engleski

IGF2 P1 Promoter Usage and Expression of IGF2AS are Regulated by IGF2-Bi Methylation and the Presence of CTCF and BORIS

The maintenance of IGF2/H19 imprinting is dependent on the methylation of H19 ICR, the presence of CTCF and/or BORIS and ADP-ribosylation. The transcription of IGF2 is regulated by four 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, CTCF and/or BORIS expression 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 methylation (HT-29 being 2.2 X more methylated than Cal-27), level of expression of CTCF and BORIS (HT-29: BORIS+, Cal-27: BORIS-) and the ADP-ribosylating capacity (lower in HT-29). DNA methylation was quantified using an ELISA-like reaction. Expression of CTCF, BORIS, IGF2 and IGF2AS was analyzed by RT-PCR. ADP-ribosylating capacity was estimated based on the elevation of NAD level in the cells after treatment with PARP-1 inhibitor, 3-aminobenzamide (3-AB). Cells were treated with 3-AB to modify expression of CTCF and BORIS or 5-azacytidine (5-aza) to induce DNA demethylation. Both untreated cell lines expressed IGF2 from P1, P3 and P4 and IGF2AS. Upon 3-AB treatment, highly methylated, BORIS positive HT-29 cells with low ADP-ribosylating activity maintained the same pattern of IGF2 promoter usage and IGF2AS expression. In Cal-27 cells, the expression of IGF2 from P1 and IGF2AS was silenced, concomitant with the appearance of BORIS (due to translocation of non-ADP-ribosylated p53 to cytoplasm and consequential loss of its negative regulation of BORIS transcription) and downregulation of CTCF. The induced expression of BORIS and its binding to the IGF2-Bi, instead of CTCF, in hypomethylated Cal-27 cells renders the insulator functional, preventing transcription of IGF2 from P1 and IGF2AS, upon 3-AB treatment. To determine whether the high methylation level of HT-29 makes the IGF2 P1 and IGF2AS promoter unresponsive to 3-AB treatment due to the inability of CTCF or BORIS to bind to the methylated IGF2-Bi, we treated them with 5-aza. Upon demethylation, the IGF2 transcription from P1 became silenced, but IGF2AS transcription was maintained, concomitant with BORIS silencing. The same treatment of Cal-27 resulted in BORIS expression, silencing of IGF2 from P1 and maintenance of IGF2AS transcription. Our results suggest that IGF2-Bi insulator regulates both IGF2 promoter P1 usage and IGF2AS transcription in methylation- and a CTCF/BORIS-dependent manner. Binding of CTCF or BORIS to IGF2-Bi is sufficient for silencing IGF2 transcription from P1, indicating the “ evolutionary flexibility” which is missing in the regulation of IGF2AS, as only the lack of CTCF binding appears to be mandatory for IGF2AS silencing.

IGF2 ; IGF2AS ; imprinting ; CTCF ; BORIS ; methylation

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