Volume 9, Issue 2, Pages 279-289 (February 2002) Opening of Compacted Chromatin by Early Developmental Transcription Factors HNF3 (FoxA) and GATA-4  Lisa Ann Cirillo, Frank Robert Lin, Isabel Cuesta, Dara Friedman, Michal Jarnik, Kenneth S Zaret  Molecular Cell  Volume 9, Issue 2, Pages 279-289 (February 2002) DOI: 10.1016/S1097-2765(02)00459-8

Figure 1 Assembly and Integrity of Albumin Nucleosome Arrays (A) Diagram of the MluI-PvuII DNA fragment used to reconstitute the albumin 5S nucleosome arrays. (B) Albumin array fragment mock reconstituted (lane 2) or reconstituted with core histones (lanes 3 and 4) was run on a native 1% agarose gel. λ, λ HindIII fragments. (C) Saturation of the extended nucleosome arrays was assessed by EcoRI digestion. φx, φx174 HaeIII fragments. (D) DNase I digestion analysis of extended and compacted nucleosome arrays. Lane E, partial EcoRI digest of end-labeled array fragment. Positions of nucleosomes on reconstituted arrays are indicated to left of gel. (E) Electron microscopy of extended arrays incubated under increasing salt concentrations and of arrays compacted with linker histone. (F and G) Schematic (F) and results (G) of chromatosome assays. φx, end-labeled φx174 HaeIII fragments. ∼165 bp chromatosome-sized products are indicated by asterisk to the left of bands in lanes 3 and 6. Molecular Cell 2002 9, 279-289DOI: (10.1016/S1097-2765(02)00459-8)

Figure 2 Analysis of Protein Binding to the Albumin Nucleosome Arrays Schematic (A) of DNase I footprinting experiments with internally labeled, extended nucleosome arrays (B and C) and free DNA (D). (B) and (C) depict footprinting reactions with 20 and 40 nM concentrations of HNF3, GATA-4, or NF-1; (D) is with 30 nM NF-1. Lanes labeled G denote guanosine cleavage ladder markers; XP, array DNA digested with XbaI and PleI. Enhancer positions of HNF3 (NS, eG, eH), GATA-4 (eF), and NF-1 (eH) binding sites are indicated by lines to the right of gels; dashed line in (C) indicates absence of NF-1 footprint. Dots to right of (B) indicate an ∼10 bp ladder of hypersensitive cleavages induced by HNF3 binding and positioning an underlying nucleosome (Shim et al., 1998). Molecular Cell 2002 9, 279-289DOI: (10.1016/S1097-2765(02)00459-8)

Figure 3 Lack of an Effect of HNF3 and GATA-4 Binding on Extended Chromatin in Nucleosome Arrays (A) Schematic of DNase I digestion assays. (B and C) DNase I analysis of HNF3 and GATA-4 binding to extended nucleosome arrays at higher (B) and lower (C) DNase I concentrations (in μg/ml). Positions of the nucleosomes corresponding to the albumin enhancer N1 and N2 particles are indicated. (D) DNase I analysis of GAL4-AH binding to GAL4 nucleosome arrays. Position of the nucleosome containing the five GAL4 binding sites is indicated. Lanes labeled E, partial EcoRI digests of end-labeled arrays. Molecular Cell 2002 9, 279-289DOI: (10.1016/S1097-2765(02)00459-8)

