Identification of a genomic DNA sequence that quantitatively modulates KLF1 transcription factor expression in differentiating human hematopoietic cells.

The onset of erythropoiesis is under strict developmental control, with direct and indirect inputs influencing its derivation from the hematopoietic stem cell. A major regulator of this transition is KLF1/EKLF, a zinc finger transcription factor that plays a global role in all aspects of erythropoiesis. Here, we have identified a short, conserved enhancer element in KLF1 intron 1 that is important for establishing optimal levels of KLF1 in mouse and human cells. Chromatin accessibility of this site exhibits cell-type specificity and is under developmental control during the differentiation of human CD34+ cells towards the erythroid lineage. This site binds GATA1, SMAD1, TAL1, and ETV6. In vivo editing of this region in cell lines and primary cells reduces KLF1 expression quantitatively. However, we find that, similar to observations seen in pedigrees of families with KLF1 mutations, downstream effects are variable, suggesting that the global architecture of the site is buffered towards keeping the KLF1 genetic region in an active state. We propose that modification of intron 1 in both alleles is not equivalent to complete loss of function of one allele.

Unanticipated repression function linked to erythroid Krüppel-like factor.

The erythroid cell-specific transcription factor erythroid Krüppel-like factor (EKLF) is an important activator of beta-globin gene expression. It achieves this by binding to the CACCC element at the beta-globin promoter via its zinc finger domain. The coactivators CBP and P300 interact with, acetylate, and enhance its activity, helping to explain its role as a transcription activator. Here we show that EKLF can also interact with the corepressors mSin3A and HDAC1 (histone deacetylase 1) through its zinc finger domain. When linked to a GAL4 DNA binding domain, full-length EKLF or its zinc finger domain alone can repress transcription in vivo. This repressive activity can be relieved by the HDAC inhibitor trichostatin A. Although recruitment of EKLF to a promoter is required to show repression, its zinc finger domain cannot bind directly to DNA and repress transcription simultaneously. In addition, the target promoter configuration is important for enabling EKLF to exhibit any repressive activity. These results suggest that EKLF may function in vivo as a transcription repressor and play a previously unsuspected additional role in regulating erythroid gene expression and differentiation.

Site-specific acetylation by p300 or CREB binding protein regulates erythroid Krüppel-like factor transcriptional activity via its interaction with the SWI-SNF complex.

Recruitment of modifiers and remodelers to specific DNA sites within chromatin plays a critical role in controlling gene expression. The study of globin gene regulation provides a convergence point within which to address these issues in the context of tissue-specific and developmentally regulated expression. In this regard, erythroid Krüppel-like factor (EKLF) is critical. EKLF is a red cell-specific activator whose presence is crucial for establishment of the correct chromatin structure and high-level transcriptional induction of adult beta-globin. We now find, by metabolic labeling-immunoprecipitation experiments, that EKLF is acetylated in the erythroid cell. EKLF residues acetylated by CREB binding protein (CBP) in vitro map to Lys-288 in its transactivation domain and Lys-302 in its zinc finger domain. Although site-specific DNA binding by EKLF is unaffected by the acetylation status of either of these lysines, directed mutagenesis of Lys-288 (but not Lys-302) decreases the ability of EKLF to transactivate the beta-globin promoter in vivo and renders it unable to be superactivated by coexpressed p300 or CBP. In addition, the acetyltransferase function of CBP or p300 is required for superactivation of wild-type EKLF. Finally, acetylated EKLF has a higher affinity for the SWI-SNF chromatin remodeling complex and is a more potent transcriptional activator of chromatin-assembled templates in vitro. These results demonstrate that the acetylation status of EKLF is critical for its optimal activity and suggest a mechanism by which EKLF acts as an integrator of remodeling and transcriptional components to alter chromatin structure and induce adult beta-globin expression within the beta-like globin cluster.

A SWI/SNF-related chromatin remodeling complex, E-RC1, is required for tissue-specific transcriptional regulation by EKLF in vitro.

