Role of erythroid Kruppel-like factor in human γ- to β-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 β-globin gene promoter and activates transcription. CACCC box mutations that cause severe β-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 β-globin promoter 8-fold more efficiently than to the human γ-globin promoter. Co-transfection experiments in the human fetal-like erythroleukemia cell line K562 demonstrate that over-expression of EKLF activates a β-globin reporter construct 1000-fold; a linked γ-globin reporter is activated only 3-fold. Mutation of the β-globin CACCC box severely inhibits activation. These results demonstrate that EKLF is a developmental stage-enriched protein that preferentially activates human β-globin gene expression. The data strongly suggest that EKLF is an important factor involved in human γ- to β-globin gene switching

Generation, characterization, and use of EKLF(Klf1)/CRE knock-in mice for cell-restricted analyses

IntroductionEKLF/Klf1 is a tissue-restricted transcription factor that plays a critical role in all aspects of erythropoiesis. Of particular note is its tissue-restricted pattern of expression, a property that could prove useful for expression control of a linked marker or enzymatic gene.Methods and resultsWith this in mind, we fused the CRE recombinase to the genomic EKLF coding region and established mouse lines. We find by FACS analyses that CRE expression driven by the EKLF transcription unit recapitulates erythroid-restricted expression with high penetrance in developing embryos. We then used this line to test its properties in the adult, where we found EKLF/CRE is an active and is a robust mimic of normal EKLF expression in the adult bone marrow. EKLF/CRE is also expressed in erythroblastic island macrophage in the fetal liver, and we demonstrate for the first time that, as seen during embryonic development, EKLF is also expressed in adult BM-derived erythroblastic island macrophage. Our data also support lineage studies showing EKLF expression at early stages of hematopoiesis.DiscussionThe EKLF/CRE mouse lines are novel reagents whose availability will be of great utility for future experiments by investigators in the red cell field

Genetic disarray follows mutant KLF1-E325K expression in a congenital dyserythropoietic anemia patient

C ongenital dyserythropoietic anemia type IV is caused by a heterozygous mutation, Glu325Lys (E325K), in the KLF1 transcription factor. Molecular characteristics of this disease have not been clarified, partly due to its rarity. We expanded erythroid cells from a patient\u2019s peripheral blood and analyzed its global expression pattern. We find that a large number of erythroid pathways are disrupted, particularly those related to membrane transport, globin regulation, and iron utilization. The altered genetics lead to significant deficits in differentiation. Glu325 is within the KLF1 zinc finger domain at an amino acid critical for site specific DNA binding. The change to Lys is predicted to significantly alter the target site recognition sequence, both by subverting normal recognition and by enabling interaction with novel sites. Consistent with this, we find high level ectopic expression of genes not normally present in the red cell. These altered properties explain patients\u2019 clinical and phenotypic features, and elucidate the dominant character of the mutation

The DEK oncoprotein is a critical component of the EKLF/KLF1 enhancer in erythroid cells

Understanding how transcriptional regulators are themselves controlled is important in attaining a complete picture of the intracellular effects that follow signaling cascades during early development and cell-restricted differentiation. We have addressed this issue by focusing on the regulation of EKLF/KLF1, a zinc finger transcription factor that plays a necessary role in the global regulation of erythroid gene expression. Using biochemical affinity purification, we have identified the DEK oncoprotein as a critical factor that interacts with an essential upstream enhancer element of the EKLF promoter and exerts a positive effect on EKLF levels. This element also binds a core set of erythroid transcription factors, suggesting that DEK is part of a tissue-restricted enhanceosome that contains BMP4-dependent and -independent components. Together with local enrichment of properly coded histones and an open chromatin domain, optimal transcriptional activation of the EKLF locus can be established

Critical adsorption on curved objects

A systematic fieldtheoretic description of critical adsorption on curved objects such as spherical or rodlike colloidal particles immersed in a fluid near criticality is presented. The temperature dependence of the corresponding order parameter profiles and of the excess adsorption are calculated explicitly. Critical adsorption on elongated rods is substantially more pronounced than on spherical particles. It turns out that, within the context of critical phenomena in confined geometries, critical adsorption on a microscopically thin `needle' represents a distinct universality class of its own. Under favorable conditions the results are relevant for the flocculation of colloidal particles.Comment: 52 pages, 10 figure

Shapes, contact angles, and line tensions of droplets on cylinders

Using an interface displacement model we calculate the shapes of nanometer-size liquid droplets on homogeneous cylindrical surfaces. We determine effective contact angles and line tensions, the latter defined as excess free energies per unit length associated with the two contact lines at the ends of the droplet. The dependences of these quantities on the cylinder radius and on the volume of the droplets are analyzed.Comment: 26 pages, RevTeX, 10 Figure

Mutant KLF1 in Adult Anemic Nan Mice Leads to Profound Transcriptome Changes and Disordered Erythropoiesis.

Anemic Nan mice carry a mutation (E339D) in the second zinc finger of erythroid transcription factor KLF1. Nan-KLF1 fails to bind a subset of normal KLF1 targets and ectopically binds a large set of genes not normally engaged by KLF1, resulting in a corrupted fetal liver transcriptome. Here, we performed RNAseq using flow cytometric-sorted spleen erythroid precursors from adult Nan and WT littermates rendered anemic by phlebotomy to identify global transcriptome changes specific to the Nan Klf1 mutation as opposed to anemia generally. Mutant Nan-KLF1 leads to extensive and progressive transcriptome corruption in adult spleen erythroid precursors such that stress erythropoiesis is severely compromised. Terminal erythroid differentiation is defective in the bone marrow as well. Principle component analysis reveals two major patterns of differential gene expression predicting that defects in basic cellular processes including translation, cell cycle, and DNA repair could contribute to disordered erythropoiesis and anemia in Nan. Significant erythroid precursor stage specific changes were identified in some of these processes in Nan. Remarkably, however, despite expression changes in large numbers of associated genes, most basic cellular processes were intact in Nan indicating that developing red cells display significant physiological resiliency and establish new homeostatic set points in vivo

Hard-Sphere Fluids in Contact with Curved Substrates

The properties of a hard-sphere fluid in contact with hard spherical and cylindrical walls are studied. Rosenfeld's density functional theory (DFT) is applied to determine the density profile and surface tension $\gamma$ for wide ranges of radii of the curved walls and densities of the hard-sphere fluid. Particular attention is paid to investigate the curvature dependence and the possible existence of a contribution to $\gamma$ that is proportional to the logarithm of the radius of curvature. Moreover, by treating the curved wall as a second component at infinite dilution we provide an analytical expression for the surface tension of a hard-sphere fluid close to arbitrary hard convex walls. The agreement between the analytical expression and DFT is good. Our results show no signs for the existence of a logarithmic term in the curvature dependence of $\gamma$.Comment: 15 pages, 6 figure