染色质免疫沉淀分析 HMGA2 的 DNA 结合位点。
Chromatin immunoprecipitation to analyze DNA binding sites of HMGA2.
机构信息
Centre for Human Genetics, University of Bremen, Bremen, Germany.
出版信息
PLoS One. 2011 Apr 14;6(4):e18837. doi: 10.1371/journal.pone.0018837.
BACKGROUND
HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure two consensus sequences for HMGA2 binding have been identified.
METHODOLOGY/PRINCIPAL FINDINGS: In this investigation chromatin immunoprecipitation (ChIP) experiments and bioinformatic methods were used to analyze if these binding sequences can be verified on chromatin of living cells as well.
CONCLUSION
After quantification of HMGA2 protein in different cell lines the colon cancer derived cell line HCT116 was chosen for further ChIP experiments because of its 3.4-fold higher HMGA2 protein level. 49 DNA fragments were obtained by ChIP. These fragments containing HMGA2 binding sites have been analyzed for their AT-content, location in the human genome and similarities to sequences generated by a SELEX study. The sequences show a significantly higher AT-content than the average of the human genome. The artificially generated SELEX sequences and short BLAST alignments (11 and 12 bp) of the ChIP fragments from living cells show similarities in their organization. The flanking regions are AT-rich, whereas a lower conservation is present in the center of the sequences.
背景
HMGA2 是一种结构转录因子，在胚胎和胎儿发育过程中大量表达，与恶性肿瘤的进展有关。该蛋白含有三个基本带电的 DNA 结合域和一个酸性蛋白结合的 C 末端结构域。DNA 结合诱导 DNA 构象的变化，从而导致基因表达模式的整体变化。最近，通过基于 PCR 的 SELEX（指数富集的配体系统进化）程序，已经鉴定出两种 HMGA2 结合的共有序列。
方法/主要发现：在本研究中，使用染色质免疫沉淀（ChIP）实验和生物信息学方法分析这些结合序列是否可以在活细胞的染色质上得到验证。
结论
在定量不同细胞系中的 HMGA2 蛋白后，由于其 HMGA2 蛋白水平高 3.4 倍，选择结肠癌衍生的细胞系 HCT116 进行进一步的 ChIP 实验。通过 ChIP 获得了 49 个 DNA 片段。这些含有 HMGA2 结合位点的片段已针对其 AT 含量、在人类基因组中的位置以及与 SELEX 研究生成的序列的相似性进行了分析。这些序列的 AT 含量明显高于人类基因组的平均值。人工生成的 SELEX 序列和来自活细胞的 ChIP 片段的短 BLAST 比对（11 和 12 个碱基）在其组织上显示出相似性。侧翼区域富含 AT，而序列的中心则存在较低的保守性。
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