Quantitative Analysis of Mechanisms for Prolactin Gene Transcription Dynamics in Living Pituitary Cells
Presentation Number: LB SUN 34
Date of Presentation: April 2nd, 2017
Julian Richard Davis*1, Anne Victoria McNamara1, Raheela Awais1, Lee Dunham1, Karen Featherstone1, Hiroshi Momiji2, Kirsty Hassall2, Claire Victoria Harper3, David Spiller1, Barbel Finkenstadt2, David Rand2 and Michael R White1
1University of Manchester, Manchester, United Kingdom, 2University of Warwick, 3University of Manchester, United Kingdom
Transcription of many mammalian genes occurs in short (‘on-’) bursts, interspersed with silent (‘off’-) periods. It is important to understand how these processes are regulated. We previously used bacterial artificial chromosomes (BACs) to express reporter genes (firefly luciferase or dEGFP) from a 160kb human prolactin gene construct. The hPRL gene showed noisy ~11h cycles of transcription in pituitary cell lines, primary cells and tissues. Here, we studied these transcriptional bursts using chemical and genetic manipulation, together with mathematical analysis.
We used chemical inhibition to target the bromodomain and extra-terminal domain (BET) family of epigenetic reader proteins. They reversibly inhibit transcriptional activity in individual pituitary cells, suggesting a core epigenetic mechanism. A similar effect was also observed following treatment of cells with the dopamine agonist cabergoline, implying epigenetic involvement in the response to dopamine. Mathematical analysis also suggested that cabergoline reduced the duration of transcription 'on'-periods.
Pit-1 plays a key role in pituitary-specific PRL transcription. We mutated/deleted multiple Pit-1 binding elements in the 5kb proximal promoter region in a hPRL reporter BAC construct that expresses luciferase (PitProKO). Live-cell imaging of stably transfected hPRL PitProKO (GH3) cells, coupled with mathematical analysis, showed that in the absence of Pit-1 binding, the duration of on-periods of active transcription were shorter than those from the wild-type Pit-1-regulated promoter. This was confirmed in single cell imaging experiments following siRNA knockdown of Pit-1 in wild-type cells. This implies a key role for Pit-1 in the changes in chromatin architecture that underlie transcription cycles.
Mathematical analysis of pulsatile promoter activity, using a stochastic switch model, showed that ‘on-switches’ tended to be all-or-nothing, with an immediate switch to a maximum transcription rate. By contrast, off-switches tended to involve a series of steps down in the rate of transcription. These novel data suggest that complex dynamic processes at the hPRL gene are important for accurate dopamine-dependent and pituitary-specific control of hormone gene expression.
Nothing to Disclose: JRD, AVM, RA, LD, KF, HM, KH, CVH, DS, BF, DR, MRW