C-reactive protein (CRP) is a major acute phase protein and inflammatory marker, the expression of which is largely liver specific and highly inducible. Enhancers are regulatory elements critical for the precise activation of gene expression, yet the contributions of enhancers to the expression pattern of CRP have not been well defined. Here, we identify a constitutively active enhancer (E1) located 37.7 kb upstream of the promoter of human CRP in hepatocytes. By using chromatin immunoprecipitation, luciferase reporter assay, in situ genetic manipulation, CRISPRi, and CRISPRa, we show that E1 is enriched in binding sites for transcription factors STAT3 and C/EBP-β and is essential for the full induction of human CRP during the acute phase. Moreover, we demonstrate that E1 orchestrates with the promoter of CRP to determine its varied expression across tissues and species through surveying activities of E1-promoter hybrids and the associated epigenetic modifications. These results thus suggest an intriguing mode of molecular evolution wherein expression-changing mutations in distal regulatory elements initiate subsequent functional selection involving coupling among distal/proximal regulatory mutations and activity-changing coding mutations.

C 反应蛋白 (CRP) 是一种主要的急性期蛋白和炎症标志物，其表达主要是肝脏特异性和高度可诱导的。增强子是精确激活基因表达的关键调控元件，但增强子对CRP表达模式的贡献尚未明确。在这里，我们确定了位于肝细胞中人类CRP启动子上游 37.7 kb 的组成型活性增强子 (E1) 。通过使用染色质免疫沉淀、荧光素酶报告基因检测、原位基因操作、CRISPRi 和 CRISPRa，我们发现 E1 富含转录因子 STAT3 和 C/EBP-β 的结合位点，对于完全诱导人类CRP至关重要在急性期。此外，我们通过调查 E1 启动子杂种的活性和相关的表观遗传修饰，证明 E1 与CRP的启动子协调以确定其在组织和物种中的不同表达。因此，这些结果表明了一种有趣的分子进化模式，其中远端调节元件中的表达改变突变引发了随后的功能选择，包括远端/近端调节突变和活性改变编码突变之间的耦合。