Segura-Totten M , Kowalski AK , Craigie R , Wilson KL .

GM48646 NIGMS NIH HHS , R01 GM048646 NIGMS NIH HHS

	Figure 1. BAF mutagenesis. Residues that comprise the five α-helices in BAF are indicated by bars above the amino acid sequence of human BAF (Umland et al., 2000; Cai et al., 2001). Point mutations are indicated by E (glutamic acid), Q (glutamine), and A (alanine), and numbered. Each BAF mutant contained one substituted residue. A few residues were changed to either of two mutant residues.
	Figure 2. Binding of mutant hBAF proteins to blot-immobilized emerin. Blots bearing human emerin protein (residues 1–222) were cut into strips. Each strip was probed with 35S-labeled wild-type or mutant BAF, numbered as in Fig. 1. Radiolabeled wild-type human BAF (WT) served as a positive control. Binding of each mutant BAF to emerin was scored relative to the amount of each input probe (unpublished data), and summarized in Table I.
	Figure 3. DNA binding activity of BAF mutants. Each 35S-labeled wild-type or mutant BAF protein was incubated with (+) or without (−) native DNA cellulose beads, then pelleted, washed, separated on SDS-PAGE, and detected by autoradiography.
	Figure 4. Subunit exchange assay. Each His-tagged mutant BAF protein was incubated with 35S-labeled wild-type BAF, and immunoprecipitated with anti-His antibody. As positive and negative controls, 35S-wild-type BAF was incubated with (WT) or without (−) His-tagged wild-type BAF, respectively, before immunoprecipitation and SDS-PAGE. The left and right panels are from different experiments, and had different amounts of input 35S-wild type BAF. (A) Autoradiographs showing the 35S-labeled wild-type BAF that coimmunoprecipitated with wild-type BAF (WT), or each BAF mutant (numbered as in Fig. 1). (B) Parallel Western blots probed with anti-His antibody, showing the amount of His-tagged BAF present in each reaction. All recombinant proteins migrated at their expected mass of 10 kD. (C) Densitrometric ratios of signals shown in A and B. Graphs show relative amounts of 35S-wild-type BAF that interacted with each input His-tagged BAF.
	Figure 5. Cloning, expression, and localization of Xenopus BAF. (A) Human (top) and Xenopus (bottom) BAF are 84% identical (red) and 91% similar (blue). (B) Affinity-purified rabbit antibodies (serum 3710) recognized both recombinant (R) and endogenous (E) Xenopus BAF. Recombinant BAF (calculated mass, 10.2 kD) migrated at 10 kD on SDS-PAGE, whereas endogenous BAF migrated at 40 kD. (C) Western blot of the soluble fraction of Xenopus egg extracts showing that recognition of endogenous BAF by affinity-purified 3710 antibody was specifically competed by pretreatment (+) with antigenic peptide. Pre, preimmune antibody; pep, antigenic peptide. (D and E) Indirect immunofluorescent staining of endogenous BAF in cultured Xenopus A6 cells (D) and XLK-WG cells (E) using affinity-purified immune (Imm) or preimmune (pre) 3710 antibody. xBAF localizes predominantly at the nuclear rim, but is also found in the nuclear interior and cytosol (D and E, right). Left panels show DNA in the same cells, stained by Hoechst 33258.
	Figure 6. Exogenous BAF has two distinct effects on chromatin when added to Xenopus nuclear assembly reactions. Purified recombinant Xenopus BAF (A) or human BAF (B) were added to Xenopus nuclear assembly reactions at time zero, at concentrations of 0, 0.5, 2.5, or 5 μM recombinant BAF dimers. (A–C) Upper panels show nuclei by phase contrast microscopy; corresponding lower panels show same nuclei stained for DNA with Hoechst 33258. Nuclei were imaged after 2 h of assembly. (C) Timecourse (20-min intervals) of nuclear assembly without (no addition), or with 5 μM exogenous xBAF dimers (xBAF). Bars: (A and B) 10 μm; (C) 30 μM.
	Figure 7. Transmission EM of control and wild-type xBAF-inhibited nuclei. Nuclei were assembled for 2 h with no BAF added (A) or 5 μM added xBAF dimers (B), and visualized by TEM. (A) Asterisks indicate nuclear pore complexes. (B) Nuclei assembled in 5 μM xBAF had patches of membranes at the chromatin surface. Arrow indicates chromatin emerging between membrane patches. Paired arrowheads bracket the electron-dense outer shell of chromatin in inhibited nuclei. (C) TEM cross-section of sperm chromatin before addition to assembly extracts. Bars, 500 nm.
	Figure 8. Effects of mutant hBAF proteins on nuclear assembly in Xenopus egg extracts. Mutant BAF proteins were added at time zero to the indicated final concentrations, and imaged after 2 h of assembly. Mutants fell into four phenotypes by light microscopy: (A) wild-type (decondensed-to-condensed), (B) inactive, (C) always condensed, and (D) inactive-to-condensed. The representative mutant shown for each class is bolded. Mutants are numbered according to Fig. 1. Mutant 75E behaved like wild-type BAF, but with clumps of DNA at the nuclear poles. Bar, 10 μm.
	Figure 9. TEM analysis of nuclei assembled in Xenopus extracts for 2 h with the indicated hBAF mutant. (A and B) Nuclei assembled in 0.5 μM (A) or 5 μM (B) always condensed mutant 14A. Arrows indicate the thin (A) and thick (B) shell of condensed chromatin caused by this mutant. (C and D) Nuclei assembled in 0.5 μM (C) or 5 μM (D) always condensed mutant 47E; note the normal chromatin and pore-less double membrane in C, and uniformly condensed chromatin in D. (E) Nucleus assembled in 5 μM inactive-to-condensed mutant 53E. (F–I) Higher magnifications of panels A, B, D, and E. (F and G) Mutant 14A at 0.5 μM (F) and 5 μM (G). (H) Mutant 47E at 5 μM. (I) Mutant 53E at 5 μM. Bars: (A–E) 500 nm; (F–I) 200 nm.
	Figure 10. Functional residues on the BAF dimer surface. (A–C) Corresponding ribbon diagram (A) and surface structure representation (B) of the wild-type human BAF dimer (left). In the front orientation, dsDNA molecules bind to the left and right ends, and the LEM-binding domain faces the reader. The BAF dimer at right is rotated down 90° to show the top surface. Unprimed and primed numbers (e.g., 27 and 27') indicate residues in the left and right monomers, respectively. (B) Residues essential for emerin binding are light blue: surface-exposed residues 51, 53, and 54 cluster in the valley that spans both monomers. Residues 46 and 47 (Umland et al., 2000) are buried at the dimer interface. Surface-exposed residues 6, 9, 27, and 75, in which mutations reduced (but did not eliminate) binding to emerin, are dark blue. (C) BAF residues essential for DNA binding are light blue: residues 6, 25, and 27 map to the left and right of the dimer; residue 46 is buried. Residue 25 is not visible in this front view. Surface-exposed residues 9, 51, 54, and 75, in which mutations reduced DNA binding, are dark blue. (D) Always condensed mutants mapped to the top of the dimer (residues 14 and 18), and the dimer interface (residue 47; buried). (E) Inactive mutants mapped to the left and right ends of the dimer (residues 25 and 27) and the dimer interface (residue 46; buried), as viewed from the side and bottom.

