Ciona Brachyury proximal and distal enhancers have different FGF dose-response relationships.
Ciona Brachyury proximal and distal enhancers have different FGF dose-response relationships.
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DOI：
10.1371/journal.pgen.1009305
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发表时间：
2021-01
期刊：
影响因子：
4.5
通讯作者：
Veeman MT
中科院分区：
文献类型：
--
作者：
Harder MJ;Hix J;Reeves WM;Veeman MT
许多基因受到两个或更多能驱动相似表达模式的增强子调控。进化理论表明，这些看似冗余的增强子必然存在功能上的重要差异。在简单的脊索动物文昌鱼中，转录因子Brachyury仅在谱系特异性调控因子和FGF（成纤维细胞生长因子）响应的Ets家族转录因子下游的预定脊索中被诱导。在此，我们利用MEK抑制剂U0126通过MAPK（丝裂原活化蛋白激酶）途径精细调节FGF信号传导活性的能力，来量化不同的Brachyury报告基因构建体所驱动的转录对这种直接上游调控因子的依赖性。我们发现，更强的邻近启动子的近端增强子和较弱的远端增强子对MAPK抑制具有根本不同的剂量 - 反应关系。远端增强子对MAPK抑制更敏感，但协同反应较弱，而近端增强子敏感性较低但协同性更强。包含两个增强子的较长构建体具有复杂的剂量 - 反应曲线，这支持了近端和远端增强子具有适度超加性的观点。我们表明，中等剂量的U0126导致的总体表达缺失不仅是表达这些报告基因的细胞比例的函数，还涉及单细胞水平表达的梯度下降。内源基因的表达与全长报告基因显示出可比的剂量 - 反应关系，并且我们发现不同的脊索前体细胞对MAPK抑制具有不同的敏感性。总之，这些结果表明，尽管两个Brachyury增强子具有定性相似的表达模式，但它们对FGF的反应在定量上不同，并共同作用以具有特定输入/输出关系的方式驱动高水平的Brachyury表达。这表明它们在根本上不是等效的遗传元件。
基因在何时何地表达是由被称为增强子的调控DNA区域控制的。基因通常具有多个增强子，它们控制在不同细胞类型或胚胎区域的表达，但也有一些基因具有多个控制近乎相同表达模式的增强子。这些“影子”增强子是许多动物基因组的常见特征，但它们在功能上在多大程度上真正相同尚不清楚。在这里，我们研究了简单模式脊索动物文昌鱼中脊索特异性基因Brachyury的一对影子增强子，它们都由相同的信号通路直接激活。我们用不同剂量的药物抑制剂调节该通路的活性，并在定量增强子分析中测量其影响。我们发现这两个增强子对相同的共享调控因子在敏感性和协同性方面存在显著差异，因此在定量评估时功能并不相同。我们还确定了不同脊索前体细胞对这种上游信号敏感性的细微差异。
Many genes are regulated by two or more enhancers that drive similar expression patterns. Evolutionary theory suggests that these seemingly redundant enhancers must have functionally important differences. In the simple ascidian chordate Ciona, the transcription factor Brachyury is induced exclusively in the presumptive notochord downstream of lineage specific regulators and FGF-responsive Ets family transcription factors. Here we exploit the ability to finely titrate FGF signaling activity via the MAPK pathway using the MEK inhibitor U0126 to quantify the dependence of transcription driven by different Brachyury reporter constructs on this direct upstream regulator. We find that the more powerful promoter-adjacent proximal enhancer and a weaker distal enhancer have fundamentally different dose-response relationships to MAPK inhibition. The Distal enhancer is more sensitive to MAPK inhibition but shows a less cooperative response, whereas the Proximal enhancer is less sensitive and more cooperative. A longer construct containing both enhancers has a complex dose-response curve that supports the idea that the proximal and distal enhancers are moderately super-additive. We show that the overall expression loss from intermediate doses of U0126 is not only a function of the fraction of cells expressing these reporters, but also involves graded decreases in expression at the single-cell level. Expression of the endogenous gene shows a comparable dose-response relationship to the full length reporter, and we find that different notochord founder cells are differentially sensitive to MAPK inhibition. Together, these results indicate that although the two Brachyury enhancers have qualitatively similar expression patterns, they respond to FGF in quantitatively different ways and act together to drive high levels of Brachyury expression with a characteristic input/output relationship. This indicates that they are fundamentally not equivalent genetic elements. When and where genes are expressed is controlled by regulatory DNA regions known as enhancers. Genes often have multiple enhancers that control expression in different cell types or embryonic regions, but there are also genes that have multiple enhancers that control near-identical expression patterns. These ‘shadow’ enhancers are common features of many animal genomes, but it is unclear to what extent they are truly identical in function. Here we studied a pair of shadow enhancers for the notochord-specific gene Brachyury in the simple model chordate Ciona that are both directly activated by the same signaling pathway. We titrated the activity of this pathway with graded doses of a pharmacological inhibitor and measured the effects in quantitative enhancer assays. We found that the two enhancers had significant differences in sensitivity and cooperativity to the same shared regulator and are thus not identical in function when assessed quantitatively. We also identified subtle differences in sensitivity to this upstream signal between different notochord precursor cells.
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影响因子：
64.8
作者：
通讯作者：
--
影响因子：
2.7
作者：
Howard, ML;Davidson, EH
通讯作者：
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DOI：
10.1016/j.cub.2016.02.054
发表时间：
2016-05-09
期刊：
Current biology : CB
影响因子：
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作者：
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通讯作者：
Levine M
影响因子：
2.7
作者：
Matsumoto, Jun;Kumano, Gaku;Nishida, Hiroki
通讯作者：
Nishida, Hiroki
影响因子：
64.8
作者：
Osterwalder M;Barozzi I;Tissières V;Fukuda-Yuzawa Y;Mannion BJ;Afzal SY;Lee EA;Zhu Y;Plajzer-Frick I;Pickle CS;Kato M;Garvin TH;Pham QT;Harrington AN;Akiyama JA;Afzal V;Lopez-Rios J;Dickel DE;Visel A;Pennacchio LA
通讯作者：
Pennacchio LA