Proteogenomic Reprogramming to a Functional Human Totipotent Stem Cell State via a PARP-DUX4 Regulatory Axis

Area of Science:

• Developmental Biology
• Stem Cell Biology
• Epigenetics

Background:

• Poly-ADP-ribose polymerases (PARPs), including PARP1 and Tankyrases (TNKS1/2), regulate genome and proteome during early embryonic development.
• Human pluripotent stem cells (hPSCs) represent a key model for studying totipotency and early human embryogenesis.

Purpose of the Study:

• To investigate the epigenetic reprogramming of hPSCs into totipotent-like cells.
• To explore the role of PARP1 and TNKS inhibition in achieving totipotency.
• To characterize the developmental potential and molecular profile of reprogrammed cells.

Main Methods:

• Chemical naive reversion of hPSCs using PARP1 and TNKS inhibitors.
• Single-cell RNA sequencing and proteomic analysis of reprogrammed cells (TIRN cells).
• ChIP-sequencing (ChIP-Seq) to map transcription factor binding sites.
• Interspecies chimera formation by injecting TIRN cells into murine embryos.

Main Results:

• hPSCs were epigenetically reprogrammed into clonal blastomere-like stem cells (TIRN cells) under PARP1/TNKS inhibition.
• TIRN cells expressed genes and factors characteristic of multiple early embryonic stages (4C-8C, primitive endoderm, trophectoderm, epiblast).
• TIRN cells demonstrated totipotent-like contribution to both embryonic and extra-embryonic lineages in human-murine chimeras.
• Global ADP-ribosylation shutdown and altered ubiquitination were observed, impacting PARP1/TNKS substrate levels and gene expression.

Conclusions:

• Global perturbations of post-translational modifications, specifically ADP-ribosylation and ubiquitination, can drive epigenetic reprogramming towards human totipotency.
• PARP1 and DUX4 cooperate with core pluripotency factors (NSO) to regulate epigenetic plasticity and developmental gene expression.
• Totipotent TIRN stem cells provide a novel model for studying human blastomere development and may aid in generating human organs in interspecies chimeras.