Figure 6.
Gene expression changes due to Calr haploinsufficiency and the CALRdel52 mutant. (A) Cell collection conditions in RNAseq experiments. Unpurified BM cells (1 × 106; B6-CD45.2) were transplanted into lethally irradiated recipients (B6-CD45.1; n = 9-12 in each group). At 8 to 12 weeks after transplantation, 2 × 104 CD45.2+ LSK cells were sorted from BM cells pooled from 3 recipient mice and used as a sample for RNAseq analysis. We prepared a total of 14 samples: 3 WT, 3 Calr+/−, 4 CALRdel52;Calr+/+, and 4 CALRdel52;Calr+/−. (B) Unsupervised hierarchical clustering of the global gene expression signatures. (C) Venn diagram depicting differentially expressed genes in LSK cells from Calr+/−, CALRdel52;Calr+/+, and CALRdel52;Calr+/− mice relative to WT controls (false discovery rate <0.1). Forty-seven genes were differentially expressed (23 upregulated and 24 downregulated) in Calr+/− mice. (D) Hierarchical clustering of expression profiles of these 14 samples according to the 47 genes differentially expressed in Calr+/− mice. Genes (rows) and genotypes (columns) with dendrograms are shown. Dendrograms were constructed using Pearson correlation. This analysis distinguished the 4 genotypes relatively clearly. The second cluster of genes was upregulated in Calr+/− mice and CALRdel52;Calr+/− mice and was clearly downregulated in WT and CALRdel52;Calr+/+ mice (colored with red). (E) Result of GSEA analysis showing enrichment of the stem cell signature29 in CALRdel52;Calr+/− LSK cells compared with CALRdel52;Calr+/+ LSK cells. (F) GSEA results using hallmark gene sets for CALRdel52;Calr+/− and CALRdel52;Calr+/+ LSK cells. Heatmap shows the normalized enrichment score (NES) of significantly altered gene sets in CALRdel52;Calr+/− cells compared with CALRdel52;Calr+/+ cells (false discovery rate q value <0.25 or normalized P value <.05). Positive ES (red) indicates that the gene set was enriched in CALRdel52;Calr+/− LSK cells, and negative ES (blue) indicates that the gene set was de-enriched in CALRdel52;Calr+/− LSK cells. Result of GSEA analysis gene sets highlighted in blue or red are shown

Gene expression changes due to Calr haploinsufficiency and the CALRdel52 mutant. (A) Cell collection conditions in RNAseq experiments. Unpurified BM cells (1 × 106; B6-CD45.2) were transplanted into lethally irradiated recipients (B6-CD45.1; n = 9-12 in each group). At 8 to 12 weeks after transplantation, 2 × 104 CD45.2+ LSK cells were sorted from BM cells pooled from 3 recipient mice and used as a sample for RNAseq analysis. We prepared a total of 14 samples: 3 WT, 3 Calr+/−, 4 CALRdel52;Calr+/+, and 4 CALRdel52;Calr+/−. (B) Unsupervised hierarchical clustering of the global gene expression signatures. (C) Venn diagram depicting differentially expressed genes in LSK cells from Calr+/−, CALRdel52;Calr+/+, and CALRdel52;Calr+/− mice relative to WT controls (false discovery rate <0.1). Forty-seven genes were differentially expressed (23 upregulated and 24 downregulated) in Calr+/− mice. (D) Hierarchical clustering of expression profiles of these 14 samples according to the 47 genes differentially expressed in Calr+/− mice. Genes (rows) and genotypes (columns) with dendrograms are shown. Dendrograms were constructed using Pearson correlation. This analysis distinguished the 4 genotypes relatively clearly. The second cluster of genes was upregulated in Calr+/− mice and CALRdel52;Calr+/− mice and was clearly downregulated in WT and CALRdel52;Calr+/+ mice (colored with red). (E) Result of GSEA analysis showing enrichment of the stem cell signature29  in CALRdel52;Calr+/− LSK cells compared with CALRdel52;Calr+/+ LSK cells. (F) GSEA results using hallmark gene sets for CALRdel52;Calr+/− and CALRdel52;Calr+/+ LSK cells. Heatmap shows the normalized enrichment score (NES) of significantly altered gene sets in CALRdel52;Calr+/− cells compared with CALRdel52;Calr+/+ cells (false discovery rate q value <0.25 or normalized P value <.05). Positive ES (red) indicates that the gene set was enriched in CALRdel52;Calr+/− LSK cells, and negative ES (blue) indicates that the gene set was de-enriched in CALRdel52;Calr+/− LSK cells. Result of GSEA analysis gene sets highlighted in blue or red are shown

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