Promoter-restricted histone code, not the differentially methylated DNA regions or antisense transcripts, marks the imprinting status of IGF2R in human and mouse

Thanh H. Vu 0 1 Tao Li 0 1 Andrew R. Hoffman 0 1 0 Palo Alto , CA 94304 , USA 1 Medical Service, Veteran Affairs Palo Alto Health Care System and Department of Medicine, Stanford University Imprinting of the mouse Igf2r depends upon an intronic differentially methylated DNA region (DMR) and the presence of the Air antisense transcript. However, biallelic expression of mouse Igf2r in brain occurs despite the presence of Air, and biallelic expression of human IGF2R in peripheral tissues occurs despite the presence of an intronic DMR. We examined histone modifications throughout the mouse and human Igf2r/ IGF2R using chromatin immuno-precipitation (ChIP) assays in combination with quantitative real time PCR. Methylation of Lys4 and Lys9 of histone H3 in the promoter regions marks the active and silenced alleles, respectively. We measured di- and tri-methyl Lys4 and Lys9 across the Igf2r and Air promoters. While both di- and tri-methyl Lys4 marked the active Igf2r and the active Air allele, tri-methyl Lys9, but not di-methyl Lys9, marked the suppressed Air allele. We show here that enrichment of parental allele-specific histone modifications in the promoter region, rather than the presence of DNA methylation or antisense transcription, correctly identifies the tissue- and species- specific imprinting status of Igf2r/IGF2R. We discuss these findings in light of recent progress in identifying specific components of the epigenetic marks in imprinted genes. - INTRODUCTION Genomic imprinting is a parent-of-origin epigenetic mechanism whereby one of the two parental alleles is preferentially suppressed, while the other parental allele is normally transcribed. In the mouse genome, 38 maternally imprinted (paternally expressed genes, PEG) and 35 paternally imprinted (maternally expressed genes, MEG) genes have been identified to date (http://www.mgu.har.mrc.ac.uk/imprinting/imprinting. html). In imprinted genes, the epigenetic information that is transmitted independently of DNA sequence is conveyed through alterations in nucleosome structure, resulting from covalent modifications of DNA (methylation) and of histones (e.g. acetylation and methylation). Recent studies of the epigenetic marks associated with imprinted genes have revealed that these epigenetic modifications occur with a differential preponderance on the expressed and silenced alleles (1 12). Imprinted genes often cluster on large chromosome regions, forming imprinted domains that are regulated by cis-acting imprinting control regions (reviewed in 13,14). One of the well-characterized imprinted domains contains the gene that encodes the insulin-like growth factor-II (IGF-II) receptor/ mannose-6-phosphate receptor (IGF2R ). The gene is imprinted (maternally expressed) in rodents, marsupials and artiodactyls, but it is biallelically expressed in primates, including humans (15 17). The IGF-II receptor regulates IGF-II, a potent mitogen, by binding it, internalizing it and then transporting it to lysosome for degradation. Loss of function of the IGF-II receptor by mutation in the coding regions and by loss of heterozygosity of IGF2R has been observed in numerous malignancies (18,19). It has been suggested therefore that IGF2R is a tumor suppressor gene (20). The mouse Igf 2r gene encodes two reciprocally imprinted transcripts, each of which is associated with a differentially methylated DNA region (DMR) (21). The first DMR (DMR1) includes the promoter for the sense Igf 2r transcript, whereas DMR2, which is located within the second intron of the gene, includes the promoter for an antisense transcript, Air. The paternally expressed Air RNA suppresses the expression of the sense Igf 2r as well as Slc22a2 and Slc22a3 on the paternal chromosome (22). Deletion or premature termination of Air leads to loss of Igf 2r locus imprinting (22,23). However, Igf 2r expression in the CNS does not appear to be regulated by Air. Although Air is paternally expressed in the CNS as well as in peripheral tissues, Igf 2r sense transcripts are biallelically expressed in brain (24,25). The human IGF2R gene contains a single DMR in intron 2 (26), but no antisense transcripts have been detected and the gene is biallelically expressed in all tissues including Wilms tumors (27). Thus, it appears that although the DNA is marked for imprinting, the putative imprint is never read. To understand this lack of epigenetic readout, we used a chromatin immuno-precipitation (ChIP) assay in combination with quantitative real time (Q)-PCR (Q-PCR) to scan for enrichment of various histone modifications throughout the mouse and human Igf 2r/IGF2R. We have found that the human DMR lacks enrichment of acetylated and methylated histones. The absence of differentially modified histones (DMH) in the human DMR may account for the epigenetic readout failure. We show here that enrichment of parental allele-specific histone modifications in the promoter region, rather than the presence of DNA methylation or antisense transcription, correctly identifies the tissue- and species-specific imprinting status of Igf 2r/IGF2R. Histone acetylation and Lys4 methylation are enriched in the human IGF2R promoter exon and are absent in the DMR We ran triplicate Q-PCR assays (PCR primers shown in Table 1) on ChIP preparations of human embryonic fibroblast cells that maintain the normal imprinting of all tested imprinted genes (28 and unpublished data). We used a panel of antibodies against acetyl lysines (H3 and H4) and methyl lysines (Lys4 and Lys9), as reported previously (11,12). Relative enrichment compared to input chromatin DNA was calibrated with GAPD (measured 0.8 kb downstream of the GAPD transcription site; Fig. 1C, right panel), calculated as previously described (11) and plotted on the same graph (under appropriate scales for comparison) in Figure 1A. We observed a specific and symmetric distribution of acetylated histones (H4-Ac and H3-Ac) across the 3 kb region of the IGF2R promoter exon (Fig. 1A, green lines). The enrichment of acetylated histones near the IGF2R transcription site was often .10-fold when compared with the enrichment of acetylated histones at 1.5 kb upstream or at 2 kb downstream of the transcription site. Low levels of acetylated histones were found in the intronic DMR or in other exons. Methyl Lys4 of histone 3 (H3 K4-Me) was also enriched in the promoter exon and was essentially absent in the DMR (Fig. 1A, blue line), whereas methyl Lys9 (H3 K9-Me) was depleted near the IGF2R transcription site (Fig. 1A, red line). Histone acetylation and H3-Lys4 methylation have been found in transcriptionally active genes (29,30); the absence of these activating histone modifications in the human DMR correlates with the absence of a potential human AIR antisense transcript (27). Histone H3 Lys4 and Lys9 methylation are enriched in mouse Igf2r DMR1 and DMR2 We scanned the distribution of methyl Lys4 and methyl Lys9 of histone H3 across the mouse Igf 2r in cul (...truncated)