Candidate downstream regulated genes of HOX group 13 transcription factors with and without monomeric DNA binding capability.
Candidate downstream regulated genes of HOX group 13 transcription factors with and without monomeric DNA binding capability.
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DOI：
10.1016/j.ydbio.2004.12.015
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发表时间：
2005-03
影响因子：
2.7
通讯作者：
Thomas M. Williams;Melissa E. Williams;R. Kuick;David E. Misek;K. McDonagh;S. Hanash;J. Innis
中科院分区：
文献类型：
--
作者：
Thomas M. Williams;Melissa E. Williams;R. Kuick;David E. Misek;K. McDonagh;S. Hanash;J. Innis
Hox 基因编码调节发育胚胎形态发生的转录因子。在哺乳动物中，对 HOX 蛋白调节的遗传途径（包括可能的直接或间接靶标）的了解极为有限。为了鉴定后 HOX 蛋白调节的下游基因，我们使用双顺反子 HOXA13/EGFP 逆转录病毒载体在缺乏旁系同源组 13 表达的小鼠胚胎成纤维细胞中表达 HOXA13。微阵列分析鉴定出稳定表达 HOXA13 的细胞中 68 个基因具有显着、可重复的 RNA 表达变化（50 个激活；18 个抑制）。带有 GO 注释术语“细胞外基质”和“基底膜”的基因被严重过度代表，并且在其他研究中，有几个基因被证明受到 HOX 蛋白的调节。 HOXA13 强烈激活的基因包括 Enpp2，这是一种双功能酶，已知可调节肿瘤和正常细胞运动，并在软骨前凝结中表达； Fhl1，一种参与肌肉细胞分化和发育的转录因子； M32486，一种在女性生殖道中表达的推定的完整膜分子。使用半定量 RT-PCR 证实了表达 HOXA13 的细胞中选定的下游基因的表达差异，并且许多人在体内与肢体指间充质中的 Hoxa13 共表达。对于两个候选者 Igfbp4 和 Fstl，Hoxa13 突变体中指间肢芽表达减少。为了探索旁系同源和非旁系同源 HOX 蛋白是否可以调节相同的基因，我们创建了新的 HOX 细胞系，并检查了 HOXA13 筛选所鉴定的选定基因的表达。 HOXD13 类似地激活/抑制 6 个测试候选者，证明多个下游遗传途径可能受到旁系同源 HOX 蛋白的调节。相比之下，HOXA9 只能抑制某些基因靶标的表达。 HOXD13 突变体 HOXD13IQN >AAA 无法与单体 DNA 结合，激活 5 个 HOXA13 上调基因的表达；但不能抑制 Ngef 和 Casp8ap2 的表达。我们的结果表明，没有直接 HOX DNA 结合的 HOX 蛋白质-蛋白质相互作用可能在 HOX 转录调控中发挥比一般假设更大的作用，并且 DNA 结合似乎对于抑制至关重要。
Hox genes encode transcription factors that regulate the morphogenesis of developing embryos. In mammals, knowledge of the genetic pathways, including the possible direct or indirect targets, regulated by HOX proteins is extremely limited. To identify the downstream genes regulated by posterior HOX proteins, we expressed HOXA13 in mouse embryonic fibroblasts lacking paralog group 13 expression using a bicistronic HOXA13/EGFP retroviral vector. Microarray analysis identified 68 genes with significant, reproducible RNA expression changes (50 activated; 18 repressed) in stable HOXA13-expressing cells. Genes with the GO annotation terms “extracellular matrix” and “basement membrane” were greatly overrepresented, and several were shown to be regulated by HOX proteins in other studies. Among the genes strongly activated by HOXA13 were Enpp2, a bifunctional enzyme known to modulate tumor and normal cell motility and which is expressed in precartilaginous condensations; Fhl1, a transcription factor implicated in muscle cell differentiation and development; and M32486, a putative integral membrane molecule expressed in the female reproductive tract. Expression differences in the HOXA13-expressing cells were confirmed for selected downstream genes using semi-quantitative RT-PCR, and in vivo coexpression with Hoxa13 in the limb interdigital mesenchyme was demonstrated for many. For two candidates, Igfbp4 and Fstl, interdigital limb bud expression was reduced in Hoxa13 mutants. To explore whether paralogous and nonparalogous HOX proteins could regulate the same genes, we created new HOX cell lines and examined the expression of selected genes identified by the HOXA13 screen. HOXD13 similarly activated/repressed 6 tested candidates, demonstrating that multiple downstream genetic pathways may be regulated by paralog HOX proteins. In contrast, HOXA9 was only able to repress expression of some gene targets. A HOXD13 mutant, HOXD13IQN >AAA, incapable of monomeric DNA-binding, activated the expression of 5 HOXA13-upregulated genes; but was incapable of repressing the expression of Ngef and Casp8ap2. Our results suggest that HOX protein–protein interactions without direct HOX DNA-binding may play a larger role in HOX transcriptional regulation than generally assumed, and DNA-binding appears critical for repression.