DLX5 and DLX6 expression is biallelic and not modulated by MeCP2 deficiency

Abstract

Mutations in MECP2 and Mecp2 (encoding methyl-CpG binding protein 2 [MeCP2]) cause distinct neurological phenotypes in humans and mice, respectively, but the molecular pathology is unclear. Recent literature claimed that the developmental homeobox gene DLX5 is imprinted and that its imprinting status is modulated by MeCP2, leading to biallelic expression in Rett syndrome and twofold overexpression of Dlx5 and Dlx6 in Mecp2-null mice. The conclusion that DLX5 is a direct target of MeCP2 has implications for research on the molecular bases of Rett syndrome, autism, and genomic imprinting. Attempting to replicate the reported data, we evaluated allele-specific expression of DLX5 and DLX6 in mouse x human somatic cell hybrids, lymphoblastoid cell lines, and frontal cortex from controls and individuals with MECP2 mutations. We identified novel single-nucleotide polymorphisms in DLX5 and DLX6, enabling the first imprinting studies of DLX6. We found that DLX5 and DLX6 are biallelically expressed in somatic cell hybrids and in human cell lines and brain, with no differences between affected and control samples. We also determined expression levels of Dlx5 and Dlx6 in forebrain from seven male Mecp2-mutant mice and eight wild-type littermates by real-time quantitative reverse-transcriptase polymerase chain reaction assays. Expression of Dlx5 and Dlx6, as well as of the imprinted gene Peg3, in mouse forebrain was highly variable, with no consistent differences between Mecp2-null mutants and controls. We conclude that DLX5 and DLX6 are not imprinted in humans and are not likely to be direct targets of MeCP2 modulation. In contrast, the imprinting status of PEG3 and PEG10 is maintained in MeCP2-deficient tissues. Our results confirm that MeCP2 plays no role in the maintenance of genomic imprinting and add PEG3 and PEG10 to the list of studied imprinted genes.

Figure 1.

Absence of imprinting of DLX5 and DLX6 in SCH lines. RT-PCR analysis of human DLX5 and DLX6 expression in mouse × human SCHs containing a single copy of human chromosome 7 shows that both genes are expressed from the maternally (M) as well as the paternally (P) derived chromosome 7. One SCH line in set 1 had apparently lost the maternal chromosome 7, and one line in set 2 had retained the maternal chromosome 7 at a low copy number. Primers used do not amplify the mouse (Mus) orthologues in C57BL/6 cortex. Mouse Snca expression is shown as a loading control. NTC = no template control; -RT= first-strand synthesis without reverse transcriptase.

Figure 2.

Absence of imprinting of DLX5 in LCLs. A, Biallelic expression revealed by DLX5 genotyping (of gDNA) and expression analysis (of cDNA) of LCLs from three females with RTT and common MECP2 mutations, as indicated, and three normal controls. All six LCLs are heterozygous for the DLX5 c.*163dupC polymorphism (rs5886002) (left panel), and all show that both alleles are expressed (right panel). The sequence shown is the sense strand. Arrows indicate the positions of nucleotide changes. B, Novel DLX5 SNP c.*142T→C detected in a normal LCL, denoted by an arrow in the gDNA. Comparison with the reference sequence (Ref. seq. [GenBank accession number NM_005221.5) shows that the SNP is located upstream of rs5886002. LCLs from control 4 are heterozygous for both SNPs, and both alleles are expressed in cDNA.

Figure 3.

Absence of imprinting of DLX5 in frontal cortex. DLX5 genotyping (of gDNA) and expression analysis (of cDNA) of frontal cortex brain samples from unaffected controls revealed biallelic expression. All six donors (age and sex indicated) were heterozygous for the c.*163dupC (rs5886002) polymorphism (left panel). In all brain samples, both alleles are expressed at a similar level (right panel). Arrows indicate the positions of nucleotide changes.

Figure 4.

Absence of imprinting of DLX6 in fetal brain. DLX6 genotyping (of gDNA) and expression analysis (of cDNA) in two fetal brain samples revealed biallelic expression. The 21-wk fetus (left panel) was heterozygous for the known c.*9A→G SNP (rs3213654). The 17-wk fetus (right panel) was heterozygous for a novel SNP, c.*775dupC. The upper trace displays the reference sequence, GenBank accession number NM_005222.2. Arrows indicate the positions of nucleotide changes.

Figure 5.

Imprinting of PEG3 and PEG10 in MECP2 mutants. A, Maintenance of imprinting of PEG3 in cell lines and brains from males and females with MECP2 mutations. Samples on the left are heterozygous for the PEG3 c.42C→T SNP (rs1860565) (gDNA); samples on the right are heterozygous for the c.*703A→G SNP (rs1055359) (gDNA). Arrows indicate the locations of variant nucleotides. As shown in the right-hand traces (cDNA), all samples express PEG3 from only one allele. B, Maintenance of imprinting of PEG10 in frontal cortex from one male and two females with MECP2 mutations who are heterozygous for the c.*2923C→T SNP (rs13073). RT-PCR analyses reveal strictly monoallelic expression (cDNA). Arrows indicate the positions of variant nucleotides.

Figure 6.

No increase in Dlx5 and Dlx6 expression in Mecp2-mutant mouse brain. A, Expression of Dlx5 and Dlx6 genes in frontal cortex of Mecp2 mutants (mut) compared with that of wild-type (wt) males from seven different litters, at age 7 wk (litters 1–5) or 9 wk (litters 6 and 7). For litters with more than one wild type or more than one mutant, individual animals are identified by different symbols (designated wt1 and wt2 or mut1 and mut2, respectively). Real-time qRT-PCR data from each mouse were converted to relative fold changes by comparison with a normalized sample, a wild-type mouse whose level of gene expression was closest to the mean value for all wild-type data—that is, wt1 in litter 3 for Dlx5 and wt1 in litter 6 for Dlx6. P values calculated by two-tailed, unequal variance are P=.4984 for Dlx5 and P=.5134 for Dlx6. P values calculated by one-tailed, unequal variance are P=.2492 for Dlx5 and P=.2567 for Dlx6. The plots illustrate the wide variability within and between litters. B, Expression of the imprinted gene Peg3, studied in the same forebrain samples used in fig. 6A, by qRT-PCR with two different primer sets (Peg3A and Peg3B). Expression levels were normalized to the same housekeeping gene, Rps28. The wide range of expression levels in individual mice and the similarity of patterns obtained with Peg3A and Peg3B primer sets indicate biological noise rather than technical variation.