Identification of CBFA1 Targets in Osteoblasts

成骨细胞中 CBFA1 靶标的鉴定

• 批准号: 6662548
• 负责人: BARUCH FRENKEL
• 金额: $ 8.13万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-23 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6662548
• 关键词: bone morphogenetic proteins; cell differentiation; fibroblasts; gel electrophoresis; gene targeting; immunoprecipitation; messenger RNA; method development; molecular cloning; nucleic acid purification; nucleic acid quantitation /detection; nucleic acid sequence; open reading frames; osteoblasts; plasmids; polymerase chain reaction; restriction endonucleases; transcription factor; transfection /expression vector

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.

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中发挥着最明确的作用。然而，几乎没有任何已知的 CBFA1 靶基因，它们似乎在促进生物矿化方面发挥着预期的关键作用。迄今为止，尚未有研究报道描述对 CBFA1 靶基因的公正追求。该申请提出在成骨细胞中克隆新的 CBFA1 靶标，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）会将来自具有正常、低或高水平 CBFA 1 的细胞的 mRNA 进行比较。然而，这种方法通常会产生一长串基因，其中许多基因的表达改变是次要的，对转录因子和感兴趣的生物过程并不重要。此外，这些方法通常需要将 CBFA1 过度表达至超生理水平，可能导致鉴定出生物学意义有限的靶标。我们已经开始开发一种新颖的方法，通过该方法 CBFA1 目标基因将根据它们与活成骨细胞中的 CBFA1 的物理相互作用进行克隆。然后，将从使用 CBFA1 抗体进行染色质免疫沉淀 (ChIP) 获得的基因组 DNA 片段库中分离出 CBFA1 靶标。我们的初步实验表明，使用最优化的 ChIP 条件，已知的 CBFA1 靶标仅富集 50 倍。就其本身而言，这不足以分离 CBFA1 靶标，因为大量片段会与真正的 CBFA1 靶标发生非特异性共沉淀。为了建立纯化真正 CBFA1 靶标的方法，我们首先证明可以使用限制性酶消化而不是超声处理来进行有效的 ChIP 染色质片段化。这将有利于 CBFA1 靶基因的浓缩， 现在，使用聚丙烯酰胺凝胶电泳，每个片段都由独特大小的片段表示。非特异性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身份将 ChiPped DNA 分离成片段家族，将进一步促进真正 CBFA1 靶标的鉴定。此步骤通过使用锚定引物对扩增切碎片段来实现，将进一步增加代表真实 CBFA1 片段的背景条带的强度。最后，最强烈的条带将从凝胶中洗脱、克隆并测序。将鉴定遗传上相邻的 ORF，并通过 CBFA1 验证它们的调节。