Figure 1
Figure 1. The ID1 gene expression is regulated by the JAK2-STAT5 signaling pathway. (A) Schematic representation of the human ID1 gene locus with a sequence conservation plot for alignments of the human, mouse, and dog genomes. Peaks of sequence conservation in coding regions of the genome are shown in purple, those in transcribed but not translated regions (3′UTR and 5′UTR) are shown in pale blue, and those in nontranscribed regions are shown in pink. The location of a STAT5 consensus binding sequence 5.5 kb downstream of the ID1 promoter is indicated by an arrowhead. (B) Local alignment of the human, mouse, and dog genomes at the +5.5 element. Nucleotides conserved between all 3 species are highlighted in black; those conserved between 2 of the 3 species are highlighted in gray. A conserved STAT5 consensus binding sequence is highlighted in yellow. (C) Chromatin immunoprecipitation (ChIP) assays performed at the ID1 +5.5 element in HEL cells using antibodies against STAT5A/B (α-STAT5) and acetylated histone H3 lysine 9 (α-H3AcK9). A typical result from 2 biologic replicates is shown. (D) Effect of the JAK kinase inhibitor JAK inhibitor I on ID1 transcript levels in HEL cells. (E) A similar experiment to that performed in panel D but using AT9383. (F) Transient and stable transfection of HEL cells with ID1 +5.5 luciferase reporter constructs. A 300-bp length of DNA corresponding to the ID1 +5.5 element was inserted downstream of a luciferase reporter gene under the transcriptional control of the human ID1 promoter and the effect of +5.5 element on the transcriptional activity of the ID1 promoter examined. A similar construct containing a scrambled version of the STAT5 binding site within the +5.5 element (+5.5ΔSTAT5) was tested in a similar way. Shown are the mean and standard error of the mean for 2 independent transfections (each performed in triplicate). (G) Relative ID1 transcript levels in JAK2 wild-type and JAK2V617F heterozygous colonies in 10 individual PV patients. (H) Relative ID1 transcript levels in JAK2 wild-type and JAK2V617F homozygous colonies in 4 PV patients. (I) Summary of the relative expression of ID1 in JAK2V617F-negative, heterozygous, and homozygous BFU-E colonies grown from a total of 13 PV patients. Relative expression in JAK2V617F heterozygous and JAK2V617F homozygous colonies for each individual patient was normalized against that in JAK2V617F-negative colonies from the same patient. Each bar represents the mean and SD of the fold increase in ID1 transcript levels in heterozygous and homozygous colonies relative to that in wild-type colonies for each patient (**P < .01; see “Results” for details).

The ID1 gene expression is regulated by the JAK2-STAT5 signaling pathway. (A) Schematic representation of the human ID1 gene locus with a sequence conservation plot for alignments of the human, mouse, and dog genomes. Peaks of sequence conservation in coding regions of the genome are shown in purple, those in transcribed but not translated regions (3′UTR and 5′UTR) are shown in pale blue, and those in nontranscribed regions are shown in pink. The location of a STAT5 consensus binding sequence 5.5 kb downstream of the ID1 promoter is indicated by an arrowhead. (B) Local alignment of the human, mouse, and dog genomes at the +5.5 element. Nucleotides conserved between all 3 species are highlighted in black; those conserved between 2 of the 3 species are highlighted in gray. A conserved STAT5 consensus binding sequence is highlighted in yellow. (C) Chromatin immunoprecipitation (ChIP) assays performed at the ID1 +5.5 element in HEL cells using antibodies against STAT5A/B (α-STAT5) and acetylated histone H3 lysine 9 (α-H3AcK9). A typical result from 2 biologic replicates is shown. (D) Effect of the JAK kinase inhibitor JAK inhibitor I on ID1 transcript levels in HEL cells. (E) A similar experiment to that performed in panel D but using AT9383. (F) Transient and stable transfection of HEL cells with ID1 +5.5 luciferase reporter constructs. A 300-bp length of DNA corresponding to the ID1 +5.5 element was inserted downstream of a luciferase reporter gene under the transcriptional control of the human ID1 promoter and the effect of +5.5 element on the transcriptional activity of the ID1 promoter examined. A similar construct containing a scrambled version of the STAT5 binding site within the +5.5 element (+5.5ΔSTAT5) was tested in a similar way. Shown are the mean and standard error of the mean for 2 independent transfections (each performed in triplicate). (G) Relative ID1 transcript levels in JAK2 wild-type and JAK2V617F heterozygous colonies in 10 individual PV patients. (H) Relative ID1 transcript levels in JAK2 wild-type and JAK2V617F homozygous colonies in 4 PV patients. (I) Summary of the relative expression of ID1 in JAK2V617F-negative, heterozygous, and homozygous BFU-E colonies grown from a total of 13 PV patients. Relative expression in JAK2V617F heterozygous and JAK2V617F homozygous colonies for each individual patient was normalized against that in JAK2V617F-negative colonies from the same patient. Each bar represents the mean and SD of the fold increase in ID1 transcript levels in heterozygous and homozygous colonies relative to that in wild-type colonies for each patient (**P < .01; see “Results” for details).

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