Erythroid Krüppel-like factor (EKLF) is necessary for stage-specific expression of the human beta-globin gene. We show that EKLF requires a SWI/SNF-related chromatin remodeling complex, EKLF coactivator-remodeling complex 1 (E-RC1), to generate a DNase I hypersensitive, transcriptionally active beta-globin promoter on chromatin templates in vitro. E-RC1 contains BRG1, BAF170, BAF155, and INI1 (BAF47) homologs of yeast SWI/SNF subunits, as well as a subunit unique to higher eukaryotes, BAF57, which is critical for chromatin remodeling and transcription with EKLF. E-RC1 displays functional selectivity toward transcription factors, since it cannot activate expression of chromatin-assembled HIV-1 templates with the E box-binding protein TFE-3. Thus, a member of the SWI/SNF family acts directly in transcriptional activation and may regulate subsets of genes by selectively interacting with specific DNA-binding proteins.

Transcription factor erythroid Krüppel-like factor (EKLF) is essential for the erythropoietin-induced hemoglobin production but not for proliferation, viability, or morphological maturation.

The erythroid Krüppel-like factor (EKLF) is essential for the transcription of betamaj globin in erythroid cells. We show here that RNA for this transcription factor did not alter during erythropoietin-induced differentiation of J2E cells; however, EKLF protein content decreased and was inversely related to globin production. This unexpected result was also observed during chemically induced maturation of two murine erythroleukemia cell lines. To explore the role of EKLF in erythroid terminal differentiation, an antisense EKLF construct was introduced into J2E cells. As a consequence EKLF RNA and protein levels fell by approximately 80%, and the cells were unable to manufacture hemoglobin in response to erythropoietin. The failure to produce hemoglobin was due to reduced transcription of not only globin genes but also key heme enzyme genes. However, numerous other genes, including several erythroid transcription factors, were unaffected by the decrease in EKLF. Although hemoglobin synthesis was severely impaired with depleted EKLF levels, morphological maturation in response to erythropoietin continued normally. Moreover, erythropoietin-induced proliferation and viability were unaffected by the decrease in EKLF levels. We conclude that EKLF affects a specific set of genes, which regulates hemoglobin production and has no obvious effect on morphological changes, cell division, or viability in response to erythropoietin.

Chromatin structure and transcriptional control elements of the erythroid Krüppel-like factor (EKLF) gene.

Erythroid Krüppel-like factor (EKLF) is a red cell-specific transcription factor whose activity is critical for the switch in expression from fetal to adult beta-globin during erythroid ontogeny. We have examined its own regulation using a number of approaches. First, the EKLF transcription unit is in an open chromatin configuration in erythroid cells. Second, in vivo transfection assays demonstrate that the more distal of the two erythroid-specific DNase-hypersensitive sites behaves as an enhancer. Although this conserved element imparts high level transcription to a heterologous promoter in all lines examined, erythroid specificity is retained only when it is fused to the proximal EKLF promoter, which contains an important GATA site. Third, extensive mutagenesis of this enhancer element has delimited its in vivo activity to a core region of 49 base pairs. Finally, in vitro footprint and gel shift assays demonstrate that three distinct DNA binding activities in erythroid cell extracts individually interact with three short sequences within this core enhancer element. These analyses reveal that high level erythroid expression of EKLF relies on the interplay between conserved proximal and distal promoter elements that alter chromatin structure and likely provide a target for genetic control via extracellular induction pathways.

Acetylation and modulation of erythroid Krüppel-like factor (EKLF) activity by interaction with histone acetyltransferases.

Erythroid Krüppel-like factor (EKLF) is a red cell-specific transcriptional activator that is crucial for consolidating the switch to high levels of adult beta-globin expression during erythroid ontogeny. EKLF is required for integrity of the chromatin structure at the beta-like globin locus, and it interacts with a positive-acting factor in vivo. We find that EKLF is an acetylated transcription factor, and that it interacts in vivo with CBP, p300, and P/CAF. However, its interactions with these histone acetyltransferases are not equivalent, as CBP and p300, but not P/CAF, utilize EKLF as a substrate for in vitro acetylation within its trans-activation region. The functional effects of these interactions are that CBP and p300, but not P/CAF, enhance EKLF's transcriptional activation of the beta-globin promoter in erythroid cells. These results establish EKLF as a tissue-specific transcription factor that undergoes post-translational acetylation and suggest a mechanism by which EKLF is able to alter chromatin structure and induce beta-globin expression within the beta-like globin cluster.

