Probing the Formation and Function of Transcription Hubs

ABSTRACT Transcription hubs are hotspots in the genome with elevated transcription activities. Concentrating proteins and nucleic acids, transcription hubs are essential for cells to transcribe genes important for cell identity and cell type-specific functions, while their dysregulation leads to many diseases such as neurodegeneration and cancer. Although there is growing interest in understanding transcription hubs, a lot remains unknown. The goal of our research is to address two fundamental questions about transcription hubs, using advanced imaging and proteomics techniques. The first of these questions is: how are transcription hubs formed? Transcription hubs appear as distinct foci in the nucleus, and it is hypothesized that liquid-liquid phase separation is responsible for transcription hubs formation. We will use Yes-associated Protein (YAP) transcription hub, an ideal system established in my postdoctoral work to understand how transcription hubs form. YAP is a transcription coactivator activating genes important in cell proliferation and survival. After hyperosmotic stress, an inducible signal to activate YAP target gene transcription, we have found that YAP forms hubs with different components over time, coincident with their different functions (clustering accessible chromatins first, and activating transcription second). We will use candidate and unbiased approaches to identify protein components of YAP hubs with live-cell imaging, and then test the effect of modulating these proteins in accessible chromatin organization and transcription activation. We will also use single particle tracking to probe the biophysical nature of YAP transcription hubs, to understand if they form by phase separation or alternative mechanisms. The second question regarding transcription hubs is: how do transcription hubs activate transcription? We hypothesize that transcription hub can mediate enhancer-promoter interaction to activate transcription. We will use multicolor live- cell imaging to visualize localization of enhancer and promoter of Myc (a target gene of YAP), and see how their relative position to YAP transcription hub lead to Myc transcription activation. Together, these studies will elucidate molecular mechanisms of transcription hub formation and function. A better understanding of these mechanisms will have implications for the formation and function of other transcription hubs, and shed light on mechanisms of diseases of impaired transcription hub formation.