Specific repression of β-globin promoter activity by nuclear ferritin

Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult β-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the β-globin promoter at −153 to −148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress β-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the β-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant β-globin promoter for the −150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the −150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human β-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases.

发育性血红蛋白转换涉及珠蛋白基因的依次激活与沉默，但其具体机制尚未完全阐明。本文所述的早期观测结果促使我们提出假说：核铁蛋白可抑制胚胎红细胞中成体β-珠蛋白基因的表达。我们的研究数据显示，K562细胞核提取物中的铁蛋白家族蛋白可特异性结合β-珠蛋白启动子中距转录起始位点（cap site）-153至-148 bp处一段高度保守的CAGTGC基序；竞争型凝胶迁移实验（gel-shift assays）结果表明，对该CAGTGC基序进行突变可使结合强度降低20倍。富集铁蛋白H链（ferritin-H chains）的纯化人源铁蛋白亦可结合携带该CAGTGC基序的启动子片段。铁蛋白H链的表达克隆可在共转染的CV-1细胞中显著抑制β-珠蛋白启动子驱动的报告基因表达；该细胞系内的β-启动子已通过转录激活因子红细胞样Krüppel样因子（EKLF）完成激活。我们构建了分别携带-150位CAGTGC基序野生型或突变型β-珠蛋白启动子的氯霉素乙酰转移酶（CAT）报告质粒，并在共转染实验中比较了这些质粒对铁蛋白H链介导的基因沉默的敏感性。实验结果显示，突变型启动子仍可保留共转染EKLF介导的激活效应，但铁蛋白H链的抑制作用则完全丧失。由此可见，-150位CAGTGC基序的突变不仅可显著降低核铁蛋白的体外结合活性，还可在本细胞转染实验中消除表达的铁蛋白H链对该启动子的抑制能力，这为DNA结合活性与功能之间的关联提供了有力证据，也为我们提出的“核铁蛋白H链是人源β-珠蛋白基因的抑制因子”这一假说提供了强有力的支持。这类抑制因子有望用于治疗镰状细胞病及其他遗传性血液疾病。