Genomic Imprinting Controls Matrix Attachment Regionsin the Igf2Gene

Abstract

Genomic imprinting at the Igf2/H19 locus originates from allele-specific DNA methylation, which modifies the affinity of some proteins for their target sequences. Here, we show that AT-rich DNA sequences located in the vicinity of previously characterized differentially methylated regions (DMRs) of the imprinted Igf2 gene are conserved between mouse and human. These sequences have all the characteristics of matrix attachment regions (MARs), which are known as versatile regulatory elements involved in chromatin structure and gene expression. Combining allele-specific nuclear matrix binding assays and real-time PCR quantification, we show that retention of two of these Igf2 MARs (MAR0 and MAR2) in the nuclear matrix fraction depends on the tissue and is specific to the paternal allele. Furthermore, on this allele, the Igf2 MAR2 is functionally linked to the neighboring DMR2 while, on the maternal allele, it is controlled by the imprinting-control region. Our work clearly demonstrates that genomic imprinting controls matrix attachment regions in the Igf2 gene.On the distal part of mouse chromosome 7, the Igf2 and H19 genes are separated by 72 kbp. At this locus, imprinting originates from paternal germ line-specific methylation that impairs the binding of a protein factor (CTCF) to the intergenic imprinting-control region (ICR). On the maternal allele, binding of CTCF results in activation of an insulator, which prevents Igf2 expression by blocking promoter access to a set of enhancers located downstream of H19 (2, 14). Thus, Igf2 gene expression is restricted to the paternal allele, whereas H19 is maternally expressed. However, Igf2 imprinting also involves additional differentially methylated regions (DMR0, DMR1, and DMR2) that control regional epigenetic modifications in a hierarchical and tissue-specific fashion (19). DMR1, located upstream of the fetal Igf2 promoters, is preferentially methylated on the paternal allele and acts as a methylation-sensitive silencer in several mesodermal tissues (5). DMR2 maps to exons 5 to 6 and augments transcription on the methylated paternal allele (24). Finally, DMR0 is maternally methylated in the placenta and overlaps the placenta-specific P0 promoter (23). All these DMRs display tissue-specific methylation patterns, and we have recently proposed that their methylation profiles are regionally coordinated through long-range chromatin interactions (19).Here, we identify intragenic AT-rich DNA sequences in the Igf2 gene that are contiguous to previously identified DMRs. These sequences have all the characteristics of matrix attachment regions (MARs) and are conserved in humans. MARs are operationally defined in MAR assays by their ability to associate to a nuclear matrix or scaffold (17) and have been implicated in the regulation of chromatin structure and gene expression. They are frequently associated with enhancers (4, 10, 11) and promote chromatin accessibility and histone acetylation (7,9,15,20,26). MARs may also sometimes act as boundaries, preventing the spreading of p...

Genomic Matrix Attachment Region and Chromosome Conformation Capture Quantitative Real Time PCR Assays Identify Novel Putative Regulatory Elements at the Imprinted Dlk1/Gtl2 Locus

“…Furthermore, it becomes increasingly clear that A/T-rich sequences and factors binding such regions play important roles in tissue-specific higher order organization (22). Therefore, MARs, as other HRS, appear as versatile regulatory elements that could combine with differentially methylated regions and other imprinting control regions to confer cell-specific higher order chromatin organization and to control monoallelic expression (3,23). As proposed previously, it may well be that such higher order chromatin architecture corresponds to a real "genome format" that specifies the transcriptional status of the genes in a tissue-and developmental stage-specific manner (24).…”

“…The genomic MAR assay, which we propose to rename the "high salt recovered sequence assay" (HRS assays: see "Discussion"), was performed as previously described (3). Briefly, the nuclei isolated from tissue samples were extracted in high salt conditions (2 M NaCl), and the resulting "nuclear halos " were digested by XbaI, HindIII, and BamHI.…”

“…Amplifications were performed as previously described (3), and the enrichments were calculated as the ratio of the amount of target DNA in the MAR fraction versus the loop fraction. To standardize each assay, the ratio values were normalized against the ratio obtained for a negative control (NC) located 41 kb upstream of the Gtl2 gene and lacking predicted MAR features (see Fig.…”

“…Using a "genomic MAR" assay based on high salt treatment of purified nucleus preparations (2, 3), we previously showed that parental genomic imprinting controls MAR activities at the mouse Igf2 (insulin-like growth factor 2) locus. That work indicated that tissue-specific MARs and differentially methylated regions (DMRs) may act as bipartite elements controlling the long range activity of other regulatory elements such as enhancers (3), thus contributing to the regulation of gene expression. Here, we investigate MAR activities at the Dlk1/Gtl2 imprinted domain located on mouse chromosome 12 (human chromosome 14).…”

“…In this context, it becomes crucial to elucidate the genomic architecture associated with imprinted genes as well as to identify DNA sequences and factors involved in such higher order chromatin organization. In the present work, we examine the potential role of the so-called matrix attachment regions (MARs) 3 in imprinting and gene regulation. MARs have been operationally defined in "in vitro MAR assays" by their ability to "attach" to a purified nuclear matrix or scaffold.…”