Estudo da cromatina nos sítios de fixação à matriz nuclear no domínio do gene TP53 e das modificações epigenéticas e no modelo de progressão tumoral mamária 21T

Abstract

Breast cancer is the most common aggressive cancer type in women. Inherited and acquired mutations as well as epigenetic alterations act together in breast carcinogenesis and tumor progression. P53 is a tumor suppressor protein critical for genome integrity. Although its control at the protein level is well known, the transcriptional regulation of the TP53 gene is still unclear. The 21T series, a series of 4 breast cell lines originating from the same patient and representative of the breast tumor progression stages, is a suitable model to investigate epigenetic alterations and their influences upon gene expression during breast tumor progression. We have analyzed the organization of the TP53 gene domain using DNA arrays in several breast cancer and control cell lines and we made an attempt to characterize these DNA elements in breast non-cancerous cell lines HB2 and MCF-10, and cancerous MCF-7, MDA-MB-231 and T47D, through the determination of epigenetic markers of euchromatin, H4Ac, and heterochromatin, H3K9me3. We further analyzed the matrix attachment region (MAR), named MAR 2, protein binding, and possible MAR 2 binding of the important MAR binding protein (MARBP), PARP-1, by Electrophoretic mobility Shift Assay (EMSA). We have found that in the control breast epithelial cell line, HB2, the TP53gene is positioned within a relatively small DNA domain, encompassing 50 kb, delimited by two nuclear matrix attachment sites. Interestingly, this domain structure was found to be radically different in the studied breast cancer cell lines, MCF7, T47D, MDA-MB-231 and BT474, in which the domain size was increased and TP53 transcription was decreased. H4Ac and H3K9me3, chromatin epigenetic markers enrichment are differentially distributed through MARs in cell lines. Surprinsingly, MAR 2 presented a defined band-shift, which could represent trans-acting factor(s), involved in chromatin organization. By bioinformatics software, we found interesting transcription factors putative binding sites, such as for c/EBP-beta and c-myb, which could be cis-acting elements regulating TP53 and neighboring genes expression. We propose a model for the chromatin organization of the TP53 gene domain with neighboring genes. Through 21T cell line series, we detected a global genomic hypomethylation profile in the cancerous 21NT and 21MT1. An important global decrease of the active chromatin mark H4Ac in the metastatic 21MT1 relative to other cell lines was detected. mRNA levels of key enzymes linked to epigenetic modifications are consistent with the observed genomic hypomethylation and hypoacetylation. By confocal immunofluorescent assay we observed that H4Ac is mostly located at periphery, and the repressive mark H3K9Me3 located pericentric, in tumorigenic cells nuclei. TP53 P1 promoter in 21T series was found to be in an open state and TP53 transcription level was found to be similar in all 21T cell lines.