Elucidating the Diverse Biochemical Potency of Human OCT4

我们的建议是基于我们最近的发现，由我们的小组率先定义直接人类细胞的命运改变从一个体细胞到另一个。我们的计划现在使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OCT 4，它代表了最多样化的自我调节因子控制转录激活/抑制。OCT 4被描述为仅限于植入前胚胎和生殖细胞，并且在胚胎干细胞和从激活天然OCT 4的成熟细胞诱导多能性（称为诱导多能干细胞）中起关键作用。** 我们的小组已经定义了OCT 4依赖性细胞命运转换过程，其使用原代人类体细胞建立独特的分子状态，允许首次形成替代细胞类型（Nature 2010，Stem Cells 2014，Cell Stem Cell，修订版）。这个过程是独立的诱导多能性，并代表了一种新的重编程路径，我们称之为“OCT 4诱导的可塑性”或（OIP）。人细胞中的OIP允许细胞外环境的进一步指令，以促进向替代谱系特异性祖细胞的直接转化。使用人OIP，我们将成人皮肤成纤维细胞转化为多能造血或神经祖细胞，而没有多能性。我们的数据表明，OIP的OCT 4的添加不同于天然OCT 4的活化，表明生化修饰和蛋白质相互作用形成其在OIP与诱导多能性中的不同作用的基础。此外，OCT 4的后修饰亚型改变支持从染色质重塑到DNA修复的重编程的能力，并且是细胞环境特异性过程。我们现在的目标是在NSERC的支持下了解调节人类OCT 4的生物化学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OIP中人OCT 4蛋白的更完整的生化和化学调控理解：**1.使用允许OCT 4的表达和生物素标记的新型慢病毒载体系统，定义OIP特异性人皮肤成纤维细胞中异位表达OCT 4的后修饰形式。这将通过质谱法进行纯化和分析。** 2.工程肽和定义蛋白质-蛋白质相互作用网络在人类OIP。将标记纯化的蛋白质，并与共纯化蛋白质质谱分析一起沿着回收。3.鉴定激活内源性OCT 4表达的小分子，并使用我们现有的基于OCT 4调控的高通量化学筛选以及不同的化学文库来评估它们靶向参与人OIP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. *****