Nucleosome Mapping in S. pombe Centromeres. Schizosaccharomyces pombe

A key element for defining the centromere identity is the incorporation of a specific histone H3, CENP-A, known as Cnp1p in S. pombe. Previous studies have suggested that functional S. pombe centromeres lack nucleosome arrays and may involve chromatin remodeling as a key step of kinetochore assembly. We used tiling microarrays to show that nucleosomes are in fact positioned in regular intervals in the core of centromere 2, providing the first high resolution map of regional centromere chromatin. Nucleosome locations are not disrupted by mutations in kinetochore proteins cnp1, mis18, mis12, nuf2, mal2, overexpression of Cnp1p, or deletion of ams2. Bioinformatic analysis of the centromere sequence indicates certain enriched motifs in linker regions between nucleosomes and reveals a sequence-bias in nucleosome positioning. We conclude that centromeric nucleosome positions are stable and may be derived from the underlying DNA sequence. In addition, sequence analysis of nucleosome-free regions identifies novel binding sites for the GATA-like protein Ams2p, which participates in CENP-A incorporation. Keywords: Nucleosome Mapping Study Overall design: Entire cnt regions and histone-related genes were tiled at 1-5 bp spacing using 60-mer probes.

定义着丝粒（centromere）身份的关键要素是一种特异性组蛋白H3（histone H3），即着丝粒蛋白A（CENP-A）；在粟酒裂殖酵母（S. pombe）中，该蛋白被称为Cnp1p。既往研究表明，功能正常的粟酒裂殖酵母着丝粒缺乏核小体阵列（nucleosome arrays），且染色质重塑（chromatin remodeling）可能是动粒（kinetochore）组装的关键步骤。本研究借助平铺式微阵列（tiling microarray）证实，核小体（nucleosome）实际上在2号着丝粒核心区域以规则间隔排布，首次获得了区域型着丝粒染色质的高分辨率图谱。核小体定位并未因动粒蛋白cnp1、mis18、mis12、nuf2、mal2发生突变，或Cnp1p过表达、ams2基因缺失而受到干扰。对着丝粒序列进行的生物信息学分析显示，核小体间连接区存在特定富集基序，并揭示了核小体定位的序列偏好性。本研究结论指出，着丝粒核小体的定位具有稳定性，且可能由其所在的基因组DNA序列所决定。此外，对无核小体区域（nucleosome-free regions）的序列分析还鉴定出了类GATA蛋白Ams2p的新型结合位点，该蛋白参与CENP-A的掺入过程。关键词：核小体定位研究 整体实验设计：采用60聚体探针，以1-5 bp的间距对全部cnt区域及组蛋白相关基因进行平铺覆盖。