Department of Medicine, Division of Medical Genetics, University of Washington , Seattle, WA 98195, USA.
Hum Gene Ther. 2012 Feb;23(2):231-7. doi: 10.1089/hum.2010.177. Epub 2011 Dec 14.
Concerns surrounding the oncogenic potential of recombinant gammaretroviral vectors has spurred a great deal of interest in vector integration site (VIS) preferences. Although gammaretroviral vectors exhibit a modest preference for integration near transcription start sites (TSS) of active genes, such associations only account for about a third of all VIS. Previous studies suggested a correlation between gammaretroviral VIS and DNase hypersensitive sites (DHS), which mark chromatin regions associated with cis-regulatory elements. In order to study this issue directly, we assessed the correlation between 167 validated gammaretroviral VIS and a deep genome-wide map of DHS, both determined in the same cell line (the human fibrosarcoma HT1080). The DHS map was developed by sequencing individual DNase I cleavage sites using massively parallel sequencing technologies. These studies revealed an overwhelming preference for integrations associated with DHS, with a median distance of only 238 bp between individual VIS and the nearest DHS for the experimental dataset, compared to 3 kb for a random dataset and 577 to 1457 bp for two unrelated cell lines (p<0.001). Indeed, nearly 84% of all VIS were found to be located within 1 kb of a DHS (p=10(-43)). Further, this correlation was statistically independent from the association with TSS. The preference for DHS far exceeds that seen for other hallmarks of gammaretroviral VIS, including TSS, and may help explain several aspects of gammaretroviral vector biology, including the mechanism of VIS selection, as well as the relative frequency and underlying biology of gammaretroviral vector-mediated genotoxicity.
人们对重组γ逆转录病毒载体的致癌潜能感到担忧，这极大地激发了人们对载体整合位点（VIS）偏好的兴趣。虽然γ逆转录病毒载体表现出对靠近活性基因转录起始位点（TSS）的整合有适度的偏好，但这种关联仅占所有 VIS 的约三分之一。先前的研究表明，γ逆转录病毒 VIS 与 DNA 酶超敏位点（DHS）之间存在相关性，DHS 标记了与顺式调控元件相关的染色质区域。为了直接研究这个问题，我们评估了在相同细胞系（人纤维肉瘤 HT1080）中确定的 167 个已验证的γ逆转录病毒 VIS 与 DHS 的全基因组深度图谱之间的相关性。DHS 图谱是通过使用大规模平行测序技术对单个 DNA 酶 I 切割位点进行测序来开发的。这些研究表明，与 DHS 相关的整合具有压倒性的偏好，对于实验数据集，个体 VIS 和最近的 DHS 之间的中位数距离仅为 238bp，而对于随机数据集为 3kb，对于两个不相关的细胞系为 577 到 1457bp（p<0.001）。事实上，几乎 84%的所有 VIS 都位于 DHS 内 1kb 范围内（p=10(-43)）。此外，这种相关性与与 TSS 的关联在统计学上是独立的。这种对 DHS 的偏好远远超过了其他γ逆转录病毒 VIS 的标志，包括 TSS，这可能有助于解释γ逆转录病毒载体生物学的几个方面，包括 VIS 选择的机制，以及γ逆转录病毒载体介导的遗传毒性的相对频率和潜在生物学。
