title: Role of Estrogen Response Element in the Human Prolactin Gene: Transcriptional Response and Timing.
abstract: The use of bacterial artificial chromosome (BAC) reporter constructs in molecular physiology enables the inclusion of large sections of flanking DNA, likely to contain regulatory elements and enhancers regions that contribute to the transcriptional output of a gene. Using BAC recombineering, we have manipulated a 160-kb human prolactin luciferase (hPRL-Luc) BAC construct and mutated the previously defined proximal estrogen response element (ERE) located -1189 bp relative to the transcription start site, to assess its involvement in the estrogen responsiveness of the entire hPRL locus. We found that GH3 cell lines stably expressing Luc under control of the ERE-mutated hPRL promoter (ERE-Mut) displayed a dramatically reduced transcriptional response to 17β-estradiol (E2) treatment compared with cells expressing Luc from the wild-type (WT) ERE hPRL-Luc promoter (ERE-WT). The -1189 ERE controls not only the response to E2 treatment but also the acute transcriptional response to TNFα, which was abolished in ERE-Mut cells. ERE-WT cells displayed a biphasic transcriptional response after TNFα treatment, the acute phase of which was blocked after treatment with the estrogen receptor antagonist 4-hydroxy-tamoxifen. Unexpectedly, we show the oscillatory characteristics of hPRL promoter activity in individual living cells were unaffected by disruption of this crucial response element, real-time bioluminescence imaging showed that transcription cycles were maintained, with similar cycle lengths, in ERE-WT and ERE-Mut cells. These data suggest the -1189 ERE is the dominant response element involved in the hPRL transcriptional response to both E2 and TNFα and, crucially, that cycles of hPRL promoter activity are independent of estrogen receptor binding.
authors.affiliation: Systems Microscopy Centre (A.V.M., A.D.A., D.G.S., M.R.H.W.), Faculty of Life Sciences, and Faculty of Medical and Human Sciences (L.S.S.D., J.R.E.D.), Centre for Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester M13 9PT, United Kingdom; and The Molecular Physiology Group (S.S., J.J.M.), Centre for Cardiovascular Science, and Medical Research Council Human Reproductive Sciences Unit (A.S.M.), Centre for Reproductive Biology, Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom.
authors.forename: Anne V
authors.initials: AV
authors.lastname: McNamara
authors.affiliation: Systems Microscopy Centre (A.V.M., A.D.A., D.G.S., M.R.H.W.), Faculty of Life Sciences, and Faculty of Medical and Human Sciences (L.S.S.D., J.R.E.D.), Centre for Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester M13 9PT, United Kingdom; and The Molecular Physiology Group (S.S., J.J.M.), Centre for Cardiovascular Science, and Medical Research Council Human Reproductive Sciences Unit (A.S.M.), Centre for Reproductive Biology, Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom.
authors.forename: Antony D
authors.initials: AD
authors.lastname: Adamson
authors.affiliation: Systems Microscopy Centre (A.V.M., A.D.A., D.G.S., M.R.H.W.), Faculty of Life Sciences, and Faculty of Medical and Human Sciences (L.S.S.D., J.R.E.D.), Centre for Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester M13 9PT, United Kingdom; and The Molecular Physiology Group (S.S., J.J.M.), Centre for Cardiovascular Science, and Medical Research Council Human Reproductive Sciences Unit (A.S.M.), Centre for Reproductive Biology, Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom.
authors.forename: Lee S S
authors.initials: LS
authors.lastname: Dunham
authors.affiliation: Systems Microscopy Centre (A.V.M., A.D.A., D.G.S., M.R.H.W.), Faculty of Life Sciences, and Faculty of Medical and Human Sciences (L.S.S.D., J.R.E.D.), Centre for Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester M13 9PT, United Kingdom; and The Molecular Physiology Group (S.S., J.J.M.), Centre for Cardiovascular Science, and Medical Research Council Human Reproductive Sciences Unit (A.S.M.), Centre for Reproductive Biology, Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Semprini
authors.affiliation: Systems Microscopy Centre (A.V.M., A.D.A., D.G.S., M.R.H.W.), Faculty of Life Sciences, and Faculty of Medical and Human Sciences (L.S.S.D., J.R.E.D.), Centre for Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester M13 9PT, United Kingdom; and The Molecular Physiology Group (S.S., J.J.M.), Centre for Cardiovascular Science, and Medical Research Council Human Reproductive Sciences Unit (A.S.M.), Centre for Reproductive Biology, Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom.
authors.forename: David G
authors.initials: DG
authors.lastname: Spiller
authors.affiliation: Systems Microscopy Centre (A.V.M., A.D.A., D.G.S., M.R.H.W.), Faculty of Life Sciences, and Faculty of Medical and Human Sciences (L.S.S.D., J.R.E.D.), Centre for Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester M13 9PT, United Kingdom; and The Molecular Physiology Group (S.S., J.J.M.), Centre for Cardiovascular Science, and Medical Research Council Human Reproductive Sciences Unit (A.S.M.), Centre for Reproductive Biology, Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom.
authors.forename: Alan S
authors.initials: AS
authors.lastname: McNeilly
authors.affiliation: Systems Microscopy Centre (A.V.M., A.D.A., D.G.S., M.R.H.W.), Faculty of Life Sciences, and Faculty of Medical and Human Sciences (L.S.S.D., J.R.E.D.), Centre for Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester M13 9PT, United Kingdom; and The Molecular Physiology Group (S.S., J.J.M.), Centre for Cardiovascular Science, and Medical Research Council Human Reproductive Sciences Unit (A.S.M.), Centre for Reproductive Biology, Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom.
authors.forename: John J
authors.initials: JJ
authors.lastname: Mullins
authors.affiliation: Systems Microscopy Centre (A.V.M., A.D.A., D.G.S., M.R.H.W.), Faculty of Life Sciences, and Faculty of Medical and Human Sciences (L.S.S.D., J.R.E.D.), Centre for Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester M13 9PT, United Kingdom; and The Molecular Physiology Group (S.S., J.J.M.), Centre for Cardiovascular Science, and Medical Research Council Human Reproductive Sciences Unit (A.S.M.), Centre for Reproductive Biology, Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom.
authors.forename: Julian R E
authors.initials: JR
authors.lastname: Davis
authors.affiliation: Systems Microscopy Centre (A.V.M., A.D.A., D.G.S., M.R.H.W.), Faculty of Life Sciences, and Faculty of Medical and Human Sciences (L.S.S.D., J.R.E.D.), Centre for Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester M13 9PT, United Kingdom; and The Molecular Physiology Group (S.S., J.J.M.), Centre for Cardiovascular Science, and Medical Research Council Human Reproductive Sciences Unit (A.S.M.), Centre for Reproductive Biology, Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom.
authors.forename: Michael R H
authors.initials: MR
authors.lastname: White
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004958:Estradiol / Q000494:pharmacology; D004967:Estrogens / Q000235:genetics* / Q000494:pharmacology*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011388:Prolactin / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D011960:Receptors, Estrogen / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D004967:Estrogens; D050505:Mutant Proteins; D011960:Receptors, Estrogen; D014409:Tumor Necrosis Factor-alpha; D004958:Estradiol; D011388:Prolactin; D008156:Luciferases