CGGBP1-dependent CTCF-binding sites restrict ectopic transcription

Binding sites of the chromatin regulator protein CTCF function as important landmarks in the human genome. The recently characterized CTCF-binding sites at LINE-1 repeats depend on another repeat-regulatory protein CGGBP1. These CGGBP1-dependent CTCF-binding sites serve as potential barrier elements for epigenetic marks such as H3K9me3. Such CTCF-binding sites are associated with asymmetric H3K9me3 levels as well as RNA levels in their flanks. The functions of these CGGBP1-dependent CTCF-binding sites remain unknown. By performing targeted studies on candidate CGGBP1-dependent CTCF-binding sites cloned in an SV40 promoter-enhancer episomal system we show that these regions act as inhibitors of ectopic transcription from the SV40 promoter. CGGBP1-dependent CTCF-binding sites that recapitulate their genomic function of loss of CTCF binding upon CGGBP1 depletion and H3K9me3 asymmetry in immediate flanks are also the ones that show the strongest inhibition of ectopic transcription. By performing a series of strand-specific reverse transcription PCRs we demonstrate that this ectopic transcription results in the synthesis of RNA from the SV40 promoter in a direction opposite to the downstream reporter gene in a strand-specific manner. The unleashing of the bidirectionality of the SV40 promoter activity and a breach of the transcription barrier seems to depend on depletion of CGGBP1 and loss of CTCF binding proximal to the SV40 promoter. RNA-sequencing reveals that CGGBP1-regulated CTCF-binding sites act as barriers to transcription at multiple locations genome-wide. These findings suggest a role of CGGBP1-dependent binding sites in restricting ectopic transcription.

染色质调控蛋白CCCTC结合因子（CTCF）的结合位点是人类基因组中的重要标志性位点。新近被表征的、位于LINE-1重复序列（LINE-1 repeats）上的CTCF结合位点，依赖于另一重复序列调控蛋白CGGBP1。此类依赖CGGBP1的CTCF结合位点可作为H3K9me3等表观遗传标记的潜在屏障元件。这类CTCF结合位点与其侧翼区域的不对称H3K9me3水平以及RNA表达水平显著相关。目前，这类依赖CGGBP1的CTCF结合位点的功能仍不明确。通过对克隆至SV40启动子-增强子游离型表达系统（episomal system）中的候选依赖CGGBP1的CTCF结合位点开展靶向研究，我们发现这些区域能够抑制SV40启动子介导的异位转录。那些能够重现其基因组功能的依赖CGGBP1的CTCF结合位点——即CGGBP1缺失时CTCF结合丧失、且紧邻侧翼区域存在H3K9me3不对称性——同时也是对异位转录抑制作用最强的位点。通过开展一系列链特异性逆转录PCR（strand-specific reverse transcription PCR）实验，我们证实该异位转录会以链特异性方式，由SV40启动子合成与下游报告基因转录方向相反的RNA。SV40启动子双向活性的解除以及转录屏障的破坏，似乎依赖于CGGBP1的缺失以及SV40启动子近端CTCF结合的丧失。RNA测序（RNA-sequencing）分析显示，受CGGBP1调控的CTCF结合位点在全基因组多个位点处均可作为转录屏障。本研究结果提示，依赖CGGBP1的CTCF结合位点在限制异位转录过程中发挥功能。