Deletion of a silencer element in Igf2 results in loss of imprinting independent of H19

Abstract

Igf2 and H19 are closely linked, reciprocally imprinted genes on mouse distal chromosome 7. The paternally expressed Igf2 encodes a potent fetal growth factor and the maternally expressed H19 encodes a non-coding RNA (refs 1,2). Shared endoderm-specific enhancers 3' to H19 are necessary for transcription of the maternal copy of H19 and the paternal copy of Igf2 (ref. 3), a chromatin boundary upstream of H19 preventing access of the enhancers to the maternal Igf2 promoters. Mesoderm-specific control elements have not been identified, and the role of differentially methylated regions (DMRs) in Igf2 has not been addressed. Two DMRs in Igf2 are methylated on the active paternal allele, suggesting that they contain silencers. Here we have deleted the DMR1 region in Igf2. Maternal transmission of the deletion results in biallelic expression of Igf2 in most mesodermally derived tissues without altering H19 imprinting or expression. Paternal or maternal transmission leads to continued postnatal transcription of Igf2, in contrast to the wild-type allele, which is silenced soon after birth. These results reveal a mesodermal silencer, which may be regulated by methylation and which has a major role in H19-independent expression and imprinting control of Igf2. Our results establish a new mechanistic principle for imprinted genes whereby epigenetically regulated silencers interact with enhancers to control expression, and suggest a new mechanism for loss of imprinting (LOI) of Igf2, which may be important in a number of diseases.

CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2

“…5) is exclusively based on neonatal liver, the major Igf2 expressing tissue in which the endoderm enhancers are active. Deletion or point mutation of DMR1 in liver reactivates the maternal Igf2 allele only marginally (18), whereas deletion of DMR1 in mesodermal tissues results in substantial reactivation (19). These observations suggest that there may be considerable differences of higher-order chromatin organization between embryonic lineages and tissues.…”

“…The mechanism underlying this function has been proposed to involve a CTCF-dependent chromatin insulator located within the ICR (13)(14)(15)(16), which is continuously required for Igf2 repression in somatic cells (17). However, the Igf2 gene itself also contains differentially methylated regions (DMRs), with DMR1 being a methylation-sensitive silencer (18,19) and DMR2 being a methylation sensitive activator (20). Additional enhancer sequences located 5Ј of the ICR (21,22) suggest that a simple insulator function of the ICR is not sufficient to explain all aspects of maternal silencing of Igf2.…”

Placental-specific Igf2 knockout mice exhibit hypocalcemia and adaptive changes in placental calcium transport

“…Animals (Scientific Procedures) Act of 1986. Placental specific Igf2 (Delta U2 P0) knockout mice, which had deletion of the U2 exon of the Igf2 gene, were generated as previously described (44) and were a kind gift from W. Reik (Babraham Institute, Cambridge, UK).…”

Reprogramming of genome function through epigenetic inheritance

“…These imprinting control regions are often complex with multiple functions acting to repress genes when methylated, or serving as boundary elements when unmethylated (the boundary element 21,22 indirectly affects expression of neighbouring genes). Some DMRs also function as silencer elements when unmethylated 23 , a function that is apparently abolished when the DMR is methylated. In other instances, a DMR is associated with the expression of an antisense transcript whose expression in turn ensures repression of the upstream gene 24 .…”