Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1–HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.

中文翻译：

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六氨基酸基序是HOX1蛋白功能进化的主要决定因素

基因复制和分歧是进化新事物出现的主要驱动力。氨基酸序列的变化如何导致祖先活性的丧失和蛋白质功能多样化的了解很少。我们使用果蝇唇及其小鼠HOX1直系同源物（HOXA1，HOXB1和HOXD1）的跨物种功能分析作为解决此问题的范例。小鼠HOX1蛋白与果蝇的序列相似性低（30％）唇唇 但是，用小鼠蛋白质替代内源性唇唇动物表明，HOXA1保留了唇唇动物的基本祖先功能，而HOXB1和HOXD1却有所不同。全基因组分析表明，HOXA1和Labial在小鼠细胞中具有相似的DNA结合模式，而HOXB1结合至不同的靶标。与HOXB1相比，HOXA1在目标位点上与PBX蛋白共存，并且与染色质上的PBX处于同一复合物中。HOXA1-HOXB1嵌合蛋白的功能分析揭示了位于同源域两侧的新型六氨基酸C端基序（CTM），它是祖先活动的主要决定因素。体外DNA结合实验和结构预测表明，CTM为HOXA1蛋白与PBX的相互作用提供了重要的域。