Farman Farman Ullah, Iqbal Mehwish, Azam Muhammad, Saeed Muhammad
Cancer Genetics and Epigenetics Lab, Department of Biosciences, COMSATS Institute of Information Technology (CIIT), Islamabad, Pakistan.
Department of Statistics & Computer Science, University of Veterinary & Animal Sciences, Lahore, Pakistan.
Mol Biol Rep. 2018 Apr;45(2):185-194. doi: 10.1007/s11033-018-4151-6. Epub 2018 Feb 7.
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.
核小体的动态定位对于确定基因表达水平至关重要，尤其是在基因转录起始位点（TSS）或其周围。本研究的目的是确定Rbl2/p130基因TSS周围的核小体位置。我们使用微球菌核酸酶（MNAse）消化试验和ChIP-PCR分析，研究了乳腺肿瘤及其相邻正常对照组织（ANCT）中Rbl2/p130的表达与TSS周围核小体位置的关系。与对照组织相比，这些肿瘤组织中Rbl2/p130的表达降低了三倍。从MNAse消化的染色质中获得的DNA用作PCR模板。使用3对引物（P1 = -137至+69；P2 = -90至+69；P3 = -33至+140）扫描TSS周围-137至+140的区域。约66%的乳腺肿瘤和约26%的ANCT样本P1呈阳性。差异具有统计学意义（p = 0.000），优势比（OD）为9.143，表明该区域在肿瘤样本中形成核小体的可能性约为9倍。约73%的肿瘤和60%的ANCT样本P2呈阳性，虽然具有统计学意义（p = 0.035），OD = 3.250，但不如P1理想。然而，未发现P3是核小体占据的优选区域（p = 0.670；OD = 1.2）。尤其对于P1，观察到核小体位置呈负相关。我们的结果表明，常规情况下核小体位于TSS的稍下游，而在乳腺癌发生时，核小体向TSS上游滑动55个碱基，使核小体中心位于+1位置，从而阻碍转录机制的进入。
