Codon adaptation by synonymous mutations impacts the functional properties of the estrogen receptor-alpha protein in breast cancer cells.

Oestrogen receptor-alpha (ERα) positivity is intimately associated with the development of hormone-dependent breast cancers. A major challenge in the treatment of these cancers is to understand and overcome the mechanisms of endocrine resistance. Recently, two distinct translation programmes using specific transfer RNA (tRNA) repertoires and codon usage frequencies were evidenced during cell proliferation and differentiation. Considering the phenotype switch of cancer cells to more proliferating and less-differentiated states, we can speculate that the changes in the tRNA pool and codon usage that likely occur make the ERα coding sequence no longer adapted, impacting translational rate, co-translational folding and the resulting functional properties of the protein. To verify this hypothesis, we generated an ERα synonymous coding sequence whose codon usage was optimized to the frequencies observed in genes expressed specifically in proliferating cells and then investigated the functional properties of the encoded receptor. We demonstrate that such a codon adaptation restores ERα activities to levels observed in differentiated cells, including: (a) an enhanced contribution exerted by transactivation function 1 (AF1) in ERα transcriptional activity; (b) enhanced interactions with nuclear receptor corepressor 1 and 2 [NCoR1 and NCoR2 (also known as SMRT) respectively], promoting repressive capability; and (c) reduced interactions with SRC proto-oncogene, non-receptor tyrosine kinase (Src) and phosphoinositide 3-kinase (PI3K) p85 kinases, inhibiting MAPK and AKT signalling pathway.

Repression of the estrogen receptor-alpha transcriptional activity by the Rho/megakaryoblastic leukemia 1 signaling pathway.

Although involved in processes leading to the emergence and development of hormone-dependent breast cancers, the estrogen receptor alpha (ERalpha) also prevents transformed cells from progressing toward a more aggressive phenotype. The transcriptional activity of ERalpha is mediated through two transactivation functions, called activation function 1 and 2, whose respective involvement varies in a cell-specific manner. Here, we identify the Rho/megakaryoblastic leukemia 1 (MKL1) signaling pathway as a main actor in controlling the cell-specific activity of both transactivation functions of ERalpha. Notably, we show that, when the coregulator MKL1 is sequestered in an inactive form by unpolymerized actin, the transcriptional activity of ERalpha mainly relies on the activation function 1. The activation of MKL1, which results from its dissociation from unpolymerized actin, promoted by the ability of Rho to support polymeric actin accumulation, silences the activation function 1 of ERalpha and allows the receptor to mainly act through its activation function 2. Importantly, this switch in the respective contribution exerted by both transactivation functions is correlated with an impaired ability of ERalpha to efficiently transactivate estrogen-regulated reporter genes. MKL1 is further shown to be present on estrogen-responsive genes in vivo. Interestingly, the Rho/MKL1 signaling pathway is activated during the epithelial-mesenchymal transition. A reduced transactivation efficiency of ERalpha, resulting from the activation of this pathway, may therefore suppress the protective role exerted by ERalpha toward tumor progression and invasiveness.

Cyclical DNA methylation of a transcriptionally active promoter.

Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.

Regulation of the intronic promoter of rat estrogen receptor alpha gene, responsible for truncated estrogen receptor product-1 expression.

We have characterized the intronic promoter of the rat estrogen receptor (ER) alpha gene, responsible for the lactotrope-specific truncated ER product (TERP)-1 isoform expression. Transcriptional regulation was investigated by transient transfections using 5'-deletion constructs. TERP promoter constructs were highly active in MMQ cells, a pure lactotrope cell line, whereas a low basal activity was detected in alphaT3-1 gonadotrope cells or in COS-7 monkey kidney cells. Serial deletion analysis revealed that 1) a minimal -693-bp region encompassing the TATA box is sufficient to allow lactotrope-specific expression; 2) the promoter contains strong positive cis-acting elements both in the distal and proximal regions, and 3) the region spanning the -1698/-1194 region includes repressor elements. Transient transfection studies, EMSAs, and gel shifts demonstrated that estrogen activates the TERP promoter via an estrogen-responsive element (ERE1) located within the proximal region. Mutation of ERE1 site completely abolishes the estradiol-dependent transcription, indicating that ERE1 site is sufficient to confer estrogen responsiveness to TERP promoter. In addition, ERalpha action was synergized by transfection of the pituitary-specific factor Pit-1. EMSAs showed that a single Pit-1 DNA binding element in the vicinity of the TATA box is sufficient to confer response by the TERP promoter. In conclusion, we demonstrated, for the first time, that TERP promoter regulation involves ERE and Pit-1 cis-elements and corresponding trans-acting factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells.

Estrogen receptor alpha mRNA in human skeletal muscles.

INTRODUCTION/PURPOSE: To explain the effect of estrogen on skeletal muscle, the presence of estrogen receptor alpha mRNA (ERalpha mRNA) was investigated in human skeletal muscle. METHODS: The highly sensitive technique of nested reverse transcriptase-polymerase chain reaction (nested RT-PCR) was applied on a variety of tissue samples of both sexes: women (deltoid, pectoral, and uterus muscles) (N= 3) and men (deltoid muscle) (N= 3). The total ribonucleic acid was isolated from each tissue sample, reverse transcribed in a thermocycler, and nested PCR was then performed with specific primers. The by-products were analyzed by agarose gel electrophoresis. Internal standard 28S was simultaneously amplified. The ERalpha mRNA level was quantitated by using the ERalpha mRNA/28S mRNA ratio. RESULTS: The expected 204-bp product corresponding to ERalpha was amplified in all tested tissue samples, i.e., deltoid, pectoral, and uterine muscles from women and deltoid muscle from men. The ERalpha mRNA/28S mRNA ratios indicating the receptor expression levels in deltoid muscle from men and women were 0.945 +/- 0.393 (mean +/- SD) (N= 3) and 0.973 +/- 0.136 (mean +/- SD) (N= 2), respectively. CONCLUSIONS: In conclusion, the nested RT-PCR technique identified the presence of transcript encoding ERalpha mRNA in human skeletal muscles. Semi-quantification did not reveal gender difference.

Expression of estrogen receptor subtypes in rat pituitary gland during pregnancy and lactation.

The aim of this study was to examine whether the expression levels of mRNA of the three estrogen receptor (ER) subtypes, ERalpha, ERbeta, and truncated ER product-1 (TERP-1) found in the rat pituitary gland were modified during gestation, lactation, and postlactation periods. By using relative quantitative RT-PCR, we found that ERalpha mRNA significantly peaked in midpregnancy. However, the ERalpha protein level remained constant. ERbeta gene expression did not change throughout pregnancy, suggesting that it was not related to estradiol levels during this reproductive period. In contrast, both TERP-1 mRNA and protein levels dramatically increased throughout the second half of gestation, being faintly detectable in early pregnancy. TERP-1 expression was rapidly reversed by lactation, whereas neither pituitary ERalpha nor ERbeta relative levels were significantly altered. In addition, pup removal for 24-96 h on d 9 postpartum significantly reduced the expression of both ERalpha and ERbeta mRNA compared with that in lactating animals, but the expression of TERP-1 mRNA was no longer detected. Collectively, our data indicate that 1) TERP-1, ERalpha, and ERbeta expression levels are differentially regulated in the pituitary; 2) TERP-1 is variably expressed depending on the hormonal environment related to the estrous cycle, pregnancy, and lactation; and 3) TERP-1/ERalpha ratios dramatically change depending on reproductive periods, suggesting a critical role for TERP-1 in reproductive events.