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Runt domain-related (Runx) transcription factors are essential for early T cell development in mice from uncommitted to committed stages. Single and double Runx knockouts via Cas9 show that target genes responding to Runx activity are not solely controlled by the dominant factor, Runx1. Instead, Runx1 and Runx3 are coexpressed in single cells; bind to highly overlapping genomic sites; and have redundant, collaborative functions regulating genes pivotal for T cell development. Despite stable combined expression levels across pro-T cell development, Runx1 and Runx3 preferentially activate and repress genes that change expression dynamically during lineage commitment, mostly activating T-lineage genes and repressing multipotent progenitor genes. Furthermore, most Runx target genes are sensitive to Runx perturbation only at one stage and often respond to Runx more for expression transitions than for maintenance. Contributing to this highly stage-dependent gene regulation function, Runx1 and Runx3 extensively shift their binding sites during commitment. Functionally distinct Runx occupancy sites associated with stage-specific activation or repression are also distinguished by different patterns of partner factor cobinding. Finally, Runx occupancies change coordinately at numerous clustered sites around positively or negatively regulated targets during commitment. This multisite binding behavior may contribute to a developmental “ratchet” mechanism making commitment irreversible.

RUNX 1 and RUNX3 drive Nf1 neurofibromagenesis by mediating the promoter usage and inducing levels of protein expression of PMP22., Patients with neurofibromatosis type 1 (NF1) are predisposed to develop neurofibromas, but the underlying molecular mechanisms of neurofibromagenesis are not fully understood. We showed dual genetic deletion of Runx1 and Runx3 in Schwann cells (SCs) and SC precursors delayed neurofibromagenesis and prolonged mouse survival. We identified peripheral myelin protein 22 (Pmp22/Gas3) related to neurofibroma initiation. Knockdown of Pmp22 with short hairpin RNAs increased Runx1fl/fl;Runx3fl/fl;Nf1fl/fl;DhhCre tumor-derived sphere numbers and enabled significantly more neurofibroma-like microlesions on transplantation. Conversely, overexpression of Pmp22 in mouse neurofibroma SCs decreased cell proliferation. Mechanistically, RUNX1/3 regulated alternative promoter usage and induced levels of protein expression of Pmp22 to control SC growth. Last, pharmacological inhibition of RUNX/core-binding factor β (CBFB) activity significantly reduced neurofibroma volume in vivo. Thus, we identified a signaling pathway involving RUNX1/3 suppression of Pmp22 in neurofibroma initiation and/or maintenance. Targeting disruption of RUNX/CBFB interaction might provide a novel therapy for patients with neurofibroma.

SummaryThe t(8;21) and inv(16) chromosomal aberrations generate the oncoproteins AML1-ETO (A-E) and CBFβ-SMMHC (C-S). The role of these oncoproteins in acute myeloid leukemia (AML) etiology has been well studied. Conversely, the function of native RUNX1 in promoting A-E- and C-S-mediated leukemias has remained elusive. We show that wild-type RUNX1 is required for the survival of t(8;21)-Kasumi-1 and inv(16)-ME-1 leukemic cells. RUNX1 knockdown in Kasumi-1 cells (Kasumi-1RX1-KD) attenuates the cell-cycle mitotic checkpoint, leading to apoptosis, whereas knockdown of A-E in Kasumi-1RX1-KD rescues these cells. Mechanistically, a delicate RUNX1/A-E balance involving competition for common genomic sites that regulate RUNX1/A-E targets sustains the malignant cell phenotype. The broad medical significance of this leukemic cell addiction to native RUNX1 is underscored by clinical data showing that an active RUNX1 allele is usually preserved in both t(8;21) or inv(16) AML patients, whereas RUNX1 is frequently inactivated in other forms of leukemia. Thus, RUNX1 and its mitotic control targets are potential candidates for new therapeutic approaches.

The oncogenic transcription factor Runx1 is required for the specification of definitive hematopoietic stem cells (HSC) in the developing embryo. The activity of this master regulator is tightly controlled during development. The transcription factors that upregulate the expression of Runx1 also upregulate the expression of Smad6, the inhibitory Smad, which controls Runx1 activity by targeting it to the proteasome. Here we show that Runx1, in conjunction with Fli1, Gata2, and Scl, directly regulates the expression of Smad6 in the aorta-gonad-mesonephros (AGM) region in the developing embryo, where HSCs originate. Runx1 regulates Smad6 activity via a novel upstream enhancer, and Runx1 null embryos show reduced Smad6 transcripts in the yolk-sac and c-Kit-positive fetal liver cells. By directly regulating the expression of Smad6, Runx1 sets up a functional rheostat to control its own activity. The perturbation of this rheostat, using a proteasomal inhibitor, results in an increase in Runx1 and Smad6 levels that can be directly attributed to increased Runx1 binding to tissue-specific regulatory elements of these genes. Taken together, we describe a scenario in which a key hematopoietic transcription factor controls its own expression levels by transcriptionally controlling its controller.

