Overexpression of recombinant putative HCRa binding proteins. Extracts of strains producing His₆-Nhp6 (A), Gcf1p-His₆ (B), Htb1-His₆ (C), and Hta1-His₆ (D) were analyzed on a Coomassie blue-stained SDS gel. Lane 1, preinduction; lane 2, postinduction; lane 3, soluble fraction; lane 4, insoluble fraction; lane 5, flowthrough; lane 6, wash; lane 7, 0.1 M imidazole elution; lane 8, 0.2 mM imidazole; lane 9, 0.5 M imidazole. MW, molecular weight (in thousands).

Isolation of Hbp1. STE was precipitated with 60% ammonium sulfate and further fractionated with heparin-Sepharose chromatography using a 20 to 1,000 mM NaCl gradient. γ-³³P-labeled HCRa (0.1 ng) was mixed with 50 ng of each fraction in the presence of 200 ng sheared salmon sperm DNA (A) or 250 ng of each fraction in the presence of 500 ng of sheared salmon sperm DNA (B). Lane D contains radiolabeled probe only. (C) Coomassie blue-stained 16% SDS-polyacrylamide gel to visualize Hbp1 (see arrow and dot next to band) and Hbp5/6. Each lane contained a 5-μg sample from the indicated fraction following TCA precipitation.

Competition assay in the presence of 50- and 200-fold molar excesses of unlabeled DNA fragments from HCR and ENO1. Recombinant proteins (100 ng) His₆-Nhp6 (A) and Gcf1p-His₆ (B) were incubated with γ-³³P-labeled subregions of HCRa designated HCRa1 and HCRa2 (positions −1410 to −1249 and positions −1322 to −1162, respectively) or with a 160-bp region of ENO1 (positions +1250 to +1405) in the presence of 1,000 ng salmon sperm DNA. Lane D, probe only; lane 1, protein only; lanes 2 and 4, 50- and 200-fold unlabeled DNA from HCR; lanes 3 and 5, 50- and 200-fold ENO1 DNA.

Proposed model for the developmental regulation of the HWP1 gene. (A) Under yeast growth conditions, tightly compacted DNA-histone octamers (white disks, H2A/H2B; gray disks, H3/H4) and relatively strong binding of Gcf1p (Hbp1) (circles) and Hbp3 (squares) to the HCR region inhibit occupation of DNA by activators. (B) Under germ tube-inducing conditions, a putative cFACT containing Nhp6 (oval) functions in facilitating the removal of ubiquitinized H2A and/or H2B (dotted disk) and relaxes DNA on nucleosomes, thereby permitting an enhancer (Hbp4) (triangles) to gain access to the promoter region to activate the HWP1 gene. The location of HCR (positions −1410 to −1042) is indicated on the top of each model.

Comparison of recombinant proteins Hbp1, Hbp2, Hbp5, and Hbp6 with proteins from STE to bind to HCRa. (A) Eluate from the 1.0 M NaCl-75% ammonium sulfate precipitation fraction shown in Fig. 3C compared to purified N-His₆-Nhp6. (B) Fraction 34 shown in Fig. 4A to C compared to purified Gcf1p-His₆. (C) Fraction 49 shown in Fig. 4A to C compared to purified Hta1- and Htb1-His₆. Protein-HCRa complexes and free DNA are indicated by arrows. Protein amounts used were as follows: lane 1, 0 ng; lane 2, 2 ng; lane 3, 10 ng; lane 4, 50 ng; lane 5, 250 ng.

Analysis of the HWP1 promoter using GFP as a reporter. (A) Time course of GFP expression driven by various promoters including HWP1 (strain HB-12) (black), −1410 HWP1 (strain HGFP3) (gray), and promoterless (0GFP3) (lined). Fluorescence was quantified according to the procedures outlined in Materials and Methods. (B) Kinetics of activation of the −1410 region (dotted line) compared to the complete HWP1 promoter (HB-12) (solid line). The values represent the ratios of the fluorescence at each time point relative to the 7-h time point. (C and D) Dissection of the HWP1 promoter in strains containing external truncations from the 5′ end (C) and internal deletions (D). Strains were grown in M199 medium at 37°C for 2.5 h. Values represent the percent fluorescence of strain −1902, which is indicated by an arrow in C. In D, deleted DNA is indicated by a dashed line. The arrow indicates the transcription start site, and the diamond just upstream of the arrow signifies a TATA element. The locations of HCR and HCRa are shown as black bars. Putative NRE and E-box sites are indicated by open circles and black squares, respectively. (E) Developmental regulation of a heterologous promoter by HCR. HCR alone activated the truncated ENO1 promoter, leading to GFP expression in germ tubes but not in yeast using strain HCRENO. The diamond represents the TATA box in the ENO1 promoter.