Regulation of erythroid Krüppel-like factor (EKLF) transcriptional activity by phosphorylation of a protein kinase casein kinase II site within its interaction domain.

Erythroid Krüppel-like factor (EKLF) is a red cell-specific activator whose presence is crucial for establishing high levels of adult beta-globin expression in definitive cells during erythroid ontogeny. However, its simple presence within the erythroid lineage is not sufficient to activate the beta-globin promoter. One explanation that may account for this is that post-translational modification of EKLF differs within erythroid cell populations and regulates its activity. We have therefore addressed whether phosphorylation plays a role in modulating EKLF action. First, in vivo analyses implicate serine/threonine kinases as important players in the terminal differentiation of MEL cells, and demonstrate that EKLF is phosphorylated at serine and threonine residues within its transactivation region. Second, directed disruption of a protein kinase casein kinase (CK) II site, located within the EKLF interaction domain, abolishes EKLF transactivation and in vivo competition activity. Third, in vitro assays demonstrate that CKIIalpha interacts with EKLF, and that the EKLF interaction domain is phosphorylated by CKII only at Thr-41; however, the CKII-site mutant is not phosphorylated. Finally, the transactivation capability of EKLF is augmented by co-transfection of CKIIalpha. We conclude that EKLF is a phosphoprotein whose ability to transcriptionally activate an adjacent promoter is critically dependent on the phosphorylation status of a specific site located within the EKLF interaction domain, and that serine/threonine kinases play an important role in this process.

Transcriptional factors for specific globin genes.

Correct temporal control of the beta-like globin cluster is generated in part by the binding of tissue-restricted transcriptional regulators to their cognate sites. Erythroid Krüppel-like Factor (EKLF) is one of these red cell-specific activators that is particularly important for switching on adult beta-globin gene expression. However, its simple presence is not sufficient to activate the beta-globin promoter, as primitive erythroid cells and a number of erythroid cell lines express EKLF yet do not express adult beta-globin. One explanation that may account for these observations is that post-translational modification of EKLF differs within these cell populations. To address this issue, we are investigating whether phosphorylation plays a role in modulating EKLF activity. In vitro and in vivo approaches have been used to demonstrate that EKLF is a phosphoprotein whose ability to bind DNA and transcriptionally activate an adjacent promoter is critically dependent on its phosphorylation status. Of particular interest is a casein kinase II site within the EKLF minimal transactivation domain.

Erythroid-specific transcription.

Over the past year, the range of research related to erythroid cell-specific transcription has expanded beyond isolation of its transcriptional players to studies that address more complex yet still fundamental questions: first, whether these factors form part of a larger protein-protein structure; second, how factor interactions with DNA affect the surrounding chromatin configuration; third, how this affects the mechanism of gene switching within the beta-like globin cluster; and finally, how these regulators are themselves transcriptionally controlled.

A shortened life span of EKLF-/- adult erythrocytes, due to a deficiency of beta-globin chains, is ameliorated by human gamma-globin chains.

Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF-/- ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF-/- colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of beta h1-globin, which continued to be expressed at a very low level. The ratio of adult beta-globin/alpha-globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF-/- cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF-/- erythrocytes in adult animals must be short-lived, apparently due to the imbalance of beta-versus alpha-globin chains, leading to the precipitation of excess alpha-globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF-/- ES cells of a human LCR/gamma-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals.

Erythroid Krüppel-like factor (EKLF) contains a multifunctional transcriptional activation domain important for inter- and intramolecular interactions.