Figure 4 HNF3 and GATA-4 Binding Opens H1-Compacted Nucleosome Arrays (A and B) DNase I digestion analysis of designated transcription factors binding to H1-compacted albumin nucleosome arrays. Location of the nucleosomes corresponding to the albumin enhancer N1 and N2 particles are indicated. The arrow to the right of the gel denotes the position of the major hypersensitive site induced upon HNF3 and GATA-4 opening of the H1-compacted arrays. (C) Indirect end-label analysis of EcoRI digested chromatin samples prepared from DNase- and MNase-treated nuclei. Liver nuclei were digested with 8 μg/ml of DNase (lane 1) or 1.6 and 0.8 U/ml MNase (lanes 2 and 3, respectively). Kidney nuclei were digested with 2.4 and 1.2 U/ml MNase (lanes 5 and 6, respectively). Position of the albumin enhancer N1 and N2 nucleosome particles is indicated. (D) DNase I analysis of GAL4-AH binding to H1-compacted GAL4 nucleosome arrays. Location of the nucleosome corresponding to the five GAL4 sites is indicated to the right of the gel. (E) XbaI accessibility assay of HNF3 and GATA-4 binding to nucleosome arrays. The arrow to the right of the gel denotes the position of the expected XbaI digestion product. (F) MNase assay. Extended (−) and compacted (+H1) nucleosome arrays, the latter with and without HNF3, were digested with the designated units of MNase to mono- (1N) and dinucleosome (2N) (or chromatosomes, when +H1) sized fragments. The resulting DNA was run on a gel, blotted to a filter, and probed with an N1-N2 DNA fragment to visualize nucleosomes at the albumin enhancer. The data show no loss of nucleosomes in the region when HNF3 binds to compacted chromatin. Molecular Cell 2002 9, 279-289DOI: (10.1016/S1097-2765(02)00459-8)

Figure 5 The HNF3 C Terminus Binds Histones H3 and H4 (A) Deletion mutant series of HNF3α. Numbers refer to amino acid position. DBD, DNA binding domain. (B) SDS-PAGE analysis of 3 μg of each protein, stained with Commassie blue. (C) Amino acid composition near a highly conserved region (boxed) that is deleted in the Δ3 construct. Acidic amino acids are indicated by asterisks. (D–F) Core histone affinity assay with full length HNF3 (D); HNF3 N-terminal, C-terminal, or DNA binding domains (E); or with HNF3 C terminus or indicated HNF3 C-terminal deletion mutants (F) covalently attached to Sepharose beads. Beads were subjected to an initial wash with 75 mM NaCl (W1, unbound fractions), followed by a more stringent wash of 0.3 M or 0.6 M NaCl (B, bound fractions). The bound fractions and half of the W1 fractions were subjected to SDS-PAGE. The gels were stained with silver. Input, input core histones. Locations of core histones are shown at the sides. Molecular Cell 2002 9, 279-289DOI: (10.1016/S1097-2765(02)00459-8)

Figure 6 Chromatin Opening Ability of HNF3 Conferred by the C-Terminal Domain (A) DNase I digestion analysis of the binding of the HNF3 and mutant proteins to H1-compacted albumin nucleosome arrays. E, partial EcoRI digest of end-labeled albumin array fragment. Arrow indicates hypersensitive region induced by wild-type HNF3. (B) Analysis of footprinting data. Footprinting gels (as in [C]) were scanned with a Fuji BAS2500 Phosphorimager and analyzed using Image Gauge software (version 3.46). Top panels: percentage of occupancy of the eH and eG HNF3 sites was determined relative to an internal control band in the same lane (out of the image). Average and SEM values of percentage of occupancy were determined from at least three independent footprinting experiments. Open and closed circles indicate the mean percentage of occupancy of the DNase footprints at eH and eG, respectively. Vertical bars indicate SEM. Bottom panels: Scatchard plots drawn from linear regression analysis of relevant data points. Dissociation constant values were estimated from the negative inverse of the slopes of Scatchard plot analysis of the footprinting data. (C) DNase I footprinting analysis of HNF3 and C- and N-terminal mutants on free DNA. 2.5 fmol of a 428 bp end-labeled probe corresponding to the N1 and N2 regions of the albumin enhancer were incubated with the indicated concentrations of HNF3. A portion of the Phosphorimage of representative gels is shown. The eG and eH HNF3 sites are marked by brackets to the right. Molecular Cell 2002 9, 279-289DOI: (10.1016/S1097-2765(02)00459-8)