Thus far, only one major form of vertebrate DNA (cytosine-5) methyltransferase (CpG MTase, EC 2.1.1.37) has been identified, cloned, and extensively studied. This enzyme, dnmt1, has been hypothesized to be responsible for most of the maintenance as well as the de novo methylation activities occurring in the somatic cells of vertebrates. We now report the discovery of another abundant species of CpG MTase in various types of human cell lines and somatic tissues. Interestingly, the mRNA encoding this CpG MTase results from alternative splicing of the primary transcript from the Dnmt1 gene, which incorporates in-frame an additional 48 nt between exons 4 and 5. Furthermore, this 48-nt exon sequence is derived from the first, or the most upstream, copy of a set of seven different Alu repeats located in intron 4. The ratios of expression of this mRNA to the expression of the previously known, shorter Dnmt1 mRNA species, as estimated by semiquantitative reverse transcription-PCR analysis, range from two-thirds to three-sevenths. This alternative splicing scheme of the Dnmt1 transcript seems to be conserved in the higher primates. We suggest that the originally described and the recently discovered forms of CpG MTase be named dnmt1-a and dnmt1-b, respectively. The evolutionary and biological implications of this finding are discussed in relation to the cellular functions of the CpG residues and the CpG MTases.In vertebrates, including mammals, chromosomal DNAs are modified by C-methylation at a limited number of CpG dinucleotides, resulting in methylation at the 5Ј position of the C residues. Many studies have indicated that this methylation process and its product, m
EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation (IP) qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation (ChIP) assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells (MEL) in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase II, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE (HS26). This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.
NF-E2 is a transcription activator for the regulation of a number of erythroid-and megakaryocytic lineagespecific genes. Here we present evidence that the large subunit of mammalian NF-E2, p45, is sumoylated in vivo in human erythroid K562 cells and in mouse fetal liver. By in vitro sumoylation reaction and DNA transfection experiments, we show that the sumoylation occurs at lysine 368 (K368) of human p45/NF-E2. Furthermore, p45 sumoylation enhances the transactivation capability of NF-E2, and this is accompanied by an increase of the NF-E2 DNA binding affinity. More interestingly, we have found that in K562 cells, the -globin gene loci in the euchromatin regions are predominantly colocalized with the nuclear bodies promyelocytic leukemia protein (PML) oncogenic domains that are enriched with the PML, SUMO-1, RNA polymerase II, and sumoylatable p45/NF-E2. Chromatin immunoprecipitation assays further showed that the intact sumoylation site of p45/ NF-E2 is required for its binding to the DNase I-hypersensitive sites of the -globin locus control region. Finally, we demonstrated by stable transfection assay that only the wild-type p45, but not its mutant form p45 (K368R), could efficiently rescue -globin gene expression in the p45-null, erythroid cell line CB3. These data together point to a model of mammalian -like globin gene activation by sumoylated p45/NF-E2 in erythroid cells.
Erythropoiesis is a highly regulated process during which BFU-E are differentiated into RBCs through CFU-E, Pro-E, PolyCh-E, OrthoCh-E, and reticulocyte stages. Uniquely, most erythroid-specific genes are activated during the Pro-E to Baso-E transition. We show that a wave of nuclear import of the erythroid-specific transcription factor EKLF occurs during the Pro-E to Baso-E transition. We further demonstrate that this wave results from a series of finely tuned events, including timed activation of PKCθ, phosphorylation of EKLF at S68 by P-PKCθ(S676), and sumoylation of EKLF at K74. The latter EKLF modifications modulate its interactions with a cytoplasmic ankyrin-repeat-protein FOE and importinβ1, respectively. The role of FOE in the control of EKLF nuclear import is further supported by analysis of the subcellular distribution patterns of EKLF in FOE-knockout mice. This study reveals the regulatory mechanisms of the nuclear import of EKLF, which may also be utilized in the nuclear import of other factors.
Erythroid Krüppel-like factor (EKLF) is an essential transcription factor for mammalian -like globin gene switching, and it specifically activates transcription of the adult  globin gene through binding of its zinc fingers to the promoter. It has been a puzzle that in the mouse, despite its expression throughout the erythroid development, EKLF activates the adult  maj globin promoter only in erythroid cells beyond the stage of embryonic day 10.5 (E10.5) but not before. We show here that expression of the mouse  maj globin gene in the aorta-gonad-mesonephros region of E10.5 embryos and in the E14.5 fetal liver is accompanied by predominantly nuclear localization of EKLF. In contrast, EKLF is mainly cytoplasmic in the erythroid cells of E9.5 blood islands in which  maj is silenced. Remarkably, in a cultured mouse adult erythroleukemic (MEL) cell line, the activation of the  maj globin gene by dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) induction is also paralleled by a shift of the subcellular location of EKLF from the cytoplasm to the nucleus. Blockage of the nuclear import of EKLF in DMSO-induced MEL cells with a nuclear export inhibitor repressed the transcription of the  maj globin gene. Transient transfection experiments further indicated that the full-sequence context of EKLF was required for the regulation of its subcellular locations in MEL cells during DMSO induction. Finally, in both the E14.5 fetal liver cells and induced MEL cells, the -like globin locus is colocalized the PML oncogene domain nuclear body, and concentrated with EKLF, RNA polymerase II, and the splicing factor SC35. These data together provide the first evidence that developmental stage-and differentiation state-specific regulation of the nuclear transport of EKLF might be one of the steps necessary for the switch-on of the mammalian adult  globin gene transcription.In mammals, the -like and ␣-like families are individually clustered on different chromosomes. Functional members of both gene clusters are arranged in the order of their expression during erythroid development (globin switch). In the mouse, the -like globin locus is arranged as follows: 5Ј-ε y (embryonic)-h1 (embryonic)- maj (fetal and adult)- min (fetal and adult, minor species)-3Ј (21, 2a) (top diagram in Fig. 1B). The hematopoietic cells, including the erythroid ones in the embryo, initially arise in the blood island that forms at embryonic day 7.5 (E7.5) (17). Within the primitive erythroblasts derived from the yolk sac are the embryonic genes, including ε y and h1, as transcribed previously (26). The adult  maj globin gene, on the other hand, is silenced in E9.5 yolk sac, but it starts to be expressed on E10.5 in the erythroblasts in the aorta-gonadmesonephros (AGM) region and the fetal liver of the embryo (26,38,54,56). During development, the major site of the erythropoiesis shifts to the fetal liver and then again to the bone marrow at the time of birth. The erythroid cells generated in the latter-named two tissues represent a def...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.