Expression of GATA-1 in a non-hematopoietic cell line induces beta-globin locus control region chromatin structure remodeling and an erythroid pattern of gene expression

Abstract

GATA-1 is a hematopoietic transcription factor expressed in erythroid, megakaryocytic, mast cell and eosinophil lineages. It is required for normal erythroid differentiation, the expression of erythroid-specific genes and for the establishment of an active chromatin structure throughout the beta-globin gene locus. GATA-1 is also necessary for the formation and function of the locus control region DNase I hypersensitive site (HS) core elements. To determine whether GATA-1 was sufficient to direct formation of the locus control region (LCR) and an erythroid pattern of gene expression, we expressed GATA-1 in the non-hematopoietic HeLa cell line that does not express other hematopoietic transcription factors but does express GATA-2, GATA-3, and GATA-6. We found that production of the GATA-1 protein resulted in the formation of LCR DNase I HSs 1-4 in their normal locations, and that histones became hyperacetylated within these regulatory elements. Transcription of several erythroid-specific genes was activated in HeLa cells expressing GATA-1, including those coding for alpha-globin, beta-globin, the erythropoietin receptor, the erythroid krüpple-like factor and p45 NF-E2. Despite increased expression of these genes at the mRNA level, their protein products were not detected. These results imply that GATA-1 is sufficient to direct chromatin structure reorganization within the beta-globin LCR and an erythroid pattern of gene expression in the absence of other hematopoietic transcription factors.

Figure 1

Forced expression of GATA-1 in Hela cells. A) RT PCR analysis shows that mGATA-1 is expressed in three Hela clones (HG1, HG2 and HG3) stably transfected with a mGATA-1 expression plasmid. Levels are comparable to those seen in the MEL control cell line. No GATA-1 mRNA is detected in the wild-type Hela cells (HWT). B) Western analysis of mGATA-1 expression. GATA-1 protein is expressed in each transfected clone at levels similar to those seen in MEL and K562 control cell lines.

Figure 2

Human β-globin LCR HS formation in Hela cells expressing GATA-1. A. DNase I HS assays for β-globin LCR HSs 1-4. K562 and Hela wild-type cells serve as positive and negative controls. Arrows indicate DNase I HSs. B. DNA and fragments used in DNase I HS assays. Arrows below the maps indicate the expected size of HS bands on the Southern blots. C. Quantitative PCR based DNase I sensitivity assay used to analyze the region of LCR HS3. B = Bam HI, Sa = Sac I, Sp = Sph I, H = Hind III.

Figure 3

Histone H3 acetylation and GATA-1 binding at the β-globin LCR HSs in Hela cells expressing GATA-1. A. Levels of H3 acetylation in the LCR HS3 region. B. Levels of H3 acetylation in the LCR HS4 region. C. GATA-1 binding to the regions of LCR HS4 and HS3. K562 and Hela wild-type cells are positive and negative controls for these experiments. IgG is used as a non-specific control for immunoprecipitation reactions. HG1 = GATA-1 expressing Hela cells.

Figure 4

Expression analysis of erythroid GATA-1 target genes in Hela cells expressing GATA-1 using reverse transcriptase-PCR. A. Expression of the human β-globin locus genes. B. Expression of non-β-globin locus genes. RNA from unfractionated human bone marrow and K562 cells are positive controls. EpoR, erythropoietin receptor gene. HG1, Hela cells expressing GATA-1.

Figure 5

EKLF and p45 NF-E2 expression in Hela cells producing GATA-1. A. RT-PCR showing that message for both genes is detectible. B. Western blot showing that neither NF-E2 or EKLF protein is detectible in any of three GATA-1 expressing Hela cell clones.

Figure 6

Expression of GATA factors in Hela cells. A. Western blots show production of GATA-2, GATA-3 and GATA-6 in wild-type Hela cells. B. Gata-6 is produced in Hela cells at levels equivalent to those seen in the COLO 320 cell line.