A mouse N-ethyl-N-nitrosourea (ENU) mutagenesis screen was established to identify genes involved in the establishment and maintenance of epigenetic state in mammals. Mice used in this study carry a multi-copy green fluorescent protein (GFP) transgene array linked to an erythroid specific α-globin promoter and a HS40 enhancer. Transgenes have been shown to undergo silencing via epigenetic mechanisms, resulting in variegated expression of GFP in the red blood cells. The mouse mutants produced from the screen are a powerful tool to dissect the role of epigenetics in both development and disease.
The majority of the mutant lines produced from the screen show homozygous embryonic lethality, consistent with the important role of epigenetics during development. A number of epigenetic modifiers have been reported to play key roles in cardiac maturation and differentiation, including mutant lines from the screen e.g. Baz1b and Pbrm1. Here we describe heart defects in mouse mutants of Rlf, a novel epigenetic modifier.
Three mouse lines with mutations in Rlf MommeD8, MommeD28 and MommeD34, were each found to have a decrease in GFP expression suggesting Rlf is an epigenetic modifier. Homozygous perinatal embryonic lethality was observed in Rlf null lines. Histological analysis of hearts from homozygous MommeD28 mid-gestation embryos showed thinning of the heart wall. This morphology was also observed in MommeD34 homozygousembryos. Wild-type and mutant placentas were examined to ask whether the heart defect may be secondary to an underlying placental defect. No effect of loss of Rlf was observed in total weight or basic histology.
RNA-seq analysis of mid-gestation MommeD28 fetal liver revealed a number of significantly differentially expressed genes. Ingenuity Pathway Analysis identified many of these genes as being implicated in cardiovascular system development and function. Further work is being undertaken to examine the role of Rlf in regulation of gene expression in the heart and heart development.