(A) Promoter occupancy by the Gal4 activator and LexA-Hairy repressor measured by chromatin immunoprecipitation. In the top group, no detectable Gal4 or LexA-Hairy protein was detected with chromatin prepared from a line containing only the lacZ reporter gene, as expected. The gel on the second line of the first group contains a strong signal for the Gal4 protein, from chromatin containing the activated reporter. These embryos were heat shocked and recovered to parallel the treatment used for LexA-Hairy-containing strains. In the bottom group, results are from lines containing all three transgenes prior to and directly after the induction of the LexA-Hairy repressor. Prior to induction, a signal for Gal4 is visible at the promoter, as well as a weak LexA-Hairy signal due to low-level expression. After a 20-min heat shock and 30-min recovery, a strong signal for LexA-Hairy is visible, in addition to the Gal4 signal. Both signals remain visible after 60 min of recovery. LexA-Hairy signals drop by 120 min. No signals are seen at the intergenic region on chromosome X. Hs and hs, heat shock; da, daughterless. (B) Sequential chromatin immunoprecipitation of embryos expressing LexA-Hairy and Gal4. Low levels of chromatin were recovered in immunoprecipitations in which anti-LexA- or anti-Gal4-enriched material was reprecipitated by nonspecific IgG. As expected, double immunoprecipitations with anti-Gal4 or anti-LexA antibodies recovered significantly more material, as did the sequential LexA/Gal4 immunoprecipitation. For panels A and B, input titrations are shown for each chromatin preparation (2%, 1%, 0.5%, 0.25%, 0.125%, and 0.0625%). At left, results of quantitative PCRs are shown, and at right, heat shock and recovery conditions are shown (heat shock was done at 37°C for 20 min, with recovery at room temperature). The presence or absence of the Gal4 protein and the heat shock-inducible LexA-Hairy transgene is noted by + or −, respectively. (C) Quantitation of LexA-Hairy and Gal4 levels at promoters before and after repression. Shown are percentages of recovery from three or more chromatin immunoprecipitation experiments (mean values and standard deviations are indicated). Black bars represent chromatin from strains lacking LexA-Hairy and Gal4, white bars represent chromatin from strains containing both LexA-Hairy and Gal4 (with only Gal4 expressed), and gray bars represent strains with both Gal4 and LexA-Hairy expressed.

Occupancy of the promoter and transcribed regions of the lacZ gene by Groucho and Rpd3 corepressors during repression by LexA-Hairy. (A) Schematic diagram of a LexA-Hairy-regulated gene, showing the portions amplified. w+, mini-white gene. (B) PCR analysis of formaldehyde-cross-linked chromatin immunoprecipitated from embryos that were induced to express the LexA-Hairy repressor for 20 min and aged for 30 min. As expected, no signal was seen for the nonspecific IgG precipitation. A strong promoter-localized signal was detected for LexA-Hairy, with weaker signals detected for distal regions of the gene. Signals for the Rpd3 histone deacetylase (results with 1, 2, and 4 μl of Rpd3 antibody are shown) and the Groucho corepressor (results with 50 and 100 μl of Groucho antibody are shown) were detected at +1 kbp and +2 kbp, with a weak signal for Groucho (Gro) at +4 kbp. No cross-linking was detected for an intergenic region on chromosome 3. Levels of input titration are shown at the right (2%, 1%, 0.5%, 0.25%, 0.125%). (C) PCR analysis of immunoprecipitated DSP-formaldehyde-cross-linked chromatin. LexA and Gal4 were detected at the promoter, with weaker signals detected in 3′ regions. In contrast to the results shown in panel B, a strong Groucho signal was detected at the start of the white gene (−350 bp), at the promoter, and throughout the transcribed region. No cross-linking was detected for an intergenic region on chromosome X. Groucho was not detected on chromatin samples derived from embryos that were not heat shock induced for LexA-Hairy or that were heat shocked but lacked the lexA-hairy gene (not shown). Levels of input titration are shown at the right (10%, 2%, 1%, 0.5%, 0.25%).

Chromatin changes occurring on the transcribed region of the gene during activation and repression as assayed by chromatin immunoprecipitation. The statistical significance of chromatin changes was determined by a one-tailed t test with an alpha of 0.05. (A) Relative histone H3 K9/14 acetylation levels (normalized to that of H3) at +1, +2, and +4 kbp. A strong increase in relative H3 acetylation at +1 kbp is observed with the Gal4 activator present (P value = 0.027), and this acetylation decreases after the induction of the repressor (P value = 0.016). No large changes at +2 and +4 kbp were observed during activation (P values = 0.091 and 0.13) or repression (P values = 0.29 and 0.37). (B) Relative histone H4 K5/8/12/16 acetylation levels (normalized to that of H3) at +1, +2, and +4 kbp. Relative H4 acetylation at +1 kbp was observed to increase after activation (P value = 0.026). However, no statistically significant change in relative H4 acetylation at +1 kbp was observed during repression (P value = 0.059). Similarly, no changes in relative H4 acetylation were observed during repression at +2 and +4 kb (P value = 0.17 and 0.22). A slight increase in relative H4 acetylation during activation was detected at +4 kbp (P value = 0.014) but not at +2 kbp (P value = 0.095). (C) Total relative histone H3 occupancy on the reporter during activation and repression. Overall levels decreased in the activated state relative to those of the unactivated gene throughout the open reading frame (P values at +1, +2, and +4 kbp were 0.0041, 0.035, and 0.016, respectively) and are not much further affected during repression (P values at +1, +2, and +4 kbp were 0.054, 0.50, and 0.29, respectively). Shown are the average values and standard deviations from at least three biological replicates. Signals have been normalized to those of the unactivated state.

