The GATA-E box-GATA motif in the EKLF promoter is required for in vivo expression.

The erythroid Krüppel-like factor (EKLF) is a key regulatory protein in globin gene expression. This zinc finger transcription factor is required for expression of the adult beta globin gene, and it has been suggested that it plays an important role in the developmental switch from fetal gamma to adult beta globin gene expression. We have previously described a sequence element in the distal promoter region of the mouse EKLF gene that is critical for the expression of this transcription factor. The element consists of an E box motif flanked by 2 GATA-1 binding sites. Here we demonstrate that mutation of the E box or the GATA-1 consensus sequences eliminates expression from the EKLF promoter in transgenic mice. These results confirm the importance of this activator element for in vivo expression of the EKLF gene. (Blood. 2000;95:1652-1655)

Multiple proteins binding to a GATA-E box-GATA motif regulate the erythroid Krüppel-like factor (EKLF) gene.

Erythroid Krüppel-like factor (EKLF) is a zinc finger transcription factor required for beta-globin gene expression and is implicated as one of the key factors necessary for the fetal to adult switch in globin gene expression. In an effort to identify factors involved in the expression of this important erythroid-specific regulatory protein, we have isolated the mouse EKLF gene and systematically analyzed the promoter region. Initially, a reporter construct with 1150 base pairs of the EKLF 5'-region was introduced into transgenic mice and shown to direct erythroid-specific expression. We continued the expression studies in erythroid cells and have identified a sequence element consisting of two GATA sites flanking an E box motif. The three sites act in concert to elevate the transcriptional activity of the EKLF promoter. Each site is essential for EKLF expression indicating that the three binding sites do not work additively, but rather function as a unit. We further show that GATA-1 binds to the two GATA sites and present evidence for binding of another factor from erythroid cell nuclear extracts to the E box motif. These results are consistent with the formation of a quaternary complex composed of an E box dimer and two GATA-1 proteins binding at a combined GATA-E box-GATA activator element in the distal EKLF promoter.

Analysis of developmental switching in transgenic mice with 5' and 3' deletions in the human beta globin gene.

Our interest in the cis-acting elements that promote the up-regulation of the beta globin gene has led to a systematic deletion analysis of portions of the beta globin gene in the context of the HS2 and gamma globin gene using transgenic mice. In constructs that delete the 5' region to only 265 bp, high-level, erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels. A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased beta globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between -122 and -265. In addition, a construct in which the entire beta globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3' region of the beta globin gene was deleted in order to examine the effect of a previously defined 3' enhancer region. With deletion of this region, the expression of the human beta globin gene in transgenic mice is unchanged relative to the parental constructs.

Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Krüppel-like factor: identification of a new multigene family.

We have identified and characterized the gene for a novel zinc finger transcription factor which we have termed lung Krüppel-like factor (LKLF). LKLF was isolated through the use of the zinc finger domain of erythroid Krüppel-like factor (ELKF) as a hybridization probe and is closely related to this erythroid cell-specific gene. LKLF is expressed in a limited number of tissues, with the predominant expression seen in the lungs and spleen. The gene is developmentally controlled, with expression noted in the 7-day embryo followed by a down-regulation at 11 days and subsequent reactivation. A high degree of similarity is noted in the zinc finger regions of LKLF and EKLF. Beyond this domain, the sequences diverge significantly, although the putative transactivation domains for both LKLF and EKLF are proline-rich regions. In the DNA-binding domain, the three zinc finger motifs are so closely conserved that the predicted DNA contact sites are identical, suggesting that both proteins may bind to the same core sequence. This was further suggested by transactivation assays in which mouse fibroblasts were transiently transfected with a human beta-globin reporter gene in the absence and presence of an LKLF cDNA construct. Expression of the LKLF gene activates this human beta-globin promoter containing the CACCC sequence previously shown to be a binding site for EKLF. Mutation of this potential binding site results in a significant reduction in the reporter gene expression. LKLF and EKLF can thus be grouped as members of a unique family of transcription factors which have discrete patterns of expression in different tissues and which appear to recognize the same DNA-binding site.

Regulated expression of the human beta globin gene in transgenic mice requires an upstream globin or nonglobin promoter.

Transgenic mice have been used extensively to study elements governing the erythroid-specific developmental switch from human fetal gamma to human adult beta globin. Previous work demonstrated that a small construct composed of hypersensitive site 2 (HS2) of the locus control region (LCR) linked to the gamma and beta globin genes (HS2-gamma-beta) is sufficient for correct tissue and temporal expression of these genes, whereas HS2-beta alone is inappropriately expressed in the embryo. Two models, which are not mutually exclusive, have been proposed to explain these results and those of other constructs in transgenic mice. One model emphasizes the conserved polarity in the globin locus and suggests a distance effect whereby the beta globin gene must be removed from the LCR/HS2 to prevent an early and incorrect activation of this gene in the embryonic compartment. A second hypothesis proposes a competition between the gamma and beta globin gene promoters for interaction with the LCR/HS2. The active gamma globin gene promoter positioned between the LCR/HS2 and the beta globin gene thereby interacts with the HS2 elements early in erythroid development and is expressed until a change in putative stage-specific nuclear factors makes an interaction with the adult beta globin gene more favorable. In an effort to test the competition model, a construct has been prepared in which a small deletion was produced in the promoter region of the gamma globin gene while in the context of the HS2-gamma-beta plasmid. Analysis of this construct in transgenic mice reveals a constitutive unregulated expression of the human beta globin gene during erythroid development. To determine if this competition effect is specific for globin genes, a heterologous reporter gene has been substituted for the gamma globin gene in the construct HS2-gamma-beta. In this case, the beta globin gene exhibits correct developmental expression. This data is consistent with a model in which transcription from a promoter upstream of the beta globin gene in some manner protects this adult gene from activation by the LCR/HS2 during early development.

Human gamma- to beta-globin gene switching using a mini construct in transgenic mice.

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.