Direct activation of hematopoietic transcription factors

 描述（由申请人提供）：造血，即从共同祖细胞分化出不同谱系的血细胞，在转录水平上受到复杂的控制。转录因子及其分子相互作用的详细知识是必不可少的正常造血以及一系列的疾病，所产生的失调转录控制的完整理解。此外，造血中的转录调控 是重新编程多能干细胞的范例，因此也是生物工程研究的关键领域。目前，造血转录因子通过遗传方法，如转基因、工程敲除/下调动物模型和RNA干扰，通过其表达水平间接调节。这些技术，这是缓慢的开始，持续的时间，和全球的影响，是不适合解剖转录途径的探针瞬时相互作用，典型的转录控制。目前，绝大多数造血调节因子无法直接化学控制转录活性，即在蛋白质/DNA水平上。此外，转录因子家族成员之间的强结构保守性混淆了针对这些转录因子的化学文库的简单筛选。在这里，我们探讨了一种物理化学策略，以分离激活剂的结构同源成员的ETS家族转录因子PU.1和Ets-1，发挥不同的作用，在造血细胞的命运决定。具体而言，我们将在可变的条件下（正常和升高的渗透压的交替条件）筛选针对同源PU.1/DNA和Ets-1/DNA复合物的噬菌体展示文库，以分离选择性地稳定PU.1或Ets-1与其同源结合位点的结合的肽。我们的建议是基于我们最近的研究表明，同源位点结合PU.1是非常敏感的渗透压，但Ets-1是不敏感的，即使这两种蛋白质的DNA结合结构域的结构重叠。在使用组合文库进一步优化候选序列后，我们将确定前导候选序列定位于造血祖细胞核并特异性激活PU.1或Ets-1靶基因的能力。鉴于在广泛的造血实验模型中对PU.1和Ets-1的新兴趣，预计从这项研究中产生的肽将成为用于询问PU.1和Ets-1靶基因的有用试剂以及血液学，风湿病和感染性疾病的靶向诊断和治疗的模板。

 DESCRIPTION (provided by applicant): Hematopoiesis, the differentiation of distinct lineages of blood cells from a common progenitor, is intricately controlled at the transcriptional level. Detailed knowledge of the transcription factors and their molecular interactions is essential to a complete understanding of normal hematopoiesis as well as an array of disorders that arise from de-regulated transcriptional control. In addition, transcriptional regulation in hematopoiesis is a paradigm for reprogramming pluripotent stem cells and is therefore a key area of inquiry in bioengineering as well. Currently, hematopoietic transcription factors are modulated indirectly through their expression levels by genetic approaches, such as transgenes, engineered knock-out/down animal models and RNA interference. These techniques, which are slow in onset, persistent in duration, and global in effect, are ill-suited to dissect transcriptional pathways an probe transient interactions that typify transcriptional control. Direct chemical control of transcriptional activity i.e. at the protein/DNA level, is currently unavailable for the vast majorty of hematopoietic regulators. Additionally the strong structural conservation among members of transcription factor families confounds simple screening of chemical libraries against these transcriptional factors. Here, we explore a physicochemical strategy to isolate activators of structurally homologous members of the ETS-family transcription factors PU.1 and Ets-1, which play distinct roles in hematopoietic cell-fate determination. Specifically, we will screen a phage display library against cognate PU.1/DNA and Ets-1/DNA complexes under osmotically variable conditions (alternating conditions of normal and elevated osmolality) to isolate peptides that selectively stabilize the binding of PU.1 or Ets-1 to their cognate binding sites. Our proposal is based on our recent studies showing that cognate site binding by PU.1 is exquisitely sensitive to osmotic stress, but Ets-1 is insensitive, even though the DNA binding domains of the two proteins are structurally superimposable. After further optimizing sequence candidates using combinatorial libraries, we will determine the ability of lead candidates to localize in the nucleiof hematopoietic progenitor cells and specifically activate PU.1 or Ets-1 target genes. Given the emerging interest in PU.1 and Ets-1 in a broad range of experimental models of hematopoiesis, the peptides generated from this research are expected to become useful reagents for interrogating PU.1 and Ets-1 target genes as well as templates for targeted diagnostics and therapeutics in hematologic, rheumatologic, and infectious diseases.