John J. Mullins

Dynamic Analysis of Stochastic Transcription Cycles

Cycling of gene expression in individual cells is controlled by dynamic chromatin remodeling.

Real-Time Visualization of Human Prolactin Alternate Promoter Usage in Vivo Using a Double-Transgenic Rat Model

We have generated a humanized double-reporter transgenic rat for whole-body in vivo imaging of endocrine gene expression, using the human prolactin (PRL) gene locus as a physiologically important endocrine model system. The approach combines the advantages of bacterial artificial chromosome recombineering to report appropriate regulation of gene expression by distant elements, with double reporter activity for the study of highly dynamic promoter regulation in vivo and ex vivo. We show first that this rat transgenic model allows quantitative in vivo imaging of gene expression in the pituitary gland, allowing the study of pulsatile dynamic activity of the PRL promoter in normal endocrine cells in different physiological states. Using the dual reporters in combination, dramatic and unexpected changes in PRL expression were observed after inflammatory challenge. Expression of PRL was shown by RT-PCR to be driven by activation of the alternative upstream extrapituitary promoter and flow cytometry analysis pointed at diverse immune cells expressing the reporter gene. These studies demonstrate the effective use of this type of model for molecular physiology and illustrate the potential for providing novel insight into human gene expression using a heterologous system.

Peritonitis Activates Transcription of the Human Prolactin Locus in Myeloid Cells in a Humanized Transgenic Rat Model

Prolactin (PRL) is mainly expressed in the pituitary in rodents, whereas in humans, expression is observed in many extrapituitary sites, including lymphocytes. Due to the lack of adequate experimental models, the function of locally produced PRL in the immune system is largely unknown. Using transgenic rats that express luciferase under the control of extensive human PRL regulatory regions, we characterized immune cell responses to thioglycollate (TG)-induced peritonitis. Resident populations of myeloid cells in the peritoneal cavity of untreated rats expressed barely detectable levels of luciferase. In contrast, during TG-induced peritonitis, cell-specific expression in both neutrophils and monocytes/macrophages in peritoneal exudates increased dramatically. Elevated luciferase expression was also detectable in peripheral blood and bone marrow CD11b(+) cells. Ex vivo stimulation of primary myeloid cells showed activation of the human extrapituitary promoter by TNF-α, lipopolysaccharide, or TG. These findings were confirmed in human peripheral blood monocytes, showing that the transgenic rat provided a faithful model for the human gene. Thus, the resolution of an inflammatory response is associated with dramatic activation of the PRL gene promoter in the myeloid lineage.

Role of Estrogen Response Element in the Human Prolactin Gene: Transcriptional Response and Timing.

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.