We are acquiring real time dynamics data from a sensitive laser scanning microscope system with a built-in long-term tissue incubation capability. A postdoctoral fellow Erik Martin has characterized RelA and c-Rel dynamics in two different cell types with a panel of Toll-like receptor ligands using quantitative time lapse imaging. We used RNA-seq to identify gene clusters which are governed by specific dynamic features of RelA or c-Rel in the two cell types. We have also developed two novel knock-in mouse strains. Each strain expresses a fluorescent Rel (among the NF-B family of subunits) fusion from the native genomic locus, by CRISPR-Cas genome engineering. We screened pups for correct expression and integration of the targeting constructs. NF-B dynamics will be monitored alone or in parallel with cytokines or lineage TFs by crossbreeding the Rel knock-in mice with existing mice such as fluorescent reporters of cytokines or transcription factors associated with distinct phenotypes. Many of these factors are co-activated during immune cell differentiation. Moreover, simultaneous live cell imaging of two live reporters will reveal unprecedented insight about how separate dynamic networks interact with each other to achieve co-regulation of gene expression programs in immunity and cell differentiation. In a collaborative study (Chen G et al. 2018), a postdoctoral fellow Sayantan Chakraborty acquired live cell imaging data on mouse CD8+ T cells, supporting the general conclusion about a role of Ezh2 in cell cycle regulation in these primary lymphocytes.
| Chen, Guobing; Subedi, Kalpana; Chakraborty, Sayantan et al. (2018) Ezh2 Regulates Activation-Induced CD8+ T Cell Cycle Progression via Repressing Cdkn2a and Cdkn1c Expression. Front Immunol 9:549 |
