Gene expression profiles at the single cell level of TNKS/PARP-inhibitor Regulated Naïve (TIRN) cells with or without transgenic inducible DUX4 (iDUX4) activation.

Proteogenomic Reprogramming to a Functional Human Blastomere-like Stem Cell State via a PARP-DUX4 Regulatory Axis; Zimmerlin et al, 2024. PARP1 (ARTD1) and Tankyrases (TNKS1/TNKS2; PARP5a/5b) are poly-ADP-ribose polymerases (PARPs) with catalytic and non-catalytic functions that regulate both the genome and proteome during zygotic genome activation (ZGA), totipotent, and pluripotent embryonic stages. Here, we show that primed, conventional human pluripotent stem cells (hPSC) cultured continuously under non-specific TNKS1/TNKS2/PARP1-inhibited chemical naïve reversion conditions underwent epigenetic reprogramming to clonal blastomere-like stem cells. TIRN stem cells concurrently expressed hundreds of gene targets of the ZGA-priming pioneer factor DUX4, as well as a panoply of four-cell (4C)-specific (e.g., TPRXL, HOX clusters), eight-cell (8C)-specific (e.g., DUXA, GSC, GATA6), primitive endoderm-specific (e.g., GATA4, SOX17), trophectoderm-specific (e.g., CDX2, TFAP2C), and naïve epiblast-specific (e.g., DNMT3L, NANOG, POU5F1(OCT4)) factors; all in a hybrid, combinatorial single-cell manner. Mapping of proteomic and single-cell expressions of TIRN cells against human preimplantation embryo references identified them as relatively homogenous 4C-8C stage populations. The bulk RNA-seq and proteomics analyses of TIRN cells (+/- iDUX4) revealed acquisition of pre-lineage embryonic transcriptional programs. To determine if TIRN +/- iDUX4 cells were mixed populations or clonal blastomere-like cells, single cell (sc)RNA-seq was performed to investigate transcriptional heterogeneity at a single cell level. Overall, single, unmodified TIRN cells shared high transcriptional identity with single TIRN+iDUX4 cells, and single primed hPSC and TIRN +/- iDUX4 cells expressed comparable and uniformly homogenous levels of core pluripotency factors (e.g., NANOG, POU5F1 (OCT4), SOX2, KLF4, CMYC). However, single TIRN and TIRN+iDUX4 populations homogenously co-expressed not only higher levels of naïve ICM-specific genes (e.g., DNMT3L, SP5, ERVH48-1, GDF3, and IFITM1) (Park et al., 2020; Zimmerlin et al., 2016), but also hundreds of diverse and antagonistic lineage-specifying genes (e.g., HOX-PRD, T-BOX, FOX, GATA families) that are co-expressed simultaneously only during 4C-to-morula human embryonic stages (Petropoulos et al., 2016; Zou et al., 2022). Transcript levels of 4C-specific (i.e., TPRXL, HOXA1, EBF2), 8C-specific (i.e., GATA6, FOXB1), trophectoderm lineage-specific (i.e., CDX2, TFAP2C), and endoderm lineage-specific (i.e., GATA4, SOX17) pioneer factors were co-expressed in the same single cell populations; with expressions that were further augmented following iDUX4 activation. TIRN cells were originally reported as possessing a naive epiblast-like pluripotent stem cell molecular phenotype, but also simultaneously expressed cohorts of transcriptional factors and a transcriptional program defining the human cleavage embryonic stage. We used single cell RNA sequencing (scRNA-seq) to examine cellular heterogeneity in TIRN cells, with or without transgenic activation of the pioneer factor DUX4. The hESC line RUES02 was cultured in parallel in primed (E8) or TIRN (LIF-3i) condition. RUES02 with transgenic doxycycline-inducible DUX4 expression (pCW57.1-DUX4-CA) were cultured in TIRN (LIF-3i + 24 hours 2ug/mL doxycycline). Viable cells were enriched and processed for single cell analysis.