Jensen Christina L, Chen Liang-Fu, Swigut Tomek, Crocker Olivia J, Yao David, Bassik Mike C, Ferrell James E, Boettiger Alistair N, Wysocka Joanna
Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA.
Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA; Department of Developmental Biology, Stanford University, Stanford, CA 94305, USA.
Mol Cell. 2025 Jan 16;85(2):347-361.e7. doi: 10.1016/j.molcel.2024.10.021. Epub 2024 Dec 2.
Although critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here, we bypass the need for sequence-specific transcription factors (TFs) and recruit activators directly using a chimeric array of gRNA oligos to target dCas9 fused to the activator VP64-p65-Rta (CARGO-VPR). We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mouse embryonic stem cells (mESCs), where neither gene is expressed. Although activator recruitment to any tested region results in the transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence, or the presence of repressive chromatin marks at the locus.
尽管增强子与远端启动子之间的通讯对于调节转录的时间和水平至关重要，但目前对其了解尚少。在此，我们绕过了对序列特异性转录因子（TFs）的需求，而是使用gRNA寡核苷酸的嵌合阵列直接招募激活因子，以靶向与激活因子VP64-p65-Rta（CARGO-VPR）融合的dCas9。我们表明，这种方法能够将激活因子有效地招募到任意基因组位点，即使是那些用单个向导靶向时无法接近的位点。我们在小鼠胚胎干细胞（mESCs）的Prdm8-Fgf5基因座上使用了CARGO-VPR，该基因座上的两个基因均未表达。尽管将激活因子招募到任何测试区域都会导致至少一个基因的转录诱导，但其表达水平在很大程度上取决于启动子与激活因子招募位点之间的基因组距离。然而，每个基因的表达-距离关系以一种无法归因于三维接触频率、启动子DNA序列或该基因座上抑制性染色质标记的存在差异的方式明显不同。
