CGGBP1-dependent CTCF-binding sites restrict ectopic transcription from SV40 early promoter

ABSTRACTBinding sites of the chromatin regulator protein CTCF function as important landmarks in the human genome. The recently characterized CTCF-binding sites at LINE-1 repeats depend on another repeat-regulatory protein CGGBP1. These CGGBP1-dependent CTCF-binding sites serve as potential barrier elements for epigenetic marks such as H3K9me3. Such CTCF-binding sites are associated with asymmetric H3K9me3 levels as well as RNA levels in their flanks. The functions of these CGGBP1-dependent CTCF-binding sites remain unknown. By performing targeted studies on candidate CGGBP1-dependent CTCF-binding sites cloned in an SV40 promoter-enhancer episomal system we show that these regions act as inhibitors of ectopic transcription from SV40 promoter. CGGBP1-dependent CTCF-binding sites that recapitulate their genomic function of loss of CTCF binding upon CGGBP1 depletion and H3K9me3 asymmetry in immediate flanks are also the ones which show the strongest inhibition of ectopic transcription. By performing a series of strand-specific reverse transcription PCRs we demonstrate that this ectopic transcription results in synthesis of RNA from the SV40 promoter in a direction opposite to the downstream reporter gene in a strand specific manner. The unleashing of the bidirectionality of the SV40 promoter activity and a breach of the transcription termination sequence required for the upstream transcription seems to depend on depletion of CGGBP1 and loss of CTCF binding proximal to the SV40 promoter. These findings suggest a role of CGGBP1-dependent binding sites in restricting ectopic transcription.

Long-Term Reversal of Renal Anemia in Rat by Erythropoietin Gene Modified Mesenchymal Stem Cell Transplantation Beneath the Renal Capsule, Which Is Effectively Regulated by Hypoxia.

INTRODUCTION: Patients with chronic renal failure usually require exogenous erythropoietin (EPO) to alleviate anaemia resulting from inadequate epo production by the kidneys. Although this treatment is effective, recombinant EPO could cause hypertension, its antibodies, and other side-effects. EPO gene therapy is more attractive since it could be more economical and more convenient for the long-term management of the disease. As targets for gene therapy, mesenchymal stem cells (MSCs) are more easily expanded, lower immunogenic, and more stable for expressing exogenous genes than other cells. Here we describe an approach of transplantation of the erythropoietin gene modified mesenchymal stem cells beneath the renal capsule to treate Wistar rats with relative erythropoietin deficiency. METHODS: we constructed an EPO-expressed vector pGenesil-HRE-EPO directed by hypoxia response element (HRE)/SV40 promoter, and transduced it into MSCs prepared by adherently culturing rat bone marrow. The EPO-gene modified MSCs (Epo+) in vitro growed normally and stably secreted high level of functional EPO in hypoxia condition, which produced from 3.23 × 10(5) to 9.98 × 10(5) mIU of EPO per 10(6) cells per 24 hr. To determine the role of these MSCs to treate renal anemia in vivo, EPO+, EPO (vector pGenesil-1 or normal) MSCs were injected into rats with renal anemia beneath the renal capsule, the skin, or in muscles (4 × 10(7) cells/rat). Renal anemia rats had been induced by feeding with adenine, whose hematocrit levels were at 33.5 +/− 4.1%, the plasma Epo concentration was 6.5 ± 1.7 mU/ml. EPO concentration was determined by ELISA. During whole experiment, we measured rat body weight and blood pressure, and obtained blood samples regularly. RESULTS: 2wk after transplantation, the hematocrit levels in anemia rats injected Epo+ MSCs beneath the renal capsule, the skin, or in muscles markedly increased to 67.2 ± 5.9%, 61.5 ± 5.7%, and 53.8 ± 5.2%, and 10wk after transplantation to 68.0 ± 5.6%, 52.7 ± 5.8%, and 40.6 ± 5.5%, respectively. Moreover, the plasma Epo concentration of anemic rats injected Epo+MSCs beneath the renal capsule, the skin, or in muscles was considerably increased to 158 ± 35 mU/ml, 136 ± 36 mU/ml, and 114 ± 42 mU/ml at 2 wk, whereas that of anemic rats injected EPO MSCs was not increased significantly. All rats injected Epo+MSCs did not have polycythemia or severe hypertension. Survival time of anemic rats injected Epo+MSCs beneath the renal capsule is longest among anemic rats. Histological examination by fluorescent microscopy showed that implanted MSCs beneath the renal capsule survived best. CONCLUSIONS: EPO gene-modified mesenchymal stem cells can be transplanted in renal anemia rats, and can produce sufficiently active EPO to correct anemia. Transplantation of the cells beneath the renal capsule is best.

