Glucocorticoid receptor activation of the I kappa B alpha promoter within chromatin

Abstract

The glucocorticoid receptor (GR) is a ligand-activated transcription factor that induces expression of many genes. The GR has been useful for understanding how chromatin structure regulates steroid-induced transcription in model systems. However, the effect of glucocorticoids on chromatin structure has been examined on few endogenous mammalian promoters. We investigated the effect of glucocorticoids on the in vivo chromatin structure of the glucocorticoid-responsive I kappa B alpha gene promoter, the inhibitor of the ubiquitous transcription factor, nuclear factor kappa B (NF kappa B). Glucocorticoids inhibit NF kappa B activity in some tissues by elevating the levels of I kappa B alpha. We found that glucocorticoids activated the I kappa B alpha promoter in human T47D/A1-2 cells containing the GR. We then investigated the chromatin structure of the I kappa B alpha promoter in the absence and presence of glucocorticoids with the use of micrococcal nuclease, restriction enzyme, and deoxyribonuclease (DNaseI) analyses. In untreated cells, the promoter assembles into regularly positioned nucleosomes, and glucocorticoid treatment did not alter nucleosomal position. Restriction enzyme accessiblity studies indicated that the I kappa B alpha promoter is assembled as phased nucleosomes that adopt an "open" chromatin architecture in the absence of hormone. However, glucocorticoids may be required for transcription factor binding, because DNaseI footprinting studies suggested that regulatory factors bind to the promoter upon glucocorticoid treatment.

Figure 1

Glucocorticoids increase IκBα transcription in A1-2 cells. (A) Glucocorticoids increase IκBα RNA levels in A1-2 cells. Northern analysis was conducted with the use of A1-2 cells that were either untreated (lane 1) or treated with dexamethasone (10⁻⁷ M) for 2, 4, 8, and 24 h (lanes 2–5). Total cellular RNA was prepared and analyzed by Northern blot. Briefly, RNA was separated on a 1% agarose/formaldehyde gel, the RNA (10 μg) transferred to a nylon membrane, and the membrane probed with ³²P-labeled IκBα and actin cDNA probes. (B) The NFκB activator, PMA, does not affect glucocorticoid activation of IκBα. Northern analysis was conducted with the use of A1-2 cells that were either untreated (lane 1) or treated with PMA (40 ng/ml) for 45 min (lane 2). In lane 3, cells were pretreated with dexamethasone (10⁻⁷ M) for 4 h (lane 3) before treatment with PMA (40 ng/ml) for 45 min. The blot was reprobed for cyclophilin as a control. (C) Glucocorticoids repress NFκB activity in A1-2 cells. Nuclear extracts were prepared from cells treated as in A and analyzed by gel shift, with the use of 10 μg of nuclear extract with a ³²P-labeled double-stranded oligonucleotide corresponding to the NFκB consensus sequence of the IL-2 promoter. The binding reactions were analyzed on a 5% nondenaturing polyacrylamide gel, followed by autoradiography.

Figure 2

The IκBα promoter is organized into a regular array of nucleosomes. (A) Nuclei (lanes 1 and 2) and genomic DNA (lanes 3 and 4) from A1-2 cells were digested with 0 (lanes 1 and 4), 1 (lane 3), or 200 units/ml (lane 2) micrococcal nuclease (MNase), and the purified DNA recut with HincII. DNA fragments were analyzed by Southern blot, with the use of a radiolabeled HincII (−699)/SgrAI (−536) fragment of the IκBα promoter. (B) Nuclei (lanes 1–5) and genomic DNA (lane 6) were digested with 0 (lane 1), 1 (lane 6), or 20–200 units/ml (lanes 2–5) MNase, and the purified DNA was recut with EcoRI. DNA fragments were analyzed by Southern blot, with the use of a radiolabeled EcoRI (−1229)/AflII (−999) fragment of the IκBα promoter. (C) Schematic diagram of nucleosome positions on the IκBα proximal promoter in A1-2 cells.

Figure 3

Fine mapping of the −280 to −50 region of the IκBα promoter by micrococcal nuclease. (A) Fine mapping of the IκBα promoter. DNA prepared as in Figure 2 was also analyzed by reiterative primer extension. Lanes 7, 8, and 9: 0, 100, and 200 units/ml MNase digests, respectively. Lane 5: (−623/+11) IκBα-luc plasmid digested with MNase, lanes 1–4, sequencing tracks with the (−623/+11) IκBα-luc plasmid. With the use of the Molecular Dynamics Phosporimager, a line graph representing lane 9 band intensity was created. The adjacent schematic is labeled as follows: ▪, peak locations relative to +1; □, restriction enzyme sites. (B) Digestion of nuclei and reiterative primer extension analysis were carried out as in A except that two different MNase concentrations were used and cells were either untreated or treated with dexamethasone (10⁻⁷ M) for 2 h. Lane 2: G sequencing track with (−623/+11) IκBα-luc plasmid. Lane 3: (−623/+11) IκBα-luc plasmid digested with MNase. (C) Line graph comparing lanes 6 and 7.

Figure 4

Restriction enzyme hypersensitivity analysis of the IκBα promoter. (A) A1-2 cells were either untreated (lane 1), or treated for 2 h with dex (10⁻⁷ M; lane 2). Nuclei were isolated, digested in vivo with DpnII or PstI and in vitro with AvaI, and analyzed by reiterative primer extension with oligonucleotide TA-80. (B) A1-2 cells were either untreated (lane 1) or treated for 2 h with dex (10⁻⁷ M; lane 2). Nuclei were isolated, digested in vivo with AvaI or DdeI and in vitro with DpnII, and analyzed by reiterative primer extension with oligonucleotide TA-53. (C) A1-2 cells were either untreated (lanes 1 and 3) or treated for 2 h with dex (10⁻⁷ M; lanes 2 and 4). Nuclei were isolated, digested in vivo with DdeI or EcoNI and in vitro with HindIII, and analyzed by reiterative primer extension with oligonucleotide TA-53. (D) Restriction enzyme hypersensitivity profile of the IκBα promoter in the absence or presence of glucocorticoid. Dotted lines, nucleosome positions determined by low-resolution mapping; solid lines, nucleosomes mapped to base pair resolution.

Figure 5

DNaseI footprinting of the IκBα promoter (−230 to −60). (A) Nuclei (lanes 3–6) or genomic DNA (lane 2) from A1-2 cells was digested with 0 (lanes 3 and 4), 0.04 (lane 2), or 50 units/ml (lanes 5 and 6) DNaseI, and the purified DNA was recut with EcoRI. DNA fragments were analyzed by reiterative primer extension. Lane 5: G sequencing track with the (−623/+11) IκBα-luc plasmid. (B) A line graph comparing lanes 5 and 6 band intensity with locations of transcription factor binding sites indicated.

Figure 6

DNaseI footprinting of the IκBα promoter (−100 to + 7). (A) Nuclei (lanes 3–6) or genomic DNA (lane 2) from A1-2 cells was digested with 0 (lanes 3 and 4), 0.01 (lane 2), or 100 units/ml (lanes 5 and 6) DNaseI, and the purified DNA was recut with PstI. DNA fragments were analyzed by reiterative primer extension. Lane 7: C sequencing track with the (−623/+11) IκBα-luc plasmid. (B) A line graph comparing lanes 5 and 6 band intensity with locations of transcription factor binding sites indicated.

Figure 7

Schematic representation of the IκBα promoter. The region of IκBα that was mapped to bp resolution is shown. ▵, sites of glucocorticoid-induced MNase hypersensitivity; ▾, sites where glucocorticoid treatment reduced DNaseI cleavage.