Transcriptional regulation of prolactin in a euryhaline teleost: Characterisation of gene promoters through in silico and transcriptome analyses

Abstract

The sensitivity of prolactin (Prl) cells of the Mozambique tilapia (Oreochromis mossambicus) pituitary to variations in extracellular osmolality enables investigations into how osmoreception underlies patterns of hormone secretion. Through the actions of their main secretory products, Prl cells play a key role in supporting hydromineral balance of fishes by controlling the major osmoregulatory organs (ie, gill, intestine and kidney). The release of Prl from isolated cells of the rostral pars distalis (RPD) occurs in direct response to physiologically relevant reductions in extracellular osmolality. Although the particular signal transduction pathways that link osmotic conditions to Prl secretion have been identified, the processes that underlie hyposmotic induction of prl gene expression remain unknown. In this short review, we describe two distinct tilapia gene loci that encode Prl₁₇₇ and Prl₁₈₈ . From our in silico analyses of prl₁₇₇ and prl₁₈₈ promoter regions (approximately 1000 bp) and a transcriptome analysis of RPDs from fresh water (FW)- and seawater (SW)-acclimated tilapia, we propose a working model for how multiple transcription factors link osmoreceptive processes with adaptive patterns of prl₁₇₇ and prl₁₈₈ gene expression. We confirmed via RNA-sequencing and a quantitative polymerase chain reaction that multiple transcription factors emerging as predicted regulators of prl gene expression are expressed in the RPD of tilapia. In particular, gene transcripts encoding pou1f1, stat3, creb3l1, pbxip1a and stat1a were highly expressed; creb3l1, pbxip1a and stat1a were elevated in fish acclimated to SW vs FW. Combined, our in silico and transcriptome analyses set a path for resolving how adaptive patterns of Prl secretion are achieved via the integration of osmoreceptive processes with the control of prl gene transcription.

Keywords: in silico; osmoreception; prolactin; promoter; salinity; tilapia; transcription factor; transcriptome.

FIGURE 1

Depicting the steps involved in the transduction of a hyposmotic stimulus into prolactin (Prl) release by the tilapia pituitary, modified from Seale et al. Prl cells of the rostral pars distalis (RPD) synthesise and release Prl₁₇₇ and Prl₁₈₈ in response to a fall in extracellular osmolality. Hyposmotic stimulation leads to an Aqp3-dependent increase in cell volume that triggers the entry of Ca²⁺ through stretch-activated Trpv4 channels. Although Ca²⁺ and cAMP secondary messengers mediate Prl release, it is unknown how these intracellular signals participate in the transcriptional regulation of prl₁₇₇ and prl₁₈₈

FIGURE 2

Comparison between predicted transcription factor modules in prl₁₇₇ (orange band) and prl₁₈₈ (green band) promoter regions up to −1.3 kb. Transcription factor-modules (TFMs) represented by white boxes are unique to either prl₁₇₇ or prl₁₈₈; TFMs represented by grey ovals are common to both prl₁₇₇ and prl₁₈₈. TFMs predicted to bind to the (−) strand are indicated by blue text; TFMs predicted to bind to the (+) stand are indicated by red text. Numbers below the promoter regions depict approximate bp positions of putative TFMs and their respective binding sites relative to the transcriptional start site (TSS). Red arrows on each promoter indicate the TSS. TFMs shown in horizontal stacks compete for binding to the same region

FIGURE 3

DNA sequence and regulatory elements of the proximal prl₁₈₈ promoter (−0.88 kb). Nucleotide positions are indicated above the sequence. Both DNA strands are shown for regions −1 to −220 and −551 to −660 bp; only the coding strand is shown for the remainder of the sequence. The transcriptional start site (TSS) is indicated by the red bent arrow; the first exon of the prl₁₈₈ gene is highlighted in yellow and the translation start site is highlighted in green. The dark purple box marks the region (−1 to −40 bp) where general transcription factors (TFs) are predicted to bind. Previously identified DNase protection regions are indicated by grey boxes. The underlined red regions represent microsatellite repeat regions (zDNA). Coloured boxes indicate putative TF-modules (TFMs) and their respective binding sites. TFMs shown above and below the sequence are predicted for the (+) and (−) strands, respectively. The purple box from −142 to −151 bp represents the overlap of the predicted sites for SORY/PAX3 and ETSF/AP1F on the (−) strand. Additional TFMs are colour coded to indicate their corresponding binding sequences

FIGURE 4

Gene expression of pou1f1 (A), stat3 (B), creb3l1 (C), pbxip1a (D) and stat1a (E) in the rostral pars distalis (RPD) of Mozambique tilapia acclimated to fresh water (FW) (solid bars) and seawater (SW) (shaded bars). mRNA levels are presented as a fold-change from the FW group. Data are the mean ± SEM (n = 12). *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t test)