Determining the Transrepression Activity of Xenoestrogen on Nuclear Factor-κB in Cos-1 Cells by Estrogen Receptor-α

Functional assays have been used to define the estrogenicity of xenoestrogens in cotransfection studies employing estrogen receptors in various cell lines. It is known that estrogen is able to affect transcription from other nuclear transcription factors, especially the nuclear factor- κB (NF- κB). The ability of selected xenoestrogens (methoxychlor [MXC], dieldrin, and o′, p′-DDT) to transrepress the NF- κB–mediated transcription in Cos-1 cells was evaluated by cotransfection of human estrogen receptor- α (hER α). These xenoestrogens have been described as comparably potent xenoestrogens, whereas their relative binding activity (RBA) has been relegated to a lower order as compare to estrogen. The two NF- κB response element–containing SV40 promoter and −242/+54 cytomegalovirus (CMV)–expressing firefly luciferase (2 × NRE-PV-Luc and 2 × NRE-CMV-Luc, respectively) were transfected into Cos-1 cells with pRL-tk, expressing the renilla luciferase as internal control. The estrogen receptor was expressed from cytomegalovirus major immediate early promoter (CMV-MIEP) (CMV5-hER α). Treatment with 1 nM estrogen (E2) (26.2%), 5 nM E2 (41.4%; p < .05), and xenoestrogens (methoxychlor [1 nM: 29.6%, p < .05; 10 nM: 22.6%), dieldrin [1 nM: 10.3%; 10 nM: 36.06%, p < .05], and o′, p′-DDT [1 nM: 17.0%; 10 nM: 7.15%]) repressed transcription from 2 × NREX-PV-Luc. The antiestrogen, ICI 182,780, failed to antagonize the effects of xenoestrogens. The effects of xenoestrogens in transrepression of NF- κB by ER α were similar when 2 × NRE-CMV-Luc was employed as reporter. Statistically significant ( p < .01) repression by 1 nM E2 (69.2%), 5 nM E2 (69.1%), 1 nM o′, p′-DDT (51.4%), 1 nM dieldrin (47.3%), and 1 nM MXC (73.3%) were observed. The effect of these xenoestrogens without ER α cotransfection on 2 × NRE-PV-Luc- and 2 × NRE-CMV-Luc-mediated NF- κB transcription was not affected by the treatment alone. It is concluded that xenoestrogens, like estrogens, are capable of producing transrepression of NF- κB by hER α.

Sequences in Intron 51 of the VWF Gene Target Promoter Activation to a Subset of Lung Endothelial Cells in Transgenic Mice.

