An Early Developmental Transcription Factor Complex that Is More Stable on Nucleosome Core Particles Than on Free DNA  Lisa Ann Cirillo, Kenneth S Zaret  Molecular Cell  Volume 4, Issue 6, Pages 961-969 (December 1999) DOI: 10.1016/S1097-2765(00)80225-7

Figure 1 Integrity of System Components (A) SDS-PAGE analysis of purified HNF3α (6 μg), GATA-4 (6 μg), and core histones (20 μg). M, protein size markers; their masses in kilodaltons (kD) are shown. The gel is 10% polyacrylamide, so histones H3 and H2B are not well resolved. The proteins were stained with Coomassie brilliant blue. (B) Uniformly labeled, purified dinucleosome templates were digested with increasing concentrations of MNase (in units/ml), then the DNA was isolated, run on a polyacrylamide gel, and autoradiographed. M, φX 174 HaeIII fragments; sizes shown in bp. The expected mononucleosomal (∼146 bp) and dinucleosomal (∼290 bp) MNase digestion products are indicated. (C) SDS-PAGE analysis of core histone proteins from purified dinucleosome templates (lane 4) compared to input histones, stained with silver. Molecular Cell 1999 4, 961-969DOI: (10.1016/S1097-2765(00)80225-7)

Figure 2 Stable HNF3-Albumin Enhancer Complexes Contain an Underlying Nucleosome Core (A) Uniformly labeled albumin enhancer dinucleosomes or free DNA were incubated with or without 45 nM HNF3 or 30 nM GATA-4, followed by cleavage with MNase for 5 min. DNA was purified, analyzed on an 8% native polyacrylamide gel, and autoradiographed. Lane 1, φX 174 HaeIII marker; lane 2, undigested control. Positions of major digestion intermediates are indicated at the right. Enzyme concentrations were calibrated to yield similar ranges of digestion for each binding reaction. MNase concentrations (U/ml) of lanes 3–12 were, respectively: 0.25, 0.5, 2, 4, 1, 2, 0.25, 0.5, 1, 4. (B) Expected MNase and AflIII cleavage products. Line at top shows relative positions of albumin enhancer dinucleosomes N1 and N2 as seen in liver chromatin (McPherson et al. 1993). Numbers beneath the ends of the line indicate nucleotide positions of the albumin enhancer (Liu et al. 1991). Numbers beneath the expected MNase and AflIII products indicate expected fragment size trimmed by both enzymes in bp. (C) Uniformly labeled DNAs shown in (A) were digested with AflIII, run on a native 10% polyacrylamide gel, and autoradiographed. Lane 1, φX 174 marker; lanes 2–8 were from (A). Molecular Cell 1999 4, 961-969DOI: (10.1016/S1097-2765(00)80225-7)

Figure 3 HNF3-Albumin Enhancer Complexes Are More Stable on Nucleosome Cores Than Free DNA (A) DNase I footprinting. Four-nanomolar concentrations of free DNA (lanes 2–7) or dinucleosomes (lanes 8–13) labeled at the 5′ position of the bottom strand were incubated overnight with or without 30 nM HNF3 as indicated. Since the dinucleosome templates have three HNF3-binding sites, the molarity of binding sites in the assays is 12 nM. Complexes were competed with a 100-fold molar excess of HNF3 eG-binding site competitor for increasing lengths of time followed by digestion with DNase I; a 3-fold higher concentration of DNase was used on the dinucleosome substrates. (Lane 1) G cleavage ladder marker. Numbers at left indicate extent of albumin enhancer sequences. Ovals at right indicate positions of the N1 and N2 particles in liver nuclei. (B) Magnified view of bracketed region in (A). The positions of the HNF3-binding sites on free and nucleosomal DNA are indicated; NS-A1 is a nucleosome-specific binding site. Time of incubation with HNF3-binding site competitor DNA is indicated above each lane. Filled dots to the right of lane 3 denote hypersensitive cleavages induced by HNF3 binding on free DNA; filled dots to the right of lanes 9 and 13 denote those on nucleosomal DNA. Open dots denote DNase protections specific to HNF3-occupied nucleosome cores. Molecular Cell 1999 4, 961-969DOI: (10.1016/S1097-2765(00)80225-7)

Figure 4 GAL4 Is Rapidly Competed from Nucleosome Cores (A) SDS-PAGE of purified Gal4-AH. (B) DNase footprinting with Gal4-AH as in Figure 3. Black dots to right of lane 5 denote 10–20 bp repeat pattern of DNase cleavage, indicative of nucleosome positioning. Vertical bars to right of lane 5 indicate position of the five Gal4-binding sites, corresponding to the repeating pattern of G residues in lane 1. Molecular Cell 1999 4, 961-969DOI: (10.1016/S1097-2765(00)80225-7)

Figure 5 Histone Acetylation Does Not Enhance HNF3 Binding to Chromatin (A) Triton-acid-urea (TAU) gel analysis of purified histones. Acetylated forms are denoted at the side of the Coomassie-stained, 15% polyacrylamide TAU gel. (B) Dinucleosome templates were assembled with HeLa cell histones that were either hyperacetylated or hypoacetylated. Binding assays were performed as for Figure 2, except that we also tested the designated subsaturating concentrations of HNF3 protein. Footprinted regions (exclusive of hypersensitive sites), relative to control bands, were quantitated with a PhosphorImager. The graphs show the average and standard deviation of percent site occupancy from two independent assays. Molecular Cell 1999 4, 961-969DOI: (10.1016/S1097-2765(00)80225-7)

Figure 6 HNF3 Binding Stabilizes GATA-4 Binding to Nucleosome Cores (A) DNase I footprinting assay of 180 bp segment of the albumin enhancer, with the designated concentrations of HNF3 and GATA-4 proteins. Reactions employed DNase I at 15 μg/ml. (B) Dinucleosome templates as in Figure 3; only a portion of the gel is shown. (A and B) Arrows designate a nuclease-hypersensitive site induced when GATA-4 binds the enhancer. (C) MNase/AflIII assay as in Figure 2. MNase concentrations in lanes 2 and 3 were 2 and 8 U/ml, respectively. Molecular Cell 1999 4, 961-969DOI: (10.1016/S1097-2765(00)80225-7)

Figure 7 Regulated Nucleosome Positioning by Stable Nucleosome Binding (A) System components. The upper bar represents the albumin enhancer DNA; positions of the HNF3-binding sites are shown. (B) The upper set of nucleosome core particles represent the mobility of the histone octamer on the albumin enhancer DNA. When all three HNF3 sites occur within the core particle (middle structure), HNF3 binding is more stable than to free DNA, and it results in a positioned nucleosome. Cirillo et al. 1998 have shown that the NS-A1 and eG/eH sites, which together span about 85 bp of DNA, are necessary for stable HNF3 binding to nucleosome core particles. Molecular Cell 1999 4, 961-969DOI: (10.1016/S1097-2765(00)80225-7)