Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF.

Abstract

Globin genes are subject to tissue-specific and developmental stage-specific regulation. A switch from human fetal ('y)-to adult (,B)-globin expression occurs within erythroid precursor cells of the adult lineage. Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Kruippel-like factor (EKLF), is required for expression of the f3-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter. To examine the role of EKLF in the developmental regulation of the human y-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human ,B-globin yeast artificial chromosome transgene. We find that in the absence of EKLF, while human f-globin expression is dramatically reduced, 'y-globin transcripts are elevated -5-fold. Impaired silencing of y-globin expression identifies EKLF as the first transcription factor participating quantitatively in the y-globin to f8-globin switch. Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the f3-globin gene promoter and the locus control region that excludes the y-globin gene.

Inhibition of β protein 1 expression enhances β-globin promoter activity and β-globin mRNA levels in the human erythroleukemia (K562) cell line

“…EKLF knockout mice die of severe anemia at the fetal liver stage, due to failure of adult β-globin gene activation. EKLF Ϫ/Ϫ mice bearing a complete human β-globin locus transgene have reduced levels of β-globin but elevated levels of γ-globin expression, compared with wild-type mice bearing the same transgene [41,42]. Our RT-PCR experiments showed that EKLF is expressed in K562 cells, but no significant difference in EKLF expression was found between cells transfected with non-siRNA and siRNA against BP1.…”

Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the β-globin locus control region in vivo

“…Deletion of 5ЈHS3 in the endogenous mouse locus results in a 30% reduction of ␤ gene expression in adult mice (Hug et al 1996). Natural mutations in the CACC box of the ␤-globin promoter result in a less severe reduction of ␤ gene expression than observed in EKLF knockout mice (Thein 1993;Perkins et al 1996;Wijgerde et al 1996). Thus, it appears that EKLF sites are necessary in both the promoter and the LCR and that these sites act independently of each other.…”

Non-random subcellular distribution of variant EKLF in erythroid cells

“…EKLF mRNA is highly restricted in its expression pattern, limited to hematopoietic organs such as the yolk sac, fetal liver, adult bone marrow, and the red pulp of the spleen [8,9]. Molecular and genetic ablation studies demonstrate that EKLF is absolutely required for β-globin transcription and plays a prominent role in the final developmental switch to adult β-globin in definitive erythroid cells [10][11][12][13][14]. EKLF protein can interact with coactivators such as p300/CBP histone acetyltransferases as well as chromatin remodelers such as SWI/SNF to maximally transactivate the β-globin gene [15,16].…”