Novel Transactivation Domain in Erythroid Kruppel-like Factor (EKLF)

Erythroid Kruppel-like Factor (EKLF) is an erythroid-specific transcription factor that plays a critical role in γ- to β-globin gene switching during development. To identify essential domains required for EKLF transactivation function, we cotransfected a human erythroleukemia cell line (K562) with a locus control region γ/Luc-β/Cat reporter and an EKLF expression vector. In this assay EKLF mediates a 500-fold induction of β/CAT expression compared with controls. To map essential transactivation domains, progressive NH2-terminal and internal deletion mutants of EKLF were constructed. All EKLF mutants were expressed at wild-type levels, localized to the nucleus, and bound DNA. When mutant EKLF proteins were tested for β/CAT activation, a novel transactivation domain was identified. This novel domain, encompassing amino acids (aa) 140-358, is sufficient for maximal β/CAT activation. An 85-amino acid subdomain within this region (aa 140-225) is essential for its activity. Interestingly, this central transactivation subdomain is functionally redundant with the amino-terminal domain (aa 1-139). Thus, EKLF possesses at least two potent transactivation domains that appear to function in a redundant manner

Activation of δ-globin gene expression by erythroid Krupple-like factor: A potential approach for gene therapy of sickle cell disease

Hemoglobin A2 (HbA2; α2δ2) is a powerful inhibitor of HbS (α2β2/(S) polymerization. However, HbA2 levels are normally low in sickle cell patients. We show that a major reason for low δ-globin gene expression is the defective CACCC box at -90 in the δ-globin promoter. When the CACCC box defect in δ is corrected, expression of an HS2 δ/Luciferase reporter is equivalent to HS2 β/Luciferase. Erythroid Krupple-like factor (EKLF), which binds to the CACCC box of the β-globin gene and activates high-level expression, does not bind to the normal δ-globin promoter. Our goal is to design a modified EKLF that binds to the defective δ-globin promoter and enhances δ-globin gene expression. To test the feasibility of this strategy, we inserted the β-globin CACCC box at -90 of the δ-globin gene promoter to produce an HS2 δ(CAC)-β construct and quantitated human δ- and β-globin mRNA in stably transformed murine erythroleukemia (MEL) cells. δ-Globin mRNA in these cells was 22.0% ± 9.0% of total human globin mRNA (δ/δ + β) as compared with 3.0% ± 1.3% in the HS2 δ-β control. In a second set of experiments a GAL4 DNA-binding site was inserted at -90 of the δ-globin gene to produce an HS2 δ(GAL4)-β construct. This construct and a GAL4((1-147))/EKLF expression vector were stably transfected into MEL cells. δ-Globin mRNA in these cells was 27.8% ± 7.1% of total human globin mRNA as compared with 9.9% ± 2.5% in the HS2 δ(GAL4)-β plus GAL4((1-147)) control. These results show that δ-globin gene expression can be significantly increased by a modified EKLF. Based on these results, we suggest that modified EKLFs, which contain zinc fingers designed to bind specifically to the defective δ-globin CACCC box, may be useful in gene therapy approaches to increase HbA2 levels and inhibit HbS polymerization

Multiple elements in human β-globin locus control region 5′ HS 2 are involved in enhancer activity and position independent, transgene expression

The human β-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human ε-, γ- and β-giobin genes and, secondly, these sequences function as a powerful enhancer of ε-, γ- and β-globin gene expression. Erythrold-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5′ HS 2 site confers position-independent expression in transgenic mice and enhances human β-giobin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease β-globin gene expression. In contrast, no single site is required for position- independent transgene expression; all mice with site- specific mutations in 5′ HS 2 express human β-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5′ HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression. © 1994 Oxford University Press

Hemopexin Therapy Improves Cardiovascular Function by Preventing Heme-Induced Endothelial Toxicity in Mouse Models of Hemolytic Diseases