The Runt domain proteins are eukaryotic transcription factors that regulate major developmental pathways. All members of this family contain a highly-conserved sequence-specific DNA binding domain: the Runt domain (RD). Structural and biochemical studies have shown that the Runt domain undergoes a conformational transition upon binding to DNA and that this process is regulated by an unrelated partner protein CBFbeta that enhances the DNA binding affinity of RD. Most of the reported studies on the Runt domain transcription factors were performed on proteins from mammals and Drosophila whereas very little has been known about the C. elegans RD protein, RUN, which provides the simplest model system for understanding the function of this class of transcription factors. We performed computational studies on RD domains from various species including C. elegans, Drosophila, and human, using the atom-atom contact surface area scoring method. The scoring analysis indicates that the DNA binding regulation of the C. elegans RD protein (CeRD) occurs via its interaction with a CBFbeta-like partner, as found for the human proteins, whereas a different mode of regulation may occur in the Drosophila system. Sequence, secondary structure and fold analyses of a putative CBFbeta protein identified in the C. elegans genome, CeCBFbeta, sharing a 22% identity with the human protein, predict a similar structure of this protein to that of the human CBFbeta protein. We produced the C. elegans proteins CeRD and CeCBFbeta in bacteria and confirmed their physical interaction as well as cross interactions with the corresponding human proteins. We also confirmed the structural similarity of CBFbeta and CeCBFbeta by circular dichroism analysis. The combined results suggest that a similar mechanism of regulation operates for the human and the C. elegans RD proteins despite the low sequence identity between their CBFbeta proteins and the evolutionary distance between the two systems.

The RUNX transcription factors are important regulators of lineage-specific gene expression. RUNX are bifunctional, acting both as activators and repressors of tissue-specific target genes. Recently, we have demonstrated that Runx3 is a neurogenic transcription factor, which regulates development and survival of proprioceptive neurons in dorsal root ganglia. Here we report that Runx3 and Runx1 are highly expressed in thymic medulla and cortex, respectively, and function in development of CD8 T cells during thymopoiesis. Runx3-deficient (Runx3 KO) mice display abnormalities in CD4 expression during lineage decisions and impairment of CD8 T cell maturation in the thymus. A large proportion of Runx3 KO peripheral CD8 T cells also expressed CD4, and in contrast to wild-type, their proliferation ability was largely reduced. In addition, thein vitrocytotoxic activity of alloimmunized peritoneal exudate lymphocytes was significantly lower in Runx3 KO compared with WT mice. In a compound mutant mouse, null for Runx3 and heterozygous for Runx1 (Runx3-/-;Runx1+/-), all peripheral CD8 T cells also expressed CD4, resulting in a complete lack of single-positive CD8+T cells in the spleen. The results provide information on the role of Runx3 and Runx1 in thymopoiesis and suggest that both act as transcriptional repressors ofCD4expression during T cell lineage decisions.

We describe the expression of three Runt-related RUNX genes (previously termed AML, Cbfa, or Pebp2alpha) Runx1 and Runx3 during the development of teeth and other craniofacial tissues and compare them to Runx2 expression reported earlier. All three genes were expressed in mesenchymal condensates. Runx1 was expressed in several cartilage primordia earlier than Runx3, and Runx2 was intense in all mesenchymal condensations of bones and teeth. Only Runx1 was expressed in epithelia, and in tooth germs transcripts were detected in outer dental epithelium. Runx1 was also intensely expressed in the midline epithelium of palatal shelves. In early tooth morphogenesis Runx3 was coexpressed with Runx2 in a thin layer of mesenchymal cells underlying dental epithelium. Unlike Runx2, Runx3 was expressed in odontoblasts. However, Runx3 mutant mice did not show obvious tooth phenotype or deviations of Runx1 and Runx2 expression patterns in the tooth.

Summary ping of the RD/CBF dimerization region and of theRD/DNA interface [28–30]. The crystallographic studies The Runt domain proteins are transcription regulators coupled with DNA binding and transcriptional assaysof major developmental pathways. Here we present providedinsightintothestructuralbasisofthedetrimen- the crystal structures of the Runt domain (RD) of the tal effects of specific point mutations in the two proteins human protein RUNX1 and its DNA binding site in their that are associated with the development of human dis- freestatesandcomparethemwiththepublishedcrys- eases [29–31]. However, our understanding of the struc- tal structures of RD bound to DNA and to the partner tural basis of specificity of interaction in this system is protein CBF . We demonstrate that (1) RD undergoes not complete without visualizing the structures of the an allosteric transition upon DNA binding, which is various components in their free states and comparing further stabilized by CBF , and that (2) the free DNA

The human RUNX3/AML2 gene belongs to the ‘runt domain’ family of transcription factors that act as gene expression regulators in major developmental pathways. Here, we describe the expression pattern of Runx3 during mouse embryogenesis compared to the expression pattern of Runx1. E10.5 and E14.5–E16.5 embryos were analyzed using both immunohistochemistry and β-galactosidase activity of targeted Runx3 and Runx1 loci. We found that Runx3 expression overlapped with that of Runx1 in the hematopoietic system, whereas in sensory ganglia, epidermal appendages, and developing skeletal elements, their expression was confined to different compartments. These data provide new insights into the function of Runx3 and Runx1 in organogenesis and support the possibility that cross-regulation between them plays a role in embryogenesis.

