GATA-1 binding sites mapped in the beta-globin locus by using mammalian chIp-chip analysis

Abstract

The expression of the beta-like globin genes is intricately regulated by a series of both general and tissue-restricted transcription factors. The hemapoietic lineage-specific transcription factor GATA-1 is important for erythroid differentiation and has been implicated in regulating the expression of the erythroid-specific genes including the genes of the beta-globin locus. In the human erythroleukemic K562 cell line, only one DNA region has been identified previously as a putative site of GATA-1 interaction by in vivo footprinting studies. We mapped GATA-1 binding throughout the beta-globin locus by using chIp-chip analysis of K562 cells. We found that GATA-1 binds in a region encompassing the HS2 core element, as was previously identified, and an additional region of GATA-1 binding upstream of the gammaG gene. This approach will be of general utility for mapping transcription factor binding sites within the beta-globin locus and throughout the genome.

Figure 1

(a) GATA-1 antibodies, anti-GATA-1(C20), anti-GATA-1(N6), and anti-GATA-1(amino acids 67–78) recognize a protein that migrates at approximately 45 kDa in human K562 nuclear extracts by immunoblot analysis, whereas sera raised against c-jun and Nrf1 recognize proteins of 50 kDa and 35 kDa, respectively, in similar extracts. (b) PCR assays of the core HS3, HS2, and HS1 regions with chromatin that was mock immunoprecipitated (noAb lanes) or immunoprecipitated with one of three GATA-1 antibodies [GATA-1(C20), GATA-1(N6), and GATA-1(amino acids 67–78) lanes]. As positive controls, 10 ng, 100 ng, and 1 μg of whole lysate DNA was amplified in parallel.

Figure 2

(a) GATA-1 chIp-chip analysis from K562 cells across the β-globin locus shows anti-GATA-1 enrichment in regions 009BG and 032BG. The bars for each region on the microarray are in the order they appear within the β-globin sequence and are in the approximate position shown relative to the schematic of the locus drawn in c. Each bar represents the median value for eight experiments, with the error bars to the 75th percentile value of all median ratios determined for that fragment. (b) ChIp-chip analysis of mock immunoprecipitated DNA from K562 cells shows no peaks in enrichment. Each bar represents the median value for three experiments with error bars to the 75th percentile value. (c) The number of GATA-1 consensus sites within each segment of the β-globin locus are presented in the order that they appear within the locus. A schematic of the β-globin locus also is shown to indicate the relative position of each of the array segments to relevant features along the locus.

Figure 3

(a) GATA-1 chIp-PCR assays for subdivisions of region 009BG containing the HS2 element show specific enrichment of the core sequence (Middle) but not of the flanking sequences (Top and Bottom). The lanes are described in Fig. 1. (b) The sequence of the core HS2 region amplified is shown, and the consensus GATA-1 site is boxed in black.

Figure 4

(a) GATA-1 chIp-PCR assay of subdivisions of region 032BG and the promoter region of the γG genes shows two regions of enrichment in GATA-1 immuno-precipitates represented in the second and fourth panels. The lanes are described in Fig. 1. (b) The sequence of enriched region represented in the second panel above is shown with GATA-1 consensus motifs boxed in black. (c) The sequence of enriched region represented in the fourth panel above is shown.