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.

Transcription Factor Pit-1 Affects Transcriptional Timing in the Dual-Promoter Human Prolactin Gene

“…Induction was markedly greater in hPRL-WT cells where both promoters were present (Supplemental Figure S2A ( 39) ). The PitProKO promoter was nonresponsive to both estradiol (E2) and tumor necrosis alpha treatment (Supplemental Figure S2B ( 39) ), supporting our previous studies demonstrating that the hPRL transcriptional response to these stimuli is mediated by an estrogen response element in the proximal promoter (−1189 bp from the transcription start site), which is not present in the PitProKO construct ( 17 , 40 ).…”

“…Pulsatile cycles of prolactin transcription have previously been shown in single cells from pituitary cell lines and in primary pituitary cells ( 17-20 , 29 ,34- 37 ). hPRL promoter directed reporter gene expression has also been observed in primary cultures of bone marrow myeloid cells (taken from transgenic rats expressing Luc under the control of the hPRL WT BAC), where it is driven by the alternative promoter.…”

“…We have previously shown the pulsatile nature of hPRL transcription in pituitary cells using both 5 kb and BAC constructs in pituitary cell lines and in normal pituitary tissue in transgenic rats ( 17 , 20 , 25 , 27-32 ). The recombinant BAC constructs allowed us to test whether this bursting transcriptional behavior was specific to activity directed by the proximal promoter and whether Pit-1 binding elements were required.…”

“…It has therefore been important to develop strategies for studying the transcription control function of the complete genomic region. bacterial artificial chromosome (BAC)-based reporter systems permit the inclusion of far-distant regulatory elements and can prove particularly beneficial in analyzing function of promoters that comprise a complex array of cis-acting regulatory elements ( 7 , 17 , 18 ). We previously generated a BAC reporter construct that spanned 163 Kb of the hPRL genomic locus including 115 kb upstream and 38 kb downstream of the PRL gene and expressed firefly luciferase (Luc) under the control of the entire hPRL gene [referred to as hPRL wild-type (WT) BAC] ( 18 ).…”

Asymmetry between Activation and Deactivation during a Transcriptional Pulse

“…High rates of transcriptional activity lasted for the shortest periods of time, while low rates of transcription could be maintained for longer. The continuous distribution of rates as modeled by the SSM approach supported the view that rather than this being a discrete binary on/off system, transcription occurs along a spectrum of possible rates ( Molina et al., 2013 , Zhang and Zhou, 2014 , Featherstone et al., 2016 , Corrigan et al., 2016 , McNamara et al., 2016 ).…”