UCSF

The acquisition of cell fate is dependent on gene regulatory networks that are regulated spatiotemporally by cell type specific transcription factors (TFs). In binding to a class of cis-regulatory elements known as enhancers, TFs stimulate transcription at target genes. Studies on enhancers have revealed that enhancers exist in multiple different states and interconvert between them. The establishment of an active, transcriptionally promoting enhancer state from an inactive state is a stepwise process initiated by TFs and then facilitated by chromatin regulators. Here, we investigate the molecular processes required to establish an active, transcriptionally promoting enhancer state. To study enhancer activation, we utilize an in vitro embryonic stem cell differentiation system known as the naive to formative transition that recapitulates gene regulatory events that occur in vivo during mouse early embryogenesis. We first investigate the generalizability of current molecular models of enhancer activation in Chapter 2. Using the naive to formative transition and genetic deletions we find that the homologous enzymes MLL3/4 (KMT2C/D) are required for activation of some but not all enhancers as previously thought. Moreover, surprisingly, there is an underwhelming impact on gene expression changes during the transition despite loss of MLL3/4 and the loss of “active” molecular signatures at many enhancers. This work demonstrates the existence of multiple modes of enhancer activation and suggests a more cell-context specific role for the key chromatin regulators MLL3/4 than known before. In ongoing work in Chapter 3, we are investigating the current prevailing molecular model of enhancer activation more deeply. We built a new synthetic system using the TF Grhl2 for rapid and conditional induction of enhancer activation. We are currently employing this system to identify the mechanistic relationships between Grhl2, MLL3/4, other key chromatin regulators, and transcription. Our discoveries on the molecular fundamentals of enhancer state dynamics serve as a foundation on which to better understand drivers of human development and disease.