酵母核糖体蛋白基因组合转录调控位点统计分析

真核基因的转录调控是后基因组时代研究的主要问题之一,其基础是认识DNA上转录因子结合位点(模体)及分布状况。基于马尔可夫链模型对酵母核糖体蛋白基因上游启动子序列中模体出现次数进行统计,利用Z-score统计量抽提出过表达和低表达的模体,其中95%的模体与实验得到的转录因子结合位点相符合。然后将抽提出的模体两两配对,通过与背景序列比较,找出酵母核糖体蛋白基因中出现概率及距离分布均具有统计显著性的模体对,这些非随机出现的模体对具有潜在的组合转录调控功能,其中一些模体对的组合调控作用已有实验支持。对提取出的模体对在序列中的位置分布进行分析,发现近94%的模体对位于转录起始位点上游,超过半数的模体对两模体之间的最短距离在0～100bp之间,距离小于30bp的模体对接近30%,这样的短距离间隔有利于两模体的相同作用。这些结果将有助于对酵母核糖体蛋白基因转录调控机制的深入认识。     

Inhibition of SIRT1 reactivates silenced cancer genes without loss of promoter DNA hypermethylation

The class III histone deactylase (HDAC), SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs) in which 5' CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively), had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA)-mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression.     

Genome-Wide Maps of Mononucleosomes and Dinucleosomes Containing Hyperacetylated Histones of Aspergillus fumigatus

It is suggested that histone modifications and/or histone variants influence the nucleosomal DNA length. We sequenced both ends of mononucleosomal and dinucleosomal DNA fragments of the filamentous fungus Aspergillus fumigatus, after treatment with the histone deacetylase inhibitor trichostatin A (TSA). After mapping the DNA fragments to the genome, we identified >7 million mononucleosome positions and >7 million dinucleosome positions. We showed that the distributions of the lengths of the mononucleosomal DNA fragments after 15-min and 30-min treatments with micrococcal nuclease (MNase) showed a single peak at 168 nt and 160 nt, respectively. The distributions of the lengths of the dinucleosomal DNA fragments after 15-min- and 30-min-treatment with MNase showed a single peak at 321 nt and 306 nt, respectively. The nucleosomal DNA fragments obtained from the TSA-treated cells were significantly longer than those obtained from the untreated cells. On the other hand, most of the genes did not undergo any change after treatment. Between the TSA-treated and untreated cells, only 77 genes had ≥2-fold change in expression levels. In addition, our results showed that the locations where mononucleosomes were frequently detected were conserved between the TSA-treated cells and untreated cells in the gene promoters (lower density of the nucleosomes). However, these locations were less conserved in the bodies (higher density of the nucleosomes) of genes with ≥2-fold changes. Our findings indicate that TSA influences the nucleosome positions, especially of the regions with high density of the nucleosomes by elongation of the nucleosomal DNA. However, most of the nucleosome positions are conserved in the gene promoters, even after treatment with TSA, because of the low density of nucleosomes in the gene promoters.     

HOP/OB1/NECC1 promoter DNA is frequently hypermethylated and involved in tumorigenic ability in esophageal squamous cell carcinoma

Promoter DNA hypermethylation with gene silencing is a common feature of human cancer, and cancer-prone methylation is believed to be a landmark of tumor suppressor genes (TSG). Identification of novel methylated genes would not only aid in the development of tumor markers but also elucidate the biological behavior of human cancers. We identified several epigenetically silenced candidate TSGs by pharmacologic unmasking of esophageal squamous cell carcinoma (ESCC) cell lines by demethylating agents (5-aza-2'-deoxycitidine and trichostatin A) combined with ESCC expression profiles using expression microarray. HOP/OB1/NECC1 was identified as an epigenetically silenced candidate TSG and further examined for (a) expression status, (b) methylation status, and (c) functional involvement in cancer cell lines. (a) The HOP gene encodes two putative promoters (promoters A and B) associated with two open reading frames (HOPalpha and HOPbeta, respectively), and HOPalpha and HOPbeta were both down-regulated in ESCC independently. (b) Promoter B harbors dense CpG islands, in which we found dense methylation in a cancer-prone manner (55% in tumor tissues by TaqMan methylation-specific PCR), whereas promoter A does not harbor CpG islands. HOPbeta silencing was associated with DNA methylation of promoter B in nine ESCC cell lines tested, and reactivated by optimal conditions of demethylating agents, whereas HOPalpha silencing was not reactivated by such treatments. Forced expression of HOP suppressed tumorigenesis in soft agar in four different squamous cell carcinoma cell lines. More convincingly, RNA interference knockdown of HOP in TE2 cells showed drastic restoration of the oncogenic phenotype. In conclusion, HOP is a putative TSG that harbors tumor inhibitory activity, and we for the first time showed that the final shutdown process of HOP expression is linked to promoter DNA hypermethylation under the double control of the discrete promoter regions in cancer.     

Mapping tumor-suppressor genes with multipoint statistics from copy-number-variation data

Array-based comparative genomic hybridization (arrayCGH) is a microarray-based comparative genomic hybridization technique that has been used to compare tumor genomes with normal genomes, thus providing rapid genomic assays of tumor genomes in terms of copy-number variations of those chromosomal segments that have been gained or lost. When properly interpreted, these assays are likely to shed important light on genes and mechanisms involved in the initiation and progression of cancer. Specifically, chromosomal segments, deleted in one or both copies of the diploid genomes of a group of patients with cancer, point to locations of tumor-suppressor genes (TSGs) implicated in the cancer. In this study, we focused on automatic methods for reliable detection of such genes and their locations, and we devised an efficient statistical algorithm to map TSGs, using a novel multipoint statistical score function. The proposed algorithm estimates the location of TSGs by analyzing segmental deletions (hemi- or homozygous) in the genomes of patients with cancer and the spatial relation of the deleted segments to any specific genomic interval. The algorithm assigns, to an interval of consecutive probes, a multipoint score that parsimoniously captures the underlying biology. It also computes a P value for every putative TSG by using concepts from the theory of scan statistics. Furthermore, it can identify smaller sets of predictive probes that can be used as biomarkers for diagnosis and therapeutics. We validated our method using different simulated artificial data sets and one real data set, and we report encouraging results. We discuss how, with suitable modifications to the underlying statistical model, this algorithm can be applied generally to a wider class of problems (e.g., detection of oncogenes).