Rsf-1 sequence. (A) Amino acid sequence of Rsf-1. Rsf-1 is a 1,441-amino-acid protein. The peptide sequences obtained by ion trap mass spectrometry are underlined. The acidic amino acids aspartic acid (D) and glutamic acid (E) found in the Rsf-1 sequence are shown in red. The arrow indicates the first methionine of HBXAP, located at amino acid 253. (B) Schematic representation of Rsf-1. The diagram shows the position of the domains found based on the amino acid sequence of Rsf-1: the PHD and the C-terminal acidic region. MM, molecular mass. (C) Northern blots with various tissues and the Rsf-1 cDNA. Northern blots of different human tissues were analyzed with an Rsf-1 probe (upper panel). After exposure to the Rsf-1 probe, the membrane was stripped and reprobed with β-actin (lower panel). The migration of RNA markers is shown on the left side of the figure. The migrations of the different mRNA species detected with the Rsf-1 probe are denoted with asterisks. (D) Phosphatase treatment. Silver staining of an SDS-polyacrylamide gel containing native RSF treated with alkaline phosphatase. Molecular markers (lane 1), p300 (lane 2), RSF (lane 3), and alkaline phosphatase-treated RSF (lane 4) are shown.

Recombinant RSF. (A) Coomassie blue staining of purified fractions of native RSF, recombinant RSF obtained by coexpression of the subunits, recombinant Rsf-1, and recombinant hSNF2H. The migration positions of the molecular markers (M) are shown on the left side of the gel. The migration of Rsf-1 and hSNF2H is indicated on the right side of the gel. (B) Western blot of recombinant RSF and its subunits. (C) ATPase activity. The graph shows the quantification of phosphate released during the ATPase reaction with increasing amounts of native and recombinant RSF obtained by coexpression of the subunits, as indicated in the figure. (D) Chromatin assembly. An agarose gel of a micrococcal nuclease digestion of a chromatin assembly reaction carried out with 0.4 μg of native RSF (lane 1), without RSF (lane 2), and with increasing amounts of recombinant RSF obtained by coexpression of the subunits (between 0.1 and 0.8 μg) (lanes 3 to 6).

DNA-binding activity. (A) Electron microscopic visualization of the binding of RSF subunits to DNA. The experiment was carried out as shown on the top of the figure. hSNF2H or Rsf-1 were incubated with EcoRI-linearized plasmid. The products of the reactions were first incubated with antibodies against hSNF2H or Rsf-1. This was followed by incubation with a secondary antibody conjugated with 10-nm gold, as described in Materials and Methods. In each image, the white dot found in the middle of the large protein complex corresponds to the gold particle. The top panels show the products of the reaction with hSNF2H visualized by electron microscopy in the presence (left) or absence (right) of histones. The bottom panel shows the products of the reaction with Rsf-1 in the presence of histones. Bar, 100 nm. (B) DNA filter-binding assay. Increasing amounts of recombinant hSNF2H, Rsf-1, and recombinant RSF obtained by coexpression of the subunits were incubated with radiolabeled DNA at the indicated molar ratio. The figure shows the quantification of DNA bound to the nitrocellulose filters as explained in Materials and Methods. This figure represents three independent experiments.

Immunofluorescence microscopy of RSF. (A) Western blots with RSF antibodies. Partially purified fractions of RSF derived from HeLa nuclear pellet were Western blotted with monoclonal antibodies against the RSF subunits Rsf-1 and hSNF2H. (B) Immunofluorescence microscopy. HeLa cells were fixed and permeabilized, followed by incubation with antibodies (Ab) against the RSF subunits Rsf-1 and hSNF2H. The antibody against Rsf-1 is shown in green, the antibody against hSNF2H is shown in red, and DAPI (4′,6′-diamidino-2-phenylindole) is shown in blue. The bottom panel shows the merge between all of them. Two cells are shown on the field. The cell to the left is in mitosis (metaphase), whereas the cell to the right is in interphase. (C) Interaction between Rsf-1 and hSNF2H during mitosis. Western blot analysis of immunoprecipitation analysis carried out with hSNF2H (top panel) and Rsf-1 (bottom panel) antibodies, as indicated in the left side of the figure. The input for the immunoprecipitation (IP) is indicated on top of the figure: interphasic (left side) or mitotic (right side) nuclear extract. As a negative control, the extract was incubated only with protein G-agarose and processed like the other samples. We used antibodies against histone H3 and phosphorylated S28 as a marker for mitosis and β-actin as a loading marker.

Functions of the two RSF subunits. (A) ATPase activity. The graph shows the quantification of phosphate released during the ATPase reaction. Lanes show the following: no factor added (lane 1), increasing amounts of recombinant hSNF2H (between 0.2 and 0.9 μg, lanes 2 to 4), increasing amounts of recombinant Rsf-1 (0.2 and 0.4 μg, lanes 5 and 6, respectively), and the same amount of recombinant hSNF2H used in lane 3 plus increasing amounts of Rsf-1 (0.2 and 0.4 μg, lanes 7 and 8, respectively). (B) Chromatin assembly. An agarose gel shows a micrococcal nuclease digestion of chromatin assembly reactions carried out without factor (lane 1), with recombinant RSF obtained by coexpression of the subunits (lane 2), with recombinant Rsf-1 plus recombinant hSNF2H (lane 3), with recombinant hSNF2H alone (lane 4), and with recombinant Rsf-1 alone (lane 5).

Comparison of Rsf-1 and HBXAP. (A) Coomassie blue staining of recombinant RSF obtained by coexpression of the subunits and HBXAP. The arrow indicates the migration position of the RSF subunits Rsf-1 and hSNF2H and of HBXAP. M, molecular marker. (B) Interaction between hSNF2H and HBXAP. Western blot analysis of the immunoprecipitation carried out as outlined at the top of the figure. Flag-tagged HBXAP or Flag-tagged Rsf-1 was incubated with hSNF2H and immunoprecipitated with antibody against Flag. The input shows the starting material for the immunoprecipitation before the subunits were mixed. (C) Chromatin assembly. An agarose gel of a micrococcal nuclease digestion of a chromatin assembly reaction carried out with HBXAP, as follows: without factor (lane 1), with recombinant RSF obtained by coexpression of the subunits (lane 2), with recombinant Rsf-1 and hSNF2H together (lane 3), with recombinant Rsf-1 alone (lane 4), with recombinant hSNF2H alone (lane 5), and with recombinant hSNF2H plus increasing amounts of HBXAP (between 0.16 and 1 μg) (lanes 6 to 8).