Figure 4.
HMGA1 transcriptional networks are involved in cell fate and cell cycle progression. (A) Heat map showssubsets of differentially expressed genes that are upregulated or downregulated by HMGA1 in DAMI and SET-2 JAK2V617F AML cell lines. (B) HMGA1 transcriptional networks are enriched for gene sets involved in cell cycle progression (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) and cell fate decisions (GATA2 networks and GATA2). The left column was derived from DAMI and the right column from SET-2 . (C) GSEA performed from transcriptomes from DAMI and SET-2 , with or without HMGA1 silencing (control vs HMGA1-sh1). Adjusted P values and normalized enrichment scores (NES) are shown. (D) Relative GATA2 expression (mean ± standard deviation [SD]) assessed from 2 independent experiments performed in triplicate (quantitative polymerase chain reaction) in DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2). GAPDH was used to control for loading. (E) Western blots for HMGA1, GATA2, and β-actin (loading control) protein levels from DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2). Western blots were performed 3 times with similar results; a representative blot is shown. Size markers (kDa) are indicated. (F) Integrative Genomics Viewer browser view of HMGA1 ChIPseq (top, red), H3 ChIPseq (middle, blue), and overlay (bottom) across the GATA2 locus from DAMI WT cells. (G) Schematic representation of the human GATA2 region with 6 regions (red) that include in silico–predicted HMGA1 DNA binding motifs (upper part of panel). GATA2-1S and GATA2-1G promoters are shown with black arrows to indicate the transcriptional start sites (TSS). The black box represents the untranslated region, and the orange boxes are coding exons. Numbers indicate the nucleotide positions relative to the GATA2-IS TSS. Schematic representation of region with 4 putative HMGA1 binding motifs within a 230-bp fragment of the GATA2 +9.5 enhancer area (lower part of panel). The 4 predicted HMGA1 binding motifs (A-D) are marked with red arrowheads. The +9.5 enhancer (yellow) with 3 DNA binding motifs (E-box, green; GATA, pink; and erythroblast transformation specific [ETS], gray) are also shown. (H) HMGA1 enrichment at the +9.5 enhancer and +9.7 regions within the GATA2 locus by ChIP-PCR in DAMI cells, with or without HMGA1 silencing, performed in triplicate from 3 independent experiments. Results (mean ± SD) are the percentage recovered from the total input DNA (% input). GAPDH promoter (left lane) was used as a negative control for HMGA1 occupancy. (I, left) HMGA1 ChIP-PCR assays comparing occupancy at +9, +9.5, and +9.7 regions using DAMI cells, with or without HMGA1 silencing (control vs HMGA1-sh1). Results (mean ± SD) are presented as the percentage recovered from the total input DNA (% input) performed in triplicate in 3 independent experiments. (I, center and right) HMGA1 recruits active histone marks at +9, +9.5, and +9.7 regions with enrichment for H3K4me1 (Middle) and H3K4me3 (Right), by ChIP-PCR in control DAMI cells with abundant HMGA1; active marks are depleted with HMGA1 silencing (control vs HMGA1-sh1). ChIP-PCR was performed in triplicate in 3 independent experiments. (J) HMGA1 DNA binding activity to probe sequences from 4 putative binding sites near the +9.5 enhancer by electrophoretic mobility shift assay. HMGA1-DNA complexes from 3 independent experiments (mean ± SD) were compared quantitatively in the bar graph. A GC-rich probe was used as a negative control. (K) HMGA1 induces GATA2 enhancer sequences linked to a luciferase reporter gene in control DAMI cells, with decreased reporter gene activation with HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2). Constructs include GATA2-1S promoter, GATA2-1S promoter plus HMGA1 binding motif A, and GATA2-1S promoter plus HMGA1 binding motifs A and B. Results (mean ± SD) represent 2 experiments performed with 4 replicates. *P < .05, **P < .01,***P < .001, ****P < .0001, 2-tailed Student t test (D,I), 1-way ANOVA, followed by Tukey’s multiple-comparison test (H,J-K).