Hairy-regulated gene system in the Drosophila embryo. (A) Three transgenes were combined onto a single chromosome to create a regulated on/off system: a lacZ reporter containing Gal4 sites, the Gal4 activator driven by the daughterless (Da) enhancer for broad expression in the embryo, and a heat-inducible LexA-Hairy construct. Gro, Groucho. (B) Repression by the LexA-Hairy protein expressed in a central domain under the Kruppel enhancer. (Upper images) lacZ reporter construct shown in panel A; (lower images) reporter activated by the rho and twi enhancers, with LexA binding sites at the promoter. In both cases, a central swathe of repression demonstrates the effect of the LexA-Hairy chimera. (C) Expression of lacZ and LexA-Hairy in blastoderm embryos. Embryos containing the transgenes shown in panel A were heat shocked (HS) for various times and fixed for in situ analysis of lacZ expression (top panel) or antibody staining of the LexA-Hairy protein (middle panel). As LexA protein accumulates, lacZ mRNA decreases. Heat shock has no effect on lacZ in embryos lacking the LexA-Hairy protein (two lower images of embryos). (D) Repression and reactivation of transgenes in embryos after heat shocks of various durations. A 5-min induction of the LexA-Hairy protein is sufficient to cause a significant loss of lacZ mRNA within 30 min. RNA levels remain low at 60 min and show recovery after 120 min (at this point, most embryos have aged to the germ band extended stage). Longer heat shocks (10 and 20 min) show similar recovery kinetics. Embryos are shown anterior to the left, with the dorsal surface up.

Promoter occupancy by the SAGA coactivator constituents Gcn5 and Ada3 as measured by chromatin immunoprecipitation. (A) Protein occupancy of the lacZ reporter promoter region in different states. The top gel shows no signal for these coactivators on the promoter lacking Gal4 activators, as expected. In the second gel, both proteins are detected on the promoter in a Gal4 activator-containing strain. Subsequent panels show continued SAGA component occupancy in strains in which the LexA-Hairy repressor was induced. No signals are seen in the intergenic region on chromosome X. Input titrations are shown for each chromatin preparation (2%, 1%, 0.5%, 0.25%, 0.125%, and 0.0625%). At left, results of quantitative PCRs are shown, and at right, heat shock and recovery conditions (heat shock was done at 37°C for 20 min, with recovery at room temperature). The presence or absence of the Gal4 protein and the heat shock-inducible LexA-Hairy transgene is noted by + or −, respectively. Hs and hs, heat shock; da, daughterless. (B) Quantitation of Gcn5 and Ada3 levels at promoters before and after repression. Shown are percentages of recovery from three or more chromatin immunoprecipitation experiments for Ada3 or from four or more experiments for Gcn5 (mean values and standard deviations are indicated). Black bars represent chromatin from strains lacking LexA-Hairy and Gal4, white bars represent chromatin from strains containing both LexA-Hairy and Gal4 (with only Gal4 expressed), and gray bars represent strains with both Gal4 and LexA-Hairy expressed.

(A) Chromatin remodeling, assayed by chromatin immunoprecipitation. Total histone levels, acetylation, and methylation were assayed on the reporter gene in unactivated, activated, and repressed states, as well as with an unactivated reporter gene with the repressor bound. The top two panels demonstrate the effect of Gal4 activators on the promoter; in the presence of Gal4 (second gel), overall histone H3 levels drop, as do H3 K27 methylation levels, while relative acetylated histone H4 and H3 levels increase. In the presence of the LexA-Hairy repressor (compare the second and third gels), 30 min after induction, total H3 levels increase modestly and relative H3 and H4 acetylation levels drop. In this experiment, H4 acetylation levels were more affected. Relative levels of H3 K27 methylation remained low in the repressed state. The binding of the repressor to the nonactivated gene (compare the first and fourth gels) did not affect total histone H3 occupancy or H3 K27 methylation and had only modest effects on relative acetylation. Below, relative levels of histone modifications at an intergenic locus on chromosome X are unchanged by the treatments. Immunoprecipitations were carried out with antibodies that recognize total histone H3, K9/14 acetylation of histone H3, K5/8/12/16 acetylation of histone H4, or K27 dimethylation of histone H3. Levels of input titration are shown at the right (10%, 2%, 1%, 0.5%, 23 0.25%, 0.125%). Hs and hs, heat shock; da, daughterless. (B) Quantitation of chromatin modifications at the promoter during activation and repression. Levels of acetylation, methylation, and histones were determined using densitometry analysis. Signals were normalized to the unactivated state. Shown are the average values and standard deviations of results from at least three biological replicates. Signals for AcH3, AcH4, and H3 K27 methylation (K27 me) are divided by the signal value of H3 to take into account differences in total histone levels. The statistical significance of chromatin changes was determined by a one-tailed t test with an alpha of 0.05. Activation is associated with a strong increase in relative H3 and H4 acetylation levels (P values = 8.1E−04 and 1.6E−04), as well as a decrease in relative K27 methylation levels and H3 occupancy (P values = 0.0033 and 2.5E−06). Repression is associated with a significant loss of H3 and H4 acetylation (P values = 1.3E−05 and 1.6E−06), as well as a slight increase in H3 occupancy (P value = 6.2E−04). No changes in relative K27 methylation levels were observed during repression (P value = 0.43).