IDENTIFICATION OF NOVEL DNA-BINDING TARGETS AND REGULATORY DOMAINS OF A MURINE TINMAN HOMEODOMAIN FACTOR, NKX-2.5
IDENTIFICATION OF NOVEL DNA-BINDING TARGETS AND REGULATORY DOMAINS OF A MURINE TINMAN HOMEODOMAIN FACTOR, NKX-2.5
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DOI：
10.1074/jbc.270.26.15628
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发表时间：
1995-06-30
影响因子：
4.8
通讯作者：
SCHWARTZ, RJ
中科院分区：
文献类型：
--
作者：
CHEN, CY;SCHWARTZ, RJ
一种名为csx的鼠心脏特异性同源结构域基因（Komuro，I.，和Izumo，S，（1993）Proc.Natl.美国科学院90，8145-8149）和nkx-2.5（Lints，T，J，，Parsons，L，M，Hartley，L，，里昂，I，和Harvey，R. P，（1993）Development 119，419-431）被鉴定为果蝇tinman的潜在脊椎动物同源物，一种昆虫心脏形成所需的中胚层决定因子（Bodmer，R，（1993）Development 118，719-729）。nkx-2.5同源结构域的细菌表达允许我们从随机产生的寡核苷酸文库中鉴定下游DNA靶标。高亲和力nkx-2.5 DNA结合位点，5 ′-TNAGTG-3 ′，代表新的结合序列，而中等和较弱亲和力位点，5 ′-C（APT）TTAATTN-3 ′，含有大多数同源结构域因子DNA结合所需的典型5 ′-TAAT-3 ′核心，我们还观察到nkx-2.5在10 T1/10 T2细胞中进行的转染试验中充当适度的转录激活剂。nkx-2.5的功能解剖揭示了主要由丙氨酸和脯氨酸簇组成的COOH末端抑制结构域，其似乎掩盖了由疏水性和高电荷氨基酸组成的有效激活结构域。
A murine cardiac-specific homeodomain gene named csx (Komuro, I., and Izumo, S, (1993) Proc. Natl. Acad, Sci, U, S, A. 90, 8145-8149) and nkx-2.5 (Lints, T, J,, Parsons, L, M,, Hartley, L,, Lyons, I,, and Harvey, R. P, (1993) Development 119, 419-431) was identified as a potential vertebrate homologue of Drosophila tinman, a mesoderm determination factor required for insect heart formation (Bodmer, R, (1993) Development 118, 719-729), Bacterial expression of the nkx-2.5 homeodomain allowed us to identify downstream DNA targets from a library of randomly generated oligonucleotides. High affinity nkx-2.5 DNA binding sites, 5'-TNNAGTG-3', represented novel binding sequences, whereas intermediate and weaker affinity sites, 5'-C(APT)TTAATTN-3', contained the typical 5'-TAAT-3' core required by most homeodomain factors for DNA binding, We also ob served that nkx-2.5 served as a modest transcription activator in transfection assays done in 10T1/2 fibroblasts with multimerized binding sites linked to a luciferase reporter gene, Functional dissection of nkx-2.5 revealed a COOH-terminal inhibitory domain composed mainly of clusters of alanines and prolines, which appeared to mask a potent activation domain composed of hydrophobic and highly charged amino acids.