Overexpression of the liver subunit of 6-phosphofructo-1-kinase in Chinese hamster ovary K1 cells was shown to increase the steady-state level of the enzyme's product, fructose 1,6-bisphosphate, and to produce a small but significant decrease in the concentration of fructose 2,6-bisphosphate, which is an allosteric activator of the enzyme. However, overexpression of the enzyme had no effect on glycolytic flux under a variety of different substrate conditions. This latter observation is consistent with similar studies in fungi and in potato tubers which indicate that 6-phosphofructo-1-kinase has very little control over flux in glycolysis.

The over-expressed Cu/Zn-superoxide dismutase (Cu/Zn-SOD) gene has been found in some circumstances phenotypically deleterious and associated with oxidative injury-mediated aberrations while in other studies it was considered neuroprotective. In this work we examine a number of biochemical markers in fetal and adult brain from transgenic (tg) mice expressing the human Cu/Zn-SOD gene, which may determine this dual characteristic. These markers include the polyunsaturated fatty acid (PUFA) profile in discrete phospholipid species, the K-tocopherol levels, a marker for lipid antioxidant status, and thiobarbituric acid reactive substance (TBARS), a marker for the tissue oxidative status. The PUFA profile in choline- and ethanolamine-phosphoglycerides was similar in tg and nontransgenic (ntg) animals of either fetal or adult brain. Serine-phosphoglycerides, however, showed a marked decrease from 20.07 ˛ 0.53 to 14.92 ˛ 0.87 wt% and 14.52 ˛ 1.15 wt% in docosahexaenoic acid (DHA; 22:6 n3), in the tg 51 and tg 69 fetal brains, respectively, but not in the comparable adult tissues. The K-tocopherol levels were significantly higher in the fetal compared to the adult brain. There were no differences in the anti-oxidant levels between the ntg and tg fetal brains, but there were differences in the adult animals; the tg mice were higher by at least two-fold than the control animals. The basal TBARS in the tg 51 fetal brain was 35% lower than that of ntg mouse and in the presence of Fe 2a , brain slices from the former released less TBARS (57% reduction) into the medium than the latter. These results suggest that higher dosages of Cu/Zn-SOD gene are compatible with increased K-tocopherol levels, reduced basal TBARS levels and a DHA deficiency in the fetal, but not the adult, tg brain. fl 2000 Elsevier Science B.V. All rights reserved.

Copper/zinc superoxide dismutase (CuZnSOD) catalyses the conversion of O 2 • 2 into H2O2. Constitutive overexpression of CuZnSOD in cells and animals creates an indigenous oxidative stress that predisposes them to added insults. In this study, we used transgenic CuZnSOD (Tg-CuZnSOD) mice with elevated levels of CuZnSOD to determine whether overexpression of CuZnSOD affected the susceptibility of these mice to plasmodium infection. Acute malaria is associated with oxidative stress, mediated by redox-active iron released from the infected RBC. Two independently derived Tg-CuZnSOD lines showed higher sensitivity than control mice to infection by Plasmodium berghei (P. berghei), reflected by an earlier onset and increased rate of mortality. Nevertheless, while Tg-CuZnSOD mice were more vulnerable than control mice, the levels of parasitemia were comparable in both strains. Moreover, treatment of infected red blood cells (RBC) with oxidative stress inducers, such as ascorbate or paraquat, reduced the viability of parasites equally in both transgenic and control RBC. This further confirms that increased CuZnSOD does not support plasmodia development. The data are consistent with the possibility that the combination of increased redox-active iron and elevated H 2O2 in the plasmodium-infected Tg-CuZnSOD mice, led to an enhanced Fenton's reaction-mediated HOproduction, and the resulting oxidative injury renders the transgenic mice more vulnerable to parasite infection. © 1998 Elsevier Science Inc. Keywords—Transgenic-CuZnSOD mice, Malaria, Oxidative stress, Plasmodium, Fenton's reaction, Redox-active iron, Free radical

The "runt domain" (RD) is a 128 amino acid region of the Drosophila pair-rule gene runt. This highly conserved region delineates the DNA-binding domain of a new family of transcription factors; the RD proteins. The family includes genes from Drosophila, chicken and mammals that are involved in a wide range of developmental processes, from sex determination and neurogenesis in Drosophila to hematopoiesis and osteoblast differentiation in mouse and human. The RD confers DNA binding ability and mediates the interaction of mammalian RD proteins with the beta-subunit (CBFbeta), which enhances the DNA binding. The primary sequence of RD shows no similarity to other known DNA-binding motifs and its three-dimensional (3D) structure is not known. We employed molecular modeling-based mutagenesis to generate a 3D model of RD. Fold recognition programs identified the palm subdomain of rat DNA polymerase beta as the most likely fold for RD. In the predicted model, the RD region which interacts with DNA contains two arginine residues, R130 and R135, which appear to be in close contact with the major groove of the DNA and to interact with the three essential guanine bases of the core DNA motif PyGPyGGT. We mutated these two R residues and demonstrated that mutations markedly reduced the binding of RD to DNA with no effect on RD interaction with CBFbeta. The data provide important clues about the possible 3D structure of the RD and its interaction with the core DNA motif.

