Exploring trophoblast-specific Tead4 enhancers through chromatin conformation capture assays followed by functional screening
Exploring trophoblast-specific Tead4 enhancers through chromatin conformation capture assays followed by functional screening
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DOI：
10.1093/nar/gkz1034
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发表时间：
2019-11
影响因子：
14.9
通讯作者：
Junko Tomikawa;S. Takada;K. Okamura;Miho Terao;Hiroko Ogata-Kawata;H. Akutsu;Satoshi Tanaka;K. Hata;K. Nakabayashi
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文献类型：
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作者：
Junko Tomikawa;S. Takada;K. Okamura;Miho Terao;Hiroko Ogata-Kawata;H. Akutsu;Satoshi Tanaka;K. Hata;K. Nakabayashi
摘要Tead 4在胚胎发育和滋养层细胞分化中起重要作用。我们在小鼠胚胎干细胞（ES）和滋养层干细胞（TS）中检测了Tead 4启动子的长距离染色体相互作用。使用荧光素酶报告基因检测与ES和TS细胞的34个候选增强子区域，我们确定了5个基因组片段，增加Tead 4启动子活性在TS特异性的方式。这5个位点由3个染色体内和2个染色体间位点组成，相对于6号染色体上的Tead 4。我们建立了五个小鼠品系，其中五个增强子元件删除，并评估了每个删除对Tead 4在囊胚中表达的影响。通过定量RT-PCR，我们测量了Tead 4表达在19号染色体上具有1.5 kb基因组间隔的纯合缺失的囊胚中（n = 14）比在野生型囊胚中降低42%。通过进行RNA-seq分析，我们证实了该增强子缺失对Tead 4的反式效应，而对其邻近基因的顺式效应至少在1.7 Mb距离内没有显著影响。我们的研究结果表明，染色体19上的基因组间隔所需的适当水平的Tead 4在囊胚中的表达，并建议，染色体间的增强子-启动子相互作用可能是潜在的机制。
Abstract Tead4 is critical for blastocyst development and trophoblast differentiation. We assayed long-range chromosomal interactions on the Tead4 promoter in mouse embryonic stem (ES) cells and trophoblast stem (TS) cells. Using luciferase reporter assays with ES and TS cells for 34 candidate enhancer regions, we identified five genomic fragments that increased Tead4 promoter activity in a TS-specific manner. The five loci consisted of three intra- and two inter-chromosomal loci relative to Tead4 on chromosome 6. We established five mouse lines with one of the five enhancer elements deleted and evaluated the effect of each deletion on Tead4 expression in blastocysts. By quantitative RT-PCR, we measured a 42% decrease in Tead4 expression in the blastocysts with a homozygous deletion with a 1.5 kb genomic interval on chromosome 19 (n = 14) than in wild-type blastocysts. By conducting RNA-seq analysis, we confirmed the trans effect of this enhancer deletion on Tead4 without significant cis effects on its neighbor genes at least within a 1.7 Mb distance. Our results demonstrated that the genomic interval on chromosome 19 is required for the appropriate level of Tead4 expression in blastocysts and suggested that an inter-chromosomal enhancer-promoter interaction may be the underlying mechanism.