Elucidating the Diverse Biochemical Potency of Human OCT4

我们的建议是基于我们团队最近的发现，这些发现定义了人类细胞命运从一个体细胞到另一个体细胞的直接改变。我们的计划现在扩展和过渡到生化和化学方法，以使用自然科学方法控制和设计这一过程。我们专注于单个转录因子，人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被描述为仅限于植入前的胚胎和生殖细胞，在胚胎干细胞和从激活天然OCT4的成熟细胞诱导多能性中发挥关键作用，被称为诱导多能干细胞。*我们的团队定义了一种依赖于OCT4的细胞命运转换过程，使用原代人类体细胞建立了一种独特的分子状态，首次允许形成不同的细胞类型(自然2010，干细胞2014，细胞干细胞，修订中)。这一过程独立于诱导多能性，代表了一种新的重新编程途径，我们称之为OCT4诱导可塑性(OIP)。在人类细胞中，OIP允许细胞外环境的进一步指导，以促进直接转化为替代的谱系特异性祖细胞。使用人OIP，我们将成人皮肤成纤维细胞转化为多能造血祖细胞或神经前体细胞，而不是多能干细胞。我们的数据表明，OCT4对OIP的添加不同于天然OCT4的激活，这表明生化修饰和蛋白质相互作用形成了OIP与诱导多能性的不同作用的基础。此外，OCT4的翻译后修饰亚型改变了能力，支持从染色质重塑到DNA修复的重新编程，是细胞环境特有的过程。在NSERC的支持下，我们现在的目标是了解调控人类OCT4的生化和潜在的化学相互作用。*我们的目标是通过以下目标对人OIP中的人OCT4蛋白进行更完整的生化和化学调控了解：*1.利用一种新型慢病毒载体系统，在人皮肤成纤维细胞中确定针对OIP的异位表达的翻译后修饰形式的OCT4，该系统允许OCT4的表达和生物素标记。这将被提纯并用质谱仪进行分析。2.设计多肽并确定人OIP过程中的蛋白质-蛋白质相互作用网络。纯化的蛋白质将被标记和回收，并进行共纯化蛋白质的质谱分析。*3.鉴定激活内源性OCT4表达的小分子，并使用我们现有的基于OCT4调控的高通量化学筛选以及各种化学文库评估它们靶向人类OIP中涉及的特定蛋白质-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. *****