Involvement of DNA methylation in regulating rat Prop1 gene expression during pituitary organogenesis

Abstract

PROP1 is a pituitary specific transcription factor that plays a crucial role in pituitary organogenesis. The Prop1 shows varied expression patterns that promptly emerge and then fade during the early embryonic period. However, the regulatory mechanisms governing Prop1 expression remain unclear. Here, we investigated whether Prop1 was under epigenetic regulation by DNA methylation. Bisulfite sequencing was performed on DNA obtained from the pituitary glands and livers of rats on embryonic days (E) 13.5 and E14.5, and postnatal days (P) 4 and P30. The methylation of CpG sites in seven regions from 3-kb upstream of the Prop1 transcription start site through to its second intron were examined. Certain differences in CpG-methylation levels were observed in Region-1 (-2772 b to -2355 b), Region-4 (-198 b to +286 b), Region-5 (+671 b to +990 b), and Region-6 (+1113 b to +1273 b) based on comparisons between pituitary and liver DNA on E13.5. DNA methylation in pituitary glands on E14.5, P4, and P30 was generally similar to that observed in in the pituitary gland on E13.5, whereas the anterior and intermediate lobes of the pituitary gland on P4 and P30 showed only small differences. These results indicate that Prop1 is under regulation by CpG methylation during the early period of pituitary primordium development around E13.5.

Fig. 1.

Immunohistochemistry and RT-PCR analysis of the rat pituitary gland during development. Immunohistochemical staining was performed on rat pituitary tissues obtained on E13.5 (A, composed of almost all stem/progenitor cells), E14.5 (B, initiating commitment), P4 (C, prior to the postnatal growth wave of the anterior lobe), and P30 (D, after the postnatal growth wave of the anterior lobe), and PROP1 and nuclei were visualized with Cy3 (red) and DAPI (blue), respectively. Dotted lines indicate the outline of Rathke′s pouch (RP) (A and B) and the anterior (AL), intermediated (IL), and posterior (PL) lobes (C and D). The marginal cell layer (MCL) of the anterior lobe, which is a known stem/progenitor niche, is facing the cleft. Scale bars indicate 100 μm. RT, rostral tip. The populations of PROP1-positive cells were counted, and their proportions are indicated as percentages of the total DAPI-stained cells in (E), where n indicates the number of tissue samples analyzed in each case. RT-PCR analysis of Prop1 in the rat pituitary gland and liver during development (F) was performed using RNAs prepared from tissues on E13.5, P4, and P30. RT-PCR products of Prop1 (42 cycles) and Tbp (34 cycles) were analyzed on 3% agarose gels.

Fig. 2.

G/C content, CpG islands, and exons near the 5′ region of the rat Prop1 gene. CpG site frequency as a ratio of observed to expected (A) is mapped across the 5′ region of Prop1, and the average GC content in this region is shown (B). The structure of rat Prop1 (C) is shown with open and closed boxes indicating the untranslated region and the coding regions, respectively. Enlarged region of the rat Prop1 gene (D), with shaded boxes indicating regions that are evolutionarily conserved among several mammals. Vertical lines in (C) and (D) indicate the positions of CpG sites, and the numbers indicate nucleotide positions, with the putative transcription start site indicated at +1 b. Boxed nucleotide numbers in (D) indicate sites that are hypomethylated in pituitary DNA as compared with liver DNA on E13.5, where the difference in methylation is > 30%.

Fig. 3.

Analysis of CpG methylation in the Prop1 region during pituitary development. DNA methylation status of each PCR clone was determined by sodium bisulfite sequencing of genomic DNA samples prepared from rat pituitary glands on E13.5, E14.5, P4, and P30 and rat livers on E13.5, P4, and P30. Anterior and intermediate/posterior lobes were separately collected from postnatal pituitary glands on P4 and P30. The open and closed circles on the grids indicate unmethylated and methylated CpG sites, respectively, and numbers indicate the percentage of the CpG sites methylated in each region. Boxed CpG sites are hypomethylated in E13.5 pituitary glands, DNA samples as compared with E13.5 liver DNA samples, where the difference is > 30%.

Fig. 4.

Comprehensive map of CpG sites and putative transcription factor binding sites in Prop1. Predicted binding sites for pituitary transcription factors (left) [17] and CpG sites within Prop1 are indicated with boxes and vertical dashed lines, respectively. Numbers indicate the positions of the features relative to the Prop1 transcription start site. Closed boxes indicate binding sites within 20 bases of CpG sites that are hypomethylated in the pituitary gland as compared with the liver on E13.5 (vertical solid lines).