Cases of familial amyotrophic lateral sclerosis (fALS; a neurodegenerative disorder) have been reported in which the gene for Cu/Zn superoxide dismutase (CuZnSOD) was mutated. Several studies with the fALS mutant CuZnSOD in transgenic mice and cells showed that the fALS mutations act through an as yet undefined dominant gain-of-function mechanism. Wild-type CuZnSOD catalyzes the dismutation of superoxide (O 2⨪ ) but also produces hydroxyl radicals ( • OH) with H 2 O 2 as substrate. Two laboratories have recently demonstrated that the • OH production ability was preferentially enhanced by the fALS mutant CuZnSOD, suggesting that this might be the function gained in fALS. In this study, we used transgenic CuZnSOD (Tg-CuZnSOD) mice with elevated levels of CuZnSOD to determine whether overexpression of wild-type CuZnSOD was also associated with increased • OH production and impaired muscle function. Enhanced formation of • OH was detected, by spin trapping, in brain and muscle extracts of the Tg-CuZnSOD mice. Three independently derived Tg-CuZnSOD lines showed muscle abnormalities, reflected by altered electromyography (EMG) and diminished performance in the rope grip test. After treatment with paraquat (PQ), a widely used herbicide and O 2⨪ -generating compound, muscle disability significantly deteriorated in Tg-CuZnSOD mice but not in control mice. The results indicate that elevated levels of CuZnSOD cause indigenous long-term oxidative stress leading to impairment of muscle function. These findings may provide valuable clues about the concurred role of indigenous oxidative stress and exogenous agents in the etiology of sporadic ALS and several other neurodegenerative diseases in which a specific subset of neurons is affected.

RUNX1 transcription factor (TF) is a key regulator of megakaryocytic development and when mutated is associated with familial platelet disorder and predisposition to acute myeloid leukemia (FPD-AML). We used mice lacking Runx1 specifically in megakaryocytes (MK) to characterized Runx1-mediated transcriptional program during advanced stages of MK differentiation. Gene expression and chromatin-immunoprecipitation-sequencing (ChIP-seq) of Runx1 and p300 identified functional Runx1 bound MK enhancers. Runx1/p300 co-bound regions showed significant enrichment in genes important for MK and platelet homeostasis. Runx1 occupied genomic regions were highly enriched in RUNX and ETS motifs and to a lesser extent in GATA motif. Megakaryocytic specificity of Runx1/P300 bound enhancers was validated by transfection mutagenesis and Runx1/P300 co-bound regions of two key megakaryocytic genes Nfe2 and Selp were tested by in vivo transgenesis. The data provides the first example of genome wide Runx1/p300 occupancy in maturating primary FL-MK, unravel the Runx1-regulated program controlling MK maturation in vivo and identify a subset of its bona fide regulated genes. It advances our understanding of the molecular events that upon RUNX1mutations in human lead to the predisposition to familial platelet disorders and FPD-AML.

Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene resides on chromosome 21 and is overexpressed in patients with Down syndrome. Cultured neurons of transgenic Cu/Zn SOD (Tg-Cu/Zn SOD) mice with elevated activity of Cu/Zn SOD were used to determine whether constitutive overexpression of Cu/Zn SOD creates an indigenous oxidative stress that predisposes the Tg-Cu/Zn SOD neurons to added insults. Neurons from three independently derived Tg-Cu/Zn SOD strains showed higher susceptibility than nontransgenic neurons to kainic acid (KA)-mediated excitotoxicity, reflected by an earlier onset and enhanced apoptotic cell death. This higher susceptibility of transgenic neurons to KA-mediated apoptosis was associated with a chronic prooxidant state that was manifested by reduced levels of cellular glutathione and altered [Ca2+]i homeostasis. The data are compatible with the thesis that overexpression of Cu/Zn SOD creates chronic oxidative stress in the transgenic neurons, which exacerbates their susceptibility to additional insults such as KA-mediated excitotoxicity.

Runx transcription factor family proteins have essential roles during T-cell development by either activating or repressing target genes. For instance, lineage- and stage-specific expression of Cd4 and ThPOK is controlled by a transcriptional silencer embedded in each locus, whose activity requires bindings of Runx complexes. The evolutionarily conserved VWRPY penta-peptide sequences in Runx proteins have been shown to be responsible for repressive function as a platform to recruit Groucho/TLE transcriptional corepressors. However, it remains elusive whether requirement for the VWRPY motif differs among Runx target genes. By examining mice lacking VWRPY motifs in both Runx1 and Runx3 proteins, here, we show a full and partial derepression of Cd4 and ThPOK in CD8-linegae T cells, respectively. Thus, whereas Cd4 silencing completely depends on the VWRPY motif, both VWRPY-dependent and -independent mechanisms operate to repress ThPOK gene. These results indicate that Runx proteins utilize different modes to repress expression of different target genes.

Background Alternative promoters usage is an important paradigm in transcriptional control of mammalian gene expression. However, despite the growing interest in alternative promoters and their role in genome diversification, very little is known about how and on what occasions those promoters are differentially regulated. Runx1 transcription factor is a key regulator of early hematopoiesis and a frequent target of chromosomal translocations in acute leukemias. Mice deficient in Runx1 lack definitive hematopoiesis and die in mid-gestation. Expression of Runx1 is regulated by two functionally distinct promoters designated P1 and P2. Differential usage of these two promoters creates diversity in distribution and protein-coding potential of the mRNA transcripts. While the alternative usage of P1 and P2 likely plays an important role in Runx1 biology, very little is known about the function of the P1/P2 switch in mediating tissue and stage specific expression of Runx1 during development. Results We employed mice bearing a hypomorphic Runx1 allele, with a largely diminished P2 activity, to investigate the biological role of alternative P1/P2 usage. Mice homozygous for the hypomorphic allele developed to term, but died within a few days after birth. During embryogenesis the P1/P2 activity is spatially and temporally modulated. P2 activity is required in early hematopoiesis and when attenuated, development of liver hematopoietic progenitor cells (HPC) was impaired. Early thymus development and thymopoiesis were also abrogated as reflected by thymic hypocellularity and loss of corticomedullary demarcation. Differentiation of CD4/CD8 thymocytes was impaired and their apoptosis was enhanced due to altered expression of T-cell receptors. Conclusion The data delineate the activity of P1 and P2 in embryogenesis and describe previously unknown functions of Runx1. The findings show unequivocally that the role of P1/P2 during development is non redundant and underscore the significance of alternative promoter usage in Runx1 biology.

