Ecotropic viral integration site 1 (EVI1) is an important transcription factor for leukemogenesis. EVI1 is a member of a group of transcription factors with C-terminal binding protein (CtBP)-binding motifs that act as transcriptional co-repressors; however, we recently found that EVI1 directly activates GATA2 transcription, which is an important gene for the maintenance of hematopoietic stem cells. We show here that EVI1-activated GATA2 transcripts derive from exon 1S of GATA2, which is specifically activated in neural and hematopoietic cells. EVI1 was acetylated by the histone acetyltransferase p300/CBP association factor (P/CAF) in myeloid leukemia cells and hematopoietic progenitor cells. Acetylation at Lys 564 , which is adjacent to the CtBP-binding consensus sequence of EVI1, was found to be important for transcriptional activation of GATA2. Mutation of Lys 564 to alanine (K564A) markedly reduced the ability of EVI1 to bind DNA and activate transcription of GATA2. Furthermore, we confirmed that Lys 564 in EVI1 was specifically acetylated in leukemia and primary hematopoietic cells by using an antibody directed against an acetylated Lys 564 EVI1 peptide. Moreover, co-transfection of P/CAF with EVI1 overcame the suppressive effect of the CtBP co-repressor and resulted in GATA2 transcriptional activation; nonetheless, CtBP2 was still included in the protein complex with EVI1 and P/CAF on the EVI1-binding site in the GATA2 promoter region. Thus, acetylation of EVI1 at Lys 564 by P/CAF enhances the DNA binding capacity of EVI1 and thereby contributes to the activation of GATA2.Ecotropic viral integration site 1 (Evi1) was first identified as a common retroviral integration site in AKXD murine myeloid tumors (1). The human homologue EVI1 is transcriptionally activated by specific chromosomal abnormalities at 3q26 such as t(3;3)(q21;q26), inv(3)(q21q26), t(3,21), or others (2, 3). These abnormalities lead to the aberrant expression of EVI1 and are associated with human acute myelogenous leukemia, myelodysplastic syndrome, and chronic myelogenous leukemia. High expression of EVI1 is detectable in around 8% of myeloid leukemia cases and is a poor prognostic indicator in myeloid leukemia (4, 5).In EVI1, two DNA binding domains with seven and three zinc finger repeats bind DNA through specific conserved GATA-like or ETS-like sequence motifs, and they have the potential to interact with both co-repressors and co-activators as a dual transcriptional factor (6 -8). EVI1 has been shown to interact directly with the known transcriptional repressor C-terminal binding protein (CtBP) 2 via two CtBP-binding consensus motifs at amino acids 544 -607 (9, 10). Although CtBP binding to EVI1 has been suggested to recruit histone deacetylase complexes (HDACs) and lead to transcriptional repression via chromatin remodeling, specific target genes repressed by EVI1 have not yet been found. On the other hand, the interaction of EVI1 with cAMP-responsive element-binding proteinbinding protein (CBP) and p300/CBP-associated factor (P/CAF) ...
Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of 18O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging 18O atom into the phosphate group during the oxidation step of the synthetic cycle by using 18O water as the oxygen donor. The 18O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The 18O/16O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of 18O-labeled RNA, and this technique was used to determine the blood concentration of 18O-labeled RNA after administration to mice. 18O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.
Age-related macular degeneration (AMD) is a vision-threatening disease characterized by choroidal fibrovascular membrane (FVM) formation, choroidal neovascularization (CNV) and choroidal fibrosis. No safe and effective therapeutic method has been developed for the choroidal fibrosis, although anti-vascular endothelial growth factor therapy can partially shrink the CNV. We recently reported that periostin (POSTN), which is produced by retinal pigment epithelial cells, has an important role in the formation of preretinal FVMs, but its role in choroidal FVMs has not been determined. In this study, we used Postn knockout mice to investigate the role played by POSTN in choroidal FVM formation. In addition, we used a new class of RNA interference (RNAi) agent (NK0144) that targets POSTN and determined its effect on choroidal FVM development. Genetic ablation of Postn had an inhibitory effect not only on CNV formation but also on choroidal fibrosis in a mouse CNV model. NK0144 also had a greater inhibitory effect on both the CNV and choroidal fibrosis than control RNAi with no apparent adverse effects. These findings suggest a causal relationship between POSTN and choroidal FVM formation, and also a potential therapeutic role of intravitreal NK0144 for AMD.
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