基于DNA变形能的核小体定位预测方法研究进展

真核细胞中,作为染色质基本结构单元的核小体参与调控基因的转录、DNA复制、重组以及RNA剪接等诸多生物学过程。阐明核小体定位机制并准确预测核小体在染色体上的位置对解读染色质结构与功能有重要生物学意义。在过去30多年时间里,研究人员发展了多种预测核小体位置的方法。最理想的方法应考虑DNA序列、组蛋白修饰和染色质重塑等影响核小体定位的诸多因素,然而现实中,捕捉主要因素的模型也往往具有很高的鲁棒性和实用价值。DNA序列偏好性是在全基因组尺度上影响核小体定位的最重要因素之一,因此基于DNA序列的核小体定位预测方法也最常见。这种方法可大致分为两类,即基于DNA序列信息的生物信息学模型和基于DNA变形能的生物物理学模型。本文重点介绍生物物理学模型近些年取得的主要进展。     

核小体定位的转录调控功能研究进展

核小体是真核生物染色质的基本组成单位,组蛋白八聚体在DNA 双螺旋上精确位置称为核小体定位．核小体定位已被证实在基因转录调控、DNA复制与修复、调控进化等过程中扮演着重要的角色．随着染色质免疫共沉淀-芯片(ChIP-chip)与染色质免疫共沉淀-测序(ChIP-seq)等高通量技术的出现,已测定了多种模式生物全基因组核小体定位图谱,掀起了一股核小体定位及其功能的研究热潮,并取得了一定的成果．本文介绍了核小体定位的概念,总结了核小体在启动子与编码区域内定位的基本模式．在此基础上,综述了核小体定位在转录起始、转录延伸、基因表达模式多样化以及可变剪接等方面的功能研究进展．     

基于遗传算法酵母核小体定位性质预测

在DNA序列上,定位模糊的特殊核小体与定位良好的普通核小体同时存在于染色体区域内,但由于二者的化学性质差异不明显,区分较为困难。本文针对实验核小体在真核基因转录起始位点周围的分布规律和保守性建立了一个核小体分布模型,并在前人所做的预测核小体位置的工作基础上,利用遗传算法寻找模型上不同性质核小体的分布中心,构建核小体定位性质判别准则,最终确定了转录起始位点上、下游定位良好和模糊核小体的位置。     

酿酒酵母核小体定位理论模型的体外实验验证

核小体定位对真核生物基因表达调控发挥着重要作用。前期基于核小体核心及连接区域的k-mer频次分布偏好性,构建了位置权重矩阵算法,并在酿酒酵母基因组内较好地预测了核小体占据率。利用该理论模型,以1 bp碱基为步长、147 bp碱基为窗口,用该算法计算了酵母1号、3号、14号染色体上核小体形成能力强、中、弱各3条长度为147 bp的DNA序列,将这些片段克隆到重组质粒中,大量扩增回收9条标记biotin分子的目的序列。同时分别表达纯化了组蛋白H2A、H2B、H3和H4,复性后装配形成组蛋白八聚体结构。利用盐透析方法将9条DNA序列在体外组装形成核小体结构,经biotin标记检测后计算了反应过程的吉布斯自由能,对比了9条目的序列形成核小体的亲和力大小。研究发现,9条序列中有5条序列与理论预测完全符合,4条序列与理论预测不完全一致。实验结果与该算法预测的核小体定位结果基本一致,表明该理论模型能够有效预测酿酒酵母基因组核小体占据水平。     

多样性增量结合支持向量机方法预测酵母核小体定位

作为目前遗传学热点领域表观遗传学的重要研究课题,核小体定位参与多种生物学过程并起着非常关键的作用,因此用理论方法预测核小体定位具有重要的生物学意义。该工作以酵母基因组为研究对象,根据其核心DNA与连接DNA序列的组分特征差异,分别统计其核苷酸k联体出现的频数,计算其多样性增量,并以此为特征值输入支持向量机构建模型。对酵母核小体核心DNA和连接DNA序列进行分类预测,整体准确率和相关系数分别达到93.10%和0.862。此方法在人类和果蝇的核小体核心DNA和连接DNA序列分类预测中也取得了较为理想的效果。将模型用于预测核小体在基因组上的位置取得初步成功。     

