Genomic imprinting at a boundary element flanking the SDHD locus - PubMed (original) (raw)
. 2011 Nov 15;20(22):4452-61.
doi: 10.1093/hmg/ddr376. Epub 2011 Aug 23.
Affiliations
- PMID: 21862453
- PMCID: PMC3196894
- DOI: 10.1093/hmg/ddr376
Genomic imprinting at a boundary element flanking the SDHD locus
Bora E Baysal et al. Hum Mol Genet. 2011.
Abstract
Germline mutations in SDHD, a mitochondrial complex II (succinate dehydrogenase) subunit gene at chromosome band 11q23, cause highly penetrant paraganglioma (PGL) tumors when transmitted through fathers. In contrast, maternal transmission rarely, if ever, leads to tumor development. The mechanism underlying this unusual monogenic tumor predisposition pattern is poorly understood. Here, we describe identification of imprinted methylation within an alternative promoter for a large intergenic non-coding RNA located at a distant gene desert boundary flanking SDHD. Methylation at this site primarily occurs within two consecutive HpaII restriction enzyme sites in a tissue-specific manner, most commonly in the adrenal gland. Informative fetal tissues and PGL tumors demonstrate maternal allelic hypermethylation. While a strong binding site for the enhancer-blocking protein CTCF within the alternative promoter shows no evidence of methylation, hyper-methylated adrenal tissues show increased binding of the chromatin-looping factor cohesin relative to the hypo-methylated tissues. These results suggest that the differential allelic methylation we observe at this locus is associated with altered chromatin architectures. These results provide molecular evidence for imprinting at a boundary element flanking the SDHD locus and suggest that epigenetic suppression of the maternal allele is the underlying mechanism of the imprinted penetrance of SDHD mutations.
Figures
Figure 1.
(A) Gene-rich SDHD domain is separated from a flanking gene desert by UPGL, a large non-coding RNA. The non-coding UPGL gene (CR616845) is located at the telomeric boundary of a gene-rich region containing SDHD on chromosome band 11q23. The locations of SDHD and the gene desert are indicated by arrows. (B) UPGL primary transcript is composed of two alternatively spliced exons. The first exon originates from the CGI major promoter, although rare transcripts originate from an alternative CGI promoter. Many truncated transcripts downstream of UPGL suggest alternative transcript processing at 3′-end of UPGL.
Figure 2.
Expression and alternative-promoter methylation of UPGL (A). Northern blot analyses of the SDHD and UPGL genes. SDHD is expressed ubiquitously at a single major transcript size, whereas UPGL is expressed predominantly in the heart, skeletal muscle and brain and at multiple transcript sizes. Database analysis of expressed sequence tags (ESTs) suggests that the most common UPGL transcript (∼1 kb) is composed of two exons and originates from the major promoter. The ESTs show variation in the 3′ termini, including a third alternatively spliced 3′-exon. These transcript variations might account for the longer bands of weaker intensity observed in the UPGL northern blot. (B) Methylation-sensitive southern blot analysis (right panel) of genomic DNA shows partial methylation of _Eco_RI (E) and _Hpa_II (H) sites in the upstream CGI (the UPGL alternative promoter) in fetal brain (Br) but not in lung (lu) and lymphoblastoid DNAs (ly). Partial methylation of _Hpa_II sites in the fetal brain in UPGL alternative promoter is confirmed (left panel) by first digesting with _Dra_I, a methylation-insensitive enzyme, followed by _Hpa_II and _Msp_I (M). (See
Supplementary Material, Fig. S1
for a more detailed interpretation of the Southern blots).
Figure 3.
