Elucidating the Diverse Biochemical Potency of Human OCT4

点击翻译按钮获取中文摘要

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.