果蝇胚胎期不同表达模式基因转录起始位点上游核小体的定位

在研究果蝇胚胎期核小体定位时,发现不同表达模式基因上的核小体定位特征有着很大的差异。与以往研究结果不同的是,在果蝇胚胎期限制性表达基因的转录起始位点上游-1核小体位置上存在着H2A.Z核小体。有趣的是,与单细胞酵母基因上的核小体定位相比较发现,果蝇胚胎期限制性表达基因上的核小体排列与酵母极其相似。     

核小体定位模式及其与DNA甲基化位点分布的关系

核小体定位是指DNA双螺旋相对于组蛋白八联体的位置.核小体定位通过限制蛋白结合位点参与基因转录调控.本文利用实验检测的人类CD4+ T细胞核小体定位数据,研究了核小体定位在转录因子结合位点(TFBS)和转录起始位点(TSS)附近的分布模式,并分析了在TFBS和TSS周围,核小体定位与DNA甲基化之间的关系.结果表明,在休眠和激活的人类CD4+ T细胞中,部分TFBS和TSS周围的核小体定位在动态改变,即在定位和缺失两种状态之间切换.在TFBS周围,核小体定位和DNA甲基化存在一种互补模式,核小体定位与DNA低甲基化相联系;而在TSS周围,两者呈现同步模式,DNA高甲基化伴随高核小体水平.而且,在TFBS和TSS周围,DNA甲基化位点的分布呈周期模式.CD4+ T细胞被激活时,较少的转录因子启动了较多的基因.     

核小体定位与RNA剪接

根据核小体定位序列和缺失序列的碱基分布特征,应用多样性增量二次判别方法(IDQD)构建模型对这两类序列进行了区分,受试者操作特性曲线下的面积达到了0.958．应用这一模型研究了核小体在人类基因组剪接位点(GT/AG)邻近序列中的分布方式,发现外显子所对应的DNA序列通常倾向参与核小体的形成,并且由它所转录的RNA统计上具有较强的刚性,而剪接位点及其邻近的内含子对应的DNA序列则避免参与核小体的形成,所转录的RNA统计上具有较强的柔性．进一步还发现,DNA序列的核小体定位/缺失和RNA的刚性/柔性具有统计相关性,为从机制上解释为何前体RNA剪接事件与DNA序列中的核小体定位信息有关提供了依据．     

压力条件下真核模式生物核小体移动模式研究

细胞在面对外界压力(热冲击)时会通过重塑核小体以应对环境变化。本文通过对热冲击前后酵母核小体位置实验数据、体外实验数据进行统计、比较、分析后,发现在没有任何生理扰动下,核小体的定位跟其DNA偏好性有关。但在环境压力下,对热冲击敏感的上表达基因上的核小体位置呈现出一种发散模式,而下表达基因上的核小体位置则呈现一种收敛模式,此外,保守基因上的核小体位置几乎不发生移动。总体上,在热冲击下处于基础表达状态的基因则呈现出一种弱收敛模式。     

果蝇核小体定位研究进展

基因组上核小体位置的确定涉及DNA、RNA聚合酶、转录因子、后转录修饰与组蛋白变异、组蛋白修饰酶和染色质重塑复合体之间的相互作用。真核状态基因组的DNA是被包裹在核小体中,故理解控制核小体沿DNA定位的原理对于进一步理解组蛋白结合所执行的基因功能是非常必要的。而核小体的定位在诸多细胞过程中起着重要重要,比如转录调控、DNA复制和修饰等。因此核小体定位已逐渐成为目前遗传学研究的热点以及表观遗传学的重要研究内容并且也将在未来生物学研究中占据相当重要的位置。本综述以果蝇为例,全面介绍核小体定位的研究现状和未来方向。     