Down's syndrome (DS), the phenotypic manifestation of trisomy 21, involves overexpression of chromosome 21-encoded genes. The gene for amyloid precursor protein (APP), known to be involved in AD pathology, resides on chromosome 21 along with the gene for Cu/Zn superoxide dismutase (SOD1), a key enzyme in the metabolism of oxygen free radicals. We investigated the consequences of a combined increase in APP and SOD1, in a double-transgenic (tg)-APP-SOD1 mouse. These mice expressed severe impairment in learning, working and long-term memory. Expression of long-term potentiation in hippocampal slices was impaired in both tg-SOD and tg-APP-SOD mice, but not in tg-APP mice, indicating that increased APP by itself did not affect in vitro synaptic plasticity. In tg-APP-SOD mice, membrane-bound high molecular weight APP species accumulated while APP cleavage products did not increase and levels of secreted APP were unchanged. Severe morphological damage, including lipofuscin accumulation and mitochondria abnormalities, were found in aged tg-APP-SOD but not in the other mice. Thus, a combined elevation of the two chromosome 21 genes in tg-APP-SOD mice induced age-dependent alterations in morphological and behavioural functions.

10 Figures. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734., The CD11a/CD18 (leukocyte function-associated antigen 1 [LFA-1]) integrin mediates critical leukocyte adhesive interactions during immune and inflammatory responses. The CD11a promoter directs CD11a/CD18 integrin expression, and its activity in lymphoid cells depends on a functional RUNX1/AML-1–binding site (AML-110) within the MS7 sequence. We now report that MS7 contains a C/EBP-binding site (C/EBP-100), which overlaps with AML-110 and is bound by C/EBP factors in myeloid cells. C/EBP and RUNX/AML factors compete for binding to their respective cognate elements and bind to the CD11a promoter MS7 sequence in a cell lineage- and differentiation-dependent manner. In myeloid cells MS7 is primarily recognized by C/EBP factors in proliferating cells whereas RUNX/AML factors (especially RUNX3/AML-2) bind to MS7 in differentiated cells. RUNX3/AML-2 binding to the CD11a promoter correlates with increased RUNX3/AML-2 protein levels and enhanced CD11a/CD18 cell surface expression. The relevance of the AML-110 element is underscored by the ability of AML-1/ETO to inhibit CD11a promoter activity, thus explaining the low CD11a/CD18 expression in t(8;21)–containing myeloid leukemia cells. Therefore, the expression of the CD11a/CD18 integrin in myeloid cells is determined through the differential occupancy of the CD11a proximal promoter by transcription factors implicated in the pathogenesis of myeloid leukemia., From the Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; Clínica Universitaria, Universidad de Navarra, Spain; Institute of Human Genetics, Aarhus, Denmark; Hospital Universitario Gregorio Maranón, Madrid, Spain; University of Colorado Health Sciences Center, Denver; and Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot Israel. Supported by grants 08.3/0026/2000.1 from Comunidad Auto´noma de Madrid, 01/0063-01 from Fondo de Investigaciones Sanitarias, and SAF2002-04615- C02-01 from Ministerio de Ciencia y Tecnología (A.L.C.). We gratefully acknowledge Drs Ana Aranda and Aurora Sánchez-Pacheco for their very generous help with ChIP assays.

AIM To examine the effect of overexpression of human intracellular copper-zinc superoxide dismutase (CuZnSOD1) gene on fetal mice brain exposed to in-utero ischemic reperfusion injury. DESIGN Transient in-utero ischemia (7 min) was induced in pregnant transgenic mice overexpressing human CuZnSOD1 and wild-type mice by occluding the blood supply to the uterine artery on day 17 of pregnancy, followed by 24 hours of reperfusion. The level of lipid peroxidation in fetal mice brains was compared between the transgenic and non-transgenic (control) fetal mice. Motor and coordination skills of transgenic and control adult mice (six to eight months old) which were exposed to ischemic reperfusion injury in-utero were compared by the rope grip test and visible platform task. RESULTS We first measured CuZnSOD1 activity in the brains of the transgenic fetal mice and confirmed that the enzyme activity is 4.2-fold higher than control. We also established that ischemia reperfusion on day 17 of pregnancy led to increased level of TBARS (Thiobarbituric acid reactive substance) in brains of wild-type fetal mice when compared to sham operated mice (72.5 +/- 3.4 vs. 49.4 +/- 1.5 nmol/mg. p < 0.001). The increase was markedly accentuated in the CuZnSOD1 transgenic mice, and significantly higher compared to control mice exposed to ischemia-reperfusion (85.6 +/- 4.0 vs. 69.5 +/- 2.3 nmol/mg, p < 0.001). Moreover, we found that the transgenic mice that were subjected to in-utero ischemia reperfusion exhibited a significantly higher rate of failures in the rope grip test and poorer performance in the visible platform task, when compared to non-transgenic mice exposed to identical insult. CONCLUSIONS Oxygen free radicals play an important role in the pathogenesis of perinatal hypoxia. Overexpression of the enzyme CuZnSOD1 in transgenic mice exposed their brains to increased damage during ischemic-reperfusion insult.

