Distinct roles of INO80 in Naive and Primed state of Pluripotency

Authors

• A Shaukat
• X Wang
• B Lackford
• G Hu

Abstract

Pluripotent stem cells (PSCs) can self-renew and differentiate into all cell types in the
adult body. They exist in different pluripotent states, corresponding to discrete embryonic
development stages. Among those, the Naïve and Primed pluripotent states are widely
studied, representing the epiblast cells in the pre- and post-implantation embryos,
respectively.
Our previous work showed that the INO80 chromatin remodeler plays an important role
in the Primed state of pluripotency, and its deletion has little impact on the Naïve state. We hypothesize that additional factors may contribute to the unique roles of INO80 between the Naïve and Primed state.
To test the hypothesis, we endogenously tagged INO80 with the Turbo-ID biotin ligase
and carried out proximity labeling followed by mass spectrometry to identify INO80-
interacting factors in the Naïve vs. Primed state. This strategy identified many chromatin factors, such
as chromatin remodelers, transcription factors, and histone modifying enzymes. To
characterize the function of these identified factors in the Naïve and Primed state, we
synthesized a custom lentiviral CRISPR-KO library targeting these genes. We are
carrying out drop-out screens in the Naïve or Primed state using the custom library to
search for genes that impair cell maintenance in either or both states. We will also test the interaction of shortlisted hits with INO80 by co-immunoprecipitation, and investigate their roles in
INO80 function by utilizing various genomic (ChIP-seq and RNA-seq) approaches.
Together, we hope our studies will provide new mechanistic insights to the function of
INO80 in chromatin regulation and PSC maintenance.

Scientific Focus Area: Stem Cell Biology

This page was last updated on Tuesday, August 6, 2024