An in vivo screen of noncoding loci reveals that Daedalus is a gatekeeper of an Ikaros-dependent checkpoint during haematopoiesis

Abstract

Haematopoiesis relies on tightly controlled gene expression patterns as development proceeds through a series of progenitors. While the regulation of hematopoietic development has been well studied, the role of noncoding elements in this critical process is a developing field. In particular, the discovery of new regulators of lymphopoiesis could have important implications for our understanding of the adaptive immune system and disease. Here we elucidate how a noncoding element is capable of regulating a broadly expressed transcription factor, Ikaros, in a lymphoid lineage-specific manner, such that it imbues Ikaros with the ability to specify the lymphoid lineage over alternate fates. Deletion of the Daedalus locus, which is proximal to Ikaros, led to a severe reduction in early lymphoid progenitors, exerting control over the earliest fate decisions during lymphoid lineage commitment. Daedalus locus deletion led to alterations in Ikaros isoform expression and a significant reduction in Ikaros protein. The Daedalus locus may function through direct DNA interaction as Hi-C analysis demonstrated an interaction between the two loci. Finally, we identify an 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. These findings represent a key illustration of how a transcription factor with broad lineage expression must work in concert with noncoding elements to orchestrate hematopoietic lineage commitment.

Keywords: Ikaros; hematopoiesis; lymphocytes; noncoding.

Fig. 1.

An in vivo CRISPR screen identifies a noncoding locus regulating lymphoid development. (A) Heatmap of differentially expressed noncoding transcripts identified in RNA-seq of CD4 effector T lymphocytes. The Daedalus locus was originally termed as Locus #7 and is denoted here by the number 7 and highlighted with a green box. (B) Representative plots from splenic FACS data show peripheral lymphocyte populations largely unaffected in noncoding RNA locus-deficient mice. CD4 T cell populations gated off CD4+ TCRb+ splenocytes: Naive CD4: CD62L+CD44–; Effector CD4: CD44+CD62L–; CD25hi: CD4+ CD25+. B-cells: gated on TCRb- NK1.1– CD19+ splenocytes; n = 3, representative of more than three pooled experiments. (C) Representative plots from thymic screening data showing early thymic progenitor populations (ETP: lineage-negative [lineage mixture: B220, CD11b, CD11c, CD19, Gr1, NK1.1, TCRgd, Ter119], CD4–, CD8–, CD25–, CD44+, cKit+). (D) Representative FACS plot of wild-type and Daedalus^−/− littermates showing relative ETP populations. (E) Representative FACS plots and bar graphs of WT and Daedalus^−/− littermate mice showing the LMPP population (Lineage negative [lineage mixture: B220, CD4, CD8, CD11b, CD11c, CD19, Gr1, NK1.1, TCRgd, Ter119], cKit+, Sca1+ Flt3hi); n = 3, representative of 10+ experiments. In this figure, unless otherwise specified, all statistics result from comparisons to WT using Student’s t test. *P < 0.05, ***P < 0.001.

Fig. 2.

The locus Daedalus controls lymphoid development through modulation of Ikaros protein levels. (A) WT and Daedalus^−/− littermate mixed chimera studies, representative plots of chimerism in Mature B and DN4 cells, plots of percentage chimerism vs. CD45.1 competitor bone marrow in B and T lymphocyte developmental pathways; n = 3 representative of three experiments. (B) Representative pictures and bar graphs from B-cell methocult colony-forming assays; n = 3, representative of five or more experiments. (C) Representative FACS plots and bar graphs of LMPP populations from Ikaros heterozygous, Daedalus^−/−, and Ikaros dF4^−/− mice and WT littermates. LMPP: lineage-negative (lineage mixture: B220, CD4, CD8, CD11b, CD11c, CD19, Gr1, NK1.1, TCRgd, Ter119), cKit+, Sca1+ Flt3hi; n = 3, representative of three experiments. (D) qPCR experiment of Ikzf1 isoforms from mouse lineage-negative cKit+, Sca1+ hematopoietic progenitor cells; n = 1; data representative of multiple experiments with different sets of Ikzf1 primers (SI Appendix, Table S3 and Fig. S7). (E) Intracellular staining of Ikzf1 in Ikzf1 KO escape mutant (n = 1) and Daedalus^−/− and WT littermate mice; Daedalus^−/− data are representative of more than six experiments. In this figure, unless otherwise specified, all statistics result from comparisons to WT using Student’s t test. *P < 0.05.

Fig. 3.

Daedalus controls Ikaros-dependent lymphoid erythromyeloid checkpoint. (A) Heatmap and dendrogram of total gene expression across hematopoietic progenitor subsets from whole-transcriptome analysis. (B) Fragments per kilobase of transcript per million mapped reads (Fpkm) expression levels of Ikaros target genes in WT and KO Flt3+ LSK progenitors. (C) Fpkm expression levels of key developmental regulators in WT and KO Flt3+ LSK progenitors. (D) Dot plots of marker genes across all cells for lymphoid-associated genes. The size of the dot illustrates the percentages of cells expressing a given gene, while the color indicates the level of expression. (E) Dot plots of marker genes across all cells in the clusters containing early progenitor and lymphoid progenitor markers for nonlymphoid and lymphoid genes. The size of the dot illustrates the percentages of cells expressing a given gene, while the color indicates the level of expression. (F) FACS plots of erythromyeloid progenitors from WT and Ikaros KO escape mutant littermates. (Top) Gated-off lineage-negative (lineage mixture: B220, CD4, CD8, CD11b, CD11c, CD19, Gr1, NK1.1, TCRgd), cKit+, Sca1–, and CD16/32– cells. (Bottom) Gated-off lineage-negative cKit+, Sca1–, CD16/32–, CD105+, and CD150 cells. (G) Bar graphs of total colonies from erythroid colony-forming assay results from equal numbers (2 × 106) of bone marrow from Daedalus^–/– and WT littermate mice (Left); n = 3; data are representative of two experiments from Ikaros heterozygous mice and WT littermates (n = 2) (Right). (H) qRT-PCR experiment of Ikzf1 and Daedalus RNA expression in LSK progenitors, GMP progenitors (lineage-negative cKit+, Sca1–, CD16/32+), and erythroid progenitors (lineage-negative cKit+, Sca1–, CD16/32–, CD105+). (I) Intracellular Ikaros protein levels by median fluorescence intensity (MFI) in subsets of lymphoid, myeloid, and erythroid progenitors; n = 4, representative of two experiments. In this figure, unless otherwise specified, all statistics result from comparisons to WT using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.