Figure 2
Figure 2. Differential methylation of the upstream of DLK1 insulator. (A) Map of the CpG-rich areas analyzed. The D1, D2, and D3 regions are located at −18 210 to −17 139 bp, −306 to +203 bp, and +7287 to +7704 bp relative to the DLK1 start site, respectively. The IG area represents the putative IG-DMR, located approximately 15 kb upstream of the MEG3 start site. The M1, M2 and M3 regions are located −2014 to −1516 bp, −299 to +289 bp, and +1219 to +1919 bp relative to the MEG3 start site, respectively. The bold boxes represent the exons in DLK1 and MEG3 genes. (B) Cluster analysis of the methylation of the region D1 in 6 NBMs (blue), 7 AML patient samples with DLK1 monoallelic expression (yellow), 22 AML patient samples with biallelic (olive green), and the K562 cell lines (purple). Pink arrows indicate the site of predicted CTCF-binding sites (detailed in supplemental Figure 14). Horizontal black arrows indicate the location of primers used for DNA amplification after ChIP. (C) This figure shows allele-specific methylation of an assessable region within the D1 region (located between CpG40 and CpG60) in one NBM (NBM1), one AML case with monoallelic DLK1 expression (part #8042), and one AML case with biallelic DLK1 expression (part #8101). The relatively hypomethylated allele was designated as allele A, whereas the hypermethylated allele was designated as allele B. The different alleles are differentially methylated with the CTCF-binding site completely unmethylated in allele A and partially methylated in allele B in the NBM sample (NBM1) and in the monoallelic DLK1 patient (part #8042). In contrast, there is increased methylation of both alleles in the patient with biallelic DLK1 expression (part #8101) with acquisition of methylation in the CTCF site of the hypomethylated allele. (D) ChIP with CTCF antibody showing that the CTCF antibody was able to precipitate the D1 region in one NBM (NBM1) and 2 AML samples with monoallelic DLK1 expression (indicated as Mono 1 and 2 with the parts #8042 and #8107, respectively), whereas there was no precipitation in 2 patient samples with biallelic DLK1 expression (indicated as Bi 1 and 2 with the parts #8145 and #8101, respectively) and the K562 cell line. Two DNA areas were amplified after ChIP as shown in panel B, and similar results were obtained from both areas.

Differential methylation of the upstream of DLK1 insulator. (A) Map of the CpG-rich areas analyzed. The D1, D2, and D3 regions are located at −18 210 to −17 139 bp, −306 to +203 bp, and +7287 to +7704 bp relative to the DLK1 start site, respectively. The IG area represents the putative IG-DMR, located approximately 15 kb upstream of the MEG3 start site. The M1, M2 and M3 regions are located −2014 to −1516 bp, −299 to +289 bp, and +1219 to +1919 bp relative to the MEG3 start site, respectively. The bold boxes represent the exons in DLK1 and MEG3 genes. (B) Cluster analysis of the methylation of the region D1 in 6 NBMs (blue), 7 AML patient samples with DLK1 monoallelic expression (yellow), 22 AML patient samples with biallelic (olive green), and the K562 cell lines (purple). Pink arrows indicate the site of predicted CTCF-binding sites (detailed in supplemental Figure 14). Horizontal black arrows indicate the location of primers used for DNA amplification after ChIP. (C) This figure shows allele-specific methylation of an assessable region within the D1 region (located between CpG40 and CpG60) in one NBM (NBM1), one AML case with monoallelic DLK1 expression (part #8042), and one AML case with biallelic DLK1 expression (part #8101). The relatively hypomethylated allele was designated as allele A, whereas the hypermethylated allele was designated as allele B. The different alleles are differentially methylated with the CTCF-binding site completely unmethylated in allele A and partially methylated in allele B in the NBM sample (NBM1) and in the monoallelic DLK1 patient (part #8042). In contrast, there is increased methylation of both alleles in the patient with biallelic DLK1 expression (part #8101) with acquisition of methylation in the CTCF site of the hypomethylated allele. (D) ChIP with CTCF antibody showing that the CTCF antibody was able to precipitate the D1 region in one NBM (NBM1) and 2 AML samples with monoallelic DLK1 expression (indicated as Mono 1 and 2 with the parts #8042 and #8107, respectively), whereas there was no precipitation in 2 patient samples with biallelic DLK1 expression (indicated as Bi 1 and 2 with the parts #8145 and #8101, respectively) and the K562 cell line. Two DNA areas were amplified after ChIP as shown in panel B, and similar results were obtained from both areas.

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