Abstract
Although the H19 gene was discovered in 1984, its precise function is still relatively poorly understood. lt is located on mouse chromosome 7, close to the Jg/2 (insulin-like growth factor 2) gene, in an imprinted locus. The maternal allele expresses a 2.3kb non coding RNA, as weil as a micro RNA, the miR-675. We have shawn that H19 acts as a transregulator of an imprinted gene network (ION) involved in growth control of the embryo. A recent study described that the miR-675 is exclusively expressed in the placent a during development, where it controls growth probably by downregulating the expression of lgflr. However, this micro RNA is unable to control the IGN in the embryo, controlthat appears therefore to be exerted by the full-length form of the H19 lncRNA. The main goal of our work was to understand molecular mechanisms that drive this control of the ION by H19.
We first investigated the imprinting status of the ION and observed that H19 controls by a trans mechanism the imprint of the /g/2 gene, but not that of other genes of the IGN. We performed RIP experiments in MEF and identified the MBDI protein as a partner of the H19 RNA. We also observed that sorne genes of the ION, including the /g/2 gene, are overexpressed both in Mbdr and H19 MEF. This suggests that H19 may act on this network because of its interaction with the MBDI protein. By ChiP experiments, we showed that MBDI indeed binds to the /g/2 gene and to other common targets of both H19 and MBDI. Interestingly, this binding is lost in H19-r. MEF. This indicates that H19 is necessary for the recruitment of MBDI to its targets. This protein is a repressor, involved in the recruitment of H3K9 methyl-transferases. We further observed that H3K9me3 enrichment is indeed strongly decreased in H19 MEFs compare to wt MEFs on the ION targets.
These results strongly suggest that the HI9 long non-coding RNA transcriptionally represses several genes of the IGN, including the /g/2 gene, through its interact ion with the MBDI protein.
