RESUMO
Estrogen receptor alpha (ER) is a member of the nuclear hormone receptor family, which upon binding estrogen shows increased apparent affinity for nuclear components (tight nuclear binding). The nuclear components that mediate this tight nuclear binding have been proposed to include both ER-DNA interactions and ER-protein interactions. In this paper, we demonstrate that tight nuclear binding of ER upon estrogen occupation requires ER-DNA interactions. Hormone-bound ER can be extracted from the nucleus in low-salt buffer using various polyanions, which mimic the phosphate backbone of DNA. The importance of specific ER-DNA interactions in mediating tight nuclear binding is also supported by the 380-fold lower concentration of the ERE oligonucleotide necessary to extract estrogen-occupied ER from the nucleus compared to the polyanions. We also demonstrate that estrogen-induced tight nuclear binding requires both the nuclear localization domain and the DNA binding domain of ER. Finally, enzymatic degradation of nuclear DNA allows us to recover 45% of tight nuclear-bound ER. We further demonstrate that ER-AIB1 interaction is not required for estrogen-induced tight nuclear binding. Taken together, we propose a model in which tight nuclear binding of the estrogen-occupied ER is predominantly mediated by ER-DNA interactions. The effects of estrogen binding on altering DNA binding in whole cells are proposed to occur through estrogen-induced changes in ER-chaperone protein interactions, which alter the DNA accessibility of ER but do not directly change the affinity of the ER for DNA, which is similar for both unoccupied and occupied ER.
Assuntos
Núcleo Celular/metabolismo , DNA/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Substituição de Aminoácidos , Ligação Competitiva/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Citosol/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Deleção de Genes , Células HeLa , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Sinais de Localização Nuclear/genética , Coativador 3 de Receptor Nuclear , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Polifosfatos/química , Polifosfatos/farmacologia , Ligação Proteica/efeitos dos fármacos , Elementos de Resposta/genética , Solubilidade , Transativadores/genética , Transativadores/metabolismo , Vanadatos/química , Vanadatos/farmacologiaRESUMO
The role of steroid hormone receptors in very early embryonic development remains unknown. Clearly, expression during organogenesis is important for tissue-specific development. However, progesterone receptor (PR) and estrogen receptors (ERalpha, ERbeta) are expressed during early development through the blastocyst stage in mice and other species, and yet are not essential for embryonic viability. We have utilized the mouse embryonic stem (mES) cell model to investigate the regulated expression of these receptors during differentiation. Surprisingly, one of the earliest changes in gene expression in response to a differentiation signal observed is PR gene induction. It parallels the time course of expression for the patterning genes Hoxb1 and Hoxa5. Unexpectedly, PR gene expression is not regulated in an estrogen-dependent manner by endogenous ERs or by transiently overexpressed ERalpha. Our results suggest a potentially novel mechanism of PR gene regulation within mES cells compared to adult tissues and the possibility of unique targets of PR action during early mES cell differentiation.
