Elucidating the Diverse Biochemical Potency of Human OCT4

我们的建议是基于我们最近的发现，我们的小组率先定义了人类细胞命运的直接改变，从一个体细胞到另一个。我们的项目现在扩展和过渡到生物化学和化学方法来控制和工程这一过程，使用自然科学的方法。我们专注于一个单一的转录因子（TF），人类OCT4，它代表了控制转录激活/抑制的最多样化的自我调节因子。OCT4被认为局限于胚胎着床前胚胎和生殖细胞，在胚胎干细胞和诱导多能性中起关键作用，成熟细胞激活天然OCT4，称为诱导多能干细胞。******我们的团队已经定义了一个OCT4依赖的细胞命运转换过程，使用原代人体细胞建立一个独特的分子状态，首次允许形成替代的细胞类型（Nature 2010, Stem cells 2014, cell Stem cell，修订版）。这个过程独立于诱导多能性，代表了一种新的重编程路径，我们称之为“oct4诱导的可塑性”或（OIP）。人类细胞中的OIP允许细胞外环境进一步指导，以促进向替代谱系特定祖细胞的直接转化。利用人OIP，我们将成人皮肤成纤维细胞转化为无多能性的多能造血祖细胞或神经祖细胞。我们的数据表明，为OIP添加OCT4与激活天然OCT4不同，这表明生化修饰和蛋白质相互作用构成了其在OIP和诱导多能性中不同作用的基础。此外，翻译后修饰的OCT4同工型改变了从染色质重塑到DNA修复的重编程能力，并且是细胞环境特异性过程。在NSERC的支持下，我们现在的目标是了解调节人类OCT4的生化和潜在化学相互作用。******我们的目标是通过以下目标获得对人类OIP中人类OCT4蛋白的更完整的生化和化学调控理解：******使用一种新的慢病毒载体系统，定义翻译后修饰的OIP特异性人皮肤成纤维细胞中异位表达OCT4的形式，该系统允许OCT4的表达和生物素标记。这将被纯化并通过质谱分析。******2。工程肽和定义蛋白蛋白相互作用网络在人类OIP。纯化蛋白将被标记并与共纯化蛋白质谱分析一起检索。* * * * * * 3。利用现有的基于OCT4调控的高通量化学筛选以及多种化学文库，鉴定激活内源性OCT4表达的小分子，并评估其靶向人类OIP中涉及的特定蛋白质相互作用的能力。＊＊＊＊＊

Our proposal is based on our recent discoveries pioneered by our group that define direct human cell fate alteration from one somatic cell to another. Our program now makes the extension and transition to biochemical and chemical methods to control and engineer this process using natural science approaches. We focus on a single transcription factor (TF), human OCT4, which represents the most diverse self-regulating factor controlling transcriptional activation/repression. OCT4 has been described as being restricted to preimplantation embryos and germ cells, and plays a critical role in embryonic stem cells and induction of pluripotency from mature cells activating native OCT4, known as induced pluripotent stem cells. ******Our group has defined an OCT4 dependent cell fate conversion process using primary HUMAN somatic cells establishing a unique molecular state allowing alternative cell types to be formed for the first time (Nature 2010, Stem Cells 2014, Cell Stem Cell, in revision). This process is independent of induced pluripotency and represents a novel reprogramming path we have termed as "OCT4-induced plasticity" or (OIP). OIP in human cells permits further instruction by the extracellular environment to facilitate direct conversion towards alternative lineage specific progenitors. Using human OIP, we converted adult human skin fibroblasts into multipotent hematopoietic or neural progenitors without pluripotency. Our data suggests the addition of OCT4 for OIP differs from activation of native OCT4, suggesting biochemical modifications and protein interactions form the basis of its distinct role in OIP vs. induced pluripotency. In addition, posttranslationally modified isoforms of OCT4 alter capacity to support reprogramming from chromatin remodeling to DNA repair and are cell context specific processes. We now aim to understand the biochemical and potential chemical interactions regulating human OCT4 with support from NSERC. ******Our goal is to gain a more complete biochemical and chemical regulatory understanding of human OCT4 protein in human OIP via the following objectives:******1. Define posttranslationally modified forms of ectopically expressed OCT4 in human skin fibroblasts specific to OIP using a novel lentiviral vector system that allows for expression and biotin labeling of OCT4. This will be purified and analyzed by mass spectrometry.******2. Engineer peptides and define the protein-protein interaction network during human OIP. Purified protein will be labeled and retrieved along with co-purifying proteins mass spec analysis. ******3. Identify small molecules that activate endogenous OCT4 expression, and evaluate their capacity for targeting specific protein-protein interactions involved in human OIP using our existing high throughput chemical screening based on OCT4 regulation, as well as diverse chemical libraries. *****