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Using artificial transcription factors to induce differentiation into cardiomyocytes
Author(s) -
Heiderscheit Evan Anthony,
Eguchi Asuka,
Wleklinski Matthew J,
Spurgat Mackenzie C,
Ansari Aseem Z
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.756.9
Subject(s) - wnt signaling pathway , induced pluripotent stem cell , biology , transcription factor , microbiology and biotechnology , zinc finger , cellular differentiation , computational biology , gene , signal transduction , genetics , embryonic stem cell
Differentiation of pluripotent cells has become an effective way to study diseases in vitro as well as to engineer tissues for regenerative medicine. However, the discovery of natural transcription factors that specifies cells to desired fates is challenging, expensive, and labor‐intensive. Instead of taking a trial and error approach toward identifying key regulatory genes, artificial transcription factors (ATFs), capable of regulating genes independently of the endogenously expressed proteins, can provide a way to study the transcriptional networks of specific cell states in an unbiased manner. By using a genome‐scale library of ATFs, each comprised of a zinc finger DNA‐binding domain, thousands of genes can be tested in parallel. We applied this forward genetic approach in the differentiation of human pluripotent stem cells into cardiomyocytes. Cardiomyocyte differentiation relies on the temporal regulation of Wnt signaling via small molecule inhibitors. First, a Wnt signaling agonist (CHIR99021) is used to specify pluripotent cells into a mesodermal lineage, followed by treatment with an antagonist (IWP2) to induce cardiomyocyte differentiation. While this method of generating cardiomyocytes is efficient, the off‐target effects of the inhibitors used in this protocol remain unknown, and therefore, the gene networks activated by the inhibitors are not completely understood. By replacing the function of these inhibitors with ATFs, the specific genes involved in differentiation can be identified. The combinations of ATFs identified in the screen were capable of replacing IWP2, an inhibitor that prevents proper Wnt secretion. After confirming that these ATFs are capable of replacing the function of IWP2, cognate site identification was used to determine the optimal binding motifs preferred by the identified ATFs. These binding motifs were then mapped to the genome to identify genes with an ATF binding site within 1 kb of the transcriptional start site. The genes identified in this screen provide further insights into the transcriptional networks controlling the differentiation of human stem cells into cardiomyocytes, and the tools used in this project can be implemented in other biological contexts where cell fate‐defining regulatory networks have been more elusive.