Targeted deletion of the MLC1f/3f downstream enhancer results in precocious MLC expression and mesoderm ablation.
Targeted deletion of the MLC1f/3f downstream enhancer results in precocious MLC expression and mesoderm ablation.
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MLC1f/3f 下游增强子的靶向缺失会导致 MLC 表达过早和中胚层消融。
DOI：
10.1006/dbio.2002.0574
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Rosenthal,Nadia
中科院分区：
文献类型：
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作者：
Jiang,Ping;Song,Jihwan;Gu,Guoqing;Slonimsky,Esfir;Li,En;Rosenthal,Nadia
骨骼肌收缩蛋白的表达在胚胎发育过程中受到严格调节。在小鼠中，肌球蛋白轻链 (MLC) 1f/3f 基因位点直到 E9.5 才被激活，仅在骨骼肌前体细胞中。 MLC1f/3f 基因座下游的有效增强子可在转基因小鼠胚胎中赋予所连接的报告基因正确的时间和空间激活。为了检查 MLC 下游增强子 (MLCE) 在 MLC1f/3f 基因位点的天然环境中的作用，我们通过定向删除从小鼠基因组中消除了包含增强子的 1.5 kb DNA 片段，在删除位点不留下外源序列。 MLCE 缺失纯合的小鼠胚胎较小且发育迟缓，到 E7.5 时未形成中胚层，并且在 E8.5 时几乎完全被吸收。对 E7.5 受影响的突变胚胎进行原位杂交和 RT-PCR 分析，揭示了异位 MLC 转录物，预计其产物会干扰决定中胚层分化的多种非肌肉细胞功能。这些结果表明，MLC下游增强子及其侧翼序列包含负调控元件，其在发育的关键窗口期间阻止中胚层前体中MLC表达的过早激活，以及随后允许分化骨骼肌中组织限制性MLC转录的正元件。
The expression of skeletal muscle contractile proteins is tightly regulated during embryonic development. In the mouse, the myosin light chain (MLC) 1f/3f gene locus is not activated until E9.5, exclusively in skeletal muscle precursor cells. A potent enhancer downstream of the MLC1f/3f locus confers correct temporal and spatial activation of linked reporter gene in transgenic mouse embryos. To examine roles of the MLC downstream enhancer (MLCE) in its native context of the MLC1f/3f gene locus, we eliminated a 1.5-kb DNA segment containing the enhancer from the mouse genome by targeted deletion, leaving no exogenous sequences at the deletion site. Mouse embryos homozygous for the MLCE deletion were smaller and developmentally delayed, formed no mesoderm by E7.5, and were resorbed almost completely at E8.5. In situ hybridization and RT-PCR analyses of affected mutant embryos at E7.5 revealed ectopic MLC transcripts, whose products would be predicted to interfere with a variety of nonmuscle cell functions determining differentiation of mesoderm. These results suggest that the MLC downstream enhancer and its flanking sequences include negative regulatory elements which block precocious activation of MLC expression in mesodermal precursors during a critical window of development, as well as positive elements which subsequently permit tissue-restricted MLC transcription in differentiating skeletal muscles.