Figure 7
Figure 7. Functional cooperation between RUNX1 and its partner TFs GATA1, AP-1, and ETS. Reporter assays in megakaryocytic cell lines demonstrate cooperation between RUNX1-GATA (A), RUNX1-AP-1 (B), and RUNX1-ETS (C) in regulation of gene expression of HEMGN, ITGB3, and ITPR1 genes. (A) Top left: RUNX1 and GATA1 ChIP-seq tracks in K562 and K562-TPA cells proximal to the HEMGN locus. The regulatory region cloned in vectors and used in transfection assays is indicated by the red rectangle, as are the evolutionary conserved RUNX and GATA binding sites (bottom left) located within this region. Top right: ChIP-quantitative PCR validation of RUNX1 and GATA1 binding to the indicated ChIP-seq region. Shown are independent ChIP assays followed by quantitative PCR using K562 and K562-TPA cells. Bottom right: Dual-luciferase reporter assays in transfected K562 cells using PGL4.73 vector alone or with intact/mutated HEMGN regulatory region (supplemental data). (B) Top left: FOS and RUNX1 ChIP-seq tracks proximal to the ITGB3 locus in K562 and K562-TPA cells. The regulatory region cloned in vectors and used in transfection assays is indicated by the red rectangle, as are the evolutionary conserved RUNX and AP-1 binding sites (bottom left) located within this region. Top right: ChIP-quantitative PCR validation of RUNX1 and AP-1 binding to the indicated ChIP-seq region. Shown are independent ChIP assays followed by quantitative PCR using K562 and K562-TPA cells. Bottom right: Dual-luciferase reporter assays in transfected K562 cells using PGL4.73 vector alone or with intact/mutated ITGB3 regulatory region (see supplemental Methods). (C) Top left: RUNX1 ChIP-seq tracks proximal to the ITRP1 locus in K562, K562-TPA, and CMK cells. The regulatory region cloned in vectors and used in transfection assays is indicated by the red rectangle, as are the evolutionary conserved RUNX and ETS binding sites (bottom left) located within this region. Top right: ChIP-quantitative PCR validation of RUNX1 binding to the indicated ChIP-seq region. Shown are independent ChIP assays followed by quantitative PCR using K562, K562-TPA, and CMK cells. Bottom right: Dual-luciferase reporter assays in transfected K562 cells using PGL4.73 vector alone or with intact/mutated ITPR1 regulatory region (see supplemental Methods). (A-C) Data of quantitative PCR and dual-reporter assays represent mean ± SE of at least 2 biologic repeats performed in triplicates.

Functional cooperation between RUNX1 and its partner TFs GATA1, AP-1, and ETS. Reporter assays in megakaryocytic cell lines demonstrate cooperation between RUNX1-GATA (A), RUNX1-AP-1 (B), and RUNX1-ETS (C) in regulation of gene expression of HEMGN, ITGB3, and ITPR1 genes. (A) Top left: RUNX1 and GATA1 ChIP-seq tracks in K562 and K562-TPA cells proximal to the HEMGN locus. The regulatory region cloned in vectors and used in transfection assays is indicated by the red rectangle, as are the evolutionary conserved RUNX and GATA binding sites (bottom left) located within this region. Top right: ChIP-quantitative PCR validation of RUNX1 and GATA1 binding to the indicated ChIP-seq region. Shown are independent ChIP assays followed by quantitative PCR using K562 and K562-TPA cells. Bottom right: Dual-luciferase reporter assays in transfected K562 cells using PGL4.73 vector alone or with intact/mutated HEMGN regulatory region (supplemental data). (B) Top left: FOS and RUNX1 ChIP-seq tracks proximal to the ITGB3 locus in K562 and K562-TPA cells. The regulatory region cloned in vectors and used in transfection assays is indicated by the red rectangle, as are the evolutionary conserved RUNX and AP-1 binding sites (bottom left) located within this region. Top right: ChIP-quantitative PCR validation of RUNX1 and AP-1 binding to the indicated ChIP-seq region. Shown are independent ChIP assays followed by quantitative PCR using K562 and K562-TPA cells. Bottom right: Dual-luciferase reporter assays in transfected K562 cells using PGL4.73 vector alone or with intact/mutated ITGB3 regulatory region (see supplemental Methods). (C) Top left: RUNX1 ChIP-seq tracks proximal to the ITRP1 locus in K562, K562-TPA, and CMK cells. The regulatory region cloned in vectors and used in transfection assays is indicated by the red rectangle, as are the evolutionary conserved RUNX and ETS binding sites (bottom left) located within this region. Top right: ChIP-quantitative PCR validation of RUNX1 binding to the indicated ChIP-seq region. Shown are independent ChIP assays followed by quantitative PCR using K562, K562-TPA, and CMK cells. Bottom right: Dual-luciferase reporter assays in transfected K562 cells using PGL4.73 vector alone or with intact/mutated ITPR1 regulatory region (see supplemental Methods). (A-C) Data of quantitative PCR and dual-reporter assays represent mean ± SE of at least 2 biologic repeats performed in triplicates.

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