Identification of Proteins Interacting with ARID3a

 描述（由申请人提供）：从造血前体产生多种细胞类型（包括红细胞谱系细胞）的能力使骨髓移植和治疗多种疾病成为可能。我们发现转录因子ARID3a是小鼠正常造血干细胞发育所必需的。缺乏ARID3a的小鼠由于红细胞生成失败而在子宫内死亡。另外的数据显示，ARID3a在体外介导人脐带血细胞中的造血谱系决定。此外，我们证明了ARID3a在调节多能性基因表达的成体分化细胞类型中具有抑制功能。总之，我们的数据表明ARID3a是造血干细胞中重要的DNA结合蛋白，也是诱导多能干细胞重编程的屏障。然而，关于ARID 3a在这些系统中介导谱系决定效应的分子机制尚不清楚。尽管我们的数据表明ARID3a可以参与抑制基因表达，以及增强基因转录，但ARID3a内没有蛋白基序表明可能介导这些功能的机制。我们假设ARID3a通过作为DNA系链与其他蛋白质相互作用来影响基因调控。为了了解ARID3a的功能，关键是要确定与它相关的蛋白质。我们的数据表明，可以被诱导分化为红系和髓系细胞的人K562骨髓单核细胞系需要ARID3a进行分化。来自ENCODE小组的现有数据确定了K562细胞中ARID3a的大量潜在基因靶点。因此，我们将使用该细胞系作为模型系统，通过质谱法鉴定与ARID3a相关的蛋白质。ARID3a敲低和对照K562细胞的转录组分析将鉴定需要ARID3a表达的基因。将检查鉴定的ARID3a基因靶标是否存在含ARID3a的蛋白复合物。我们预测ARID3a相互作用蛋白将包括表观遗传介质，如组蛋白脱乙酰酶，为ARID3a如何调节基因表达提供重要线索。未来的研究将利用这些数据来更好地了解ARID3a如何参与人类造血干细胞的谱系决定。最终，操纵造血干细胞产生选择谱系的能力可能具有治疗用途。

 DESCRIPTION (provided by applicant): The ability to generate multiple cell types, including erythrocyte lineage cells, from hematopoietic precursors enables bone marrow transplantation and treatment of a wide variety of diseases. We found that the transcription factor ARID3a is required for normal hematopoietic stem cell development in the mouse. Mice deficient in ARID3a die in utero as the result of failed erythropoiesis. Additional data show ARID3a mediates hematopoietic lineage decisions in human cord blood cells in vitro. Furthermore, we demonstrated that ARID3a has suppressive functions in adult differentiated cell types that regulate expression of pluripotency genes. Together, our data implicate ARID3a as an important DNA-binding protein in hematopoietic stem cells and as a barrier for reprogramming of induced pluripotent stem cells. However, nothing is known regarding the molecular mechanisms by which ARID3a mediates lineage decision effects in these systems. Although our data suggest ARID3a can participate in suppression of gene expression, as well as enhancement of gene transcription, no protein motifs within ARID3a suggest mechanisms which might mediate these functions. We hypothesize that ARID3a interacts with other proteins by serving as a DNA tether to affect gene regulation. In order to understand how ARID3a functions it is critical to identify te proteins with which it is associated. Our data indicate that the human K562 myelomonocytic cell line which can be induced to differentiate into both erythroid and myeloid lineage cells requires ARID3a for differentiation. Existing data from the ENCODE group identify a large number of potential gene targets for ARID3a in K562 cells. Therefore, we will use this cell line as a model system to identify proteins associated with ARID3a by mass spectrometry. Transcriptome analyses of ARID3a knockdown and control K562 cells will identify genes requiring ARID3a for expression. Identified gene targets for ARID3a will be examined for the presence of ARID3a-containing protein complexes. We predict that ARID3a-interacting proteins will include epigenetic mediators, such as histone deacetylases, providing important clues for how ARID3a regulates gene expression. Future studies will utilize these data to develop a better mechanistic understanding of how ARID3a participates in lineage decisions in human hematopoietic stem cells. Ultimately, the ability to manipulate hematopoietic stem cells to generate lineages of choice could have therapeutic use.