Erythroid Krüppel-like factor (EKLF) was originally isolated from erythroid cell RNA by differential screening and shown to be erythroid-specific, although a low level of EKLF was found in mast cell lines. EKLF contains three zinc-fingers homologous to those found in the Krüppel family of transcription factors. Because it binds the sequence CCACACCCT, EKLF may affect erythroid development as a result of its ability to bind to the CAC box in the promoter of the beta-globin gene. Mutation of this element leads to reduced beta-globin expression and it appears to mediate the effect of the globin locus control region on the promoter. Here we inactivate the EKLF gene through insertion of a lacZ reporter gene by homologous recombination in embryonic stem (ES) cells. Heterozygous EKLF+/- mice show that the reporter gene is expressed in a developmentally specific manner in all types of erythroblasts in the fetal liver and adult bone marrow. Homozygous EKLF-/- mice appear normal during the embryonic stage of haematopoiesis in the yolk sac, but develop a fatal anaemia during early fetal life when haematopoiesis has switched to the fetal liver. Enucleated erythrocytes are formed but these do not contain the proper amount of haemoglobin. We conclude that the transcription factor EKLF is essential for the final steps of definitive erythropoiesis in fetal liver.

We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human 13-globin genes in compound EKLF knockout/human I~-locus transgenic mice. EKLF affects only the adult mouse 13-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human e and 7-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of 7" and 13-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of 7" to 13-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active 13 genes with a reciprocal increase in the number of transcriptionally active 7 genes. 13-gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the 13 promoter. There is a further increase in the number of transcriptionally active 7 genes and accompanying 7 gene promoter chromatin alterations. These results indicate that EKLF plays a major role in 7-and I~-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the 13-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.

Disruption of the gene for transcription factor EKLF (erythroid Krü ppel-like factor) results in fatal anaemiacaused by severely reduced expression of the adult β-globin gene, while other erythroid-specific genes, including the embryonic ε-and fetal γ-globin genes, are expressed normally. Thus, EKLF is thought to be a stage-specific factor acting through the CACC box in the β-gene promoter, even though it is already present in embryonic red cells. Here, we show that a β-globin gene linked directly to the locus control region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF -/-embryos. Thus, embryonic β-globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the locus control region, we analysed the expression of LCR-driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5ЈHS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5ЈHS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from γ-to β-globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in β-globin gene activation.

Transcription from the late promoter, PA3, of Bacillus sublilis phage #29 is activated by the viral regulatory protein p4. A kinetic analysis of the activation process has revealed that the role of protein p4 is to stabilize the binding of RNA polymerase to the promoter as a cosed complex without significantly affecting further steps of the initiation process. Electrophoretic band-shift assays performed with a DNAfragment spanning only the protein p4 binding site showed that RNA polymerase could efficiently retard the complex formed by protein p4 bound to the DNA. Similarly, when a DNA fmraent containing only the RNA polymerase-binding region of PA3 was used, p4 greatly stimulated the binng of RNA polymerase to the DNA. These results strongly suggest that p4and RNA polymerase contact each other at the PA3 promoter.In the light of current knowledge of the p4 activation mechanism, we propose that direct contacts between the two proteins participate in the activation process. and the RNA polymerase have been proposed to participate in the activation process (3,6,18,(20)(21)(22)(23)(24)(25)(26)(27)(28). At the same time, DNA is thought to play an active role in the initiation mechanism by adopting three-dimensional structures that either directly accelerate one of the steps leading to transcription initiation or facilitate the correct stereospecific alignment of the activator and the RNA polymerase (4, [29][30][31][32][33] 11401The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.