UC San Diego

T cells are critical players in the adaptive immune system with broad roles that include activating other immune cells and mediating the response to threats such as viruses and cancer. A recent study demonstrated that ThymoD, a long non-coding RNA, directs T cell lineage determination by instructing changes in chromatin folding across a genomic region encoding for Bcl11b, a critical transcription factor in immune development. ThymoD facilitated binding of CTCF, an architecture protein, to bring the Bcl11b enhancer and promoter region close together and promote its transcription. Here, we examined the role of CTCF in regulating Bcl11b by ablating CTCF binding sites found within the ThymoD and Bcl11b genomic regions. We identified five sites in which CTCF binding was lost in ThymoD mutants. When we deleted them in a DN3-like cell line, each of these mutations significantly reduced Bcl11b expression. Bcl11b expression further diminished when pairs of CTCF sites were deleted together, demonstrating that partially redundant CTCF sites cooperatively orchestrate Bcl11b expression.We then asked whether CTCF binding sites were necessary for immune development by knocking out two of these sites individually in mice: CR4 and B4. Mild but significant changes in the CR4 -/- mice were observed in thymic double negative T cell populations. In order to control for age and size differences between mice, we employed a system of culturing bone marrow progenitors on stromal cells that present Notch ligands. There were dramatic changes to all double negative T cell compartments derived from CR4 -/- progenitors, with an increase of DN1/2 and a loss of DN3/4 cells. We further saw an increase in developmental markers for alternative immune lineages in progenitors derived from B4 -/- mice. Our genomics analysis of the CR4 -/- mice demonstrated that CTCF binding was only lost at the targeted region. This loss was associated with a significant reduction in Bcl11b expression and an overlapping set of differentially regulated genes to those found in ThymoD p(A)/p(A) and Bcl11b -/- mice. Altogether, this study establishes a requirement for CTCF binding in activating Bcl11b expression and directing T cell fate determination.