IUGR differentially alters MeCP2 expression and H3K9Me3 of the PPARγ gene in male and female rat lungs during alveolarization

Abstract

Intrauterine growth restriction (IUGR) increases the risk of postnatal lung disease, with males more affected. In rat lungs, IUGR impairs alveolarization in conjunction with altered expression of peroxisome proliferator-activated receptor gamma (PPARγ). In non-lung cells, PPARγ transcription is regulated in part by the epigenetic modifying enzyme, and the methyl CpG binding protein 2 (MeCP2). However, it is unknown if IUGR affects MeCP2 expression or its interaction with PPARγ in the rat lung during alveolarization. In this study, we hypothesized that the rat lung would be characterized by the presence of MeCP2 short and long mRNA transcripts, MeCP2 protein isoforms, and the MeCP2 regulatory micro RNA, miR132. We further hypothesized that IUGR would, in a gender-specific manner, alter the levels of MeCP2 components in association with changes in PPARγ mRNA, MeCP2 occupancy at the PPARγ promoters, and PPARγ histone 3 lysine 9 trimethylation (H3K9Me3). To test these hypotheses, we used a well-characterized rat model of uteroplacental insufficiency-induced IUGR. We demonstrated the presence of MeCP2 mRNA, protein, and miR132 in the rat lung throughout alveolarization. We also demonstrated that IUGR alters MeCP2 expression and its interaction with PPARγ in a gender-divergent manner. We conclude that IUGR induces gender-specific alterations in the epigenetic milieu in the rat lung. We speculate that in the IUGR rat lung, this altered epigenetic milieu may predispose to gender-specific alterations in alveolarization.

Figure 1

Ontogeny of MeCP2 mRNA and miR132 in rat lung at birth (d0), postnatal day 7 (d7) and day 21 (d21). Male (panel a and c) and female (panel b and d) lungs were examined. Differences in MeCP2 mRNA and miR132 between d0, d7 and d21, for each gender, were assessed with ANOVA with Fisher’s PLSD. Significant difference to d0 indicated by *, significant difference to d7 indicated by #. n=6.

Figure 2

Ontogeny of MeCP2 protein isoforms in rat lung at birth (d0), postnatal day 7 (d7) and day 21 (d21). Male (panel a) and female (panel b) lungs were examined. Inserts are representative western blots. Differences in MeCP2 protein between d0, d7 and d21, for each gender, were assessed with ANOVA with Fisher’s PLSD. Significant difference to d0 indicated by *, significant difference to d7 indicated by #. n=6.

Figure 3

IUGR alters MeCP2 mRNA and miR132 levels in a gender-specific manner in rat lung. Male (panel a and c) and female (panel b and d) control and IUGR lungs were examined at birth (d0), postnatal day 7 (d7) and day 21 (d21). Data are IUGR as a % of age and gender matched control, error bars are standard deviation. Asterisks denote significant differences between IUGR and gender-matched control determined by unpaired t-test. n=6.

Figure 4

IUGR alters MeCP2 protein isoform levels in a gender-specific manner in rat lung. Male (panel a) and female (panel b) control and IUGR lungs were examined at birth (d0), postnatal day 7 (d7) and day 21 (d21). Data are IUGR as a % of age and gender matched control, error bars are standard deviation. Asterisks denote significant differences between IUGR and gender-matched control determined by unpaired t-test. n=6.

Figure 5

IUGR decreases PPARγ variant mRNA levels at birth at d7 in rat lung. Male (panel a) and female (panel b) control and IUGR lungs were examined at birth (d0), postnatal day 7 (d7) and day 21 (d21). Data are IUGR as a % of age and gender matched control, error bars are standard deviation. Asterisks denote significant differences between IUGR and gender-matched control determined by unpaired t-test. n=6.

Figure 6

IUGR increases MeCP2 occupancy at PPARγ promoter 1 (P1) and promoter 2 (P2) in d0 rat lung. Male (panel a) and female (panel b) control and IUGR lungs were examined at birth (d0), postnatal day 7 (d7) and day 21 (d21). MeCP2 occupancy at the PPARγ promoters was not detectable at d21 in male or female lung. Data are IUGR as a % of age and gender matched control, error bars are standard deviation. Asterisks denote significant differences between IUGR and gender-matched control determined by unpaired t-test. n=6.

Figure 7

IUGR alters levels of H3K9Me3 along the PPARγ gene in a gender-specific manner. Male (panel a) and female (panel b) control and IUGR lungs were examined at birth (d0), postnatal day 7 (d7) and day 21 (d21). Positions examined were: 5′P1 (5′ of promoter 1), at P1, at promoter 2 (P2), in Exon 4 and at the 3′ end of the PPARγ gene. Data are IUGR as a % of age and gender matched control, error bars are standard deviation. Asterisks denote significant differences between IUGR and gender-matched control determined by unpaired t-test. n=6.