Calcium dynamics and chromatin remodelling underlie heterogeneity in prolactin transcription

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.

Keywords: calcium; chromatin; heterogeneity; prolactin; single cell; transcription.

Figure 1

Temporal heterogeneity in prolactin transcription and calcium profiles between pituitary cells. (A and B) GH3 cells stably expressing a 5kb prolactin-luciferase reporter gene (GH3/prolactin-luc cells) show 2 transcription patterns in unstimulated conditions; low and high (see Methods for classification), measured using time-lapse luminescence imaging. Each line represents a single cell, thick black line is experiment average. (C and D) GH3/prolactin-luc cells loaded with Fluo-4 show both inactive and active calcium patterns in unstimulated conditions measured using time-lapse fluorescence imaging. Each line represents a single cell. Scatter plots show the proportion of cells defined by each category in unstimulated conditions where each point represents a single experiment (B and D right panels). Bars in image series represent 50 µm.

Figure 2

Relationship between calcium patterns and prolactin transcription in pituitary cells in unstimulated conditions. (A and B) Resting calcium profiles and prolactin transcription were measured sequentially in the same cells. (B) Representative cells showing inactive and active calcium patterns and their subsequent transcriptional patterns. Right panels show mean prolactin transcriptional activity from all cells within an experiment that show inactive or active calcium ± s.d.. (C) Scatter plot shows the proportion of cells exhibiting low or high prolactin transcription following active or inactive calcium profiles (six experiments, 91 cells; P < 0.01) where each point represents a single experiment. Bar in image represents 20 µm.

Figure 3

Relationship between calcium patterns and prolactin transcription in pituitary cells in stimulated conditions. (A and B) Calcium profiles and subsequent prolactin transcriptional response patterns following treatment with 0.5 µM BayK8644. The calcium and transcriptional responses to 0.5 µM BayK8644 were measured in cells that showed initial (pre-stimulus) active (A) or inactive (B) resting calcium profiles. Red gene expression traces show a response and black traces show no response to the stimulus (see methods for classification). (C) Mean single cell transcriptional response patterns from cells showing initial active or inactive calcium profiles. Points show mean ± s.d. (D) The proportion of cells showing transcriptional response to stimulus following initial active or inactive calcium profiles, mean ± s.d. (five experiments, 77 cells, P < 0.01) where each point represents a single experiment.

Figure 4

Maintenance of heterogeneity between clonal cells. (A) Model showing protocol. GH3 cells stably expressing a 5 kb prolactin-destabilised EGFP reporter gene (GH3-DP1 cells) were sorted for basal prolactin expression level using FACS. The fluorescence of these sorted cell populations was then measured after 1 h and 26 h. (B) Variation in basal prolactin gene expression in clonal GH3-DP1 cells (green trace) compared to the WT GH3 cell line (black trace). Measurement of fluorescence levels in High (blue trace) and Low (red trace) expressing GH3-DP1 cells following FACS after 1 h (C) and 26 h (D). Data from one representative experiment are shown. (E) Table showing the proportion of cells ± s.d. classified as High or Low prolactin expression 1 h and 26 h post-FACS in GH3 cells (control), unsorted cells, low expressing cell population and high expressing cell population (three experiments).

Figure 5

Relationship between level of prolactin transcription and chromatin status at the prolactin promoter. (A) Location of target sites for amplification within the proximal prolactin promoter (designated P1, P2 and P3). GH3-DP1 cells expressing prolactin-eGFP were sorted by level of basal prolactin transcription using FACs (Fig. 4). Cells were classified as unsorted (Un), low transcription (Low) and high transcription (High). (B and C) The level of Acetylated histone H3 was measured using ChIP across the three amplification sites (two experiments, mean ± s.d.).

Figure 6

Schematic showing cellular heterogeneity in single pituitary cells and pituitary cells within a tissue. (A) Relationship between calcium profile, prolactin transcription and chromatin status in single pituitary cells. (B, top panel) In basal conditions a subset of cells within pituitary tissue is expressing prolactin at any one time, resulting in low, chronic basal expression of prolactin across the tissue. (B, bottom panel) In stimulated conditions, the cells showing low prolactin transcription within the tissue respond to the stimulus, mounting an acute surge of prolactin expression.