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

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. In order to assess the effect of Daedalus knockout on genome-wide chromatin accessibility, ATAC-seq was performed on sorted progenitors from Daedalus-/- mice and wild-type littermates. This assay permits assessment of changes in chromatin accessibility on a genome-wide scale, enabling determination of whether the loss of Daedalus disrupts histone remodeling at known Ikaros-regulated loci. Ikaros is known to act primarily as a part of the chromatin-modifying NuRD complex, exerting its function on gene expression through epigenetic modifications that affect locus accessibility. Since deletion of the Daedalus locus leads to apparent disruption of Ikaros function, it was hypothesized that changes in chromatin accessibility would be observed in the Daedalus knockout cells.