Maconochie M K, Nonchev S, Studer M, Chan S K, Pöpperl H, Sham M H, Mann R S, Krumlauf R
Laboratory of Developmental Neurobiology, Medical Research Council (MRC), National Institute for Medical Research (NIMR), London, UK.
Genes Dev. 1997 Jul 15;11(14):1885-95. doi: 10.1101/gad.11.14.1885.
Correct regulation of the segment-restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene.
正确调控Hox基因表达的节段限制模式对于脊椎动物后脑的正常模式形成至关重要。我们通过在转基因小鼠中进行缺失分析,从小鼠基因中鉴定出一个r4增强子,从而研究了Hoxb2在菱脑节4(r4)中限制表达的分子基础。一个二分的Hox/Pbx结合基序位于该增强子内,体外DNA结合实验表明,脊椎动物唇相关蛋白Hoxb1将以Pbx/Exd依赖的方式与该位点协同结合。Hoxb2 r4增强子可通过Hoxb1、Hoxa1和果蝇唇蛋白在转基因小鼠中的异位表达在体内被反式激活。相反,异位的Hoxb2和Hoxb4无法诱导表达,这表明在体内该增强子优先响应唇蛋白家族成员。突变分析表明,二分的Hox/Pbx基序是r4增强子活性以及对视黄酸和异位Hox表达反应所必需的。此外,三个拷贝的Hoxb2基序足以介导转基因小鼠胚胎中的r4表达和果蝇胚胎中的唇蛋白模式。这种在果蝇胚胎中的报告基因表达依赖于内源性唇蛋白和exd,这表明该Hox/Pbx位点与唇相关蛋白相互作用的能力在进化上是保守的。在Hoxb1纯合突变胚胎中,内源性Hoxb2基因在r4中不再上调。基于这些实验,我们得出结论,后脑Hoxb2的r4限制域是Hoxb1通过涉及脊椎动物Pbx蛋白作为辅因子的直接交叉调节相互作用的结果。这表明Hoxb1在维持r4特征中的部分功能作用可能由Hoxb2基因介导。