DNA binding domain of PU.1 is not sufficient to antagonize a DNase I-hypersensitive site. A, schematic representation of full-length PU.1 and PU.1ΔN. PU.1ΔN lacks residues 1–145 of the full-length protein. The black box indicates the ETS domain, and the gray box includes the N-terminal transcription activation, PEST, and interferon regulatory factor-interaction domains of PU.1. B, indirect end-labeling analysis. Aliquots of μ70 dimer plasmid assembled into chromatin were incubated as follows: lane 1, no proteins; lane 2, 100 nm full-length PU.1; lanes 3–5, 50 nm, 100 nm, and 200 nm PU.1ΔN, respectively; lane 6, 57 nm TFE3; lane 7, 57 nm TFE3 and 100 nm full-length PU.1; lanes 8 and 9, 57 nm TFE3, and 50 nm, 100 nm, and 200 nm PU.1ΔN, respectively. Chromatin was treated with DNase I, and DNA isolated from each sample was analyzed by indirect end-labeling. An arrowhead indicates the position of the DNase I HS, and asterisks indicate nucleosome boundaries. C, DNase I footprinting. DNA from the DNase I-treated samples in B was analyzed in parallel by primer extension followed by denaturing polyacrylamide gel electrophoresis. The bar indicates TFE3 protection, an arrowhead indicates PU.1 sensitivity, and μB, μE3, and μA indicate binding sites for PU.1, TFE3, and ETS-1, respectively. The lane order is the same as in B.

ACF catalyzes establishment of a DNase I-hypersensitive site by TFE3, but not erasure by PU.1. Chromatin was assembled using recombinant ACF, NAP-1, topoisomerase I fragment, and purified Drosophila histones. A, micrococcal nuclease analysis of chromatin assembled with purified proteins. Aliquots of assembled chromatin were incubated with either no protein (lane pair 1), 57 nm TFE3 (lane pair 2), 100 nm PU.1 (lane pair 3), or 57 nm TFE3 and 100 nm PU.1 (lane pair 4). Aliquots of each sample were treated with two different concentrations of micrococcal nuclease. DNA was isolated from each reaction, resolved by agarose gel electrophoresis, and visualized by staining with ethidium bromide. B and C, indirect end-labeling analysis of DNase I treated chromatin. Aliquots of chromatin incubated with the indicated combinations of TFE3, PU.1, and apyrase were digested with DNase I. DNA from each reaction was isolated and was analyzed by indirect end-labeling. An arrowhead indicates the position of the DNase I HS, and asterisks indicate nucleosome boundaries. D, DNase I footprinting analysis. DNA from the same DNase I-treated samples in B was analyzed in parallel by primer extension footprinting. The bar indicates TFE3 protection, an arrowhead indicates PU.1 sensitivity, and μB, μE3, and μA indicate binding sites for PU.1, TFE3, and ETS-1, respectively. The lane order is the same as in B.

DNA binding domain of TFE3 is not sufficient to establish a DNase I-hypersensitive site. A, μ70 dimer construct. μB, μE3, and μA indicate PU.1, TFE3, and Ets-1 binding sites, respectively. The arrow indicates the transcription start site. B, schematic representation of TFE3, TFE3ΔN, and TFE3ΔNΔC. TFE3ΔN lacks residues 1–89 of the full-length protein. TFE3ΔNΔC lacks residues 1–89 and residues 198–326 of the full-length protein. The black box indicates the basic-helix-loop-helix zip domain of TFE3. C, indirect end-labeling analysis. Aliquots of μ70 dimer plasmid assembled into chromatin were incubated as follows: lane 1, no proteins; lane 2, 57 nm full-length TFE3; lanes 3–5, 30 nm, 60 nm, and 120 nm TFE3ΔN, respectively; lanes 6–8, 30 nm, 60 nm, and, 120 nm TFE3ΔNΔC, respectively. Chromatin was treated with DNase I, and DNA isolated from each sample was analyzed by indirect end-labeling. On the left of the panel, a rectangle indicates the enhancer, ellipses indicate positioned nucleosomes, and numbers indicate the size and position of markers. On the right of the panel, an arrowhead indicates the position of the DNase I HS, and asterisks indicate nucleosome boundaries. D, DNase I footprinting. DNA from the DNase I-treated samples in C was analyzed in parallel by primer extension followed by denaturing polyacrylamide gel electrophoresis. The bar indicates TFE3 protection, an asterisk indicates increased DNase I sensitivity in the presence of TFE3, and μB, μE3, and μA indicate binding sites for PU.1, TFE3, and ETS-1, respectively. The lane order is the same as in C.

ATP is required to establish and erase changes in chromatin structure, but not to maintain chromatin structure or for transcription factor binding to chromatin. μ70 dimer plasmid was assembled into chromatin. After 4.5-h chromatin assembly, TFE3 (57 nm), PU.1 (100 nm), TSA (1 nm), or the ATPase apyrase (2 units/ml, Sigma), were added at different time points separated by 30 min, as indicated; +, simultaneous additions; arrows indicate consecutive additions, and apy indicates apyrase. Following additional incubation for 30 min, all the samples were treated with DNase I (∼6 h after initiation of chromatin assembly). A, aliquots of DNA isolated from the DNase I-treated samples were processed and analyzed by indirect end-labeling. The arrowhead indicates the position of the DNase I HS, and asterisks indicate nucleosome boundaries. B, aliquots of DNA isolated from the DNase I-treated samples in A were analyzed in parallel by primer extension followed by denaturing polyacrylamide gel electrophoresis. A bar indicates TFE3 protection, an arrowhead indicates PU.1 sensitivity, and μB, μE3, and μA indicate binding sites for PU.1, TFE3, and ETS-1, respectively. The lane order is the same as in A. C, TSA treatment. Aliquots of DNA isolated from the DNase I-treated samples were processed and analyzed by indirect end-labeling. An arrowhead indicates the position of the DNase I HS, and asterisks indicate nucleosome boundaries.

Mi2β catalyzes PU.1-dependent erasure of DNase I-hypersensitive site. A, indirect end-labeling analysis. Chromatin was assembled using recombinant ACF, NAP-1, topoisomerase I fragment, and purified Drosophila histones. 57 nm TFE3 was added to aliquots of chromatin, where indicated, followed by incubation for 30 min. Following that, 100 nm PU.1 and 2 nm human Mi2β protein, 2 nm Drosophila NURF complex, or 2 nm human SWI/SNF complex, where indicated, and the samples were further incubated for 30 min. (The concentration of nucleosomes was 40 nm, calculated with a repeat length of 170 bp, resulting in approximately one remodeling ATPase per 20 nucleosomes.) The samples were treated with DNase I, and DNA isolated from each sample was analyzed by indirect end-labeling. Aliquots of DNA isolated from the DNase I-treated samples were processed and analyzed by indirect end-labeling. The arrowhead indicates the position of the DNase I HS, and asterisks indicate nucleosome boundaries. B, DNase I footprinting analysis. DNA from the DNase I-treated samples in A was analyzed in parallel by primer extension followed by denaturing polyacrylamide gel electrophoresis. The bar indicates TFE3 protection, an arrowhead indicates PU.1 sensitivity, and μB, μE3, and μA indicate binding sites for PU.1, TFE3, and ETS-1, respectively. The lane order is the same as in A.