HMGN Proteins Act in Opposition to ATP-Dependent Chromatin Remodeling Factors to Restrict Nucleosome Mobility  Barbara P. Rattner, Timur Yusufzai, James T. Kadonaga  Molecular Cell  Volume 34, Issue 5, Pages 620-626 (June 2009) DOI: 10.1016/j.molcel.2009.04.014 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Assembly of Recombinant Human HMGN Proteins into Chromatin (A) Synthesis and purification of wild-type and mutant human HMGN1 and HMGN2 proteins. Proteins were subjected to SDS-polyacrylamide gel electrophoresis and visualized by staining with Coomassie Brilliant Blue R-250. (B) Wild-type, but not mutant, HMGN proteins bind to two sites on nucleosomes. Chromatin was assembled by using the purified ACF system in the presence of the indicated HMGN proteins (at a 10:1 ratio of HMGN molecules to histone octamers) and then digested extensively with micrococcal nuclease. The resulting mononucleosomes were subjected to nondenaturing 5% polyacrylamide gel electrophoresis. The DNA fragments were visualized by ethidium bromide staining. (C) Incorporation of HMGN2 into chromatin results in an increase in the nucleosome repeat length. Micrococcal nuclease digestion analysis was performed with chromatin assembled in the presence of the indicated amounts of purified human HMGN2. The two lanes for each reaction condition represent different micrococcal nuclease concentrations (increasing from left to right). The asterisks indicate the positions of trinucleosomes and tetranucleosomes. The DNA size markers (M) are the 123 bp ladder (Invitrogen). Molecular Cell 2009 34, 620-626DOI: (10.1016/j.molcel.2009.04.014) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 HMGN Proteins Repress Chromatin Remodeling by ACF and BRG1 Restriction enzyme accessibility assays were carried out with salt dialysis-reconstituted chromatin in the presence of wild-type or mutant HMGN1 (upper panel; 2.3 μM) or HMGN2 (lower panel; 2.6 μM), as indicated. The chromatin remodeling factors, ACF (5 nM) or BRG1 (130 nM), were included where noted. Naked DNA was used as a control/reference. After digestion with HaeIII, the samples were deproteinized, and the resulting DNA species were subjected to electrophoresis and visualized by staining with ethidium bromide. Molecular Cell 2009 34, 620-626DOI: (10.1016/j.molcel.2009.04.014) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Reversible and Dynamic Interactions between HMGN2 and ACF (A) HMGN-mediated repression of chromatin remodeling can be overcome by the presence of additional ACF. Restriction enzyme accessibility assays were carried out with salt dialysis-reconstituted chromatin in the presence of the indicated concentrations of purified ACF. Purified HMGN2 (2.6 μM) was included where noted. HMGN2 was preincubated with the chromatin for 20 min prior to the addition of ACF. (B) Preincubation of chromatin with ACF does not block the inhibition of chromatin remodeling by HMGN2. Restriction enzyme accessibility assays were carried out as in (A) with ACF (5 nM) and wild-type or mutant HMGN2 (2.6 μM). The factors were added at the indicated times, as outlined in the schematic diagram. (C) HMGN2 substantially reduces chromatin-stimulated ATP hydrolysis by ACF. The ATPase activity of ACF was determined in the presence of either plasmid DNA (left) or salt dialysis-reconstituted chromatin (right). Reactions contained the indicated concentrations of wild-type or mutant (S24,28E) human HMGN2. Error bars represent the standard deviation (n = 3). Molecular Cell 2009 34, 620-626DOI: (10.1016/j.molcel.2009.04.014) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 HMGN2 Decreases the Binding of ACF to Chromatin (A) Salt dialysis-reconstituted chromatin was subjected to glycerol gradient sedimentation in the presence or absence of HMGN2 and/or ACF, as indicated. Proteins were detected by western blot analysis, and DNA was monitored by agarose gel electrophoresis followed by staining with ethidium bromide. (B) A model in which HMGN proteins act in opposition to chromatin remodeling factors. This diagram depicts a working model in which the HMGN proteins counteract the action of the ATP-dependent chromatin remodeling factors to provide a means for restricting nucleosome mobility. Molecular Cell 2009 34, 620-626DOI: (10.1016/j.molcel.2009.04.014) Copyright © 2009 Elsevier Inc. Terms and Conditions