Lowary P T, Widom J
Department of Biochemistry Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208-3500, USA.
J Mol Biol. 1998 Feb 13;276(1):19-42. doi: 10.1006/jmbi.1997.1494.
DNA sequences that position nucleosomes are of increasing interest because of their relationship to gene regulation in vivo and because of their utility in studies of nucleosome structure and function in vitro. However, at present our understanding of the rules for DNA sequence-directed nucleosome positioning is fragmentary, and existing positioning sequences have many limitations. We carried out a SELEX experiment starting with a large pool of chemically synthetic random. DNA molecules to identify those individuals having the highest affinity for histone octamer. A set of highest-affinity molecules were selected, cloned, and sequenced, their affinities (free energies) for histone octamer in nucleosome reconstitution measured, and their ability to position nucleosomes in vitro assessed by native gel electrophoresis. The selected sequences have higher affinity than previously known natural or non-natural sequences, and have a correspondingly strong nucleosome positioning ability. A variety of analyses including Fourier transform, real-space correlation, and direct counting computations were carried out to assess non-random features in the selected sequences. The results reveal sequence rules that were already identified in earlier studies of natural nucleosomal DNA, together with a large set of new rules having even stronger statistical significance. Possible physical origins of the selected molecules' high affinities are discussed. The sequences isolated in this study should prove valuable for studies of chromatin structure and function in vitro and, potentially, for studies in vivo.
由于核小体定位的DNA序列与体内基因调控相关，且在体外核小体结构和功能研究中具有实用性，因此越来越受到关注。然而，目前我们对DNA序列指导核小体定位规则的理解尚不完整，现有的定位序列存在诸多局限性。我们开展了一项指数富集的配体系统进化（SELEX）实验，起始于大量化学合成的随机DNA分子，以鉴定那些对组蛋白八聚体具有最高亲和力的分子。挑选出一组具有最高亲和力的分子，进行克隆和测序，测定它们在核小体重组中对组蛋白八聚体的亲和力（自由能），并通过非变性凝胶电泳评估它们在体外定位核小体的能力。所选序列比先前已知的天然或非天然序列具有更高的亲和力，相应地具有更强的核小体定位能力。我们进行了包括傅里叶变换、实空间相关性和直接计数计算在内的多种分析，以评估所选序列中的非随机特征。结果揭示了在早期天然核小体DNA研究中已确定的序列规则，以及大量具有更强统计学意义的新规则。文中还讨论了所选分子高亲和力可能的物理起源。本研究中分离出的序列应会被证明对体外染色质结构和功能研究有价值，并且可能对体内研究也有价值。
