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. 2006 Jun 13;45(23):7299-306.
doi: 10.1021/bi060314k.

Flanking sequence composition differentially affects the binding and functional characteristics of glucocorticoid receptor homo- and heterodimers

Brian Morin  1 , LaNita A Nichols, Lené J Holland
Affiliations

Flanking sequence composition differentially affects the binding and functional characteristics of glucocorticoid receptor homo- and heterodimers

Brian Morin et al. Biochemistry. .

Abstract

The core binding sites for a multitude of transcription factors have been identified and characterized, but these sequences cannot fully account for the nuances of cell-specific and gene-specific control of gene transcription. Many factors may contribute to the precise responsiveness of a gene to a particular transcriptional regulatory protein, including the nucleotides in the proximity of the core binding site for that protein. Here, we examine two flanking sequences bordering a site in the gamma-fibrinogen gene regulatory region that binds a heterodimer of the Xenopus glucocorticoid receptor accessory factor (XGRAF) and the glucocorticoid receptor (GR). Mutation of the upstream flank results in a decrease in the level of XGRAF binding but little change in hormone induction. However, alteration of the downstream flank adjacent to the GR binding site causes a decrease in levels of both GR monomer binding and hormone induction. Conversion of the XGRAF-GR binding site to a full glucocorticoid response element (GRE) alters the role of the flanking sequences. A full GRE in this position requires the wild-type upstream flank to bind GR homodimer and induce transcription to maximal levels. In contrast, mutation of the downstream flank is not detrimental to either the binding or the function of the GR dimer. Thus, flanking sequence composition and dimer partner combine to influence GR function, underscoring the complexities involved in the identification of authentic transcription factor response elements.

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Figures

FIGURE 1
FIGURE 1
The Xenopus γ-fibrinogen DNA from -187 to -157 relative to the transcription start site at +1 is shown. The XGRAF binding site (white box), GRE half site (grey box), and both flanking sequences in the γ-wt sequence are displayed. The γ-GRE mutant and a consensus GRE (32) are shown in relation to the γ-wt sequence. The GRE mutation involves conversion of the base at -173 from G to C (indicated by the arrowhead) to cause the sequence to function as a full GRE.
FIGURE 2
FIGURE 2
The XGRAF:GR core binding sequence has reduced function following replacement of the flanking sequences. The diagrammatic schemes of the constructs containing the Xenopus γ-fibrinogen gene upstream DNA show the presence of the XGRAF binding site (white box), the GRE half site (grey box), and mutated sequence (×). See Table 1 for wild type and mutant sequences. Constructs were transfected into Xenopus primary hepatocytes and glucocorticoid responsiveness was determined as described in Materials and Methods. The fold hormonal induction of each construct is given as a percentage of the wt (Panel A) or mutU*D (Panel B) control. (A) The data are expressed as the average of nine separate experiments ± SEM. The fold induction of the wt construct was 2.3. (B) The data are expressed as the average of ten separate experiments ± SEM. The fold induction of the mutU*D construct was 1.6. All experimental constructs were significantly different from their controls (p < 0.05) as determined by the Wilcoxon signed-rank test (26).
FIGURE 3
FIGURE 3
The downstream flanking sequence is important for XGRAF:GR function. The diagrammatic schemes of the constructs containing the Xenopus γ-fibrinogen gene upstream DNA show the presence of the XGRAF binding site (white box), the GRE half site (grey box), and mutated sequence (×). See Table 1 for wild type and mutant sequences. Constructs were transfected into Xenopus primary hepatocytes and glucocorticoid responsiveness was determined as described in Materials and Methods. The fold hormonal induction of each construct is given as a percentage of the wt control. The data are expressed as the average of eight separate experiments ± SEM. The fold induction of the wt construct was 1.8. The mutUD and mutD constructs were significantly different from the control (p < 0.05) as determined by the Wilcoxon signed-rank test (26).
FIGURE 4
FIGURE 4
XGRAF binding to γ-fibrinogen DNA is negatively impacted by mutation of the upstream flanking sequence. (A) The gel mobility shift assay was carried out as described in Materials and Methods with a large control DNA probe (LP) present in each lane. The small experimental probes (SP) are described in Table 1. The bands are denoted to the left as XGRAF complexed with either the large or small probe (XGRAF:LP or XGRAF:SP) or as the free individual probes. (B) Data for each experimental probe were analyzed from nine independent samples run on three separate gels as described in Materials and Methods and are expressed as a percentage of XGRAF binding to the small wt control ± SEM. XGRAF binding to the mutUD and mutU probes was significantly different from the control (p < 0.05) as determined by the Wilcoxon signed-rank test (26).
FIGURE 5
FIGURE 5
Formation of XGRAF:GR:DNA complex is reduced by mutation of the upstream flanking sequence. (A) The gel mobility shift assay was carried out as described in Materials and Methods with the experimental probes described in Table 1. The bands are denoted to the left as XGRAF:GR, XGRAF, or GR complexed with probe or as free probe. (B) Data for each experimental probe were analyzed from ten independent samples run on three separate gels as described in Materials and Methods and are expressed as a percentage of XGRAF:GR binding to the wt control ± SEM. XGRAF:GR binding to the mutUD and mutU probes was significantly different from the control (p < 0.05) as determined by the Wilcoxon signed-rank test (26).
FIGURE 6
FIGURE 6
GR monomer binding to γ-fibrinogen DNA is decreased by mutation of the downstream flanking sequence. (A) The gel mobility shift assay was carried out as described in Materials and Methods with a large control DNA probe (LP) present in each lane. The small experimental probes (SP) are described in Table 1. The bands are denoted to the left as GR complexed with either the large or small probe (GR:LP or GR:SP) or as the free individual probes. (B) Data for each experimental probe were analyzed from fifteen independent samples run on three separate gels as described in Materials and Methods and are expressed as a percentage of GR monomer binding to the small wt control ± SEM. GR monomer binding to the three experimental probes was significantly different from the control (p < 0.05) as determined by the Wilcoxon signed-rank test (26).
FIGURE 7
FIGURE 7
The upstream flanking sequence is important for GR homodimer function. The diagrammatic schemes of the constructs containing the Xenopus γ-fibrinogen gene upstream DNA modified to contain a full GRE show the presence of the GRE half sites (grey boxes) and mutated sequence (×). See Table 1 for wild type and mutant sequences. Constructs were transfected into Xenopus primary hepatocytes and glucocorticoid responsiveness was determined as described in Materials and Methods. The fold hormonal induction of each construct is given as a percentage of the GRE control. The data are expressed as the average of six separate experiments ± SEM. The fold induction of the GRE construct was 14.3. The GREmutUD and GREmutU constructs were significantly different from the control (p < 0.05) as determined by the Wilcoxon signed-rank test (26).
FIGURE 8
FIGURE 8
GR homodimer binding to γ-GRE DNA is reduced by mutation of the upstream flanking sequence. (A) The gel mobility shift assay was carried out as described in Materials and Methods with the small experimental probes described in Table 1. The bands are denoted to the left as GR:GR (GR homodimer), GR (GR monomer), and free probe. (B) Data for each experimental probe were analyzed from fifteen independent samples run on three separate gels as described in Materials and Methods and are expressed as a percentage of GR dimer binding to the GRE control ± SEM. GR dimer binding to the GREmutUD and GREmutU probes was significantly different from the control (p < 0.05) as determined by the Wilcoxon signed-rank test (26).
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