster gene CG9424 (Bocksbeutel)

The LEM motif is a sequence of 40-50 amino acids that has been identified in a number of non-related proteins of the inner nuclear membrane including the lamina-associated polypeptides 2 (LAP2), emerin, MAN1 and the Drosophila protein otefin. This evolutionary conserved sequence motif can mediate via the interaction with the small protein BAF the binding of LEM-domain proteins to DNA. Taking advantage of its sequenced genome we analyzed whether Drosophila possesses beside otefin additional genes coding for proteins with a LEM motif. A putative candidate gene was the annotated gene CG9424 which we named Bocksbeutel. Of all putative Drosophila LEM-domain proteins, otefin and Bocksbeutel exhibited the highest similarity in the LEM motif (53% identical amino acids). The Bocksbeutel gene can code for two isoforms of 399 and 351 amino acids that are produced by alternative splicing. In the alpha-isoform a transmembrane domain is localized close to the carboxyterminus. This segment is absent in the shorter beta-isoform. By RT-PCR we could show that in the embryo the mRNA coding for the alpha-isoform and in significantly lower amounts the mRNA coding for the beta-isoform are expressed. When expressed in transfected cells as GFP fusion proteins, the beta-isoform is localized predominantly in the nucleoplasm and the alpha-isoform is targeted to the nuclear envelope, indicating that Bocksbeutel-alpha is localized in the inner nuclear membrane. Bocksbeutel-alpha is the predominant isoform expressed in cells, larvae, and flies. Indirect immunofluorescence with Bocksbeutel-specific antibodies on tissues and cultured cells revealed that Bocksbeutel proteins are localized in the nuclear envelope and in the cytoplasm. By RNA interference we have down-regulated the expression of Bocksbeutel, BAF, otefin, and lamin DmO in Drosophila Kc167 cells. The down-regulation of Bocksbeutel and otefin had no influence on the viability of Kc167 cells and the intracellular localization of all other nuclear and nuclear envelope proteins analyzed. In contrast, when lamin DmO was reduced by RNAi the distribution of Bocksbeutel and otefin in the nuclear envelope of Kc167 cells was significantly altered. We conclude that the two LEM-domain proteins Bocksbeutel and otefin are no limiting components for the maintenance of the nuclear architecture in cultured Drosophila cells at interphase.