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. 2009 Mar 6;284(10):6048-52.
doi: 10.1074/jbc.C800212200. Epub 2009 Jan 5.

Glucocorticoid receptor activation of the Ciz1-Lcn2 locus by long range interactions

Ofir Hakim  1 , Sam John, Jian Qun Ling, Simon C Biddie, Andrew R Hoffman, Gordon L Hager
Affiliations

Glucocorticoid receptor activation of the Ciz1-Lcn2 locus by long range interactions

Ofir Hakim et al. J Biol Chem. .

Abstract

The cellular response to glucocorticoid receptor (GR) activation involves a highly orchestrated series of regulatory actions influenced at multiple levels by a variety of mechanisms including the action of transcription factors and chromatin modifiers. Because the majority of GR binding sites (glucocorticoid-responsive elements (GREs)) are distant from promoters, it is likely that interactions at a distance play an important role in GR action. To determine whether long range chromosomal associations play a role in transcription regulation by GR, we utilized a chromosome conformation capture-based technique (associated chromosome trap) to identify unknown, remote sequences that interact with the GR-induced Lipocalin2 (Lcn2) gene. Our screen revealed that the Lcn2 GRE interacts with the Ciz1 gene, nearly 30 kb upstream. Ciz1 was subsequently found to be a novel GR-responsive gene. The GRE proximal to the Lcn2 promoter apparently functions to regulate both the Lcn2 gene and the distal Ciz1 gene. Using quantitative chromosome conformation capture, we find that a loop structure is organized between these two genes. This structure is hormone-independent and present only in cell types where the genes are active. The strong correlation between gene expression and loop structure in different cell lines suggests that high order interactions play a role in determining tissue-specific gene regulation.

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Figures

FIGURE 1.
FIGURE 1.
The ACT assay reveals Ciz1 as an Lcn2 associated partner under hormone regulation. A, PCR reactions products were run on 5% polyacrylamide electrophoresis gels and analyzed with PhosphorImager, showing a 260-bp DNA product. B, the PCR product was sequenced and revealed Ciz1 (lowercase letters), flanked by Lcn2 (uppercase letters) and a BamHI restriction site (lowercase underline letters) on one side, and the linker (uppercase letters, overline) together with a TaqI restriction site (lowercase underline letters) on the other side. C, graphical representation of the Lcn2-Ciz1 locus. Gene structures from transcription start site to end are shown with introns (lines) and exons (boxes) and 3C and ChIP loop assays primers (black arrows on top), HindIII restriction sites (vertical black lines), GRE, and the Dex-induced DNase I hypersensitivity site (HS). D–F, time course analysis of Lcn2, Ciz1, and Ptges2 transcript levels following hormone treatment. Samples were collected before DEX treatment (0) and at 0.5, 1, 2, 4, 8, 12, and 24 h after Dex induction. Lcn2 (D) and Ciz1 (E) are up-regulated in response to Dex treatment, whereas Ptges2 transcription (F) is unaffected. Results shown are the average of two independent experiments, and S.D. values are indicated (error bars).
FIGURE 2.
FIGURE 2.
Long range interactions between Lcn2 GRE and Ciz1. A, 3C analysis shows high interaction frequencies between Lcn2 GRE and Ciz1 upstream region in the presence (diamond points) or absence (square points) of hormone. The Lcn2 GRE primer was used as an anchor and paired with primers across and surrounding the Ciz1 gene. The graph presents relative cross-linking frequency on the vertical axis. The x axis indicates the position in kb relative to the Lcn2 GRE in the middle of the HindIII fragment. S.E. values are indicated. Results shown are the average of two independent biological experiments with PCR reactions from each experiment performed in duplicates for 6–8 times. Each signal was normalized to control templates to correct for primer efficiency and also to the cross-linking frequency for two close fragments of the Gapd locus as an internal control to correct for differences in 3C preparations in different conditions and repeats. B, Lcn2 GRE interacts with Ciz1 upstream region and GR protein in cells under Dex stimulation as shown by quantitative PCR analysis of GR ChIP loop DNA. Each signal was normalized to the GR ChIP loop signal of non-induced samples. Data shown are the average of three independent experiments. S.D. values are indicated. C, time course GR ChIP loop assay between Lcn2 GRE and the Ciz1 upstream region (32 kb apart) indicates a stable interaction frequency. Cells were harvested at 1, 4, and 8 h after Dex treatment. Data were normalized to GR ChIP loop signal with non-induced samples and to GR occupancy at the Lcn2 GRE. Results shown are the average of two independent experiments. S.D. values are indicated.
FIGURE 3.
FIGURE 3.
Cell-specific expression of Lcn2 and Ciz1 is correlated with loop formation. A and B, 3C analysis over the Lcn2-Ciz1 locus shows interaction frequencies between the Lcn2 GRE and the Ciz1 upstream region in the presence (diamond) or absence (square) of hormone in AtT20 cells (A) and in Hepa1 cells (B). Controls and symbols are described in the legend for Fig. 2. C–F, mRNA analysis shows that Lcn2 and Ciz1 transcription is strongly up-regulated in response to Dex treatment in Hepa1 liver cells but not in AtT20 pituitary cells. Results shown are the average of three independent experiments. S.D. values are indicated.
FIGURE 4.
FIGURE 4.
Loop formation in the Lcn2-Ciz1 domain. A long range interaction is detected between the immediate upstream regions of the Lcn2 and Ciz1 promoters, both in the presence and in the absence of hormone (Dex). This interaction is likely to result from dynamic and transient pairing between the regions occurring on a rapid time scale. The glucocorticoid receptor, although not a critical component of the loop interaction, stimulates transcriptional activity of both promoters by mechanisms related to the loop-induced proximity of the promoters. Other long range interactions between the Lcn2 GRE and intermediate regions of the Ciz1 domain are possible (– – –).
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