The H19 locus: regulatory function of an imprinted non coding RNA in embryonic development

Background

Although the H19 gene was discovered in 1984, its precise function is still relatively poorly understood. It is located on mouse chromosome 7, close to the Igf2 (insulin-like growth factor 2) gene, in an imprinted locus. The maternal allele expresses a 2.3kb non coding RNA, as well as a micro RNA, the miR-675. We showed that H19 acts as a transregulator of an imprinted gene network (IGN) involved in growth control of the embryo [1]. A recent study described that the miR-675 is exclusively expressed in the placenta during development, where it controls growth probably by downregulating the expression of Igf1r [2]. However, this micro RNA is unable to control the IGN in the embryo, control that appears therefore to be exerted by the full-length form of H19. The main goal of our work was to understand molecular mechanisms that drive this control of the IGN by H19.

Materials and methods

Loss-of-function (H19^Δ3) and gain-of-function (H19^Tg) mouse mutants were used. H19^Δ3/+;Tg females were crossed with Mus musculus molossinus (JF1) wt males. Polymorphisms between molossinus and domesticus mice allow to distinguish the maternal from the paternal allele. H19^Δ3/+mol, H19^+dom/+mol and H19^-/+mol;Tg E14.5 embryos were collected from this cross in order to dissect limb muscles, or produce primary mouse embryonic fibroblasts (MEF) for ChIP and RIP (RNA immunoprecipitation) experiments.

Results

We first investigated the imprinting status of the IGN and observed that H19 controls by a trans mechanism the imprint of the Igf2 gene, but not of other genes of the IGN. We performed RIP experiments in MEF and identified the MBD1 protein as a partner of the H19 RNA. We also observed that some genes of the IGN, including the Igf2 gene, are overexpressed both in Mbd1^-/- and H19^Δ3/+mol MEF. This suggests that H19 may act on this network because of its interaction with the MBD1 protein. By ChIP experiments, we showed that MBD1 indeed binds to the Igf2 gene and to other common targets of both H19 and MBD1. Interestingly, this binding is lost in H19^Δ3/+mol MEF. This indicates that H19 is necessary for the recruitment of MBD1 to its targets.

Conclusion

These results strongly suggest that the control of the expression of some genes of the IGN, including the Igf2 gene, could be exerted through an interaction of the H19 full-length RNA with the MBD1 protein.