Abstract The goal of this proposal is to determine how lineage-specific repressive epigenetic landscapes are established to safeguard cell identity. Current dogma is that pioneer TFs initiate lineage-specific activation processes by locally opening chromatin. In contrast, significantly less is known about how repressive epigenetic landscapes are dynamically reshaped during cell differentiation. This knowledge gap creates a major stumbling block in developing high-fidelity cell reprogramming technology. Hence, it is essential to define how alternative lineage programs are repressed and how this process can be manipulated. Intriguingly, our preliminary data unexpectedly revealed that pioneer TFs are involved in this process. We developed a new doxycycline (dox)- inducible CRISPR interference (CRISPRi) tool to target two pioneer TFs: OCT4 in human pluripotent stem cells (hPSCs) and FOXA1/A2/A3 (FOXA) in hPSC-derived endoderm differentiation. We found that OCT4 and FOXA play critical roles in repressing alternative-lineage programs. In fact, gene expression changes in OCT4/FOXA- CRISPRi cells correlate better with directional changes of the repressive H3K27me3 mark than with those of H3K4me1/3 active marks. To identify mechanisms underlying OCT4/FOXA-directed repression, we focused on the OCT4 and FOXA binding peaks that co-localize with the H3K27me3 domain. Interestingly, we found that these loci are highly enriched at key alternative-lineage genes, and unbiased motif analysis revealed significant enrichment for a PRDM14 (a key pluripotency TF) binding motif at OCT4 sites and a PRDM1 (a key germ-cell TF) binding motif at FOXA sites. This data provides the first indication that PRDM1 may play an important role in endoderm specification. Further, our ChIP-qPCR assays confirmed that OCT4-CRIPSRi hPSCs were impaired in recruiting PRDM14, PRC1/H2Aub, and PRC2 at OCT4-bound H3K27me3 sites. Intriguingly, our preliminary co-IP assay suggested that FOXA interacts with PRC1, and prior co-IP studies showed that OCT4 interacts with PRC1, and PRDM14 and PRDM1 interact with PRC25–9. Collectively, our findings led to our overarching novel hypothesis that pioneer TFs play a crucial role in establishing Polycomb repressive domains at key alternative- lineage genes by recruiting PRC1 and/or PRDM TFs, which in turn recruit PRC2. We will test this hypothesis in two Aims: Aim 1: Test the hypothesis that OCT4 and FOXA play instructive roles in recruiting PRCs and PRDM TFs to establish lineage-specific Polycomb repressive domains. Aim 2: Determine how PRDM1 impacts accurate lineage restriction during human endoderm specification. The successful completion of our Aims will provide valuable insights and reveal novel mechanisms underlying lineage-specific Polycomb repression to safeguard cell identity. In the long-term, this study will likely open new avenues to manipulate cell fate by selectively repressing alternative regulatory programs, with potential future therapeu...