Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Genome Res. 2011 Mar;21(3):377-89. doi: 10.1101/gr.111328.110. Epub 2010 Dec 20.
We have used a novel bubble-trapping procedure to construct nearly pure and comprehensive human origin libraries from early S- and log-phase HeLa cells, and from log-phase GM06990, a karyotypically normal lymphoblastoid cell line. When hybridized to ENCODE tiling arrays, these libraries illuminated 15.3%, 16.4%, and 21.8% of the genome in the ENCODE regions, respectively. Approximately half of the origin fragments cluster into zones, and their signals are generally higher than those of isolated fragments. Interestingly, initiation events are distributed about equally between genic and intergenic template sequences. While only 13.2% and 14.0% of genes within the ENCODE regions are actually transcribed in HeLa and GM06990 cells, 54.5% and 25.6% of zonal origin fragments overlap transcribed genes, most with activating chromatin marks in their promoters. Our data suggest that cell synchronization activates a significant number of inchoate origins. In addition, HeLa and GM06990 cells activate remarkably different origin populations. Finally, there is only moderate concordance between the log-phase HeLa bubble map and published maps of small nascent strands for this cell line.
我们使用了一种新颖的气泡捕获方法，从早期 S 期和对数期 HeLa 细胞以及染色体正常的淋巴母细胞系 GM06990 构建了几乎纯净和全面的人类起源文库。当这些文库与 ENCODE 平铺阵列杂交时，它们分别照亮了 ENCODE 区域中基因组的 15.3%、16.4%和 21.8%。大约一半的起始点片段聚集成区，它们的信号通常高于孤立片段的信号。有趣的是，起始事件在基因和基因间模板序列之间大致均匀分布。虽然 ENCODE 区域内的基因只有 13.2%和 14.0%在 HeLa 和 GM06990 细胞中实际转录，但 54.5%和 25.6%的区起始片段与转录基因重叠，大多数在其启动子中具有激活染色质标记。我们的数据表明细胞同步化激活了大量早期起始点。此外，HeLa 和 GM06990 细胞激活了显著不同的起始点群体。最后，对数期 HeLa 气泡图谱与该细胞系的小新生链的已发表图谱之间只有适度的一致性。
