Analysis of the mechanisms of neuronal cell-type diversification by the transcription factor UNC-86

我们提出了一种新的方法（转录因子靶基因表位标记法）来分离线虫体内转录因子的结合位点。一般来说，到目前为止，这类实验是非常困难的。我们克隆了unc-86基因的整个DNA片段。我们将GFP（绿色荧光蛋白）cDNA插入到转录因子的编码区，并利用该DNA结构挽救了unc-86突变体的表型。我们从这种菌株中分离出细胞核，将核蛋白与DNA交联。然后，细胞核分裂成核小体。UNC-86/ gfp靶DNA序列复合物用抗gfp单克隆抗体免疫纯化。用这种方法分离的DNA片段被克隆并测序。迄今为止，秀丽隐杆线虫基因组序列联盟利用该数据库已鉴定出29个独特序列，与秀丽隐杆线虫染色体上的某些基因组位点相对应。其中24个出现了特定基因的调控序列。24个基因中有4个与已知基因相对应，其中3个是unc-86基因本身，另一个是在HSN神经元分化的遗传途径中作用于unc-86下游的基因。其他基因分为两类。第一种是基因在某种程度上与已知的在神经系统中起作用的基因具有同源性。其他基因与已知基因没有任何同源性，未来将对其在神经系统中的可能功能进行分析。

We developed a novel method (epitope-tagging of target genes of transcription factors) to isolate in vivo binding sites of transcription factors in the nematode C.elegans. Generally, this kind of experiments are very difficult so far. We cloned the whole DNA fragment for unc-86 gene. We inserted GFP (green fluorescent protein) cDNA in frame to the coding region of the transcription factor, and used the DNA construct to rescue unc-86 mutant phenotypes. We isolated nuclei from such a strain, cross-linked the nuclear protein to DNA.Then, the nuclei were fragmented to nucleosomes. The UNC-86/GFP-target DNA sequence complexes were immuno-purified with an anti-GFP monoclonal antibody. The DNA fragments isolated in this way were cloned and sequenced. So far, 29 unique sequences corresponding to certain genome 1oci on the C.elegans chromosomes were identified using the database by C.elegans genome sequence consortium. Twenty-four of which appeared regulatory sequences of certain genes. Four of 24 corresponding to known genes, three of which are unc-86 gene itself and one known as a gene which works downstream of unc-86 in a genetic pathway for HSN neuron differentiation. Other genes are categorized into two. First are genes sharing somehow homologies to known genes which can work in nervous systems. Others didn't share any homology to known genes and are to be analyzed in the future on the possible functions in the nervous systems.