Ramamurthy L, Barbour V, Tuckfield A, Clouston D R, Topham D, Cunningham J M, Jane S M
Division of Experimental Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
J Biol Chem. 2001 Mar 16;276(11):7836-42. doi: 10.1074/jbc.M004351200. Epub 2000 Sep 19.
The NTF-like family of transcription factors have been implicated in developmental regulation in organisms as diverse as Drosophila and man. The two mammalian members of this family, CP2 (LBP-1c/LSF) and LBP-1a (NF2d9), are highly related proteins sharing an overall amino acid identity of 72%. CP2, the best characterized of these factors, is a ubiquitously expressed 66-kDa protein that binds the regulatory regions of many diverse genes. Consequently, a role for CP2 has been proposed in globin gene expression, T-cell responses to mitogenic stimulation, and several other cellular processes. To elucidate the in vivo role of CP2, we have generated mice nullizygous for the CP2 allele. These animals were born in a normal Mendelian distribution and displayed no defects in growth, behavior, fertility, or development. Specifically, no perturbation of hematopoietic differentiation, globin gene expression, or immunological responses to T- and B-cell mitogenic stimulation was observed. RNA and protein analysis confirmed that the nullizygous mice expressed no full-length or truncated version of CP2. Electrophoretic mobility shift assays with nuclear extracts from multiple tissues demonstrated loss of CP2 DNA binding activity in the -/- lines. However, a slower migrating complex that was ablated with antiserum to NF2d9, the murine homologue of LBP-1a, was observed with these extracts. Furthermore, we demonstrate that recombinant LBP-1a can bind to known CP2 consensus sites and form protein complexes with previously defined heteromeric partners of CP2. These results suggest that LBP-1a/NF2d9 may compensate for loss of CP2 expression in vivo and that further analysis of the role of the NTF family of proteins requires the targeting of the NF2d9 gene.
NTF 样转录因子家族在果蝇和人类等多种生物的发育调控中发挥作用。该家族的两个哺乳动物成员，CP2（LBP-1c/LSF）和 LBP-1a（NF2d9），是高度相关的蛋白质，总体氨基酸同一性为 72%。CP2 是这些因子中研究最深入的，是一种普遍表达的 66 kDa 蛋白质，可结合许多不同基因的调控区域。因此，有人提出 CP2 在珠蛋白基因表达、T 细胞对有丝分裂原刺激的反应以及其他一些细胞过程中发挥作用。为了阐明 CP2 在体内的作用，我们构建了 CP2 等位基因纯合缺失的小鼠。这些动物以正常孟德尔分布出生，在生长、行为、生育能力或发育方面没有缺陷。具体而言，未观察到造血分化、珠蛋白基因表达或对 T 细胞和 B 细胞有丝分裂原刺激的免疫反应受到干扰。RNA 和蛋白质分析证实，纯合缺失小鼠不表达全长或截短形式的 CP2。对多个组织的核提取物进行的电泳迁移率变动分析表明，-/-系中 CP2 的 DNA 结合活性丧失。然而，用针对 LBP-1a 的小鼠同源物 NF2d9 的抗血清消除的较慢迁移复合物在这些提取物中被观察到。此外，我们证明重组 LBP-1a 可以结合已知的 CP2 共有位点，并与先前定义的 CP2 异源二聚体伙伴形成蛋白质复合物。这些结果表明，LBP-1a/NF2d9 可能在体内补偿 CP2 表达的缺失，并且对 NTF 蛋白家族作用的进一步分析需要靶向 NF2d9 基因。