Prediction of nucleosome positions in the yeast genome based on matched mirror position filtering

Nucleosome positioning can affect the accessibility of the underlying DNA to the nuclear environment and as such plays an essential role in the regulation of cellular processes. Specific patterns have been found in the underlying DNA sequences of the nucleosome, and one of the most important patterns includes dinucleotides distributed every 10 to 11 base pairs. Based on this property, we propose to match each dinucleotide in the sequence against its mirror occurrences for 10 to 11 base pairs on both left-hand and right­hand sides. A large number of matches in a local region will then signify the existence of a nucleosome. In this paper, we propose the matched mirror position filters for efficient matching of periodic dinucleotide patterns and computationally predict the nucleosome positions. Experimental results on the Saccharomyces cerevisiae (yeast) genome show that the proposed algorithm can predict nucleosome positions effectively. More than 50% of our predicted nucleosomes are within 35 base pairs of those detected by biological experiments.     

The core histone tail domains contribute to sequence-dependent nucleosome positioning

The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.     

Prediction of nucleosome occupancy in Saccharomyces cerevisiae using position-correlation scoring function

Knowledge of the detailed organization of nucleosomes across genomes and the mechanisms of nucleosome positioning is critical for the understanding of gene regulation and expression. In the present work, the bias of 4-mer frequency in nucleosome and linker sequences of the S. cerevisiae genome was analyzed statistically. A novel position-correlation scoring function algorithm based on the bias of 4-mer frequency in linker sequences was presented to distinguish nucleosome vs linker sequences. Five-fold cross-validation demonstrated that the algorithm achieved a good performance with mean area under the receiver operator characteristics curve of 0.981. Next, the algorithm was used to predict nucleosome occupancy throughout the S. cerevisiae genome and relatively high correlation coefficients with experiment maps of nucleosome positioning were obtained. Besides, the distinct nucleosome depleted regions in the vicinity of regulatory sites were confirmed. The results suggest that intrinsic DNA sequence preferences in linker regions have a significant impact on the nucleosome occupancy.     

Human nucleosomes: special role of CG dinucleotides and Alu-nucleosomes

Background

The periodical occurrence of dinucleotides with a period of 10.4 bases now is undeniably a hallmark of nucleosome positioning. Whereas many eukaryotic genomes contain visible and even strong signals for periodic distribution of dinucleotides, the human genome is rather featureless in this respect. The exact sequence features in the human genome that govern the nucleosome positioning remain largely unknown.

Results

When analyzing the human genome sequence with the positional autocorrelation method, we found that only the dinucleotide CG shows the 10.4 base periodicity, which is indicative of the presence of nucleosomes. There is a high occurrence of CG dinucleotides that are either 31 (10.4 × 3) or 62 (10.4 × 6) base pairs apart from one another - a sequence bias known to be characteristic of Alu-sequences. In a similar analysis with repetitive sequences removed, peaks of repeating CG motifs can be seen at positions 10, 21 and 31, the nearest integers of multiples of 10.4.

Conclusions

Although the CG dinucleotides are dominant, other elements of the standard nucleosome positioning pattern are present in the human genome as well. The positional autocorrelation analysis of the human genome demonstrates that the CG dinucleotide is, indeed, one visible element of the human nucleosome positioning pattern, which appears both in Alu sequences and in sequences without repeats. The dominant role that CG dinucleotides play in organizing human chromatin is to indicate the involvement of human nucleosomes in tuning the regulation of gene expression and chromatin structure, which is very likely due to cytosine-methylation/-demethylation in CG dinucleotides contained in the human nucleosomes. This is further confirmed by the positions of CG-periodical nucleosomes on Alu sequences. Alu repeats appear as monomers, dimers and trimers, harboring two to six nucleosomes in a run. Considering the exceptional role CG dinucleotides play in the nucleosome positioning, we hypothesize that Alu-nucleosomes, especially, those that form tightly positioned runs, could serve as "anchors" in organizing the chromatin in human cells.