Evidence of imprinting at the UPGL alternative promoter. (A) Average CpG methylation rates in the UPGL alternative promoter in the fetal adrenal gland (_n_= 4) and fetal brain (_n_= 5) reveals highest methylation in an _Hpa_II restriction enzyme site (CpG#13). CpG graph displays mean ± standard error of the mean (horizontal bars) for average methylation rates for each of the 31 CpGs. (B) Allelic methylation profiles in four heterozygous adrenal glands indicate statistically significant allelic methylation differences at CpG#13 in four informative cases (Fisher's exact test: *P< 0.05, *****P< 10−5, ******P< 10−6). Allelic sequence variations are shown on the left. Each row demonstrates methylation rates in the CpGs in the allele defined by the sequence variation on left (Del/Wt or A/G). The del(GAA) variant removes one of the GAA triplets following CpG#13 without eliminating the CpG or the _Hpa_II restriction enzyme site. In sample 2028 AM, maternal and paternal alleles could be assigned by the availability of an informative (homozygous AA) maternal decidual tissue. In fetus 2032, the methylation profile of each allele was derived from a different paraganglionic tissue: AM, adrenal medulla/gland, OZ, organ of Zuckerkandl (peri-aortic paraganglia). N indicates the number of PCR clones sequenced for each allele. Core CTCF-binding site is shown by a thick red bar, the extended binding site is shown by a thin red bar. (C) An expressed SNP (G/T) in UPGL exon 2 in one fetus reveals marked imbalance in allelic expression levels, revealing monoallelic expression of the G allele in the fetal adrenal gland and heart, imbalanced expression in the kidney and balanced expression in the skin. Because all tissues are confirmed to be heterozygous at the genomic level by sequencing, the allelic imbalances likely stem from allelic epigenetic differences.
Figure 4.
Methylation profile of the UPGL alternative promoter in PGL tumors (A). Comparison of average CpG methylation rates in non-SDHD (_n_= 6) and SDHD PGLs (_n_= 6) shows statistically significantly more clones methylated at the UPGL alternative promoter (CpG#13) (47/378 versus 5/268 methylated clones; Fisher's exact test: P< 10−8) among non-SDHD tumors. (B) Comparison of sequence fluorograms between blood and tumor shows attenuation of the normal unmutated alleles (arrows) in the tumors consistent with LOH.
Figure 5.
CTCF and cohesin binding in the UPGL alternative promoter. (A) ChIP-PCR analysis of CTCF in adrenal gland (noted AM or A) or lung (noted LUNG or L) tissues from the 2028 sample. − and + indicate mock- or CTCF-specific immunoprecipitation. Input indicates total chromatin prior to immunoprecipitation. The ChIP-PCR is analyzed by agarose gel electrophoresis. (B) The results from ChIP-qPCR analysis of cohesin binding at the DMR using an antibody against RAD21, a component of cohesin. Three paired fetal lung and adrenal medulla tissues from three fetuses (numbers 2028, 2031, 2038) and two paired fetal brain and adrenal medulla tissues from two fetuses (numbers 138 and 27097) were analyzed by cohesin ChIP. The fold enrichment values are determined by determining the threshold cycle numbers for the ChIP DNA and input control DNA. In all experiments, the adrenal tissue shows increased RAD21 binding. Increased RAD21 binding is also noted in one of two fetal brains (number 138) consistent with frequent methylation at _Hpa_II restriction sites observed in fetal brains. (C) Restriction enzyme digestion of the RAD21 ChIP DNA with _Hpa_II or _Msp_I (which both cut DNA at CCGG sites, but _Hpa_II is blocked by CG methylation) and PCR analysis shows that Rad21 ChIP DNA is resistant to _Hpa_II digestion, while PCR failed to amplify the specific product from _Msp_I-digested ChIP DNA, suggesting that significant fraction of RAD21 bound DNA is methylated. (MW, molecular weight marker in base pairs. Lane ‘–’ was not treated by restriction enzyme.)
References
- Baysal B.E. Clinical and molecular progress in hereditary paraganglioma. J. Med. Genet. 2008;45:689–694. - PubMed
- Hensen E.F., Jordanova E.S., van Minderhout I.J., Hogendoorn P.C., Taschner P.E., van der Mey A.G., Devilee P., Cornelisse C.J. Somatic loss of maternal chromosome 11 causes parent-of-origin-dependent inheritance in SDHD-linked paraganglioma and phaeochromocytoma families. Oncogene. 2004;23:4076–4083. - PubMed
- van der Mey A.G., Maaswinkel-Mooy P.D., Cornelisse C.J., Schmidt P.H., van de Kamp J.J. Genomic imprinting in hereditary glomus tumours: evidence for new genetic theory. Lancet. 1989;2:1291–1294. - PubMed
- Mariman E.C., van Beersum S.E., Cremers C.W., Struycken P.M., Ropers H.H. Fine mapping of a putatively imprinted gene for familial non-chromaffin paragangliomas to chromosome 11q13.1: evidence for genetic heterogeneity. Hum. Genet. 1995;95:56–62. - PubMed