Probing long range genomic interactions and gene expression dynamics that instruct T cell fate

Abstract

VI newlineABSTRACT newlineCell growth, differentiation, and response to external stimulation all necessitate precise and newlinecoordinated control of gene expression. It is becoming clear that gene expression programs rely newlineon cis-regulatory interactions mediated through the binding of combinatorial transcription factors. newlineHere, we have focused on studying molecular mechanisms that underlie Bcl11b promoterenhancer newlineinteractions and the transcription dynamics of Id3 that are essential for the development newlineof diverse T cell lineages, by establishing genetically engineered animal models. We have pursued newlinethese, using high-throughput molecular approaches and single cell live imaging technology. These newlinestudies have revealed a novel CTCF binding region necessary for Bcl11b activation. Deletion of newlinethis CTCF region impeded Bcl11b expression, indicating a change in chromatin confirmation that newlineis incompatible with its expression. Consistent with these observations, multipotent progenitors newline(MPPs) isolated from CTCF-mutant animals failed to differentiate into T cells, as evidenced by a newlineblock at the DN2 cell stage. Furthermore, genome-wide expression analysis has revealed that newlinemutation of the CTCF binding region within the Bcl11b locus perturbed the expression pattern of newlinea large number of genes, indicating a key role for CTCF binding within the Bcl11b locus. In newlineparallel, single cell live imaging studies have shown that Id3 transcription is found to be newlinedynamically switching between on and off states. Furthermore, the threshold of Id3 transcription newlineis positively correlated with the externally regulated signals that drive diverse T cell lineages (and#947;and#948; newlineand and#945;and#946; TCR signaling). It is also remarkable that upon antigenic stimulation double negative T newlinecells exhibit increased Id3 burst transcription as determined by high intensity bursting activity. newlineThis was antagonized by inhibiting ERK signaling. The transcription variations could be explained newlineby changes in the proximity interaction between cis-regulatory elements and/or binding of newlinetranscription factors, suggest