Berger, The characterization and localization of the mouse thymopoietin/lamina-associated polypeptide 2 gene and its alternatively spliced products. 1996, Pubmed

Berger, The characterization and localization of the mouse thymopoietin/lamina-associated polypeptide 2 gene and its alternatively spliced products. 1996, Pubmed
Boman, GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro. 1992, Pubmed , Xenbase
Cai, Solution structure of the constant region of nuclear envelope protein LAP2 reveals two LEM-domain structures: one binds BAF and the other binds DNA. 2001, Pubmed
Cai, Solution structure of the cellular factor BAF responsible for protecting retroviral DNA from autointegration. 1998, Pubmed
Chen, The barrier-to-autointegration protein is a host factor for HIV type 1 integration. 1998, Pubmed
Clements, Direct interaction between emerin and lamin A. 2000, Pubmed
Cohen, Transcriptional repression, apoptosis, human disease and the functional evolution of the nuclear lamina. 2001, Pubmed
Dechat, Detergent-salt resistance of LAP2alpha in interphase nuclei and phosphorylation-dependent association with chromosomes early in nuclear assembly implies functions in nuclear structure dynamics. 1998, Pubmed
Dechat, Review: lamina-associated polypeptide 2 isoforms and related proteins in cell cycle-dependent nuclear structure dynamics. 2000, Pubmed , Xenbase
Dechat, Lamina-associated polypeptide 2alpha binds intranuclear A-type lamins. 2000, Pubmed
Foisner, Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation. 1993, Pubmed
Furukawa, LAP2 binding protein 1 (L2BP1/BAF) is a candidate mediator of LAP2-chromatin interaction. 1999, Pubmed
Gant, 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. 1999, Pubmed , Xenbase
Goldberg, Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA. 1998, Pubmed
Haraguchi, BAF is required for emerin assembly into the reforming nuclear envelope. 2001, Pubmed
Harris, Both the structure and DNA binding function of the barrier-to-autointegration factor contribute to reconstitution of HIV type 1 integration in vitro. 2000, Pubmed
Kasof, Btf, a novel death-promoting transcriptional repressor that interacts with Bcl-2-related proteins. 1999, Pubmed
Lee, Distinct functional domains in emerin bind lamin A and DNA-bridging protein BAF. 2001, Pubmed
Lee, A previously unidentified host protein protects retroviral DNA from autointegration. 1998, Pubmed
Lee, C. elegans nuclear envelope proteins emerin, MAN1, lamin, and nucleoporins reveal unique timing of nuclear envelope breakdown during mitosis. 2000, Pubmed
Lin, MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated polypeptide 2 and emerin. 2000, Pubmed
Lohka, Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. 1983, Pubmed , Xenbase
Meier, Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation. 1995, Pubmed , Xenbase
Newmeyer, Egg extracts for nuclear import and nuclear assembly reactions. 1991, Pubmed , Xenbase
Philpott, Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin. 1991, Pubmed , Xenbase
Shao, Common fold in helix-hairpin-helix proteins. 2000, Pubmed
Shumaker, LAP2 binds to BAF.DNA complexes: requirement for the LEM domain and modulation by variable regions. 2001, Pubmed , Xenbase
Stuurman, Nuclear lamins: their structure, assembly, and interactions. 1998, Pubmed
Umland, Structural basis of DNA bridging by barrier-to-autointegration factor. 2000, Pubmed
Vaughan, Both emerin and lamin C depend on lamin A for localization at the nuclear envelope. 2001, Pubmed
Wilson, A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro. 1988, Pubmed , Xenbase
Wolff, Structural analysis of emerin, an inner nuclear membrane protein mutated in X-linked Emery-Dreifuss muscular dystrophy. 2001, Pubmed
Woodland, The synthesis and storage of histones during the oogenesis of Xenopus laevis. 1977, Pubmed , Xenbase
Zheng, Barrier-to-autointegration factor (BAF) bridges DNA in a discrete, higher-order nucleoprotein complex. 2000, Pubmed