Evaluating the capabilities of POU (Pit-Oct-Unc) transcription factors to engage epigenetically silenced chromatin

Abstract

Pioneer transcription factors (TFs) bind to and activate epigenetically silenced chromatin, making them critical regulators of cell fate. Oct4 of the Pit-Oct-Unc (POU) TF family is essential to establishing and maintaining pluripotency. Oct4 binds to silenced chromatin, such as methylated DNA or DNA compacted by nucleosome core particles (NCPs). Significant gaps remain in accurately defining the unique traits of pioneer TFs to differentiate them from collaborating non-pioneering TFs. Furthermore, the mechanism behind how Oct4 asserts its function on methylated DNA remains elusive. In this study, Oct4's interaction with a methylated DNA element called “CpGpal” was examined. A re-analysis of genomics datasets revealed that Oct4 binds to CpGpal in pluripotent cells and during reprogramming into induced pluripotent stem cells (iPSCs). Interestingly, other POU factors, like Brn2, avoid CpGpal in cells. Remarkably, while global demethylation occurs at most Oct4-bound enhancers during iPSC reprogramming, methylation at CpGpal sites remains largely unchanged. To differentiate pioneer- and collaborating non-pioneer TFs, Oct4 and Brn2's binding to methylated DNA and nucleosomes were examined. Brn2, a homologous protein of Oct4, is a POU factor with essential neural development functions. POU factors possess a DNA binding domain comprised of two sub-domains: POU-specific (POUS) and POU-homeodomain (POUHD). Oct4 was found to bind CpGpal as a homodimer, driven by the POUHD domain, while the POUS domain remains detached from DNA. Conversely, Brn2 was found to bind CpGpal primarily as a monomer. Both Oct4 and Brn2 show non-specific binding to nucleosomes. However, Oct4 was found to specifically target its consensus binding sequence (Octamer motif) on nucleosomes when present, while Brn2 kept binding non-specifically. Collectively, the investigation revealed that Oct4, a pioneer TF, targets silenced chromatin differently from Brn2, a collaborating TF. These differences include homeodomain-driven dimerization on methylated DNA and precise binding to cognate motifs in the context of nucleosomes. The evaluation of POU factors and their partners was further explored from a molecular evolution perspective. POU and their interactions with Sox factors are crucial to regulating stemness in mammals. To investigate the evolutionary conservation of biochemical properties in the closest unicellular relatives of animals, we compared Sox and POU TFs from choanoflagellates and filastereans to their mammalian counterparts. Results revealed unicellular Sox TFs exhibiting DNA-binding specificity like mammalian Sox TFs. However, unicellular POU TFs have binding profiles different from Oct4. Notably, choanoflagellate Sox can interact with mammalian Oct4 on pluripotency enhancer DNA elements. Our findings imply that ancestral Sox genes already had the molecular capacity to participate in the pluripotency network, suggesting their potential to be repurposed as animal stem cell regulators. However, POU factors required further evolutionary innovations to partner with Sox to regulate the pluripotency network. These discoveries reveal insights into the asynchronous molecular evolution of Sox and POU factors for pluripotency and their possible role in the evolution of multicellular animals. Overall, this study deepens our understanding of how different binding patterns influence the divergent biological functions of POU paralogs. It also redefines the role of stemness factors in facilitating the evolution to multicellularity in animals.