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Nucleosomes positioning around transcriptional start site of tumor suppressor (Rbl2/p130) gene in breast cancer

Farman, FarmanUllah, Iqbal, Mehwish, Azam, Muhammad, Saeed, Muhammad
Molecular biology reports 2018 v.45 no.2 pp. 185-194
DNA, breast neoplasms, breasts, carcinogenesis, genes, hybridization probes, micrococcal nuclease, nucleosomes, polymerase chain reaction, transcription (genetics)
Dynamic positioning of nucleosomes is pivotal in determining level of genes expression especially on or around transcription start site (TSS) of a gene. Purpose of the current study was to determine nucleosome position around TSS of Rbl2/p130. We investigated Rbl2/p130 expression in connection to nucleosome positions around its TSS among breast tumors and their adjacent normal control tissues (ANCT) using micrococcal nuclease (MNAse) digestion assay and ChIP–PCR analysis. Three fold reduced Rbl2/p130 expression in these tumor tissues were noticed compared to their control tissues. DNA obtained from MNAse digested chromatin was used as PCR template. Region between − 137 to + 140 around TSS was scanned using 3 primer pairs (P1 = − 137 to + 69; P2 = − 90 to + 69; P3 = − 33 to + 140). ~ 66% breast tumors and ~ 26% ANCT samples were positive for P1. The difference was found statistically significant (p = 0.000) with an odd ratio (OD) of 9.143, suggesting that nucleosome formation in this region is ~ 9 times more probable in tumor samples. ~ 73% of the tumor and 60% ANCT were positive for P2, which although is significant (p = 0.035) with OD = 3.250, but less preferable than P1. However, P3 was not found to be a preferred area for nucleosome occupancy (p = 0.670; OD = 1.2). Negative correlations for nucleosome positions were observed especially for P1. Our results indicate that nucleosome are present slightly downstream of TSS in routine, while in case of breast carcinogenesis nucleosomes slides 55 bases upstream of the TSS, aligning + 1 position at the center of nucleosome, hence hindering access to the transcriptional machinery.