Evaluation of MYC E-box phylogenetic footprints in glycolytic genes by chromatin immunoprecipitation assays
Evaluation of MYC E-box phylogenetic footprints in glycolytic genes by chromatin immunoprecipitation assays
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DOI：
10.1128/mcb.24.13.5923-5936.2004
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发表时间：
2004-07-01
影响因子：
5.3
通讯作者：
Dang, CV
中科院分区：
文献类型：
--
作者：
Kim, JW;Zeller, KI;Dang, CV
利用系统发育足迹法预测基因调控序列已经取得了相当大的进展，但缺乏实验验证。在这里，我们报告是否可以通过染色质免疫沉淀试验验证点绘图或基于网络的Trafac分析预测的转录因子结合位点。MYC过表达增强糖酵解而不缺氧，因此可能有助于改变肿瘤代谢。因为由Myc直接调控的糖酵解基因的全部谱是未知的，所以我们选择Myc作为模型转录因子以确定其是否结合具有保守的典型Myc结合位点或E盒（5 '-CACGTG-3'）的靶糖酵解基因。ENO 1、HK 2和LDHA中保守的典型E盒出现在31至111 bp的岛屿中，具有高物种间序列同一性（>65%）。Trafac分析揭示了ENO 1中的另一个区域，该区域对应于具有非经典E盒的鼠区域。Myc在人P493-6 B淋巴细胞中与所有这些保守区域都有很好的结合。我们还确定了Myc是否可以结合在剩余的人类糖酵解基因中发现的非保守的典型E盒。Myc结合PFKM，但不显著结合GPI、PGK 1和PKM 2。未检测到与BPGM、PGAM 2和PKLR的结合。GAPD和TPI 1都不具有保守的E盒，而是通过具有非典型E盒的区域被Myc诱导和结合。我们的研究结果表明，Myc很好地结合到保守的典型E盒，但不是非保守的E盒。然而，Myc与具有非典型E盒的不可预测的基因组区域的结合揭示了系统发育足迹的局限性。总之，这些观察结果表明，Myc是糖酵解基因的重要调节因子，表明MYC在细胞增殖或肿瘤发生期间糖酵解代谢的转换中起关键作用。
Prediction of gene regulatory sequences using phylogenetic footprinting has advanced considerably but lacks experimental validation. Here, we report whether transcription factor binding sites predicted by dot plotting or web-based Trafac analysis could be validated by chromatin immunoprecipitation assays. MYC overexpression enhances glycolysis without hypoxia and hence may contribute to altered tumor metabolism. Because the full spectrum of glycolytic genes directly regulated by Myc is not known, we chose Myc as a model transcription factor to determine whether it binds target glycolytic genes that have conserved canonical Myc binding sites or E boxes (5'-CACGTG-3'). Conserved canonical E boxes in ENO1, HK2, and LDHA occur in 31- to 111-bp islands with high interspecies sequence identity (>65%). Trafac analysis revealed another region in ENO1 that corresponds to a murine region with a noncanonical E box. Myc bound all these conserved regions well in the human P493-6 B lymphocytes. We also determined whether Myc could bind nonconserved canonical E boxes found in the remaining human glycolytic genes. Myc bound PFKM, but it did not significantly bind GPI, PGK1, and PKM2. Binding to BPGM, PGAM2, and PKLR was not detected. Both GAPD and TPI1 do not have conserved E boxes but are induced and bound by Myc through regions with noncanonical E boxes. Our results indicate that Myc binds well to conserved canonical E boxes, but not nonconserved E boxes. However, the binding of Myc to unpredicted genomic regions with noncanonical E boxes reveals a limitation of phylogenetic footprinting. In aggregate, these observations indicate that Myc is an important regulator of glycolytic genes, suggesting that MYC plays a key role in a switch to glycolytic metabolism during cell proliferation or tumorigenesis.