In Vitro Reconstitution of Progesterone-Dependent RNA Transcription in Nuclear Extracts of Human Breast Carcinoma Cells
Steroid hormones, such as estrogen, progesterone, androgens, glucocorticoids, and mineralocorticoids, are well-known regulators of the expression of specific gene networks in higher eukaryotes (1, 2, 3). The hormonal action is mediated through specific intracellular receptor proteins that are functionally inactive in the absence of the hormone ligand. The hormonal response is initiated by the high-affinity binding of a steroid ligand to its cognate receptor. Hormone binding triggers a poorly understood structural activation of the receptor termed “transformation,” which enables it to bind to specific enhancer sequences known as steroid response elements at the target gene. The receptor then modulates gene expression presumably by interacting with the transcription machinery at the target promoter. The precise mechanisms underlying the key molecular events in the steroid-induced gene activation pathway, such as ligand-induced activation of receptors and transactivation of target promoters by the activated receptors, have not been clearly resolved.
KeywordsNuclear Extract T47D Cell Transcription Assay HeLa Nuclear Extract JA20 Rotor
- 9.Reinberg, D., Horikoshi, M., and Roeder, R. G. (1987) Factors involved in specific transcription by mammalian RNA polymerase II: functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site. J. Biol. Chem. 262, 3322–3330.PubMedGoogle Scholar
- 13.Klein-Hitpass, L., Tsai, S Y., Weigel, N. L., Allan, G F., Riley, D., Rodriguez, R., Schrader, W. T., Tsai, M.-J., and O’Malley, B. W. (1990) The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex. Cell 60, 247–257.PubMedCrossRefGoogle Scholar
- 17.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laborutory Manual, 2nd ed., part 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 1.38–1.46.Google Scholar