Erythroid Krüppel-like factor (EKLF) is a red cell-restricted transcriptional activator that plays a dominant role in establishing high levels of beta-globin gene expression during erythroid ontogeny. Although its DNA binding domain belongs to the well-studied class of Krüppel-like zinc fingers, its proline-rich activation region has not been thoroughly examined. We have analyzed this region by monitoring the functional effects of its mutagenesis upon EKLF activity in vivo and in vitro. First, using co-transfection assays, we find that the transactivation region contains discrete stimulatory and inhibitory subdomains. Second, in vitro binding assays indicate that the inhibitory domain exerts its effect in cis by interfering with DNA binding. Third, in vivo competition assays demonstrate that EKLF interacts with a positive-acting cellular factor, and that the domain responsible for this trans interaction lies within a 40 amino acid sequence that is coincident with the EKLF minimal transactivation domain. Finally, site-directed mutagenesis of this domain implies that conformation and/or phosphorylation status of its central core may be critical for such interactions. These results point towards post-translational steric and/or allosteric control of EKLF function that may be important not just for its DNA binding ability, but also for its potential to interact with other proteins that fully establish the correct stereospecific array leading to efficient switching of beta-globin transcription during development.

Erythroid Krüppel-like factor exhibits an early and sequentially localized pattern of expression during mammalian erythroid ontogeny.

Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific transcription factor that mediates activation via binding to a 9 base pair sequence that encompasses the CACCC element, one of a trio of evolutionarily conserved sequence motifs that are functionally important for transcription of red cell-specific genes. Molecular analyses have delineated the specificity of its interaction and activation through the CAC site at the adult beta-globin promoter. However, its expression and distribution during murine ontogeny have not been established. To address these issues, we have focused on biological aspects of EKLF expression by examining the onset and localization of its mRNA during murine development by using reverse transcription/polymerase chain reaction (RT/PCR) analysis of differentiating embryonic stem cells and in situ analyses of normal developing embryos. In addition, we have monitored the presence of EKLF protein by blot analysis of whole-cell extracts derived from circulating cells and embryonic tissue. Our studies show that EKLF mRNA is first expressed at the neural plate stage (day 7.5) within primitive erythroid cells at the very beginning of blood island formation in the yolk sac. EKLF is then expressed by day 9 in the hepatic primordia and remains high in the liver, which becomes the sole source of EKLF mRNA in the 14.5 day fetus. Concomitantly with EKLF mRNA, EKLF protein is also expressed in primitive erythroid cells and in the fetal liver. Finally, EKLF expression in the adult spleen is strictly localized to the red pulp. These studies demonstrate that EKLF is a specific, early marker of erythroid differentiation consistent with its requirement for later globin (and possibly other red cell gene-specific) expression. In addition, EKLF exhibits alternate, sequentially active sites of expression within regions known to harbor hematopoietic precursors during murine ontogeny. Thus, EKLF expression exhibits biological properties that, in addition to previous molecular and more recent genetic studies, augment the evidence in favor of its important role in erythroid cell-specific expression.

Isolation, genomic structure, and expression of human erythroid Krüppel-like factor (EKLF).

Erythroid Krüppel-like factor (EKLF) is an essential transcriptional activator that directs high-level expression of the adult beta-globin promoter by binding to its CACCC element, one of a trio of highly conserved sequences present in erythroid cell-specific promoters and enhancers. This report describes the isolation and characterization of the human homolog of murine EKLF. The human EKLF transcription unit shares a number of structural properties with its marine counterpart. Human EKLF is contained within 3 kb of genomic DNA, and its coding region is interrupted by two introns whose locations are conserved with the murine gene. The three zinc fingers share >90% sequence similarity with and are predicted to bind the same target sequence as the mouse EKLF. The rest of the protein is proline-rich and retains approximately 70% sequence similarity to the mouse gene. Human EKLF is expressed in bone marrow and HEL, JK1, and OCIM1 erythroleukemic cell lines but not in K562 nor in myeloid or lymphoid cell lines. These results indicate that the genomic structure and erythroid-restricted expression of EKLF are highly conserved between the murine and human homologues. Availability of human EKLF will enable initiation of studies to molecularly assess whether it is functionally compromised in those cases of beta-thalassemia that contain a normal beta-globin gene locus.

Functional interaction of GATA1 with erythroid Krüppel-like factor and Sp1 at defined erythroid promoters.

