Figure S4.
Genomic analysis of Helios function in HPC7 and LSK cells. (A) LSK and LK phenotype of HPC7 cells and Helios expression. Blue histogram corresponds to cells stained with the secondary Ab only. FSC, forward scatter; SSC, side scatter. (B) Helios binding across genomic regions: promoter (−3 kb from transcription start site), gene body (exons, introns, UTR), distal intergenic. (C) Enriched DNA motifs found within the 1000 regions with the strongest Helios binding (ranked by decreasing pileup score). (D) Frequency of genes bound by Helios among those from the LT-HSC, MPP4, or MkP signatures, or among genes deregulated in He KO LT-HSCs. Random sets of 500 genes were selected from genes detected in the RNA-seq experiment. Poorly defined genes (Riken genes or Gene Models, rarely bound by Helios) were excluded from the gene lists that were intersected with the list of Helios-bound genes. ***, P < 0.001 (hypergeometric test). (E) Proportion of Helios-bound sites with a significant increase in chromatin accessibility (ATAC log2FC > 0.5; adj P < 0.1) among all ATAC-seq peaks, or peaks associated with MPP4 or MkP genes. P values were calculated with the hypergeometric test. (F) Metascape analysis of the enriched pathways associated with ATAC-seq regions that were significantly increased in KO LSK cells (adj P < 0.2). (G) Integrative genome viewer screenshots of WT and KO LSK ATAC-seq signals, and Helios, Gata2, and Runx1 binding in HPC7 cells, for the representative MPP4 genes. (H) Left: Seq-miner heatmap showing Helios, Gata2, and Runx1 binding on the 10,890 Helios-bound regions that coincided with an ATAC-seq peak. Three clusters (C1–C3) were defined using K-means clustering. Right: Mean profiles of the ATAC-seq signals in peaks from clusters C1–C3, in each pair of WT/KO replicates. (I) Proportion of Helios sites bound by Gata2 and/or Runx1, based on the magnitude of ATAC-seq changes. P values were calculated with the hypergeometric test, using the frequency of Gata2/Runx1 binding among unchanged peaks (−0.1 < log2FC < 0.1) as a reference distribution.

Genomic analysis of Helios function in HPC7 and LSK cells. (A) LSK and LK phenotype of HPC7 cells and Helios expression. Blue histogram corresponds to cells stained with the secondary Ab only. FSC, forward scatter; SSC, side scatter. (B) Helios binding across genomic regions: promoter (−3 kb from transcription start site), gene body (exons, introns, UTR), distal intergenic. (C) Enriched DNA motifs found within the 1000 regions with the strongest Helios binding (ranked by decreasing pileup score). (D) Frequency of genes bound by Helios among those from the LT-HSC, MPP4, or MkP signatures, or among genes deregulated in He KO LT-HSCs. Random sets of 500 genes were selected from genes detected in the RNA-seq experiment. Poorly defined genes (Riken genes or Gene Models, rarely bound by Helios) were excluded from the gene lists that were intersected with the list of Helios-bound genes. ***, P < 0.001 (hypergeometric test). (E) Proportion of Helios-bound sites with a significant increase in chromatin accessibility (ATAC log2FC > 0.5; adj P < 0.1) among all ATAC-seq peaks, or peaks associated with MPP4 or MkP genes. P values were calculated with the hypergeometric test. (F) Metascape analysis of the enriched pathways associated with ATAC-seq regions that were significantly increased in KO LSK cells (adj P < 0.2). (G) Integrative genome viewer screenshots of WT and KO LSK ATAC-seq signals, and Helios, Gata2, and Runx1 binding in HPC7 cells, for the representative MPP4 genes. (H) Left: Seq-miner heatmap showing Helios, Gata2, and Runx1 binding on the 10,890 Helios-bound regions that coincided with an ATAC-seq peak. Three clusters (C1–C3) were defined using K-means clustering. Right: Mean profiles of the ATAC-seq signals in peaks from clusters C1–C3, in each pair of WT/KO replicates. (I) Proportion of Helios sites bound by Gata2 and/or Runx1, based on the magnitude of ATAC-seq changes. P values were calculated with the hypergeometric test, using the frequency of Gata2/Runx1 binding among unchanged peaks (−0.1 < log2FC < 0.1) as a reference distribution.

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