Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
PLoS Genet. 2021 Apr 26;17(4):e1009536. doi: 10.1371/journal.pgen.1009536. eCollection 2021 Apr.
Several distinct activities and functions have been described for chromatin insulators, which separate genes along chromosomes into functional units. Here, we describe a novel mechanism of functional separation whereby an insulator prevents gene repression. When the homie insulator is deleted from the end of a Drosophila even skipped (eve) locus, a flanking P-element promoter is activated in a partial eve pattern, causing expression driven by enhancers in the 3' region to be repressed. The mechanism involves transcriptional read-through from the flanking promoter. This conclusion is based on the following. Read-through driven by a heterologous enhancer is sufficient to repress, even when homie is in place. Furthermore, when the flanking promoter is turned around, repression is minimal. Transcriptional read-through that does not produce anti-sense RNA can still repress expression, ruling out RNAi as the mechanism in this case. Thus, transcriptional interference, caused by enhancer capture and read-through when the insulator is removed, represses eve promoter-driven expression. We also show that enhancer-promoter specificity and processivity of transcription can have decisive effects on the consequences of insulator removal. First, a core heat shock 70 promoter that is not activated well by eve enhancers did not cause read-through sufficient to repress the eve promoter. Second, these transcripts are less processive than those initiated at the P-promoter, measured by how far they extend through the eve locus, and so are less disruptive. These results highlight the importance of considering transcriptional read-through when assessing the effects of insulators on gene expression.
染色质绝缘子具有几种不同的活性和功能，可将染色体上的基因分隔成功能单元。在这里，我们描述了一种新的功能分离机制，其中绝缘子可防止基因抑制。当 homie 绝缘子从果蝇 even skipped（eve）基因座的末端缺失时，侧翼 P 元件启动子会以部分 eve 模式被激活，导致由 3' 区域增强子驱动的表达受到抑制。该机制涉及侧翼启动子的转录通读。这一结论基于以下事实。由异源增强子驱动的通读足以抑制表达，即使 homie 存在。此外，当侧翼启动子反转时，抑制作用最小。即使不产生反义 RNA 的转录通读仍能抑制表达，从而排除了 RNAi 作为这种情况下的机制。因此，当绝缘子被去除时，增强子捕获和转录通读引起的转录干扰会抑制 eve 启动子驱动的表达。我们还表明，增强子-启动子特异性和转录的连续性对绝缘子去除的后果具有决定性影响。首先，一个核心热休克 70 启动子不能很好地被 eve 增强子激活，因此不会引起足以抑制 eve 启动子的通读。其次，与从 P 启动子起始的转录相比，这些转录物的连续性较差，通过它们在 eve 基因座中延伸的距离来衡量，因此不太具有破坏性。这些结果强调了在评估绝缘子对基因表达的影响时，考虑转录通读的重要性。
