Multiple, distant Gata2 enhancers specify temporally and tissue-specific patterning in the developing urogenital system
Multiple, distant Gata2 enhancers specify temporally and tissue-specific patterning in the developing urogenital system
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DOI：
10.1128/mcb.24.23.10263-10276.2004
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发表时间：
2004-12-01
影响因子：
5.3
通讯作者：
Engel, JD
中科院分区：
文献类型：
--
作者：
Khandekar, M;Suzuki, N;Engel, JD
转录因子加塔-2在发育中的胚胎中以复杂的时间和组织特异性模式表达。在小鼠中的功能丧失研究表明，在非常早期的造血过程中首先需要加塔-2活性。我们随后表明，271 kbp的酵母人工染色体（YAC）转基因可以完全弥补Gata 2造血功能的丧失，但这些YAC拯救的Gata 2无效突变小鼠死于围产期由于泌尿生殖发育缺陷。挽救的YAC没有显示Gata 2的适当的泌尿生殖道表达，这意味着存在位于该转基因边界之外的泌尿生殖道特异性增强子。在这里，我们概述了一个耦合的调控序列发现的一般策略，将生物信息学功能基因组学的基础上，用于产生小鼠基因组序列的细菌人工染色体（BAC）库。利用这种策略，我们筛选了Gata 2周围> 1 Mbp的基因组DNA的泌尿生殖增强子活性。我们发现Gata 2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增强子元件赋予，其中两个位于Gata 2结构基因的3'端。包括使用这种策略发现的额外增强子（称为BAC陷阱）将Gata 2基因座的功能范围扩展到大于1 Mbp。
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.