AML2 is a member of the acute myelogenous leukemia, AML family of transcription factors. The biologic functions of AML1 and AML3 have been well characterized; however, the functional role of AML2 remains unknown. In this study, we found that AML2 protein expressed predominantly in cells of hematopoietic origin is a nuclear serine phosphoprotein associated with the nuclear matrix, and its expression is not cell cycle-related. In HL-60 cells AML2 expression can be induced by all three natural retinoids, all-trans-retinoic acid (RA), 13-cis-RA, and 9-cis-RA in a dose-dependent manner. A synthetic retinoic acid derivative, 4HPR, which neither activates RA receptor (RAR) alpha nor retinoic X receptor alpha was unable to induce the expression of AML2. A RAR-selective activator, TTNPB, induced AML2 expression similar to RA. Our study further showed that AGN193109, a potent RARalpha antagonist, suppressed AML2 expression induced by RA and that a retinoic X receptor pan agonist AGN194204 had no effect on its expression. Taken together, these studies conclusively demonstrated that the expression of AML2 in HL-60 cells is regulated through the RARalpha-specific signaling pathway. Our study further showed that after all-trans-retinoic acid priming, AML2 expression could be augmented by vitamin D(3). Based on these studies we hypothesize that AML2 expression is normally regulated by retinoid/vitamin D nuclear receptors mainly through the RARalpha-dependent signaling pathway and that it may play a role in hematopoietic cell differentiation.

Patients with Down's syndrome (DS) exhibit elevated activity of copper zinc superoxide dismutase (CuZnSOD) caused by the trisomy 21 state. To investigate the possible involvement of CuZnSOD gene dosage in perturbation of prostaglandin biosynthesis we analyzed transfected cells and transgenic mice that express elevated levels of human CuZnSOD. It was found that the synthesis of prostaglandin E2 (PGE2) was diminished in transfected PC12-CuZnSOD cells as well as in fibroblasts from DS patients. Primary cells derived from transgenic CuZnSOD mice showed similar reduction. Impaired biosynthesis of prostaglandins was not confined to cells grown in culture since secretion of PGE2 and PGD2 by kidney and cerebellum of transgenic CuZnSOD was significantly lower than in non-transgenic littermate mice. These findings strongly suggest that overexpression of the CuZnSOD gene induces a demotion in PGE2 and PGD2 formation and establish a connection between alteration of prostaglandin biosynthesis in trisomy 21 cells and gene dosage of CuZnSOD.

The region of the simian virus 40 genome complementary to the 5' end of the most abundant poly(A)-containing 19S and 16S mRNAs was mapped by hybridization of double-labeled RNA ([3H]methyl group and [14C]uridine) to specific DNA fragments. Chemical identification of methylated residues indicated that a common "leader" sequence adjacent to the 5' terminus of both 19S and 16S mRNA is transcribed from DNA sequences located between 0.67 and 0.76 map units. The estimated size of this "leader" RNA, which does not code for any known viral protein, is 170-200 nucleotides. Our results indicate that sequences complementary to the "leader" region and coding portion of 16S mRNA are located in separate parts of the simian virus 40 genome.

Rat PC12 cells expressing elevated levels of transfected human Cu/Zn-superoxide dismutase (CuZn-SOD) gene were generated. These transformants (designated PC12-hSOD) closely resembled the parental cells in their morphology, growth rate, and response to nerve growth factor, but showed impaired neurotransmitter uptake. The lesion was localized to the chromaffin granule transport mechanism. We found that the pH gradient (ΔpH) across the membrane, which is the main driving force for amine transport, was diminished in PC12-hSOD granules. These results show that elevation of CuZnSOD activity interferes with the transport of biogenic amines into chromaffin granules. Since neurotransmitter uptake plays an important role in many processes of the central nervous system, CuZnSOD gene-dosage may contribute to the neurobiological abnormalities of Down's syndrome.

The synthesis of carrier-free β- 32 P-ribonucleosides triphosphate in almost quantitative yields is described. The procedures are based on a polynucleotide phosphorylase-catalyzed phosphorolysis of either poly- or oligoribonucleotides with carrier-free 32 P-inorganic phosphate, coupled with a pyruvate kinase-catalyzed phosphorylation of the resulting β- 32 P-nucleoside diphosphate.

RNA tumor viruses replicate via a pathway involving both DNA synthesis and DNA-dependent RNA synthesis (6,7,14). The first step in the pathway probably involves the action of viral RNA-dependent DNA polymerase to yield covalent RNA:DNA hybrid structures. By a mechanism, presently unknown, this intermediate is converted to DNA, which is found integrated in the host chromosome. It may be that, once integrated, viral and host genes are treated identically, and subsequent steps depend upon the nuclear transcription machinery.

