Lau J S, Baumeister P, Kim E, Roy B, Hsieh T Y, Lai M, Lee A S
Department of Biochemistry and Molecular Biology and the USC/Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, Los Angeles, California 90089-9176, USA.
J Cell Biochem. 2000 Sep 7;79(3):395-406. doi: 10.1002/1097-4644(20001201)79:3<395::aid-jcb50>3.0.co;2-m.
In search for nuclear proteins that interact with the human thymidine kinase (htk) promoter, we discovered that p37AUF, a hnRNP C-like protein, and hnRNP A1, both members of the heterogeneous ribonucleoprotein family, can bind with high affinity to an ATTT sequence motif contained within the cell cycle regulatory unit (CCRU). We report here that over-expression of p37AUF stimulates gene expression mediated by the htk promoter in a promoter-sequence specific manner, whereas hnRNP A1 suppresses it. Both recombinant p37AUF and hnRNP A1 can bind the htk CCRU, suggesting that their binding to the DNA target does not require additional cellular components. We further discovered that hnRNP K is a potent suppressor of htk mediated gene activity. However, its mechanism of action is mediated through protein-protein interaction, since hnRNP K itself cannot bind the htk CCRU but can competitively inhibit the binding of other hnRNPs. The binding site for the hnRNPs on the htk CCRU is not required for S-phase induction of the htk promoter. However, in stable but not transient transfectants, the mutation of the hnRNP binding site results in 5- to 10-fold reduction of htk mediated gene activity in synchronized and exponentially growing cells. Collectively, these findings support emerging evidence that hnRNPs, in addition to their traditional role in RNA biogenesis, could be regulators of gene expression through direct DNA binding or interaction with other proteins.
为了寻找与人类胸苷激酶（htk）启动子相互作用的核蛋白，我们发现p37AUF（一种hnRNP C样蛋白）和hnRNP A1（不均一核糖核蛋白家族的两个成员）能够以高亲和力结合到细胞周期调节单元（CCRU）内包含的ATTT序列基序上。我们在此报告，p37AUF的过表达以启动子序列特异性方式刺激由htk启动子介导的基因表达，而hnRNP A1则抑制该表达。重组p37AUF和hnRNP A1都能结合htk CCRU，这表明它们与DNA靶点的结合不需要其他细胞成分。我们进一步发现hnRNP K是htk介导的基因活性的有效抑制剂。然而，其作用机制是通过蛋白质-蛋白质相互作用介导的，因为hnRNP K本身不能结合htk CCRU，但能竞争性抑制其他hnRNPs的结合。htk CCRU上hnRNPs的结合位点对于htk启动子的S期诱导不是必需的。然而，在稳定转染而非瞬时转染的细胞中，hnRNP结合位点的突变导致在同步化和指数生长的细胞中htk介导的基因活性降低5至10倍。总的来说，这些发现支持了新出现的证据，即hnRNPs除了在RNA生物合成中的传统作用外，还可能通过直接结合DNA或与其他蛋白质相互作用来调节基因表达。
