Samantha A. Morris, Ph.D. - US grants

PROJECT SUMMARY/ABSTRACT The reprogramming of abundant and accessible cells into therapeutically useful cell types holds great promise for regenerative medicine. Cellular reprogramming can be achieved by ectopically expressing transcription factors (TFs) that directly convert one differentiated cell type into another, bypassing embryonic states in an attempt to boost the speed and efficiency of target cell production. A number of different cell types have been generated by such ?direct lineage reprogramming? methods, but their practical utility has been limited because, in most protocols, only a small percentage of cells are successfully converted to the target cell type. Even then, the resulting populations are often partially differentiated or incompletely specified. Most cell engineering methods require the use of at least one pioneer factor, a unique class of TFs that are able to access their binding sites in silent chromatin. Pioneer TFs have the capacity to impart lineage competence in a context- specific manner, and play central roles in development, as reflected by their redeployment across disparate developmental programs. Our long-term goal is to understand the mechanism of pioneer factor-mediated direct lineage reprogramming. In this proposal we employ prototypical pioneer TFs, the FoxA family, to drive conversion of fibroblasts to an endoderm progenitor-like (iEP) state, representing a paradigm for direct lineage reprogramming. Based on our preliminary results and current evidence, we hypothesize that during direct lineage reprogramming, pioneer TFs re-engage developmental GRNs, depending on the chromatin state of the cells into which they are introduced. Our three independent yet related aims are directed at understanding: (Aim 1) the nature of transcriptional changes during reprogramming from their origin and their relation to developmental programs; (Aim 2) the direct targets of FoxA pioneer TFs and their cofactor, Hnf4a and their activity to drive fate change; (Aim 3) the influence of chromatin context on target accessibility of pioneer TFs and how this impacts efficiency of reprogramming and the potential of cells generated. Here we apply an innovative single-cell lineage tracing methodology and genomic technology to record TF binding history in cells undergoing reprogramming. Together, this will generate an unprecedented digital quantification of the reprogramming process, and will reveal barriers to the successful conversion of cell identity. An improved understanding of pioneer-mediated reprogramming mechanisms will facilitate enhanced conversion efficiency and fidelity across an array of reprogramming strategies, and improve knowledge of the action of this important class of transcriptional regulators.

Project Summary The reprogramming of skin or other easy to obtain cell types into therapeutically useful cells holds great promise for regenerative medicine, but the practical usefulness of this technology has been limited because typically only a small percentage of cells are successfully converted to the target cell type and these are often immature or incompletely specified. Single-cell expression analysis has revealed that most reprogrammed cells start with very similar expression profiles but then acquire a wide variety of different fates, many of which are developmental dead ends. Why does a cell population that is initially homogenous produce a myriad of different cell fates, with only a few cells achieving the targeted fate? This question is extremely difficult to answer with existing methods. The main focus of our proposal is to develop self-reporting Calling Cards, a new technology that can answer this question by simultaneously measuring transcription factor binding and genome-wide mRNA levels from thousands of single cells. We will demonstrate the utility of self-reporting Calling Cards by mapping the binding and function of the pioneer factor Foxa1 and its cofactor Hnf4a during the reprogramming of fibroblasts into induced endoderm progenitors (iEPs). We hypothesize that the direct targets of these TFs are stochastically expressed in cells undergoing lineage reprogramming, and that by forcing the expression of target genes that are usually transcribed only in successfully converted cells, we can improve overall target cell yield and maturity.

PROJECT SUMMARY/ABSTRACT The reprogramming of abundant and accessible cells into therapeutically useful cell types holds great promise for regenerative medicine. Cellular reprogramming can be achieved by ectopically expressing transcription factors (TFs) that directly convert one differentiated cell type into another, bypassing embryonic states in an attempt to boost the speed and efficiency of target cell production. A number of different cell types have been generated by such ?direct lineage reprogramming? methods, but their practical utility has been limited because, in most protocols, only a small percentage of cells are successfully converted to the target cell type. Even then, the resulting populations are often partially differentiated or incompletely specified. Most cell engineering methods require the use of at least one pioneer factor, a unique class of TFs that are able to access their binding sites in silent chromatin. Pioneer TFs have the capacity to impart lineage competence in a context- specific manner, and play central roles in development, as reflected by their redeployment across disparate developmental programs. Our long-term goal is to understand the mechanism of pioneer factor-mediated direct lineage reprogramming. In this proposal we employ prototypical pioneer TFs, the FoxA family, to drive conversion of fibroblasts to an endoderm progenitor-like (iEP) state, representing a paradigm for direct lineage reprogramming. Based on our preliminary results and current evidence, we hypothesize that during direct lineage reprogramming, pioneer TFs re-engage developmental GRNs, depending on the chromatin state of the cells into which they are introduced. Our three independent yet related aims are directed at understanding: (Aim 1) the nature of transcriptional changes during reprogramming from their origin and their relation to developmental programs; (Aim 2) the direct targets of FoxA pioneer TFs and their cofactor, Hnf4a and their activity to drive fate change; (Aim 3) the influence of chromatin context on target accessibility of pioneer TFs and how this impacts efficiency of reprogramming and the potential of cells generated. Here we apply an innovative single-cell lineage tracing methodology and genomic technology to record TF binding history in cells undergoing reprogramming. Together, this will generate an unprecedented digital quantification of the reprogramming process, and will reveal barriers to the successful conversion of cell identity. An improved understanding of pioneer-mediated reprogramming mechanisms will facilitate enhanced conversion efficiency and fidelity across an array of reprogramming strategies, and improve knowledge of the action of this important class of transcriptional regulators.