Genomic clones of human liver phosphofructokinase (PFK) were isolated by screening a gene bank enriched for chromosome 21 sequences with two synthetic oligonucleotide probes designed from peptide sequences of purified human liver PFK. A 3.3 Kb fragment derived from the genomic clones was sub-cloned and designated to pG-PFKL 3.3. It hybridized with a 3.5 Kb mRNA on Northern blots and was able to enrich selectively for liver PFK mRNA by hybrid-selection. These results demonstrated that the isolated clones contain sequences homologous to human PFKL mRNA. When hybridized to genomic DNA blots pG-PFKL 3.3 reacted with the same 3.3 Kb BamHI fragment in both human DNA and DNA of the mouse/human hybrid line WA17 which contains human chromosome 21 as the only human chromosome. These data confirm the assignment of the PFKL gene to chromosome 21.

High specific activity [beta-32P]ATP and [beta-32P]CTP were used to study in vitro transcriptional initiation and subsequent capping of simian virus 40 (SV40) early and later RNAs. More than 40% of the capped SV40 RNA synthesized in vitro was also polyadenylylated. With [beta-32P]ATP, only adenosine-containing caps were labeled and the incorporated radioactive phosphate was found exclusively in the beta position. Cap digestion patterns showed extensive qualitative and quantitative similarities between these 32P-labeled caps and caps labeled in vivo [Canaani, D., Kahana, C., Mukamel, A. & Groner, Y. (1979) Proc. Natl. Acad. Sci. USA 76, 3078--3082]. With [beta-32P]CTP, only early SV40 RNA was labeled, consistent with the absence of cytosine-containing caps in late transcripts. The [beta-32P]CTP-labeled cap was identified as m7GpppCmpU, which was previously identified as the major cap of in vivo labeled early SV40 mRNA [Kahana, C., Gidoni, D., Canaani, D. & Groner, Y. (1981) J. Virol. 37, 7--16]. This experiment provides biochemical evidence for eukaryotic RNA polymerase II initiation of transcription with CTP. The data imply that, on SV40 DNA, RNA polymerase II initiates transcription at multiple nucleotide sequences and capping occurs at the initiator nucleotide.

In vivo [methyl 3H]-labeled SV40 19S and 16S mRNA species were purified and their internal methylation as well as their capping structures analyzed. SV40 viral mRNA sedimenting in the 19S region contains approximately equal proportions of m7GpppAm and m7Gppm6Am, while the 16S mRNA contains mainly m7Gpppm6Am. N6 methyl adenosine is located internally within the RNA chains of both the 19S and 16S species.

Cycloleucine, a competitive inhibitor of methionine transferase was used to generate in vivo partially methylated mRNA in SV40-infected BSC-1 cells. Cycloleucine at 0.5 mg/ml causes more than a 30% decrease in internal m6As of late SV40 mRNA with only minor effect on the dimethyladenosine of the 5' caps m7GpppmAm. After treatment with 2 and 5 mg/ml of cycloleucine, internal m6As were reduced by 10- and 100-fold, respectively. The inhibition of BSC-1 mRNA methylations paralleled that observed for late SV40 mRNAs. In cells exposed to 2 mg/ml cycloleucine production of late SV40 mRNA was inhibited by 80% whereas the amount of SV40 nuclear RNA was only slightly reduced. Size fractionation of SV40 nuclear RNA from cycloleucine-treated cells revealed a loss of SV40 19 S RNA with a corresponding increase of fragmented RNA sedimenting between 11 to 5 S, so that the total amount of SV40 RNA in the nucleus was almost unchanged. Analysis of viral transcription complexes from cells treated with cycloleucine indicated that SV40 transcription was not affected by cycloleucine. SV40-transformed cells, in contrast to BSC-1 cells, were able to process and transport undermethylated RNA. When transformed cells were treated with 2 mg/ml cycloleucine no changes in quantities or size of cytoplasmic and nuclear RNA were detected. The data argues for a role of internal m6A moieties in modulating the processing-linked transport of mRNA from the nucleus to the cytoplasm of nontransformed cells. Transformed cells may escape these controls due to structural alterations in their perinuclear regions.

A RNase from calf thymus, which specifically cleaves native or synthetic double-stranded RNA molecules endonucleolytically, has been isolated and purified from calf thymus. For optimal activity, the enzyme requires a sulfhydryl reagent and divalent cations; over 95 per cent of the activity is inhibited by 0.5 mm ethidium bromide. The degradation of [3H]poly(C)-poly(I) by purified enzyme preparations yields labeled dinucleotides and octanucleotides; the latter oligonucleotide contained 5'-phosphate and 3'-hydroxyl termini. The enzyme cleaves high molecular weight RNAs such as RNA products formed in vitro by T3 phage-induced RNA polymerase from T3 phage DNA, heterogeneous RNA isolated from duck reticulocyte nuclei, and 45 S RNA isolated from rat liver nucleoli. The mode of degradation of RNA in vitro with the double-stranded RNase is similar to that of Escherichia coli RNase III and appears to act endonucleolytically. The degradation of 45 S RNA with the enzyme results in the production of 29 S and 19 S RNA fragments. These findings suggest that the enzyme may be involved in the processing of high molecular weight precursor RNAs to mRNA or rRNAs in a manner analogous to that reported for RNase III of E. coli.