Fractionation of DNA binding proteins in STE by ammonium sulfate precipitation (A) followed by heparin-Sepharose column chromatography of each ammonium sulfate fraction (B to D). Gel mobility shift assays were performed in the presence of 500 ng sheared salmon sperm DNA (A) or with 200 ng sheared salmon sperm DNA using the heparin-Sepharose fractions from stepwise elutions with NaCl from the 50% (B), 75% (C), and supernatant (D) ammonium sulfate fractions. Lane D, DNA only; lane CE (1,000 ng), crude extract under high-stringency conditions (see Materials and Methods); FT, flowthrough fraction; W, wash fraction; lanes 2, 3, and 4, 100 ng protein from 50% and 75% ammonium sulfate saturated fractions and the supernatant fraction, respectively; lanes 11, 12, and 13, 25 ng, 100 ng, and 500 ng protein, respectively, from the indicated fractions. Fractions that were compared by SDS-PAGE in E are marked with asterisks and diamonds to indicate the presence of Hbp1 and Hbp2, respectively, and with ovals to indicate fractions that did not show binding activities for either protein. The boxed numbers correspond to the lane numbers in E that were used for separating the indicated fractions by SDS-PAGE. (E) Silver-stained 16% SDS-polyacrylamide gel of representative Hbp1- and Hbp2-containing fractions as determined by assays shown in C and D. Each lane contains 1 μg of the appropriate fraction. The location of Hbp2 is indicated. The band indicated by the black dot was later found to contain histones (Fig. 4). MW, molecular weight (in thousands).

Identification of HCRa-protein complexes using crude protein extracts and fractionation by anion-exchange chromatography and gel mobility shift assays performed on either a 15- by 19-cm vertical gel electrophoresis system (Life Technologies, Gaithersburg, MD) at 200 V for 2 h (A and B) or a 7- by 10-cm Mini-PROTEAN 3 cell (Bio-Rad, Hercules, CA) at 120 V for 50 min (C to F). (A) Crude extracts (HE, YE, and STE) were mixed with γ-³³P-labeled HCRa in the presence of 3 μg sheared salmon sperm DNA (high-stringency reaction conditions). Protein-DNA complexes 1 and 2, as well as free DNA, are indicated by arrows. Amounts of crude extract used were as follows: lane 1, 0 ng (DNA only); lane 2, 250 ng; lane 3, 500 ng; lane 4, 1,000 ng; lane 5, 3,000 ng; lane 6, 5,000 ng. (B) Competition assay in the presence of unlabeled HCRa and ENO1 DNA. The γ-³³P-labeled fragment was incubated with 1,000 ng of crude extract from YE and HE and 3,000 ng from STE. Lane D, labeled probe only; lane 1, crude extract only; lanes 2 and 3, 200- and 50-fold molar excesses of unlabeled HCRa, respectively; lane 4, 200-fold molar excess of a region of ENO1 (see Materials and Methods). (C to F) Fractions prepared from YE and HE containing 25 ng (C and E) or 250 ng (D and F) of protein were mixed with γ-³³P-end-labeled HCRa in the presence of 200 ng (C and E) or 500 ng (D and F) sheared salmon sperm DNA except for assays in lanes labeled CE, which were performed under high-stringency conditions as in A and B. The positions of the free DNA and four protein-DNA complexes (Hbp1 to Hbp4) are indicated by arrows. White arrowheads (C and E) and diamonds (F) represent Hbp1 and Hbp4, respectively. See Results for a complete explanation. Lane D, DNA only; CE, crude extract; FT, flowthrough; W, wash; lane 5, 0.2 M NaCl; lane 6, 0.3 M; lane 7, 0.4 M; lane 8, 0.5 M; lane 9, 1.0 M.