An in-vivo screen of noncoding elements reveals that Daedalus is a gatekeeper of a novel Ikaros-dependent checkpoint during haematopoiesis I

The development of all lymphoid lineages relies on a tightly-controlled series of gene expression patterns as development proceeds through a series of progenitors1-8. These gene expression patterns are understood to be controlled by lineage-specifying transcription factors, however these factors often are found to have broad expression patterns and activities across lineages. Given their role in regulating cell-type specific gene expression, noncoding loci are thought be important in the lineage-specific regulation of this complex process6,9-16. To identify transcripts that might mark such loci, we performed transcriptomic analysis in terminally differentiated murine CD4 effector T lymphocytes. Our analysis identified a variety of loci containing dynamically expressed noncoding RNAs and we tested their role in the lymphoid lineage using an in-vivo CRISPR knockout-mouse screen. This approach revealed that a non-coding locus proximal to the haematopoietic transcription factor Ikaros17,18, named Daedalus, exerted control over the earliest fate decisions during lymphoid lineage commitment. Deletion of Daedalus led to a cell-autonomous reduction in bone marrow and thymic lymphoid progenitor populations, phenocopying Ikaros deficient strains. Strikingly, loss of Daedalus resulted in a loss of Ikaros protein expression without impacting total Ikaros transcript levels. Finally, we identify a novel Ikaros regulated erythroid-lymphoid checkpoint that is governed by Daedalus in a lymphoid-lineage specific manner. Daedalus appears to act as a gatekeeper of Ikaros's broad lineage specifying functions, selectively stabilizing Ikaros activity in the lymphoid lineage and permitting diversion to the erythroid fate in its absence. To our knowledge, these findings represent the first illustration of how a transcription factor with broad lineage expression must work in concert with non-coding elements to orchestrate haematopoietic lineage specification and commitment. Overall design: In order to uncover novel non-coding loci with the potential to regulate lymphoid cell biology, whole transcriptomic analysis of purified T-cell populations was conducted. Populations of CD4 “memory-like” (CD44hi CD62L-) and “naïve” (CD44- CD62Lhi) cells were sorted from mouse lymph nodes. Separately, naïve T-cells from wild-type mice were sorted and polarized under neutral “Th0” conditions, with stimulation from anti-CD28 and anti-CD3 antibodies, which bind the TCR and CD28 costimulatory receptor, stimulating antigen binding. In the “Th0” culture conditions, antibodies blocking Il-4 and interferon gamma (IFN?) are also added to block Th1 and Th2 differentiation, and Il2 is added to stimulate T-cell differentiation and proliferation. In addition, purified populations of “naïve” CD4 T-cells were sorted from several reporter mouse lines were isolated, polarized into effector subsets in culture and sorted using reporter expression to generate highly purified effector populations. Naïve CD4 T-cells from IFN?, Il-4, Il-9, Il-17 and FoxP3 reporter mice were sorted and polarized under Th1, Th2, Th9, Th17 and Treg conditions respectively. Purified populations were obtained by fluorescence-activated cell sorting of cells expressing the respective reporter genes to generate CD4 effector T-cell populations with a very high purity. RNA was extracted from these populations, along with from Th0-polarized effector CD4 cells and the naïve and memory populations described previously. This RNA was then used for a transcriptomic analysis by RNA-sequencing in order to identify differentially expressed RNAs between different effector T-cell populations that might mark important noncoding loci.