Conserved characteristics of heterochromatin-forming DNA at the 15q11-q13 imprinting center

Abstract

Nuclear matrix binding assays (NMBAs) define certain DNA sequences as matrix attachment regions (MARs), which often have cis-acting epigenetic regulatory functions. We used NMBAs to analyze the functionally important 15q11-q13 imprinting center (IC). We find that the IC is composed of an unusually high density of MARs, located in close proximity to the germ line elements that are proposed to direct imprint switching in this region. Moreover, we find that the organization of MARs is the same at the homologous mouse locus, despite extensive divergence of DNA sequence. MARs of this size are not usually associated with genes but rather with heterochromatin-forming areas of the genome. In contrast, the 15q11-q13 region contains multiple transcribed genes and is unusual for being subject to genomic imprinting, causing the maternal chromosome to be more transcriptionally silent, methylated, and late replicating than the paternal chromosome. We suggest that the extensive MAR sequences at the IC are organized as heterochromatin during oogenesis, an organization disrupted during spermatogenesis. Consistent with this model, multicolor fluorescence in situ hybridization to halo nuclei demonstrates a strong matrix association of the maternal IC, whereas the paternal IC is more decondensed, extending into the nuclear halo. This model also provides a mechanism for spreading of the imprinting signal, because heterochromatin at the IC on the maternal chromosome may exert a suppressive position effect in cis. We propose that the germ line elements at the 15q11-q13 IC mediate their effects through the candidate heterochromatin-forming DNA identified in this study.

Figure 1

NMBAs of EcoRI-digested phage λ48.26 and its plasmid subclones. In each panel, the left lane is unassayed probe and the second and third lanes are the results of NMBAs in the presence of 100 and 300 μg/ml of cold competitor DNA, respectively. The three EcoRI bands marked with arrowheads are the insert fragments binding more strongly than both of the phage vector arms (λ1 and λ2) or the plasmid vector (P). Densitometry was used to confirm that the adjusted relative ratio of insert to vector DNA was high, as calculated previously (17). The relative ratios for the 300 μg/ml lanes are 40.9 (L48.26III), 15.1 (L48.26II), and 39.0 (L48.26I). The region analyzed at SNURF-SNRPN therefore contains a large MAR (gray bar).

Figure 2

A summary of the results of NMBAs for (a) the human 15q11-q13 IC/SNURF-SNRPN region and (b) the mouse Snurf-Snrpn locus. Exons 1–10 of the SNURF-SNRPN gene (13) and two exons of the SNRPN U transcript (11, 15) are shown. NMBAs were performed on each of the genomic clones shown (primary data in www.pnas.org). The gray bars on the human and mouse maps show the extent of MARs identified. (a) In total, 49.1 kb of the 68.9-kb region at the human IC/SNURF-SNRPN gene region are biochemically defined as MARs by using the NMBA. (b) The organization of MARs at the homologous mouse region is similar in terms of the density and distribution of MARs. (c) Sequence comparison [percent identity plots (31)] reveals extensive divergence of sequence at these loci, except at the immediate promoter/exon 1 region of SNURF-SNRPN/Snurf-Snrpn and within intron 1. Conservation of MARs is therefore not accounted for by identity of sequence between these two species.

Figure 3

MARs are present and conserved at the orthologous (a) human ZNF127 and (b) mouse Zfp127 loci. There are upstream and downstream MARs at each locus (a and b) and DNA sequence divergence (c) similar to that found at the IC despite the conservation of MARs. Note that the ZNF127/Zfp127 loci are intronless, with a conserved antisense gene, ZNF127AS/Zfp127as, at this locus (27, 28).

Figure 4

The relationship of the 15q11-q13 IC to the nuclear matrix depends on its parental origin. The halo nuclei analyzed were those in which both copies of the chromosome 15 α satellite repeat (15cen, yellow) are present within the nuclear matrix (visible on 4′,6-diamidino-2-phenylindole counterstaining, blue), and one of the two copies of the HPRT probe (HPRT, green) is confined to the nuclear matrix with the homologue associated, at least in part, with the surrounding DNA halo. The IC probe is shown as the red signal in these images. (a) In a normal female, the majority (16/20) of analyzed halo nuclei show one IC signal solely within the nuclear matrix, the other partially within the DNA halo. The remaining DNA haloes (4/20) showed both signals to be present within the matrix. (b) In a patient with PWS caused by a deletion of the paternal 15q11-q13 region, the sole signal is from the maternal chromosome, found only in the matrix in the majority (18/20) of halo nuclei.

Figure 5

A model for the function of the 15q11-q13 IC. The locations and target chromosomes for the putative germ line imprint switch elements are represented by vertical arrows marked oogenesis-responsive element (ORE) (responsible for switching the paternal to a maternal imprint in the female germ line) and spermatogenesis-responsive element (SRE) (responsible for maternal to paternal switching in the male germ line). We propose that the maternal chromosome is inherited with a heterochromatic organization of the MAR sequences identified in this study. The function of the SRE (SNURF-SNRPN promoter) as a switch element in this model is to displace the heterochromatin constituents, whereas the ORE is directing the formation of heterochromatin at the adjacent MARs on the paternal chromosome. This model does not require imprinted expression of the SNRPN U transcript or SNURF-SNRPN during gametogenesis but does require that SNURF-SNRPN be expressed on the maternal chromosome in the male germ line, for which there is evidence in mouse (50). The presence of the heterochromatin on the maternal chromosome is likely to be associated with suppressive effects in cis, allowing the proposal of a position effect model for imprint spreading to regulate the 2-Mb domain.