Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency

Abstract

Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.

The stoichiometry and interactome of the Nucleosome Remodeling and Deacetylase (NuRD) complex are conserved across multiple cell lines

“…This indicates that these two proteins are not functionally redundant. A similar observation has been made for the GATAD2 subunit: depletion of GATAD2A but not GATAD2B leads to highly efficient induced pluripotent stem cell formation . In addition, GATAD2A‐NuRD is selectively recruited by ZMYND8 to DNA double‐stranded breaks .…”

High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency

“…We have also found that complete inhibition of Gatad2a (also known as P66a), a NuRD specific subunit, does not compromise somatic cell proliferation as previously seen upon complete Mbd3 protein elimination, and yet disrupts Mbd3/NuRD repressive activity on the pluripotent circuitry and yields 90-100% highlyefficient reprogramming within 8 days as similarly observed in Mbd3 hypomorphic donor somatic cells (Extended Data Fig. 1 and Mor N. et al 15 Under final preparation). Such systems now enable high-resolution temporal dissection of epigenetic dynamics underlying conducive naïve iPSC formation, while simultaneously eliminating 'noise' from heterogeneous populations that fail to correctly embark or successfully complete the reprogramming process.…”

“…Secondary MEF for Gatad2a -/cell line and WT-2 were obtained as described by Mor et al 15 . Shortly, iPSCs were established following primary reprogramming of cells using M2rtTA and TetO-OKSM-STEMCCA.…”

Long noncoding RNAs sustain high expression of exogenous Oct4 by sponging miRNA during reprogramming

“…In the study, we performed ribo-minus RNA-seq and small RNA-seq from cells collected at the early reprogramming stages and fully reprogrammed iPSCs which can generate "all-iPSCs" pups ( Fig 1A), to profile expression patterns of miRNAs, lincRNAs, circRNAs and mRNAs. We also took advantage of publicly available RNA-seq and small RNA-seq data from Mbd3 flox/reprogramming system, which results in near 100% efficiency of iPSCs reprogramming within 7 days by OSKM induction [28], to confirm our analysis. Our studies ought to determine the possible function of linc/circRNAs as miRNAs sponge during cellular reprogramming.…”

“…During reprogramming, genes associated with pluripotent establishment need to be activated or up-regulated, whereas developmental genes need to be silenced [28]. In order to determine the role of miRNAs in regulating the transcriptomic changes, we profiled expression of mRNAs on MEFs, rMEFs, and iPSCs as well, and then analyzed the effect of the MEFs-highly expressed miRNAs on up-and down-regulated genes during reprogramming.…”