The interaction of the estrogen receptor with mononucleosomes.

To directly activate specific gene expression, the estrogen receptor (ER) must bind to estrogen receptor response elements (EREs) in the context of nucleosomes. In order to investigate the interaction of the ER with mononucleosomes, we developed a mononucleosome gel shift assay. A 164 bp high specific activity [(32)P]probe DNA (32 bp consensus ERE with flanking regions separated by 23 nucleotides from an artificial nucleosome positioning sequence) was prepared. Nuclear extracts from MCF-7 cells or recombinant human ERalpha were incubated with the labeled ERE +/- excess ERE. A retarded band was seen which was completely obliterated with excess ERE, confirming the specificity of binding. This probe was then used to make reconstituted mononucleosomes by sequential dilution of a high salt histone preparation. The nucleosomes were purified by sucrose density gradients and footprinting analysis was performed to demonstrate that the mononucleosomes were rotationally phased as seen by a periodic digestion pattern (10 bp) of the nucleosomes versus ERE. Nucleosomes were incubated with nuclear extracts containing ER or recombinant ERalpha. Dose dependence in the shift of the mononucleosomes with increasing concentrations of ER was observed. Specificity was demonstrated in experiments with excess ERE and anti-ER antibody. Footprinting analysis was also performed. We also determined that addition of high mobility group protein-2 (HMGB-2, a protein closely related to HMGB-1) with the ER increased the interaction of ER with mononucleosomes. These studies will allow us to address the interactions of ER with core histones containing a multiplicity of variants and modifications in nucleosomal structure.

Aryl hydrocarbon receptor-mediated posttranscriptional regulation of IL-1beta.

TCDD stimulated IL-1beta gene expression in differentiating human keratinocyte cell lines in a time- and dose-dependent manner. Increases in prointerleukin-1beta (pIL-1beta) protein and IL-1beta steady state mRNA levels were observed in both SCC-12F and HaCaT cells following TCDD treatment. When pretreated with alpha-naphthoflavone, an AhR antagonist, TCDD-mediated increases in IL-1beta gene expression were attenuated, demonstrating for the first time that the environmental toxin, TCDD, can stimulate cytokine (IL-1beta) gene expression in an AhR-dependent manner. Nuclear run-on experiments were performed in SCC-12 cells to determine if the AhR-dependent increases in IL-1beta expression were due to transcriptional activation of the IL-1beta gene. Results showed high constitutive levels of IL-1beta transcriptional activity, however, TCDD treatment, which stimulated IL-1beta steady state mRNA levels, failed to potentiate IL-1beta transcription. Taken together, these results demonstrate that AhR-mediated IL-1beta regulation is occurring posttranscriptionally.