Transvection-like interchromosomal interaction is not observed at the transcriptional level when tested in the Rosa26 locus in mouse

Long-range associations between enhancers and their target gene promoters have been shown to play critical roles in executing genome function. Recent variations of chromosome capture technology have revealed a comprehensive view of intra- and interchromosomal contacts between specific genomic sites. The locus control region of the β-globin genes (β-LCR) is a super-enhancer that is capable of activating all of the β-like globin genes within the locus in cis through physical interaction by forming DNA loops. CTCF helps to mediate loop formation between LCR-HS5 and 3’HS1 in the human β-globin locus, in this way thought to contribute to the formation of a “chromatin hub”. The β-globin locus is also in close physical proximity to other erythrocyte-specific genes located long distances away on the same chromosome. In this case, erythrocyte-specific genes gather together at a shared “transcription factory” for co-transcription. Theoretically, enhancers could also activate target gene promoters at the identical loci, yet on different chromosomes in trans, a phenomenon originally described as transvection in Drosophilla. Although close physical proximity has been reported for the β-LCR and the β-like globin genes when integrated at the mouse homologous loci in trans, their structural and functional interactions were found to be rare, possibly because of a lack of suitable regulatory elements that might facilitate such trans interactions. Therefore, we re-evaluated presumptive transvection-like enhancer-promoter communication by introducing CTCF binding sites and erythrocyte-specific transcription units into both LCR-enhancer and β-promoter alleles, each inserted into the mouse ROSA26 locus on separate chromosomes. Following cross-mating of mice to place the two mutant loci at the identical chromosomal position and into active chromation in trans, their transcriptional output was evaluated. The results demonstrate that there was no significant functional association between the LCR and the β-globin gene in trans even in this idealized experimental context.

增强子与其靶基因启动子之间的远程关联，已被证实对基因组功能的执行发挥关键调控作用。近年来，染色体捕获技术的迭代改良版本，已全面揭示了特定基因组位点之间的染色体内与染色体间互作图谱。β-珠蛋白基因座控制区（β-globin locus control region, β-LCR）是一种超级增强子，可通过形成DNA环的物理互作方式，顺式激活该基因座内所有的β类珠蛋白基因。在人类β-珠蛋白基因座中，CCCTC结合因子（CTCF）可介导LCR-HS5与3’HS1之间的环结构形成，据信这有助于“染色质枢纽”的构建。β-珠蛋白基因座还可与同一条染色体上远距离分布的其他红细胞特异性基因保持紧密物理邻近，在此情形下，这些红细胞特异性基因会聚集于共享的“转录工厂”中以实现共转录。从理论上讲，增强子也可通过反式作用，在同一基因座但不同染色体上激活靶基因启动子，这一现象最初在果蝇中被描述为“转位交互（transvection）”。尽管已有研究报道，当β-LCR与β类珠蛋白基因通过反式整合至小鼠同源基因座时，二者可形成紧密物理邻近，但二者的结构与功能互作却极为罕见，这可能是由于缺乏能够促进此类反式互作的合适调控元件。因此，本研究重新评估了疑似转位交互样的增强子-启动子通信机制：将CTCF结合位点与红细胞特异性转录单元分别引入LCR增强子与β-启动子等位基因，且两个等位基因均插入至不同染色体上的小鼠ROSA26基因座中。通过小鼠杂交使两个突变基因座处于同一染色体位置，并以反式方式处于活跃染色质状态，随后对二者的转录产出进行了评估。实验结果表明，即便在这一理想化的实验环境中，LCR与β-珠蛋白基因之间也不存在显著的反式功能关联。