HMGA1 transcriptional networks are involved in cell fate and cell cycle progression. (A) Heat map showssubsets of differentially expressed genes that are upregulated or downregulated by HMGA1 in DAMI and SET-2 JAK2V617F AML cell lines. (B) HMGA1 transcriptional networks are enriched for gene sets involved in cell cycle progression (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) and cell fate decisions (GATA2 networks and GATA2). The left column was derived from DAMI and the right column from SET-2 . (C) GSEA performed from transcriptomes from DAMI and SET-2 , with or without HMGA1 silencing (control vs HMGA1-sh1). Adjusted P values and normalized enrichment scores (NES) are shown. (D) Relative GATA2 expression (mean ± standard deviation [SD]) assessed from 2 independent experiments performed in triplicate (quantitative polymerase chain reaction) in DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2). GAPDH was used to control for loading. (E) Western blots for HMGA1, GATA2, and β-actin (loading control) protein levels from DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2). Western blots were performed 3 times with similar results; a representative blot is shown. Size markers (kDa) are indicated. (F) Integrative Genomics Viewer browser view of HMGA1 ChIPseq (top, red), H3 ChIPseq (middle, blue), and overlay (bottom) across the GATA2 locus from DAMI WT cells. (G) Schematic representation of the human GATA2 region with 6 regions (red) that include in silico–predicted HMGA1 DNA binding motifs (upper part of panel). GATA2-1S and GATA2-1G promoters are shown with black arrows to indicate the transcriptional start sites (TSS). The black box represents the untranslated region, and the orange boxes are coding exons. Numbers indicate the nucleotide positions relative to the GATA2-IS TSS. Schematic representation of region with 4 putative HMGA1 binding motifs within a 230-bp fragment of the GATA2 +9.5 enhancer area (lower part of panel). The 4 predicted HMGA1 binding motifs (A-D) are marked with red arrowheads. The +9.5 enhancer (yellow) with 3 DNA binding motifs (E-box, green; GATA, pink; and erythroblast transformation specific [ETS], gray) are also shown. (H) HMGA1 enrichment at the +9.5 enhancer and +9.7 regions within the GATA2 locus by ChIP-PCR in DAMI cells, with or without HMGA1 silencing, performed in triplicate from 3 independent experiments. Results (mean ± SD) are the percentage recovered from the total input DNA (% input). GAPDH promoter (left lane) was used as a negative control for HMGA1 occupancy. (I, left) HMGA1 ChIP-PCR assays comparing occupancy at +9, +9.5, and +9.7 regions using DAMI cells, with or without HMGA1 silencing (control vs HMGA1-sh1). Results (mean ± SD) are presented as the percentage recovered from the total input DNA (% input) performed in triplicate in 3 independent experiments. (I, center and right) HMGA1 recruits active histone marks at +9, +9.5, and +9.7 regions with enrichment for H3K4me1 (Middle) and H3K4me3 (Right), by ChIP-PCR in control DAMI cells with abundant HMGA1; active marks are depleted with HMGA1 silencing (control vs HMGA1-sh1). ChIP-PCR was performed in triplicate in 3 independent experiments. (J) HMGA1 DNA binding activity to probe sequences from 4 putative binding sites near the +9.5 enhancer by electrophoretic mobility shift assay. HMGA1-DNA complexes from 3 independent experiments (mean ± SD) were compared quantitatively in the bar graph. A GC-rich probe was used as a negative control. (K) HMGA1 induces GATA2 enhancer sequences linked to a luciferase reporter gene in control DAMI cells, with decreased reporter gene activation with HMGA1 silencing (control vs HMGA1-sh1 or HMGA1-sh2). Constructs include GATA2-1S promoter, GATA2-1S promoter plus HMGA1 binding motif A, and GATA2-1S promoter plus HMGA1 binding motifs A and B. Results (mean ± SD) represent 2 experiments performed with 4 replicates. *P < .05, **P < .01,***P < .001, ****P < .0001, 2-tailed Student t test (D,I), 1-way ANOVA, followed by Tukey’s multiple-comparison test (H,J-K).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal