Twist1 deletion displays altered expression of genes associated with regulation of Ca²⁺ and mitochondrial function upon IR stress. (A) Transcriptome profiling of cKO and control (Ctrl) LT-HSCs after IR treatment. Red box displays the number of upregulated genes, and blue box displays the number of downregulated genes. (B) Gene ontology (GO) analysis of genes upregulated >1.5-fold in Twist1-deficient LT-HSCs after IR stress. Only top 10 GO terms are listed. (C) Scatterplot shows differentially accessible peaks from ATAC-seq analysis between cKO and Ctrl LT-HSCs after IR stress. (D) Venn diagram of genes with discrete Twist1 peaks compared with genes upregulated after Twist1 deletion. (E) Relative messenger RNA expression levels of calcium signaling factors in LT-HSCs from cKO and Ctrl BM (n = 3; Student t test). (F) Quantification of CACNA1B expression (green) in HSCs at 12 days after IR at 5 Gy (right) and representative confocal microscopic images (left). Rabbit immunoglobulin G (IgG) was used as a negative control. Scale bars, 10 µm (Student t test). (G) Promoter activity assay using a Twist1 expression plasmid and luciferase reporter constructs driven by various lengths of the Cacna1b 5′ flanking region in the 293T cell line (n = 3; Student t test). (H) Relative luciferase activity in 293T cells transfected with WT or mutated Cacna1b promoter–luciferase reporter constructs (right). The schematic representation of the mutated constructs of the Cacna1b promoter (left; n = 3; Student t test). (I) Binding of TWIST1 was analyzed by chromatin immunoprecipitation–polymerase chain reaction in EML cell line and LSK cells from WT mice or from Ctrl and cKO mice treated with 5-FU (n v= 3; Student t test). Column plots show means ± standard deviation. *P < .05, **P < .01, ***P < .001. ns, not significant.