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

Anne V Mcnamara,John J Mullins,Nicholas A Jones,David Rand,Michael R H White,Julian R E Davis,Raheela Awais,Karen Featherstone,David G Spiller,Bärbel F Finkenstädt,Hiroshi Momiji,Antony A Adamson,Lee Dunham,Sabrina Semprini,Claire V Harper

doi:10.1210/endocr/bqaa249

Abstract

Gene transcription occurs in short bursts interspersed with silent periods, and these kinetics can be altered by promoter structure. The effect of alternate promoter architecture on transcription bursting is not known. We studied the human prolactin (hPRL) gene that contains 2 promoters, a pituitary-specific promoter that requires the transcription factor Pit-1 and displays dramatic transcriptional bursting activity and an alternate upstream promoter that is active in nonpituitary tissues. We studied large hPRL genomic fragments with luciferase reporters, and used bacterial artificial chromosome recombineering to manipulate critical promoter regions. Stochastic switch mathematical modelling of single-cell time-lapse luminescence image data revealed that the Pit-1–dependent promoter showed longer, higher-amplitude transcriptional bursts. Knockdown studies confirmed that the presence of Pit-1 stabilized and prolonged periods of active transcription. Pit-1 therefore plays an active role in establishing the timing of transcription cycles, in addition to its cell-specific functions.

Highlights

Gene transcription occurs in short bursts interspersed with silent periods, and these kinetics can be altered by promoter structure
We studied the human prolactin gene that contains 2 promoters, a pituitary-specific promoter that requires the transcription factor Pit-1 and displays dramatic transcriptional bursting activity and an alternate upstream promoter that is active in nonpituitary tissues
Pulsatile cycles of prolactin transcription have previously been shown in single cells from pituitary cell lines and in primary pituitary cells [17,18,19,20,29,34,35,36,37]. human prolactin (hPRL) promoter directed reporter gene expression has been observed in primary cultures of bone marrow myeloid cells, where it is driven by the alternative promoter

Summary

Introduction

Gene transcription occurs in short bursts interspersed with silent periods, and these kinetics can be altered by promoter structure. Prolactin is a multifunctional mammalian hormone with a major role in lactation as well as other biological functions, including reproduction, immunomodulation and behavior [1,2] This polypeptide hormone mainly originates in the lactotroph cells of the anterior pituitary gland but is expressed at extra-pituitary sites, such as brain decidualized endometrium, myometrium, and circulating lymphocytes, and requires tissue-specific transcriptional control mechanisms to regulate its functional versatility [1,2,3]. In lymphocytes and human endometrial cells, hPRL expression has been shown to be driven by an alternative promoter, upstream of the pituitary transcriptional start site [7] This evolved from a long terminal repeat-like transposon, resulting in transcription of the additional exon 1a, giving rise to messenger RNA (mRNA) that is 150 bp longer than the pituitary mRNA [2,8]. It has been important to develop strategies for studying the transcription control function of the complete genomic region

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Conclusion