Sequence-specific binding of recombinant Zbed4 to DNA: insights into Zbed4 participation in gene transcription and its association with other proteins.
Sequence-specific binding of recombinant Zbed4 to DNA: insights into Zbed4 participation in gene transcription and its association with other proteins.
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DOI：
10.1371/journal.pone.0035317
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Farber DB
中科院分区：
文献类型：
--
作者：
Mokhonov VV;Theendakara VP;Gribanova YE;Ahmedli NB;Farber DB
Zbed4是锌指蛋白Bed亚类中的一员，表达于人视网膜的视锥感光细胞和神经胶质Müler细胞，而仅存在于小鼠视网膜的Müler细胞。为了鉴定Zbed4的结构和功能，需要足够数量的纯化蛋白。因此，在大肠杆菌中表达了重组Zbed4，并对其复性条件进行了优化，以生产均一的具有功能活性的蛋白。圆二色谱分析表明，该蛋白质含有32%的α-螺旋、18%的β-折叠、20%的转角和30%的无序结构。用铸型法确定Zbed4在DNA中的靶点。获得的大多数DNA片段含有多聚G，其中一些还具有GC盒的核心特征；少数克隆只有GC盒。通过电泳迁移率改变分析，我们证明了Zbed4不仅与DNA结合，而且以非常高的亲和力与RNA寡核苷酸结合，与至少具有5个Gs的聚-G链相互作用；它与GC-box共同序列结合。然而，后一种结合取决于GC盒两侧的核苷酸。我们还发现Zbed4在Y79视网膜母细胞瘤细胞中与核蛋白和细胞质蛋白支架附着因子B1(SFB1)、雌激素受体α(ERα)和细胞肌球蛋白9(MyH9)相互作用，免疫沉淀、质谱学研究以及凝胶覆盖分析表明。此外，免疫染色证实了Zbed4与这些蛋白的共同定位。最重要的是，使用含有引导荧光素酶基因表达的基因启动子的构建体进行的体外实验表明，Zbed4通过多聚G链反式激活这些启动子的转录。
Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4’s secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% β-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts.
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