Davis R J, Bennicelli J L, Macina R A, Nycum L M, Biegel J A, Barr F G
Department of Pathology and Labortory Medicine, University of Pennsylvania of Medicine, Phildelphia 19104-6082, USA.
Hum Mol Genet. 1995 Dec;4(12):2355-62. doi: 10.1093/hmg/4.12.2355.
The FKHR gene, which contains a forkhead DNA-binding motif, is fused to either PAX3 or PAX7 by the t(2;13) or t(1;13) translocation in alveolar rhabdomyosarcoma,respectively. These tumors express chimeric transcripts encoding the N-terminal portion of either PAX protein fused to the C-terminal portion of FKHR. To understand the structural basis and functional consequences of these translocations, we characterized the wild-type FKHR gene and its rearrangement in alveolar rhabdomyosarcomas. By isolating and analyzing phage, cosmid and YAC clones, we determined that FKHR consists of three exons spanning 140 kb and that several highly similar loci are present in other genomic regions. Exon 1 encodes the N-terminus of the forkhead domain and is embedded within demethylated CpG island. RNA analyses reveal FKHR transcripts initiate from a TATA-less promoter within this island. Exon 2 encodes the C-terminus of the forkhead domain and a transcription activation domain, whereas exon 3 encodes a large 3' untranslated region. The intron 1-exon 2 boundary precisely matches the FHKR fusion point in the chimeric transcripts found in alveolar rhabdomyosarcomas. Using pulsed-field and fluorescence in situ hybridization analyses, we demonstrate that the 130kb FKHR intron 1 is rearranged in t(2;13)-containing alveolar rhabdomyosarcomas. Our findings indicate that FKHR intron 1 provides a large target for DNA rearrangemnt. Rearrangement of this intron with PAX3 produces two important functional consequences: in-frame fusion of N-terminal PAX3 sequences to the FKHR transcriptional activation domain and disruption of the FKHR DNA binding domain.
FKHR基因含有一个叉头DNA结合基序，在肺泡横纹肌肉瘤中，通过t(2;13)或t(1;13)易位分别与PAX3或PAX7融合。这些肿瘤表达嵌合转录本，编码与FKHR C端部分融合的PAX蛋白的N端部分。为了了解这些易位的结构基础和功能后果，我们对肺泡横纹肌肉瘤中的野生型FKHR基因及其重排进行了表征。通过分离和分析噬菌体、黏粒和酵母人工染色体克隆，我们确定FKHR由跨越140 kb的三个外显子组成，并且在其他基因组区域存在几个高度相似的基因座。外显子1编码叉头结构域的N端，嵌入在去甲基化的CpG岛中。RNA分析显示FKHR转录本从该岛内一个无TATA框的启动子起始。外显子2编码叉头结构域的C端和一个转录激活结构域，而外显子3编码一个大的3'非翻译区。内含子1-外显子2边界与肺泡横纹肌肉瘤中发现的嵌合转录本中的FHKR融合点精确匹配。使用脉冲场和荧光原位杂交分析，我们证明在含有t(2;13)的肺泡横纹肌肉瘤中130kb的FKHR内含子1发生了重排。我们的研究结果表明FKHR内含子1为DNA重排提供了一个大的靶点。该内含子与PAX3的重排产生了两个重要的功能后果：N端PAX3序列与FKHR转录激活结构域的读码框内融合以及FKHR DNA结合结构域的破坏。
