Functional organization of co-regulated RNA polymerase II nuclear subcompartments activated by TNFα or TGFβ
During development or in response to external signals, the genome organizes into active or silenced chromatin subcompartments to implement cell type/state specific gene expression programs. How this segregation on the µm scale is established and relates to transcriptional activity is a long-standing question. Recent studies propose that the unmixing of soluble factors from the nucleoplasm into protein droplets around chromatin loci by a liquid-liquid phase separation (LLPS) drives the assembly of active “transcriptional condensates”. However, alternative models exist that are based on the chromatin binding of proteins and RNAs together with long- and short-range bridging interaction between these factors and looping of the intervening nucleosome chain. The project proposed here will investigate structure-function relationships underlying the assembly of endogenous promoters and enhancers into co-regulated active RNA polymerase II (Pol II) subcompartments. Transcriptional activation will be induced by stimulation of human cells with TNFα (signaling via the NFκB transcription factor) and TGFβ (signaling via SMAD2/3 transcription factors). These two cytokines cause acute and delayed induction of specific gene expression programs. In this cellular system the activation of co-regulated gene clusters has been established previously. Furthermore, p65/RelA as part of NFκB as well as SMAD3 have intrinsically disordered regions and can form liquid droplets by LLPS when ectopically expressed. Thus, stimulation with TNFα and TGFβ is ideally suited to assess a potential contribution of LLPS to the formation and/or activation of Pol II subcompartments. To dissect the underlying mechanisms, we will combine state-of-the-art single cell sequencing and fluorescence microscopy methods into a spatially resolved transcriptomics approach. By applying an integrative analysis of co-regulation, changes of RNA production, protein mobility and interactions and chromatin features we will reveal organization principles that underly the formation/activation of Pol II subcompartments upon TNFα and TGFβ induction. In this manner, we will assess the contribution of LLPS to this process and advance our understanding on how the dynamic nuclear organization of Pol II subcompartments is coupled to gene activation.
Dr. Karsten Rippe,Heidelberg
Deutsches Krebsforschungszentrum (DKFZ)
FSP Funktionelle und strukturelle Genomforschung
Im Neuenheimer Feld 267