A model system to study genomic imprinting of human genes

Abstract

Somatic-cell hybrids have been shown to maintain the correct epigenetic chromatin states to study developmental globin gene expression as well as gene expression on the active and inactive X chromosomes. This suggests the potential use of somatic-cell hybrids containing either a maternal or a paternal human chromosome as a model system to study known imprinted genes and to identify as-yet-unknown imprinted genes. Testing gene expression by using reverse transcription followed by PCR, we show that functional imprints are maintained at four previously characterized 15q11-q13 loci in hybrids containing a single human chromosome 15 and at two chromosome 11p15 loci in hybrids containing a single chromosome 11. In contrast, three gamma-aminobutyric acid type A receptor subunit genes in 15q12-q13 are nonimprinted. Furthermore, we have found that differential DNA methylation imprints at the SNRPN promoter and at a CpG island in 11p15 are also maintained in somatic-cell hybrids. Somatic-cell hybrids therefore are a valid and powerful system for studying known imprinted genes as well as for rapidly identifying new imprinted genes.

Figure 1

Imprinted-gene maps of chromosomes 15q11–q13 (a) and 11p15 (b). The gene positions and loci assayed in somatic-cell hybrids (•) are shown. Jagged lines in a represent the common deletion breakpoints in PWS and AS patients. Symbols are: +, gene expression; −, lack of gene expression. PAT, paternal; MAT, maternal; cen, centromere; tel, telomere. The −/+ in a indicates that UBE3A is not expressed from the paternal allele in certain regions of the brain. Figure adapted from refs. and .

Figure 2

DNA methylation analysis in chromosome-15 hybrids. (a) Maintenance of methylation imprints at SNRPN in somatic-cell hybrids. Normal individuals (lane 1) have both a methylated (4.3-kb) allele corresponding to the maternally inherited chromosome and an unmethylated (0.9-kb) allele corresponding to the paternally inherited chromosome. PWS deletion patients (lane 2) lack a paternal contribution, whereas AS deletion patients (lane 3) lack a maternal contribution. Most hybrids show either a completely methylated band, indicating the presence of only a maternal chromosome, or a completely unmethylated band, indicating the presence of only a paternal chromosome. Hybrids with both a methylated and an unmethylated band [15A and 55R-16 (latter data not shown)] were shown to contain both a maternal (mat) and a paternal (pat) human chromosome 15 (see Fig. 4a and Results). Lane 1, normal human lymphoblast; lane 2, lymphoblast from a PWS deletion patient; lane 3, lymphoblast from an AS deletion patient; lanes 4–11 contain DNAs from a subset of the chromosome-15 hybrid panel. (b) Methylation patterns are not maintained at D15S63, with most hybrids being hypomethylated. Hybrid 15A is hypermethylated, despite containing a proportion of cells with a paternally derived chromosome 15. Lane 1, normal human lymphoblast; lanes 2–6 contain DNAs from a subset of the chromosome-15 hybrid panel.

Figure 3

Gene expression in hybrids containing human chromosome 15. Expression of SNRPN (a), IPW (b), PAR5 (c), and NDN (e) as determined by RT-PCR correlates with the SNRPN methylation data. Only those hybrids that are unmethylated at the SNRPN promoter express each of these four transcripts. The GABRA5 (f) and GABRB3 (g) receptor subunit genes are expressed in hybrids containing either a maternal or a paternal chromosome 15. For GABRG3 (data not shown), hybrid A9+15 (containing a paternal chromosome 15) and hybrid 20L-28 (containing a maternal chromosome 15) consistently showed high levels of expression. Additionally, one paternal (A59–3az2 maz) and two maternal (ALA-8, GAR-1) hybrids showed low levels of expression, whereas expression in A15 (paternal) was not detected in multiple experiments. The control gene RPS12 (d) is expressed in all hybrids. PCR was performed with (+) or without (−) reverse transcriptase.

Figure 4

Polymorphism studies of chromosome-15 hybrids. (a) Monoallelic imprinted gene expression in hybrid 55R-16 with biallelic SNRPN DNA methylation. Primers amplify an HphI polymorphism in the IPW gene in exon 3. Digestion of amplified genomic DNA (lane 2) reveals the presence of two alleles in hybrid 55R-16, only one of which is expressed (lane 3). Lane 1 contains the 123-bp ladder size marker. (b) Microsatellite analysis of hybrid cell line GAR-1 and lineage. GAR-1 contains a maternal chromosome 15 from cell line GM04305, in accordance with complete methylation at the SNRPN promoter (see Fig. 2a).

Figure 5

Methylation and gene expression analyses of hybrids containing a single human chromosome 11. (a) DNA methylation imprints are monoallelic at an intronic KVLQT1 intronic NotI site. Normal individuals have both a methylated (6.0-kb) allele corresponding to the maternally inherited chromosome (mat) and an unmethylated allele (1.8-kb) corresponding to the paternally inherited chromosome (pat). Each hybrid shows either the presence of a methylated or an unmethylated chromosome, suggesting that methylation imprints at this site are maintained in somatic-cell hybrids. (b) Each hybrid expressed either the maternal-only H19 or paternal-only IGF2 gene but never both, consistent with maintenance of functional imprints in chromosome 11p15. The primers used for H19 RT-PCR span a small intron, giving a smaller-sized product in cDNA (667 bp) compared with amplification from genomic DNA (24). Although TAPA-1 maps within the imprinted domain in 11p15, it is expressed from both maternal and paternal chromosomes, consistent with knockout studies in the mouse. The control gene, WT1, is expressed in all hybrid cell lines. + and − are as for Fig. 3.