Figure 6
Figure 6. Altered expression of cell cycle regulatory genes in Hhex−/Δ lymphoid precursors. (A) Deletion of Hhex exons 2 and 3 Hhex−/Δ in progenitors used in RNA-sequence analyses. Read depth analysis is shown at the Hhex locus for RNA-sequence data obtained from populations of CLPs and “pro-B” cells (CD19+ Kit+ cells) from mice of the indicated Hhex genotypes. This shows a loss of reads at exon 2-3 of Hhex in Hhex−/Δ cells. Arrows at the bottom indicate the location of loxP sites in the Hhexfl allele. (B) Expression of lymphoid transcription factors in Hhex−/Δ progenitors. Data show the globally normalized exonic read counts of each of each gene in CLPs and “pro-B” cells of the indicated Hhex genotypes, as determined by RNA sequencing. Units are reads per thousand exonic bases per million mapped reads. (C-D) Gene Set Enrichment Analysis showing association of Hhex-regulated genes in CLPs and “pro-B” cells. In (C), genes significantly upregulated in Hhex−/Δ CLPs were compared in wild-type and Hhex−/Δ “pro-B” cells. In the enrichment plot, skewing to the right indicates that most genes are also upregulated in Hhex−/Δ “pro-B” cells. The heat-map (right) shows the top 30 genes most upregulated in “pro-B” cells. Arrowheads show upregulation of Cdkn2a and Cdkn2b. In (D), genes significantly downregulated in Hhex−/Δ CLPs were compared in wild-type and Hhex−/Δ “pro-B” cells. In the enrichment plot, skewing to the left indicates that most genes are also downregulated in Hhex−/Δ “pro-B” cells. The heat-map (right) shows the top 30 most downregulated genes in “pro-B” cells. Arrowhead shows downregulation of Ccnd1. (E) Loss of Ccnd1 expression in Hhex−/Δ CLPs. RNA-sequencing tracks show RNA-sequencing coverage of CLPs at the Ccnd1 locus. Units are reads per kilobase per million mapped reads. (F) Real-time PCR data showing loss of Ccnd1 expression in Hhex−/Δ progenitors. Data are mean + standard deviation of 3 independent experiments. FDR, false discovery rate; NES, normalized enrichment score; WT, wild-type; KO, knockout. *P < .05; ***P < .001 by Student t test.

Altered expression of cell cycle regulatory genes in Hhex−/Δ lymphoid precursors. (A) Deletion of Hhex exons 2 and 3 Hhex−/Δ in progenitors used in RNA-sequence analyses. Read depth analysis is shown at the Hhex locus for RNA-sequence data obtained from populations of CLPs and “pro-B” cells (CD19+ Kit+ cells) from mice of the indicated Hhex genotypes. This shows a loss of reads at exon 2-3 of Hhex in Hhex−/Δ cells. Arrows at the bottom indicate the location of loxP sites in the Hhexfl allele. (B) Expression of lymphoid transcription factors in Hhex−/Δ progenitors. Data show the globally normalized exonic read counts of each of each gene in CLPs and “pro-B” cells of the indicated Hhex genotypes, as determined by RNA sequencing. Units are reads per thousand exonic bases per million mapped reads. (C-D) Gene Set Enrichment Analysis showing association of Hhex-regulated genes in CLPs and “pro-B” cells. In (C), genes significantly upregulated in Hhex−/Δ CLPs were compared in wild-type and Hhex−/Δ “pro-B” cells. In the enrichment plot, skewing to the right indicates that most genes are also upregulated in Hhex−/Δ “pro-B” cells. The heat-map (right) shows the top 30 genes most upregulated in “pro-B” cells. Arrowheads show upregulation of Cdkn2a and Cdkn2b. In (D), genes significantly downregulated in Hhex−/Δ CLPs were compared in wild-type and Hhex−/Δ “pro-B” cells. In the enrichment plot, skewing to the left indicates that most genes are also downregulated in Hhex−/Δ “pro-B” cells. The heat-map (right) shows the top 30 most downregulated genes in “pro-B” cells. Arrowhead shows downregulation of Ccnd1. (E) Loss of Ccnd1 expression in Hhex−/Δ CLPs. RNA-sequencing tracks show RNA-sequencing coverage of CLPs at the Ccnd1 locus. Units are reads per kilobase per million mapped reads. (F) Real-time PCR data showing loss of Ccnd1 expression in Hhex−/Δ progenitors. Data are mean + standard deviation of 3 independent experiments. FDR, false discovery rate; NES, normalized enrichment score; WT, wild-type; KO, knockout. *P < .05; ***P < .001 by Student t test.

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