Figure 3.
In vivo binding of SATB1 family protein to MARs in the β-globin cluster. (A) Specific primer pairs for potential MARs for HS4, HS3, HS2 (M1-M5), Mϵ, γ5′, γ1, and γ2 in the 3′ Aγ-globin enhancer, β5′, and β-globin IVS2 are indicated. HindIII (H) and XbaI (X) restriction enzyme sites flank the HS2 core. (B) Probes corresponding to HS2-M1 (lanes 1-4) and Mϵ (lanes 5-8) MARs were incubated with K562 nuclear extract to assess in vitro SATB1-family protein binding; competitors used were SATB1-binding motif (S; lanes 2, 6), mutation of the SATB1-binding motif mut \((24)_{8}^{17}\)
(ΔS; lanes 3, 7), and anti-SATB1 antibody (αS; lanes 4, 8). NC (lanes 1, 5) indicates no competitor. (C) To assess in vivo SATB1-family protein binding, specific primers that could amplify gDNA (0.01 μg; control lanes 1-6; m indicates 100-bp ladder) were used for ChIP analysis. ChIP DNA selected using anti-SATB1 antibody (SATB; lanes 7-12) or preimmune serum (α; lanes 13-18) was amplified and produced a specific PCR product corresponding to M1 (lane 1) and Mϵ (lane 6). Dilutions of SATB1 selected ChIP DNA at 10 pg, 100 pg, and 1 ng, respectively, and primer pairs for M1 (lanes 19-21), M2 (lanes 23-25), and MϵMARs (lanes 26-28) were used for PCR amplification (genomic control Co; lane 22). (D) Quantification of anti-SATB1 antibody selected ChIP DNA is shown for M1 and MϵMARs in K562 (K; □) and K562/SATB1 (SATB; ▪) cells. (E) Primer pairs for MARs in the γ- and β-globin genes used to amplify anti-SATB1 antibody selected ChIP DNA as indicated (lanes 4-6) show no specific PCR products, in contrast to the gDNA control (lanes 1-3). (F) Primer pairs for proposed MARs in HS3 (lanes 1-4), HS4 (lanes 5-8), the γ-globin promoter (γ5′; lanes 9-12), and the β-globin promoter (β5′; lanes 13-16) were used to amplify anti-SATB1 antibody selected ChIP DNA as indicated. DNA used corresponded to genomic control (Co), and 10 pg, 100 pg, and 1 μg SATB1 selected ChIP DNA for lanes 1-4, 5-8, 9-12, and 13-16, respectively.
![Figure 3. In vivo binding of SATB1 family protein to MARs in the β-globin cluster. (A) Specific primer pairs for potential MARs for HS4, HS3, HS2 (M1-M5), Mϵ, γ5′, γ1, and γ2 in the 3′ Aγ-globin enhancer, β5′, and β-globin IVS2 are indicated. HindIII (H) and XbaI (X) restriction enzyme sites flank the HS2 core. (B) Probes corresponding to HS2-M1 (lanes 1-4) and Mϵ (lanes 5-8) MARs were incubated with K562 nuclear extract to assess in vitro SATB1-family protein binding; competitors used were SATB1-binding motif (S; lanes 2, 6), mutation of the SATB1-binding motif mut \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((24)_{8}^{17}\) \end{document} (ΔS; lanes 3, 7), and anti-SATB1 antibody (αS; lanes 4, 8). NC (lanes 1, 5) indicates no competitor. (C) To assess in vivo SATB1-family protein binding, specific primers that could amplify gDNA (0.01 μg; control lanes 1-6; m indicates 100-bp ladder) were used for ChIP analysis. ChIP DNA selected using anti-SATB1 antibody (SATB; lanes 7-12) or preimmune serum (α; lanes 13-18) was amplified and produced a specific PCR product corresponding to M1 (lane 1) and Mϵ (lane 6). Dilutions of SATB1 selected ChIP DNA at 10 pg, 100 pg, and 1 ng, respectively, and primer pairs for M1 (lanes 19-21), M2 (lanes 23-25), and MϵMARs (lanes 26-28) were used for PCR amplification (genomic control Co; lane 22). (D) Quantification of anti-SATB1 antibody selected ChIP DNA is shown for M1 and MϵMARs in K562 (K; □) and K562/SATB1 (SATB; ▪) cells. (E) Primer pairs for MARs in the γ- and β-globin genes used to amplify anti-SATB1 antibody selected ChIP DNA as indicated (lanes 4-6) show no specific PCR products, in contrast to the gDNA control (lanes 1-3). (F) Primer pairs for proposed MARs in HS3 (lanes 1-4), HS4 (lanes 5-8), the γ-globin promoter (γ5′; lanes 9-12), and the β-globin promoter (β5′; lanes 13-16) were used to amplify anti-SATB1 antibody selected ChIP DNA as indicated. DNA used corresponded to genomic control (Co), and 10 pg, 100 pg, and 1 μg SATB1 selected ChIP DNA for lanes 1-4, 5-8, 9-12, and 13-16, respectively.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/105/8/10.1182_blood-2004-08-2988/6/zh80080577250003.jpeg?Expires=1735534371&Signature=J6bPsQU3p57Ksm1XPBPql5~NRYo~InWMd-58tBh94TvxSn1cB9G3CB2wl4jE252x5z5PrDs5CivXgUCIcA7kCxxlixcTXRqQpWpLDX6a6gpsqV2DKIfFf7f~ev~DYl9XI7iOAz840iiAELJRaCG6IP4M5-1VZxv6mahQg9Zxam5WOZIJ2voDdLeNtmuwTXs21eALx76RhY~RkBPH8DkF6vvdsvoxpeZ5H-XS49aBsO1eqln3Ba7GZLPhXpE~4cg-OiYO7O-4wHxZ8JYE8d09~m1vwAu1TrgdZSfULqAMuUAYEk1-1A2mUzH22jFRHDJHxPAsbCdiSVtQDQGYssR3Qg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