The VWF gene, located on chromosome 12, is 178 kb long and contains 52 exons. VWF is synthesized exclusively by endothelial cells and megakaryocytes. We have previously characterized a region of the VWF gene spanning sequences −487 to +247 that functions as an endothelial specific promoter in vitro. Subsequently a number of transacting factors including, NF1, Oct 1, Ets, GATA6, NFY and Ebp4 that positively and negatively regulate the activity of this promoter were identified by us and others. However, in vivo analysis of the promoter demonstrated that this promoter fragment (sequences −487 to +247) targets activation of fused heterologous transgenes (LacZ and amyloid precursor proteins) specifically and exclusively to brain vascular endothelial cells of transgenic mice. A longer VWF promoter fragment, including 2182 bp of the 5′ flanking sequences, the first exon and the first intron was reported by Aird et al to activate LacZ transgene expression in endothelial cells of the heart and muscle as well as brain of transgenic mice. Considering that endogenous VWF expression is observed in almost all endothelial cells, these results suggested that additional VWF gene sequences were required for transcriptional activation of the VWF promoter in vascular endothelial cells of multiple other organs in vivo; and that distinct regions of the VWF gene are required to achieve promoter activity in endothelial cells of distinct organs. To identify additional cis acting elements within the VWF gene that may participate in its transcriptional regulation generally and/or in distinct organs, we explored the possibility that such sequences may be located in VWF chromatin regions that show hypersensitivity to DNase I. We have now identified a region within intron 51 of the VWF gene that is DNase I hypersensitive (HSS) specifically in non-endothelial cells. This region was shown to interact with YY1 transcription factor in a manner that forms endothelial and non-endothelial specific complexes. In vitro transfection analyses demonstrated that HSS sequences containing this YY1 binding site significantly increased a heterologous SV40 promoter activity specifically in endothelial cell and that this increase was dependent on the presence of an intact YY1 binding site. In contrast, the HSS sequences significantly decreased the SV40 promoter activity in non-endothelial cells. These results suggested that the HSS sequences may participate in activation of gene expression in an YY1 dependent manner in endothelial cells, while repress gene expression in non-endothelial cells. Nevertheless the HSS sequences did not significantly affect the homologous VWF promoter activity that was analyzed by in vitro transfection analyses. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (−487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene participate in organ specific regulation of VWF gene expression, an observation that could not be determined by in vitro analysis. These analyses suggest that the HSS sequences contain cis-acting elements that are specifically necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo.

244 GATA-1 PLAYS A CRITICAL ROLE IN ENDOTHELIAL CELL-SPECIFIC EXPRESSION OF THE 1.1-kb CD55 PROMOTER REGION

The complement system is composed of a complex group of soluble proteins that have important roles in the immune response against foreign cells such as xenografted tissue. Some of the cell surface regulators, also known as membrane complement regulatory proteins (CRPs), are the membrane cofactor protein (CD46), the decay-accelerating factor (CD55), and protectin (CD59). The CD55 is a 70 kDa glycolipid-anchored membrane-bound protein that has regulatory activity by preventing C3 convertase formation and is reported to be expressed in blood and vascular endothelial cells. Its high expression in leukocytes and endothelium is thought to safeguard against locally augmented C3 activation that can occur with inflammation. In this study, we originally cloned the 1.1-kb promoter region of the CD55 gene and constructed a luciferase reporter plasmid, pGL3-1.1CD55. The pGL3-1.1CD55 plasmid and its control plasmid, pGL3-control (SV40 promoter), were transfected into endothelial (MS-1 and BAEC) and epithelial (HaCaT and HEK293) cells, and the soluble fraction of the cell lysates was assayed for luciferase activity. Its promoter activity was compared with that of the promoters of endothelial-specific genes such as MCP, Flk-1, ICAM-2, and throbomodulin. Luciferase activity in each sample was normalized to the �-galactosidase activity of the same sample and the data were analyzed by Sigma Plot program (P < 0.01 versus control). Our results showed that the 1.1 kb CD55 promoter was the strongest among the endothelial cell-specific promoters. To define the important region for the strong expression, the deletion constructs containing 0.96, 0.86, and 0.74, CD55 promoter regions were prepared. Relative to the activity of the control SV40 promoter, decreased luciferase activity was obtained with these deletion constructs, suggesting that the about 0.2 kb 52-flanking region (between -1125 and -967) of the 1.1 kb CD55 promoter region was important for the strong gene expression. Interestingly, on the 52-end of the 0.2 kb region, we could detect two GATA-1 binding sites (from -1122 to -1118 and from -1111 to -1107); the deletion of the sequences between -1125 and -1100 (ΔGATA-1) significantly reduced the promoter activity. Furthermore, ectopic expression of GATA-1 dose-dependently induced the 1.1 kb CD55 promoter activity, but not the ΔGATA-1 activity, strongly implying that the GATA-1 binding site is important for the strong expression. Finally, we confirmed the binding of GATA-1 on the CD55 promoter region with the electrophoretic mobility shift assay (EMSA) using the 28 base pair oligonucleotide probes corresponding to the GATA-1 binding site and chromatin immunoprecipitation (Ch-IP) using anti-GATA-1 antibody. Taken together, these results strongly suggested that GATA-1 plays a critical role in endothelial cell-specific expression of the 1.1 kb CD55 promoter region. This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea, and by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-070-C00095).