The 5'-cap-containing leader sequence of the most abundant 19S and 16S mRNAs of simian virus 40 (SV40) was previously mapped between 0.67 and 0.76 map units. We now find that the two late mRNA species contain multiple 5' ends. Eight different RNase T2-resistant cap structures were identified:m7GpppmAmpU (47%); m7GpppmAmpUmpU (19%); m7GpppmAmpC (16%); m7GpppmAmpCmpA (5%); m7GpppmAmpG (6%); m7GpppGmpC (3%); m7GpppmAmGmpA (2%); m7GpppGmpCmpG (2%). Capped T1 oligonucleotides of 19S and 16S mRNAs have been isolated by two different procedures: (i) chromatography on a DEAE-cellulose column followed by paper electrophoresis and (ii) two-dimensional electrophoresis/homochromatography. Cap structures of the isolated 5' oligonucleotides were identified. Each of the major caps was found to be associated with a few differential 5' oligonucleotides, implying a vast heterogeneity at the termini of SV40 late mRNAs. The results suggest that on SV40 DNA, RNA polymerase II has a reportoire of initiation points. In most of the cases, initiation takes place with adenosine triphosphate followed by a pyrimidine. Alternatively, transcription may start at one specific point but a unique mechanism of processing generates heterogeneous populations of termini with a common 5' adenosine triphosphate.

The gene locus for human cytoplasmic superoxide dismutase (SOD-1; superoxide:superoxide oxidoreductase, EC 1.15.1.1) is located in or near a region of chromosome 21 known to be involved in Down syndrome. To approach the molecular biology of this genetic disease we have constructed a SOD-1 cDNA clone. Poly(A)-containing RNA enriched for human SOD-1 mRNA was isolated, used to synthesize double-stranded cDNA, and inserted into the endonuclease Pst I site of the plasmid pBR322. The chimeric molecules were used to transform Escherichia coli. Two clones containing SOD-1 cDNA inserts were identified by their ability to hybridize specifically with mRNA coding for SOD-1. Each of these clones carries a 650-base-pair insert, as was determined by restriction enzyme digestion and electron microscopic heteroduplex analysis. Hybridization of labeled cloned cDNA to RNA blots revealed two distinct SOD-1 mRNA classes of 500 and 700 nucleotides. The data suggest that both are polyadenylylated and are coded by chromosome 21.

This paper reports on the concentration of or-ganochlorine insecticides in the tissues of 35 pregnant women, 33 nonpregnant women in the fertile period of life, and 23 neonates. In pregnant women, fat tissue, placenta, uterus, and blood were examined; in nonpregnant women, fat tissue only was analyzed; and in neonates, the umbilical blood. All the samples were analyzed by gas liquid chromatography. The findings of this study indicate that in pregnancy a lower storage of the following organo-chlorine insecticides occurs in the adipose tissue: p,p′-DDT, p,p′-DDD, o,p′-DDT + o,p′-DDD, o,p′-DDE, and total o,p′-DDT. The same phenomenon is observed for BHC isomers and dieldrin. All the organochlorine insecticides found in the fat tissue of pregnant women were present both in maternal and fetal blood in all the cases studied. These findings suggest that in pregnancy the metabolism of organochlorine insecticides is enhanced, and that organochlorine insecticides pass the placental barrier.

The 'housekeeping' enzyme Cu/Zn-superoxide dismutase (SOD-1) is encoded by a gene residing on human chromosome 21, at the region 21q22 known to be involved in Down's syndrome. The SOD-1 gene and the SOD-1 cDNA were introduced into mouse L-cells and human HeLa cells, respectively as part of recombinant plasmids containing the neoR selectable marker. Human and mouse transformants were obtained that expressed elevated levels (up to 6-fold) of authentic, enzymatically active human SOD-1. This enabled us to examine the consequences of hSOD-1 gene dosage, apart from gene dosage effects contributed by other genes residing on chromosome 21. Human and mouse cell clones that overproduce the hSOD-1 had altered properties; they were more resistant to paraquat than the parental cells and showed an increase in lipid peroxidation. The data are consistent with the possibility that gene dosage of hSOD-1 contributes to some of the clinical symptoms associated with Down's syndrome.

Reduced levels of the neurotransmitter serotonin in blood platelets is a clinical symptom characteristic of individuals with Down's syndrome. To investigate the possible involvement of the Cu/Zn-superoxide dismutase (CuZnSOD) gene, which resides at the Down locus on chromosome no. 21, in the etiology of that symptom, we examined blood platelets of transgenic mice harboring the human CuZnSOD gene. It was found that platelets of transgenic CuZnSOD animals, which overexpress the transgene, contain lower levels of serotonin than nontransgenic littermate mice, due to a reduced rate of uptake of the neurotransmitter by the dense granules of the platelets. We found that the pH gradient (delta pH) across the dense granule membrane, which is the main driving force for serotonin transport, was diminished in dense granules of transgenic-CuZnSOD. Furthermore, a significantly lower than normal serotonin accumulation rate was also detected in dense granules isolated from blood platelets of Down's syndrome individuals. These findings suggest that CuZnSOD gene dosage is affecting the dense granule transport system and is thereby involved in the depressed level of blood serotonin found in patients born with Down's syndrome.