Common effects of acidic activators on large-scale chromatin structure and transcription

Abstract

Large-scale chromatin decondensation has been observed after the targeting of certain acidic activators to heterochromatic chromatin domains. Acidic activators are often modular, with two or more separable transcriptional activation domains. Whether these smaller regions are sufficient for all functions of the activators has not been demonstrated. We adapted an inducible heterodimerization system to allow systematic dissection of the function of acidic activators, individual subdomains within these activators, and short acidic-hydrophobic peptide motifs within these subdomains. Here, we demonstrate that large-scale chromatin decondensation activity is a general property of acidic activators. Moreover, this activity maps to the same acidic activator subdomains and acidic-hydrophobic peptide motifs that are responsible for transcriptional activation. Two copies of a mutant peptide motif of VP16 (viral protein 16) possess large-scale chromatin decondensation activity but minimal transcriptional activity, and a synthetic acidic-hydrophobic peptide motif had large-scale chromatin decondensation activity comparable to the strongest full-length acidic activator but no transcriptional activity. Therefore, the general property of large-scale chromatin decondensation shared by most acidic activators is not simply a direct result of transcription per se but is most likely the result of the concerted action of coactivator proteins recruited by the activators' short acidic-hydrophobic peptide motifs.

FIG. 1.

Heterodimerization system for inducible protein targeting to lac operator arrays. (A) Schematic of the system. (B) Fusion proteins are listed with their names. MCS, multiple cloning site (polylinker); NLS, nuclear localization signal. (C) Results of transient transcription assays, where an 8-lac operator-E1b TATA-luciferase reporter plasmid was cotransfected with CFP-lac rep-FKBP3 plus each expression plasmid listed below the chart into CHO-K1 cells. Error bars show standard errors of the mean (SEM).

FIG. 2.

Rapid targeting of VP16 and inducible large-scale chromatin decondensation with heterodimerization system. (A) VP16 unfolds chromatin when inducibly targeted to a heterochromatic chromatin array. A03_1 cells which contain a compact heterochromatic lac operator array were transfected with CFP-lac rep-FKBP3 (NYE73) and FRB*-YFP-VP16 (NYE82) and treated with rapamycin 48 h prior to fixation. Single deconvolved optical sections are shown. Scale bar, 1 μm. (B) Unfolding of chromatin in living 2A5a cells. Cells were transfected with CFP-lac rep-FKBP3 and FRB*-YFP or FRB*-YFP-VP16 and incubated for 48 to 72 h. Cells were then maintained live on a microscope stage and imaged in single optical sections with short exposure times. Time points indicate minutes after rapamycin was added. Scale bar, 1 μm. (C) Quantitation of recruitment and chromatin unfolding in living 2A5a cells. Two independent experiments were carried out as above (B) for each condition. In each experiment, approximately 10 cells were imaged at time points before and after rapamycin was added. Each data point shown is the mean of about 20 cells.

FIG. 3.

Transcriptional activity and large-scale chromatin decondensation activity of inducible acidic activators. (A) The transient transcription assay used an 8- or 256-lac operator-E1b TATA-luciferase reporter plasmid in CHO-K1 cells. Fold induction is shown, meaning activity in the presence of rapamycin divided by activity in the absence of rapamycin. (B) Recruitment of each FRB*-YFP fusion protein to the chromatin array; see Materials and Methods for details. The median is shown. (C) Chromatin-unfolding assay. Chromatin arrays were measured for each FRB*-YFP fusion protein in the presence and absence of rapamycin. CFP-lac rep-FKBP3 was cotransfected with all FRB*-YFP fusion proteins. Tails of the box plots mark the 5th and 95th percentiles, boxes mark the 25th and 75th percentiles, and the line in the box marks the median for each sample. See Materials and Methods for details.

FIG. 4.

Transcriptional activity and large-scale chromatin decondensation activity of acidic activator motifs, all fused to FRB*-YFP. none, FRB*-YFP with no protein fused to it. See the legend to Fig. 3 for details. Error bars show SEMs. (A) Transient transcription assay. (B) Recruitment to the chromatin array. Sample labels are the same as for panels A and C. (C) Chromatin-unfolding assay. The initial experiment is shown. A subsequent experiment yielded similar results, except that the area of Gal4(861-873)₂ was larger in the presence of rapamycin in the second experiment, and the area of VP16(437-448) was smaller in the absence of rapamycin and larger in the presence of rapamycin in the second experiment than in the first. The samples in the left and right charts were prepared separately and therefore statistically analyzed separately; note that the P values for the right side are compared to those of the positive control. The samples shown on the left (B and C) were prepared and analyzed at the same time as the samples in Fig. 3, so “none” and “VP16(413-490)” are the same data sets as shown in Fig. 3B and C.

FIG. 5.

Transcriptional activity and chromatin-unfolding ability of VP16(437-448) motifs and the F442P mutant thereof (left) and DELQPASIDP motif (right) recruited directly by the lac repressor. See the legend to Fig. 3 for details. The samples in the left and right charts were prepared separately and were therefore statistically analyzed separately. (A) Transient transcription assay. Note the log scale. Error bars show SEMs. (B) Chromatin-unfolding assay. The initial experiment is shown. A subsequent experiment yielded similar results, except that the distribution of areas showed more spread for the YFP-lac rep-dimer and less spread for the YFP-lac rep-mutant dimer in the second experiment.

FIG. 6.

Large-scale chromatin-unfolding motifs. Hydrophobic, FMILVCW (red); acidic, DE (green); basic, KR (blue) (11). Protein segments listed have all been shown to unfold large-scale chromatin structure (this study and references , , and 54). The amino acids of each segment are shown in parentheses. The regions underlined are hypothesized to be responsible for the unfolding activity. The highlighted A in BRCA1 indicates the position where the cancer-predisposing mutation A1708E unfolds chromatin more effectively than the wild type in the context of the full-length protein (54). The highlighted Y in Hap4 indicates a discrepancy between the protein we used (34) and the National Center for Biotechnology Information sequence (accession no. X16727), which indicates an N at this position.