Purification of proteins associated with Flag-tagged Ini1. (A) Purification of Flag-tagged hSWI/SNF complexes. FL-Ini1-11 nuclear extracts were incubated with anti-Flag M2 affinity gel, and after several washes with buffer containing increasing amounts of salt, the proteins retained on the affinity column were eluted with buffer containing 20-fold molar excess of Flag peptide. Eluted proteins were then fractionated on a Superose 6 sizing column. (B) hSWI/SNF subunits cofractionate with FL-Ini1. Western blot analysis was performed with 50 μg of nuclear extract (NE); 1.2 μg of affinity purified SWI/SNF fraction (M2), and 15 μl of sizing column fractions 14–23 [Superose 6 (Sup6)].

Biochemical characterization of Flag-tagged hSWI/SNF complexes. (A) Amounts of 560 ng of affinity-purified hSWI/SNF (M2, lane 1) and 465 ng of sizing column fraction Superose 6 (sup6), lane 2] were analyzed by SDS-PAGE, and proteins were visualized by silver staining. (B) Nucleosome disrupting activity. Either 280 ng (lanes 4,5) or 560 ng (lanes 6,7) of M2 fractions was incubated with mononucleosomes with or without ATP. Similarly, 155 ng (lanes 8,9) and 310 ng (lanes 10,11) of Sup6 peak fractions were incubated with core particles as indicated. As a control, naked DNA (N, lane 1) and nucleosomal DNA with or without ATP (lanes 2,3) are shown. Symbols at right indicate the changes to DNase sensitivity caused by hSWI/SNF in an ATP-dependent manner. (C) Remodeling activity on nucleosomal arrays. Increasing amounts of either M2: 19 ng (lanes 4,5), 56 ng (lanes 6,7), 84 ng (lanes 8,9), or Sup6 fractions: 10 ng (lanes 10,11), 31 ng (lanes 12,13), 62 ng (lanes 14,15), were incubated with 8 ng of assembled chromatin templates with or without ATP as indicated. (Lane 1) Linear plasmid DNA (lanes 2,3) assembled template incubated with or without ATP. The resolved DNA templates are supercoiled (Sc); relaxed and supercoiled (Sc.R); linear (L); and nicked (N).

Mitotic hSWI/SNF is phosphorylated and lacks nucleosome disrupting activity. (A) hSWI/SNF subunits are regulated differently during mitosis. Approximately 10 μg of total protein from exponentially growing cells (Asynch), cells blocked in S phase (S phase), or cells blocked in prometaphase (G₂–M) were analyzed by Western blotting with anti-Brg1, anti-hBrm, anti-hSWI3, or anti-Flag antiserum. Where indicated, proteins were incubated with 10–20 units of calf alkaline phosphatase (CIAP) for 45 min at 37°C. (B) Mitotic hSWI/SNF subunits cofractionate through Superose 6 (Sup6) sizing column. Western blot analysis of Sup6 column fractions (fr.) 15–27, with anti-Brg1, anti-hSWI3, and anti-Flag antisera. Mitotic hSWI/SNF subunits consistently (n = 4) eluted later (fr. 23) than the asynchronous complex (fr. 19). (C) Mitotic hSWI/SNF complex is intact. Equal amounts of hSWI/SNF sizing column peak fractions (∼500 ng) from either exponentially growing cells (Asynch, fr. 19), or cells blocked in early mitosis (G₂–M, fr. 23) were analyzed by SDS-PAGE and silver staining. (D) Phosphatase treatment of mitotic hSWI/SNF enables the complex to disrupt mononucleosomes. Similar amounts of Sup6 fractions from either asynchronous cells (500 ng) or cells blocked in G₂-M (560 ng) were incubated with mononucleosomes with or without ATP and CIAP as indicated.

Cell cycle-dependent reactivation of hSWI/SNF complexes. (A) Exponentially growing FL-Ini1 cells were synchronized in G₂–M by nocodazole treatment, and then released from the block by washing in medium without drug. Cells were harvested at the indicated times (T), and their DNA content was determined by FACS analysis. For comparison, the DNA content of exponentially growing cells (Asynch) is shown. Small arrowheads indicate the position of each stage of the cell cycle. (B) Cell cycle-dependent dephosphorylation of hSWI/SNF subunits. hSWI/SNF complexes were affinity purified from either asynchronous cells (Asynch), or cells blocked in G₂–M and then released (T = 0, 1, 2, 3, 5, and 24), and hSWI/SNF fractions were then examined for their protein composition and phosphorylation state. Approximately 200 ng of each fraction was analyzed by Western blotting with either anti-Brg1, anti-hBrm, or anti-hSWI3. (C) hSWI/SNF reactivation correlates with dephosphorylation of its subunits. Equal amounts (200 ng) of affinity-purified hSWI/SNF fractions from asynchronous cells (lanes 3,4), cells blocked in G₂–M (lanes 5,6), or cells that were released from the block (lanes 7–16) were tested for chromatin remodeling activity by use of an 8 ng assembled template, with or without ATP as indicated. Nicked closed circular (N); linear (L); supercoiled (Sc); and supercoiled and relaxed (Sc.R) plasmid DNA are indicated.

Active hSWI/SNF is inhibited by ERK1 in vitro. (A) Brg1, hBrm, and hSWI3 are phosphorylated in vitro by GST–ERK1. Equal amounts (0.75 μg/15 μl reaction) of either wild-type GST–ERK1 or mutant GST–ERK1 (K63M) were activated by GST–His–MEK1 (0.25 μg/15 μl reaction) as described in Materials and Methods. Approximately 200 ng of asynchronous SWI/SNF was added to the reactions containing either GST–ERK1 (lane 2), or GST–ERK1 (K63M) (lane 3). Samples were incubated at 30°C for 1 hr and analyzed by Western blotting. When PP2A was added (lane 4), the GST fusion proteins were removed by adding GST beads to the reactions, and the supernatant was incubated with 0.1 units of PP2A at 30°C for 1 hr. As a control, asynchronous hSWI/SNF subunits are shown (lanes 1,5). (B) hSWI/SNF that is phosphorylated in vitro by ERK1 is inactive. Asynchronous hSWI/SNF fractions were incubated (conditions as in A) with either MEK1-activated or inactive GST–ERK1 and GST–ERK1 (K63M) as indicated. As controls, hSWI/SNF was also tested for activity after incubation with either GST–MEK1 (lane 5), GST–ERK1 (lane 6), or GST–ERK1 (K63M) (lane 7). When PP2A was added (lanes 10–15), samples were treated as described in A. Assays are performed as in Fig. 4. (C) Brg1-containing complex is efficiently reactivated by PP2A. hSWI/SNF fractions purified from cells growing exponentially (Asynch), cells blocked in G₂–M (T = 0), or cells blocked and then released (T = 1), were incubated with supercoiled templates with or without ATP and PP2A as indicated. When PP2A was added, samples were preincubated with 0.1 units of PP2A at 30°C for 1 hr.