Identification of CBFA1 Targets in Osteoblasts

描述(由申请人提供)： CBFA1是一种转录因子，在成骨细胞分化和生物矿化过程中发挥着最重要的作用。然而，几乎没有已知的CBFA1靶基因，它们似乎在促进生物矿化方面发挥了预期的关键作用。到目前为止，还没有任何研究报道，描述了对CBFA1靶基因的公正追求。这项申请提出在成骨细胞中克隆新的CBFA1靶点，长期目标是发现在生物矿化中发挥关键作用的基因。发现CBFA1靶标的传统方法(例如，差异显示、微阵列)会比较CBFA1正常、低或高水平的细胞的mRNAs。然而，这种方法通常会导致一长串基因，其中许多基因的表达变化是次要的，对转录因子和感兴趣的生物过程并不重要。此外，这些方法通常会导致CBFA1的过度表达达到超生理水平，可能导致识别生物学意义有限的靶点。我们已经开始开发一种新的方法，通过这种方法，CBFA1 靶基因的克隆将基于它们与活的成骨细胞中CBFA1的物理相互作用。然后，将从CBFA1抗体通过染色质免疫沉淀(CHIP)获得的基因组DNA片段池中分离CBFA1靶标。我们的初步实验表明，在最优的芯片条件下，已知的CBFA1靶只浓缩了50倍。就其本身而言，这将不足以分离CBFA1靶标，因为大量过剩的片段将非特异性地与真正的CBFA1靶标一起共沉淀。为了建立一种纯化真实CBFA1靶标的方法，我们首先证明了用限制性内切酶消化代替超声裂解染色质可以进行有效的芯片。这将有助于CBFA1靶基因的浓缩， 现在每个片段都由一个唯一的大小片段代表，使用聚丙烯酰胺凝胶电泳法。非特定沉淀的污染碎片将沿着凝胶的更大区域扩散。根据每个片段末端核苷酸的同一性，将碎片DNA分离成片段家族，将进一步便利鉴定真正的CBFA1靶标。这一步骤通过用一对锚定的引物扩增片断来实现，将进一步增加代表真实CBFA1片段的条带在背景上的强度。最后，最强的条带将从凝胶中洗脱出来，进行克隆和测序。将确定基因上相邻的ORF，并验证CBFA1对它们的调控。

DESCRIPTION (provided by applicant): CBFA1 is a transcription factor with the most well established role in osteoblast differentiation and biomineralization. However, there are hardly any known CBFA1 target genes, which seem to play the anticipated critical role in promoting biomineralization. No study has been reported to date, describing an unbiased pursuit of CBFA1 target genes. This application proposes to clone new CBFA1 targets in osteoblasts, with the long-term goal of discovering genes playing critical roles in biomineralization. Traditional approaches to discover CBFA1 targets (e.g., differential display, microarrays) would compare mRNAs from cells with normal, low, or high levels of CBFA 1. However, such approaches often result in a long list of genes, for many of which the altered expression is secondary and of little importance to the transcription factor and to the biological process of interest. In addition, these approaches would normally entail over-expression of CBFA1 to supra-physiological levels, possibly leading to the identification of targets with limited biological significance. We have begun to develop a novel approach, by which CBFA1 target genes would be cloned based on their physical interaction with CBFA1 in living osteoblasts. Then CBFA1 targets will be isolated from a pool of genomic DNA fragments obtained by chromatin immunoprecipitation (ChIP) with CBFA1 antibodies. Our preliminary experiments show that, using the most optimal ChIP conditions, known CBFA1 targets are enriched by only 50-fold. By itself, this would be insufficient for isolating CBFA1 targets, due to vast excess of fragments that would non-specifically coprecipitate along with true CBFA1 targets. Towards establishing a method of purifying true CBFA1 targets, we first showed that efficient ChIP could be performed using restriction enzyme digestion instead of sonication for fragmentation of the chromatin. This will facilitate the concentration of CBFA1 target genes, each now represented by a unique size fragment, using polyacrylamide gel electrophoresis. Non-specifically precipitated, contaminating fragments will be spread along a much larger area of the gel. Identification of true CBFA1 targets will be further facilitated by segregating the ChiPped DNA into families of fragments based on the identity of nucleotides at the ends of each fragment. This step, achieved by amplification of the Chipped fragments with pairs of anchored primers, will further increase the intensity of bands representing true CBFA1 fragments over the background. Finally, the most intense bands will be eluted from the gel, cloned and sequenced. Genetically adjacent ORFs will be identified and their regulation by CBFA1 validated.