Background-Hemolytic diseases are characterized by enhanced intravascular hemolysis resulting in heme-catalyzed reactive oxygen species generation, which leads to endothelial dysfunction and oxidative damage. Hemopexin (Hx) is a plasma heme scavenger able to prevent endothelial damage and tissue congestion in a model of heme overload. Here, we tested whether Hx could be used as a therapeutic tool to counteract heme toxic effects on the cardiovascular system in hemolytic diseases. Methods and Results\u2014By using a model of heme overload in Hx-null mice, we demonstrated that heme excess in plasma, if not bound to Hx, promoted the production of reactive oxygen species and the induction of adhesion molecules and caused the reduction of nitric oxide availability. Then, we used \u3b2-thalassemia and sickle cell disease mice as models of hemolytic diseases to evaluate the efficacy of an Hx-based therapy in the treatment of vascular dysfunction related to heme overload. Our data demonstrated that Hx prevented heme-iron loading in the cardiovascular system, thus limiting the production of reactive oxygen species, the induction of adhesion molecules, and the oxidative inactivation of nitric oxide synthase/nitric oxide, and promoted heme recovery and detoxification by the liver mainly through the induction of heme oxygenase activity. Moreover, we showed that in sickle cell disease mice, endothelial activation and oxidation were associated with increased blood pressure and altered cardiac function, and the administration of exogenous Hx was found to almost completely normalize these parameters. Conclusions-Hemopexin treatment is a promising novel therapy to protect against heme-induced cardiovascular dysfunction in hemolytic disorders

ATF4 Is an Oxidative Stress–Inducible, Prodeath Transcription Factor in Neurons in Vitro and in Vivo

Oxidative stress is pathogenic in neurological diseases, including stroke. The identity of oxidative stress-inducible transcription factors and their role in propagating the death cascade are not well known. In an in vitro model of oxidative stress, the expression of the bZip transcription factor activating transcription factor 4 (ATF4) was induced by glutathione depletion and localized to the promoter of a putative death gene in neurons. Germline deletion of ATF4 resulted in a profound reduction in oxidative stress-induced gene expression and resistance to oxidative death. In neurons, ATF4 modulates an early, upstream event in the death pathway, as resistance to oxidative death by ATF4 deletion was associated with decreased consumption of the antioxidant glutathione. Forced expression of ATF4 was sufficient to promote cell death and loss of glutathione. In ATF4(-/-) neurons, restoration of ATF4 protein expression reinstated sensitivity to oxidative death. In addition, ATF4(-/-) mice experienced significantly smaller infarcts and improved behavioral recovery as compared with wild-type mice subjected to the same reductions in blood flow in a rodent model of ischemic stroke. Collectively, these findings establish ATF4 as a redox-regulated, prodeath transcriptional activator in the nervous system that propagates death responses to oxidative stress in vitro and to stroke in vivo

CubeSat Lunar and Deep Space Challenges

No abstract availabl

Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578 leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-mRNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia

Global Reorganization of Replication Domains During Embryonic Stem Cell Differentiation

DNA replication in mammals is regulated via the coordinate firing of clusters of replicons that duplicate megabase-sized chromosome segments at specific times during S-phase. Cytogenetic studies show that these “replicon clusters” coalesce as subchromosomal units that persist through multiple cell generations, but the molecular boundaries of such units have remained elusive. Moreover, the extent to which changes in replication timing occur during differentiation and their relationship to transcription changes has not been rigorously investigated. We have constructed high-resolution replication-timing profiles in mouse embryonic stem cells (mESCs) before and after differentiation to neural precursor cells. We demonstrate that chromosomes can be segmented into multimegabase domains of coordinate replication, which we call “replication domains,” separated by transition regions whose replication kinetics are consistent with large originless segments. The molecular boundaries of replication domains are remarkably well conserved between distantly related ESC lines and induced pluripotent stem cells. Unexpectedly, ESC differentiation was accompanied by the consolidation of smaller differentially replicating domains into larger coordinately replicated units whose replication time was more aligned to isochore GC content and the density of LINE-1 transposable elements, but not gene density. Replication-timing changes were coordinated with transcription changes for weak promoters more than strong promoters, and were accompanied by rearrangements in subnuclear position. We conclude that replication profiles are cell-type specific, and changes in these profiles reveal chromosome segments that undergo large changes in organization during differentiation. Moreover, smaller replication domains and a higher density of timing transition regions that interrupt isochore replication timing define a novel characteristic of the pluripotent state