(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.

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 EcoRI (E) and HpaII (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 HpaII sites in the fetal brain in UPGL alternative promoter is confirmed (left panel) by first digesting with DraI, a methylation-insensitive enzyme, followed by HpaII and MspI (M). (See Supplementary Material, Fig. S1 for a more detailed interpretation of the Southern blots).

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 HpaII 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 HpaII 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.

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.

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 HpaII restriction sites observed in fetal brains. (C) Restriction enzyme digestion of the RAD21 ChIP DNA with HpaII or MspI (which both cut DNA at CCGG sites, but HpaII is blocked by CG methylation) and PCR analysis shows that Rad21 ChIP DNA is resistant to HpaII digestion, while PCR failed to amplify the specific product from MspI-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.)