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Figure 1. Genomic map of human FAM50B locus. (A) FAM50B-AS RACE-PCR showing a 211 bp product of 5′-RACE and an 823 bp product of 3′-RACE. M, 100 bp DNA ladder. N, control reactions were performed using cDNA prepared in the absence of primers to eliminate the possibility of genomic DNA and RNA contamination. (B) Sequencing of 3′-RACE-PCR demonstrates the polyA sequence and AAUAAA motif (underlined) of FAM50B-AS. (C) The FAM50B locus spans 2 kb of genomic sequence at chromosome 6p25 and consists of two exons (E1 and E2). Horizontal arrows indicate the sense (FAM50B) and antisense (FAM50B-AS) transcripts of the gene. FAM50B-AS originates at the 3′-end of FAM50B and terminates within FAM50B intron 1. The black horizontal line indicates the position of the DMR. Vertical arrows designate positions of SNPs rs2239713 (C/T), rs6597007 (G/C), rs3196512 (G/C) and the translational start codon ATG. The position and direction of primers used for RACE is indicated by horizontal arrowheads. Primer sequences are provided in Supplementary Table S1.
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Figure 2. CpG methylation status at the FAM50B locus. (A) The percent methylation of CpG sites 1–166 was quantified by Sequenom MassARRAY using DNA from conceptus liver (blue line, n = 3) and brain (red line, n = 3). CpG site numbers correspond to the positions within the GC rich region (−1353 to +1066 relative to the FAM50B translational start codon). Arrow indicates the position of the translational start codon. The location of a DMR is shown by a horizontal line. (B) Average methylation of CpG sites (12 to 110) does not vary significantly for conceptus tissues from the three germ layers and from different gestational ages, while sperm shows low levels of methylation at this locus. B, brain; L, liver; K, kidney; P, placenta; S, sperm; B1, L1 and K1—gestational age of 81 days; B2, L2 and K2—gestational age of 110 days.
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Figure 3. Allele-specific methylation at the FAM50B locus. (A) Clones obtained from bisulfite-treated conceptus liver DNA are either predominantly methylated or unmethylated from the 18th CpG site to the 66th CpG site. (B) Clones obtained from bisulfite-treated conceptus liver DNA, representing one of three individuals heterozygous for SNP rs2239713 (arrow), while their matched maternal deciduae are homozygous G/G for the SNP sites. The allele-specific distribution of methylation with respect to the G/A polymorphism demonstrates that the maternally-derived allele is methylated while the paternally derived allele is unmethylated. (C) Clones obtained from bisulfite-treated mature sperm DNA are completely unmethylated. CpG sites are represented by circles. Unfilled circle, unmethylated CpG site; filled circle, methylated CpG site.
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Figure 4. Imprinting analysis of human FAM50B. Genomic DNA from human conceptuses (A and B) is heterozygous for polymorphism rs6597007 (G/C). Sequencing of cDNA demonstrates monoallelic expression of FAM50B transcripts in multiple conceptus tissues, including brain, liver, placenta, adrenal gland, kidney, heart and testis. In contrast, FAM50B expression is biallelic in fetal ovary. The position of SNP rs6597007 used to determine allelic expression is indicated by the arrows.
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Figure 5. Phylogenetic comparison of the genomic structure of FAM50B and flanking genes. FAM50B is flanked by C6orf145 and PRPF4B in human (chromosome 6) and mouse (chromosome 13), but is 78 Mb upstream from the SLC22A23 ortholog in the opossum (chromosome 3). FAM50B orthologs are not present in the platypus (UltraContig Ultra 352) and chicken genomes (chromosome 2).
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Figure 6. Imprinting analysis of FAM50B in the opossum and the mouse. (A) cDNA sequencing demonstrates biallelic expression of FAM50B in opossum placenta and liver. Arrows indicate the position of the SNP used to determine the allelic expression patterns. (B) Methylation at CpG dinucleotides is <20% for most of the region that encompasses the opossum FAM50B translational start codon, from −340 to 972 bp. Blue line, DNA from liver; red line, DNA from kidney. Arrow indicates the position of the translational start codon. (C) FAM50B cDNA sequencing demonstrates biallelic expression of the FAM50B in mouse brain and testis. Arrows indicate the position of the SNP used to determine the allelic expression patterns. (D) Methylation is >60% at most CpG sites in proximity to the translational start codon of murine FAM50B (from −8800 to 1120 bp relative to translational start codon). Arrow indicates the position of the translational start codon. Blue line, DNA from kidney; red line, DNA from liver.
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Figure 7. Methylation status and gene expression of FAM50B locus in TGCTs. (A) Percent methylation of the FAM50B DMR in each tumor and in normal testis controls (n = 3). (B) Relative expression levels, normalized to GAPDH, of FAM50B transcripts in TGCTs and in normal testis controls (n = 3). (C) Relative expression levels, normalized to GAPDH, of the FAM50B-AS transcripts in TGCTs and in normal testis controls (n = 3). TGCTs include: embryonic carcinoma (C1, C2), seminoma mixed with embryonic carcinoma (SC1), pure seminoma (S1, S2, S3 and S4, S5), and mixed germ cell tumor (M1, M2 and M3). Normal testis tissue (TN, n = 3) was used for comparison.
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Figure 8. Allelic expression of FAM50B in TGCTs. (A) cDNA sequencing of TGCTs demonstrates maintenance of monoallelic expression of the FAM50B transcript in embryonic carcinoma C1 and C2 and mixed germ cell tumor M2 and M3. (B) cDNA sequencing of TGCTs demonstrates biallelic expression of the FAM50B transcripts in the seminomas S2 and S3 and biallelic expression of both FAM50B and FAM50B-AS in seminoma mixed with embryonic carcinoma SC1. The SNP rs3196512 (G/C) was used to determine allelic expression of FAM50B in sample C1, C2, M2, M3, S2 and S3. The SNP rs6597007 (G/C) was used to determine allelic expression of FAM50B and FAM50B-AS in sample SC1. The position of the SNP used to determine allelic expression is indicated by the arrows.
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