engleski

IGF2 promoter usage and expression of IGF2AS are regulated by IGF2-Bi methylation, the presence CTCF and poly(ADP-ribosyl)ation

Abstract The maintenance of IGF2/H19 imprinting depends on the methylation of H19 ICR, the presence of CTCF and ADP-ribosylation. The transcription of IGF2 is regulated by five promoters in a tissue-and age-specific manner. The imprinted IGF2 has an antisense transcript originating from the promoter located within a CpG island near IGF2 promoters P2-P4. The IGF2- Bi fragment in this region has insulator properties and contains methylation-sensitive CTCF-binding sites. Our aim was to determine the influence of DNA methylation and ADP- ribosylation on the regulation of IGF2 promoter usage and IGF2AS transcription. Human tumor cell lines HT-29 and Cal-27 were chosen for this study based on different global DNA methylation (HT-29 being 2.2 x more methylated than Cal-27) and the ADP-ribosylating capacity (lower in HT-29). They were treated with PARP-1 inhibitor, 3-aminobenzamide (3-AB), to inhibit poly(ADP-ribosyl)ation and promote DNA methylation, or 5-azacytidine (5-azaC) to induce DNA demethylation. DNA methylation of DMR0 and IGF2AS promoter was analyzed by COBRA. ADP-ribosylating capacity was estimated based on the elevation of NAD level in the cells after 3-AB treatment. Expression of IGF2AS was analyzed by qPCR. The promoter specific transcripts of IGF2 were analyzed by RT-PCR. Both untreated cell lines had hypomethylated IGF2AS promoter to which CTCF could bind, but IGF2AS was expressed only in HT-29 cells with low ADP-ribosylating capacity. The IGF2 promoter usage differed: in HT-29 cells IGF2 transcription originated from promoters P1, P0, P3 and P4 but not from P2, although P2 transcripts including alternative exon 4b (P2/4b) were present. In Cal27 cells, only promoters P3 and P4 were active. The DMR0 was hemimethylated in HT-29 and hypomethylated in Cal-27 cell line. Treatment with 5-azaC, which causes DNA demethylation and inhibits poly(ADP- ribosyl)ation, downregulated IGF2AS expression in HT-29 cells by 60% most likely due to the inability of AP-1 to bind IGF2AS promoter in the complete lack of ADP-ribosylation. The same treatment had no effect on IGF2AS expression in Cal-27 cells but caused upregulation of IGF2 P2 and the appearance of P2/4b transcripts, likely due to the inhibitory effect of 5-azaC on ADP- ribosylation. The P1 and P0 promoters remained silent in Cal27 cells. No changes in methylation of IGF2AS promoter and DMR0 were observed in either cell line. Upon 3-AB treatment, the expression of IGF2AS was downregulated in HT-29 cells by 30% concomitant with emergence of very low level IGF2 P2 transcripts. Methylation status of IGF2AS promoter and transcription of IGF2 from other promoters has not changed, while DMR0 methylation has increased. The expression of IGF2AS was still undetectable in Cal27 cells upon 3-AB treatment. This treatment resulted in appearance of IGF2 P2 and P2/4b transcripts. No changes in methylation of IGF2AS promoter or DMR0 were detectd. Our results suggest that in tested cell lines: 1) IGF2AS and IGF2 P2 transcription are mutually exclusive ; 2) binding of CTCF to hypomethylated IGF2-Bi element prevents transcription from P1, P0 and P2/4b only in poly(ADP-ribosyl)ation-competent cells ; and 3) IGF2 transcription originates from P1 and P0 only in IGF2AS-expressing cells, regardless of DMR0 methylation.

IGF2AS; IGF2 promoter usage; DNA methylation; poly(ADP)ribosylation

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