GATA and CACC elements commonly are codistributed within the regulatory domains of a variety of erythroid genes. Using Drosophila S2 cells, the actions of GATA1, Sp1, and erythroid Kruppel-like factor (EKLF) at these elements within model erythroid promoters have been assessed. For each promoter studied (erythroid pyruvate kinase, glycophorin B, and a murine betamaj globin-derived construct, GCT) Sp1 and EKLF each activated transcription despite differences in CACC element sequence, orientation, and positioning. However, GATA1 acted in apparent cooperativity with Sp1 at the pyruvate kinase promoter; with EKLF at the betamaj globin-derived GCT promoter; and with either Sp1 or EKLF at the glycophorin B promoter. Thus, GATA1 may functionally interact with each of these Krüppel-like factors depending on promoter context; and at the GCT promoter, transcriptional activation by GATA1 and EKLF was > or = 10-fold higher than levels attributable to additive effects. The possibility that interactions between these activators may be direct was supported by the specific binding of baculoviral-expressed EKLF to GATA1. This report underlines the likelihood that discrete roles exist for Sp1 and EKLF in erythroid gene activation, and supports a mechanism of direct cooperativity for EKLF and GATA1 as coregulators.

The erythroid Krüppel-like factor transactivation domain is a critical component for cell-specific inducibility of a beta-globin promoter.

Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific DNA-binding protein that activates transcription from the beta-globin CACCC element, a functionally important and evolutionarily conserved component of globin as well as other erythroid cell-specific promoters and enhancers. We have attempted to elucidate the molecular role of EKLF in erythrocyte-specific transcriptional activation. First, in vivo and in vitro analyses have been used to demonstrate that the level of activation by EKLF is dependent on the orientation and number of CACCC elements, that EKLF contains separable activation and DNA-binding domains, and that the EKLF proline-rich region is a potent activator in CV-1 cells when fused to a nonrelated DNA-binding module. Second, we have established a transient assay in murine erythroleukemia cells in which reproducible levels of a reporter can be induced when linked to a locus control region enhancer-beta-globin promoter and in which induction is abolished when the promoter CAC site is mutated to a GAL site. Third, we demonstrate that the EKLF transactivation region, when fused to the GAL DNA-binding domain, can restore inducibility to this mutated construct and that this inducibility exhibits activator-, promoter-, and cell-type specificity. These results demonstrate that EKLF provides a crucial transactivation function for globin expression and further reinforce the idea that EKLF is an important regulator of CACCC element-directed transcription in erythroid cells.

Role of erythroid Kruppel-like factor in human gamma- to beta-globin gene switching.

Erythroid Kruppel-like factor (EKLF) is an erythroid-specific transcription factor that contains zinc finger domains similar to the Kruppel protein of Drosophila melanogaster. Previous studies demonstrated that EKLF binds to the CACCC box in the human beta-globin gene promoter and activates transcription. CACCC box mutations that cause severe beta-thalassemias in humans inhibit EKLF binding. Results described in this paper suggest that EKLF functions predominately in adult erythroid tissue. The EKLF gene is expressed at a 3-fold higher level in adult erythroid tissue than in fetal erythroid tissue, and the EKLF protein binds to the human beta-globin promoter 8-fold more efficiently than to the human gamma-globin promoter. Co-transfection experiments in the human fetal-like erythroleukemia cell line K562 demonstrate that over-expression of EKLF activates a beta-globin reporter construct 1000-fold; a linked gamma-globin reporter is activated only 3-fold. Mutation of the beta-globin CACCC box severely inhibits activation. These results demonstrate that EKLF is a developmental stage-enriched protein that preferentially activates human beta-globin gene expression. The data strongly suggest that EKLF is an important factor involved in human gamma- to beta-globin gene switching.

Regulation of the erythroid Kruppel-like factor (EKLF) gene promoter by the erythroid transcription factor GATA-1.

Erythroid Kruppel-like factor (EKLF) is an erythroid-specific transcription factor that binds a CACCC motif found in the human beta-globin gene promoter. We have studied the promoter of the EKLF gene and identified binding sites for the transcription factors GATA-1 and CCAAT-binding Protein 1 (CP1). We show that both types of binding sites are required for full activity, and that the GATA motif at -60 is essential. The EKLF promoter can be directly activated in nonerythroid cells in cotransfection experiments by forced expression of GATA-1. These results suggest that EKLF is dependent on GATA-1 for its expression and lies downstream of, or coincident with, GATA-1 in a regulatory hierarchy in erythroid development.