Uncovering the functional diversification mechanisms of transcription factor isoforms involved in stem cell differentiation

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SUMMARY Stem cells are unspecialized cells harboring the potential for differentiation into any cell within the body. In the landmark experiment in the Yamanaka lab, it was revealed the addition of four key transcription factors (TFs)— OCT4, SOX2, c-MYC and KLF4—can reprogram differentiated somatic cells back into induced pluripotent stem cells, demonstrating the outsize role of TF activities in controlling cell fate. A widespread mechanism by which TF activities can be modulated is alternative splicing, as nearly every TF gene expresses multiple splice isoforms. TF isoforms from the same gene can have different abilities to bind DNA targets, cofactors, or chromatin-associated proteins, resulting in complementary or opposing consequences to the gene regulatory network. Overall, alternative TF isoform usage is emerging as a major regulatory strategy in the control of differentiation and development; however, the precise isoform-specific functions of TFs in differentiation is poorly understood. The goal of the parent grant is to provide a fundamental understanding of mechanisms by which splicing modulates the function of TFs in stem cells. In the Sheynkman laboratory, we are developing a suite of approaches that enable isoform-specific detection, overexpression, and interactome mapping. Central to the parent grant's goals is to utilize exogenous overexpression of TF isoforms to interrogate their functions, to test for sufficiency of individual TF isoforms in their ability to induce differentiation outcomes. An independent, yet complementary, approach would be to test for the necessity of TF isoforms for effective differentiation outcomes, by attenuating expression of TF isoforms before and during differentiation. An array of tools exist for knockdown of genes for functional study, including RNAi and CRISPR Cas9. However, to precisely target for reduced expression of a particular isoforms is far more challenging. This is because for isoform-specific knockdown in expression, only a small region of the isoform containing a sequence-specific region can be targeted for morpholino, RNAi, or CRISPR binding, and due to variable efficacy, may not result in a robust knockdown or overexpression outcome. In the last year, Cas13-based technologies for isoform-specific transcript knockdown has shown promise. Leveraging these developments, along with the opportunity for making an impact in the candidate's career goals, we propose that the applicant develop methodology for the ability to attenuate expression of particular TF isoforms. The goal of this project is to test and apply newly emerging CRISPR Cas13-based technology within a stem cell model for experimentally testing the effect of TF isoforms, with the potential for multiplexed screening capability to test thousands of isoforms en masse. This project will provide a toolkit (isoform-specific knockdown) complementary to the overexpression screens in the parent grant, and greatly expand the space and scale of TF isoforms that can be interrogated in terms of their role in stem cell differentiation.