Khandekar Melin, Suzuki Norio, Lewton Jon, Yamamoto Masayuki, Engel James Douglas
Department of Cell and Developmental Biology, 4643 Med. Sci. II, 1335 Catherine St., Ann Arbor, MI 48109, USA.
Mol Cell Biol. 2004 Dec;24(23):10263-76. doi: 10.1128/MCB.24.23.10263-10276.2004.
Transcription factor GATA-2 is expressed in a complex temporally and tissue-specific pattern within the developing embryo. Loss-of-function studies in the mouse showed that GATA-2 activity is first required during very early hematopoiesis. We subsequently showed that a 271-kbp yeast artificial chromosome (YAC) transgene could fully complement the loss of Gata2 hematopoietic function but that these YAC-rescued Gata2 null mutant mice die perinatally due to defective urogenital development. The rescuing YAC did not display appropriate urogenital expression of Gata2, implying the existence of a urogenital-specific enhancer(s) lying outside the boundaries of this transgene. Here we outline a coupled general strategy for regulatory sequence discovery, linking bioinformatics to functional genomics based on the bacterial artificial chromosome (BAC) libraries used to generate the mouse genome sequence. Exploiting this strategy, we screened >1 Mbp of genomic DNA surrounding Gata2 for urogenital enhancer activity. We found that the spatially and tissue-specific functions for Gata2 in the developing urogenital system are conferred by at least three separate regionally and temporally specific urogenital enhancer elements, two of which reside far 3' to the Gata2 structural gene. Including the additional enhancers that were discovered using this strategy (called BAC trap) extends the functional realm of the Gata2 locus to greater than 1 Mbp.
转录因子GATA-2在发育中的胚胎内以复杂的时间和组织特异性模式表达。在小鼠中进行的功能丧失研究表明,GATA-2活性在非常早期的造血过程中首先是必需的。我们随后表明,一个271千碱基对的酵母人工染色体(YAC)转基因可以完全补充Gata2造血功能的丧失,但这些YAC拯救的Gata2基因敲除突变小鼠在围产期因泌尿生殖系统发育缺陷而死亡。拯救用的YAC没有显示出Gata2在泌尿生殖系统中的适当表达,这意味着在该转基因边界之外存在泌尿生殖系统特异性增强子。在这里,我们概述了一种用于调控序列发现的耦合通用策略,该策略基于用于生成小鼠基因组序列的细菌人工染色体(BAC)文库,将生物信息学与功能基因组学联系起来。利用这一策略,我们筛选了Gata2周围超过1兆碱基对的基因组DNA以寻找泌尿生殖系统增强子活性。我们发现,Gata2在发育中的泌尿生殖系统中的空间和组织特异性功能由至少三个独立的区域和时间特异性泌尿生殖系统增强子元件赋予,其中两个位于Gata2结构基因下游很远的位置。包括使用这种策略(称为BAC陷阱)发现的额外增强子,将Gata2基因座的功能范围扩展到大于1兆碱基对。