Calcium dynamics and chromatin remodelling underlie heterogeneity in prolactin transcription.

Claire V Harper,Julian R E Davis,David G Spiller,Jayne C Charnock,Anne V Mcnamara,Michael R H White

doi:10.1530/jme-20-0223

Claire V Harper, Julian R E Davis + Show 4 more

Open Access

https://doi.org/10.1530/jme-20-0223

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Abstract

Pituitary cells have been reported to show spontaneous calcium oscillations and dynamic transcription cycles. To study both processes in the same living cell in real time, we used rat pituitary GH3 cells stably expressing human prolactin-luciferase or prolactin-EGFP reporter gene constructs loaded with a fluorescent calcium indicator and measured activity using single-cell time-lapse microscopy. We observed heterogeneity between clonal cells in the calcium activity and prolactin transcription in unstimulated conditions. There was a significant correlation between cells displaying spontaneous calcium spikes and cells showing spontaneous bursts in prolactin expression. Notably, cells showing no basal calcium activity showed low prolactin expression but elicited a significantly greater transcriptional response to BayK8644 compared to cells showing basal calcium activity. This suggested the presence of two subsets of cells within the population at any one time. Fluorescence-activated cell sorting was used to sort cells into two populations based on the expression level of prolactin-EGFP however, the bimodal pattern of expression was restored within 26 h. Chromatin immunoprecipitation showed that these sorted populations were distinct due to the extent of histone acetylation. We suggest that maintenance of a heterogeneous bimodal population is a fundamental characteristic of this cell type and that calcium activation and histone acetylation, at least in part, drive prolactin transcriptional competence.

Highlights

In previous work using luminescent and fluorescent microscopy of reporter gene constructs we described the evidence of clearly defined prolactin transcription cycles in single cells, occurring approximately every 11–12 h (Harper et al 2011)
We show that within a clonal population of resting GH3 cells there is variability in the extent of prolactin expression, calcium dynamics and histone acetylation
The resting calcium dynamics appear to determine the transcriptional competence of the cell

Summary

Introduction

It is widely reported the that transcription of genes is not a static process and can occur in rapid bursts (with secondminute timescales (Ozbudak et al 2002, Blake et al 2003, Raser & O’Shea 2004, Golding et al 2005, Raj et al 2006, Yu et al 2006, Harper et al 2011, Fujita et al 2016)) or longer cycles (with minute–hour timescales (Wijgerde et al 1995, Zenklusen et al 2008, Degenhardt et al 2009, Harper et al 2011, Suter et al 2011, Molina et al 2013)). This supports other studies that suggest that transcription bursts/cycles can be regulated by defined periods of histone modification (Blake et al 2003, Metivier et al 2003, Raser & O’Shea 2004, Metivier et al 2006, Raj et al 2006, Kangaspeska et al 2008)

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