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

Autori
Grbeša, Ivana ; Novak-Kujundžić, Renata ; Gall-Trošelj, Koraljka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
An AACR Special Conference in Cancer Research: Cancer Epigenetics / Issa, Jean Pierre J. ; Laird, Peter W. - Boston (MA) : AACR Centennial, 2008, B16-B17

Skup
Cancer Epigenetics

Mjesto i datum
Boston (MA), Sjedinjene Američke Države, 28.05.2008. - 31.05.2008

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
IGF2 ; IGF2AS ; imprinting ; CTCF ; BORIS ; methylation

Sažetak
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.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

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