Search

Purpose: Transcriptional regulation of estrogen receptor-alpha (ERalpha) involves both epigenetic mechanisms and trans-active factors, such as TFAP2C, which induces ERalpha transcription through an AP-2 regulatory region in the ERalpha promoter. Attempts to induce endogenous ERalpha expression in ERalpha-negative breast carcinomas by forced overexpression of TFAP2C have not been successful. We hypothesize that epigenetic chromatin structure alters the activity of TFAP2C at the ERalpha promoter., Experimental Design: DNA methylation, histone acetylation, and chromatin accessibility were examined at the ERalpha promoter in a panel of breast carcinoma cell lines. TFAP2C and polymerase II binding were analyzed by chromatin immunoprecipitation. Epigenetic chromatin structure was altered using drug treatment with 5-aza-2'-deoxycytidine (AZA) and trichostatin A (TSA)., Results: The ERalpha promoter in the ERalpha-negative lines MDA-MB-231, MCF10A, and MCF7-5C show CpG island methylation, histone 3 lysine 9 deacetylation, and decreased chromatin accessibility compared with ERalpha-positive cell lines MCF7 and T47-D. Treatment with AZA/TSA increased chromatin accessibility at the ERalpha promoter and allowed TFAP2C to induce ERalpha expression in ERalpha-negative cells. Chromatin immunoprecipitation analysis showed that binding of TFAP2C to the ERalpha promoter is blocked in ERalpha-negative cells but that treatment with AZA/TSA enabled TFAP2C and polymerase II binding., Conclusion: We conclude that the activity of TFAP2C at specific target genes depends upon epigenetic chromatin structure. Furthermore, the combination of increasing chromatin accessibility and inducing TFAP2C provides a more robust activation of the ERalpha gene in ERalpha-negative breast cancer cells.

Interferon-tau (IFNT) is released by preimplantation conceptuses of ruminant species and prepares the mother for pregnancy. Although one important function is to protect the corpus luteum from the luteolytic activity of prostaglandin-F 2alpha, IFNT most likely regulates a range of other physiological processes in endometrium. Here, an immortalized cell line from ovine uterine luminal epithelial cells was treated with IFNT for either 8 or 24 h. RNA was subjected to cDNA microarray analysis, with RNA from untreated cells as the reference standard. Of 15 634 genes, 1274 (8%) were IFNT responsive at P<0.01 and 585 at P<0.001 to at least one treatment. Of the latter, 356 were up-regulated and 229 down-regulated. Increasing IFNT concentrations from 10 ng/ml to 10 microg/ml had minor effects, and most genes up- or down-regulated at 8 h were regulated similarly at 24 h. Although IFNT influences many genes implicated in antiviral activity and apoptosis, its action also likely regulates prostaglandin metabolism, growth factors and their receptors, apoptosis and the nuclear factor (NF)-kappaB cascade, extracellular matrix accretion, angiogenesis, blood coagulation, and inflammation. In particular, it increased mRNA concentrations of genes related to the vascular endothelial growth factor R2 pathway of angiogenesis and down-regulated ones associated with hypoxia. Two genes implicated in the antiluteolytic actions of IFNT (encoding cyclooxygenase-2 and the oxytocin receptor respectively) were down-regulated in response to all treatments. IFNT targets a complex range of physiological processes during the establishment of pregnancy.

In ruminants, conceptus interferon-tau (IFNT) alters maternal physiology to accommodate a pregnancy. We hypothesized that the effectiveness of IFNT on extending corpus luteum (CL) life span in nonpregnant ewes would depend upon the dose and manner of administration and would be correlated with the response in gene expression in endometrium. We anticipated that IFNT, whether administered im or by uterine infusion, would mimic changes observed in pregnancy. Ewes were assigned to five treatments: 1) uterine infusion of saline; 2) uterine infusion of ovine IFNT4 (200 microg/d); 3) saline im injection; 4) im injection of IFNT4 at low dose (200 microg/d); and 5) high dose (2 mg/d). CL life span was increased in groups 2 and 5, but not in 1, 3, and 4. Endometrial RNA extracted from groups 1-5 on d 14 and from d 14 pregnant and nonbred (cyclic) ewes was used to assess expression of 70 genes on microarrays. When pregnant and cyclic ewes were compared, 30 genes were up-regulated and nine down-regulated during pregnancy. Responses were slightly less in groups 2 and 5 but were much lower in group 4. The majority of the highly up-regulated genes were associated with antiviral responses. Those down-regulated included ones for IGF-II, hypoxia-inducible factor 1alpha, oxytocin receptor, prostaglandin F synthase, and cyclooxygenase-2. Quantitative PCR for selected genes confirmed these data and revealed that similar gene expression changes occurred in the CL of pregnant and group 2 ewes. IFNT treatment mimics pregnancy, but relatively high doses of im-injected IFNT are required to elicit a full endometrial response.