title: A Pit-1 Binding Site Adjacent to E-box133 in the Rat PRL Promoter is Necessary for Pulsatile Gene Expression Activity.
abstract: Recent evidence reveals that prolactin gene expression (PRL-GE) in mammotropes occurs in pulses, but the molecular process(es) underlying this phenomenon remains unclear. Earlier, we have identified an E-box (E-box133) in the rat PRL promoter that binds several circadian elements and is critical for this dynamic process. Preliminary analysis revealed a Pit-1 binding site (P2) located immediately adjacent to this E-box133 raising the possibility that some type of functional relationship may exist between these two promoter regions. In this study, using serum shocked GH3 cell culture system to synchronize PRL-GE activity, we determined that Pit-1 gene expression occurred in pulses with time phases similar to that for PRL. Interestingly, EMSA analysis not only confirmed Pit-1 binding to the P2 site, but also revealed an interaction with factor(s) binding to the adjacent E-box133 promoter element. Additionally, down-regulation of Pit-1 by siRNA reduced PRL levels during pulse periods. Thus, using multiple evidences, our results demonstrate clearly that the Pit-1 P2 site is necessary for PRL-GE elaboration. Furthermore, the proximity of this critical Pit-1 binding site (P2) and the E-box133 element coupled with the evidences of a site-to-site protein interactions suggest that the process of PRL-GE pulse activity might involve more dynamic and intricate cross-talks between promoter elements that may span some, or all, of the proximal region of the PRL promoter in driving its pulsatile expression.
authors.affiliation: Amity Institute of Biotechnology, Amity University, Gautam Buddha Nagar, Sector-125, Noida, UP, 201313, India. skbose1@amity.edu.|Laboratory of Molecular Dynamics, Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, 29425, USA. skbose1@amity.edu.
authors.forename: Sudeep
authors.initials: S
authors.lastname: Bose
authors.affiliation: Institute of Molecular Medicine, 254 Okhla Industrial Estate, Phase III, New Delhi, 110020, India.
authors.forename: Surajit
authors.initials: S
authors.lastname: Ganguly
authors.affiliation: Amity Institute of Biotechnology, Amity University, Gautam Buddha Nagar, Sector-125, Noida, UP, 201313, India.
authors.forename: Sachin
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Laboratory of Molecular Dynamics, Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, 29425, USA.
authors.forename: Fredric R
authors.initials: FR
authors.lastname: Boockfor
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D024721:E-Box Elements / Q000502:physiology*; D005786:Gene Expression Regulation; D011388:Prolactin / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology*; D051381:Rats; D050611:Sodium-Phosphate Cotransporter Proteins, Type III / Q000235:genetics / Q000378:metabolism*
chemical_list: C493588:Slc20a1 protein, rat; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D011388:Prolactin
keywords: E-box; Pit-1; Pituitary; Prolactin; Pulses; Transcription