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 cellspecific 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 ␤-globin reporter gene in the absence and presence of an LKLF cDNA construct. Expression of the LKLF gene activates this human ␤-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.

The developmental regulation of the human globin genes involves a key switch from fetal (y-) to adult (13-) 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 13-globin locus. High-level, correct developmental regulation of 1-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 13-globin locus is required to silence the 13-globin gene in embryonic life. The results presented here clearly show that the presence of the 'y-globin gene (3.3 kb) alone is sufficient to down-regulate the 1,-globin gene in embryonic transgenic mice made with an LCR--1B-globin mini construct.The results also show that the y-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-y-globin constructs not including the 13-globin gene, i.e., that the y-tglobin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the -y-globin gene may be important for down-regulation in the adult. The 5'HS2yenl3 construct described is a suitable model for further study of the mechanism of human y-to 13-globin gene switching in transgenic mice.

Erythroid Krü ppel-like factor (EKLF) is a zinc finger transcription factor required for ␤-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.

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