Identification of a negative regulatory element that inhibits c-mos transcription in somatic cells.

We have used transient expression assays to identify a cis-acting region in the 5' flanking sequence of murine c-mos which, when deleted, allows expression from the c-mos promoter in NIH 3T3 cells. This negative regulatory sequence, located 400 to 500 nucleotides upstream of the c-mos ATG, also inhibited expression from a heterologous promoter. In addition to NIH 3T3 cells, the c-mos negative regulatory sequence was active in BALB/3T3 cells, PC12 rat pheochromocytoma cells, and A549 human lung carcinoma cells. Site-specific mutagenesis identified three possibly interacting regions that were involved in negative regulatory activity, located around -460, -425, and -405 with respect to the ATG. RNase protection analysis indicated that once the negative regulatory sequences were deleted, transcription in NIH 3T3 cells initiated from the same transcription initiation sites normally utilized in spermatocytes, approximately 280 nucleotides upstream of the ATG. Deletions beyond the spermatocyte promoter, however, allowed transcription initiation from progressively downstream c-mos sequences. Deletion or mutation of sequences surrounding the oocyte promoter at -53 also had little effect on expression of c-mos constructs in NIH 3T3 cells. Therefore, the major determinant of c-mos expression in NIH 3T3 cells was removal of the negative regulatory sequence rather than the utilization of a unique promoter. The c-mos negative regulatory sequences thus appear to play a significant role in tissue-specific c-mos expression by inhibiting transcription in somatic cells.

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273 SELECTION OF PLURIPOTENT STEM CELLS INDUCED BY XENOPUS EGG EXTRACTS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab273 ◽

2009 ◽

Vol 21 (1) ◽

pp. 234 ◽

Cited By ~ 1

Author(s):

C.-Y. Chiang ◽

P.-C. Tang

Keyword(s):

Somatic Cells ◽

Egfp Expression ◽

3T3 Cells ◽

Xenopus Egg Extract ◽

Egg Extract ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Nih 3T3 ◽

Nih 3T3 Cells

It has been reported that Xenopus egg extracts contain molecules that are capable of reprogramming mammalian somatic cells. The reprogrammed somatic cells, which are called extract treated cells (ETC), possess the potential for clinical therapy as embryonic stem (ES) cells do. Therefore, in addition to establishment of an efficient method to reprogram mouse NIH/3T3 cells by Xenopus egg extracts, the aim of this study was to select the ETC cells by the expression of Oct4. In Experiment 1, two methods, electroporation or permeabilization, were conducted to treat mouse NIH/3T3 cells with Xenopus egg extracts. 2 × 105 cells in 200 μL reprogramming mixture containing Xenopus egg extracts were stimulated by a direct current (DC) pulse (80 V mm–1 for 3 msec) three times followed by a pause of incubation at 37°C for 5 min and a single DC pulse (170 V mm–1, for 0.4 msec) subsequently. The electroporated cells were then incubated at 22°C for 1 h. In the other treatment group, NIH/3T3 cells (5 × 105) were permeabilized by streptolysin O (SLO, 500 ng mL–1 in PBS) for 50 min at 37°C before mixed with Xenopus egg extracts at 22°C for 2 h. Cells were cultured in DMEM supplemented with 10% FBS for the first 4 days and then changed to ES medium (DMEM supplemented with 15% FBS, 0.1 mm β-mercaptoethanol, 1000 unit mL–1 mLIF, 0.5% nonessential amino acids, 2 mm L-glutamine) for the last 6 days after Xenopus egg extract treatment. Cell colonies were found in both treatment groups at the end of culture. Examination by immunocytochemical staining, results showed that the extract-treated cell colonies expressed pluripotent marker proteins, such as alkaline phosphatase, Oct4, Nanog and Sox2. In Experiment 2, an enhanced green fluorescent protein (EGFP) expression vector was constructed and EGFP was driven by Oct4 enhancer and promoter (Oct4-EGFP). Mouse NIH/3T3 cells were then transfected with Oct4-EGFP plasmids and selected for stable clone by G418 screening. After 6 passages, the NIH/3T3-Oct4-EGFP cells were treated with egg extracts to induce reprogramming as Experiment 1, and monitored pluripotency based on the expression of EGFP. Results showed that some of the cells or cell colonies expressed green fluorescence driven by Oct4 regulatory element at the 8th day of culture after extract treatment. Our results demonstrated that both methods of electroporation and reversible permeabilization could introduce reprogramming molecules in Xenopus egg extract to the mammalian somatic cells and generate ETCs cells in vitro. Also, with the establishment of NIH/3T3-Oct4-EGFP cell line, the potentially reprogrammed colonies could be easily selected by EGFP expression. The changes of epigenetic modifications in the ETC cells would be investigated in the short future.

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Human erythropoietin gene expression in transgenic mice: multiple transcription initiation sites and cis-acting regulatory elements

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.930-938.1990 ◽

1990 ◽

Vol 10 (3) ◽

pp. 930-938

Author(s):

G L Semenza ◽

R C Dureza ◽

M D Traystman ◽

J D Gearhart ◽

S E Antonarakis

Keyword(s):

Transgenic Mice ◽

Dna Sequences ◽

Transcription Initiation ◽

Cobalt Chloride ◽

Fetal Liver ◽

Regulatory Element ◽

Regulatory Elements ◽

Flanking Sequence ◽

Rnase Protection ◽

Cis Acting

Erythropoietin (EPO) is the primary humoral regulator of mammalian erythropoiesis. The single-copy EPO gene is normally expressed in liver and kidney, and increased transcription is induced by anemia or cobalt chloride administration. To identify cis-acting DNA sequences responsible for regulated expression, transgenic mice were generated by microinjection of a 4-kilobase-pair (kb) (tgEPO4) or 10-kb (tgEPO10) cloned DNA fragment containing the human EPO gene, 0.7 kb of 3'-flanking sequence, and either 0.4 or 6 kb of 5'-flanking sequence, respectively. tgEPO4 mice expressed the transgene in liver, where expression was inducible by anemia or cobalt chloride, kidney, where expression was not inducible, and other tissues that do not normally express EPO. Human EPO RNA in tgEPO10 mice was detected only in liver of anemic or cobalt-treated mice. Both tgEPO4 and tgEPO10 mice were polycythemic, demonstrating that the human EPO RNA transcribed in liver is functional. These results suggest that (i) a liver inducibility element maps within 4 kb encompassing the gene, 0.4 kb of 5'-flanking sequence, and 0.7 kb of 3'-flanking sequence; (ii) a negative regulatory element is located between 0.4 and 6 kb 5' to the gene; and (iii) sequences required for inducible kidney expression are located greater than 6 kb 5' or 0.7 kb 3' to the gene. RNase protection analysis revealed that human EPO RNA in anemic transgenic mouse liver and hypoxic human hepatoma cells is initiated from several sites, only a subset of which is utilized in nonanemic transgenic liver and human fetal liver.

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RelB-p50 NF-κB Complexes Are Selectively Induced by Cytomegalovirus Immediate-Early Protein 1: Differential Regulation of Bcl-xL Promoter Activity by NF-κB Family Members

Journal of Virology ◽

10.1128/jvi.76.11.5737-5747.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5737-5747 ◽

Cited By ~ 34

Author(s):

H. Y. Jiang ◽

Constantinos Petrovas ◽

Gail E. Sonenshein

Keyword(s):

Promoter Activity ◽

Regulatory Element ◽

Immediate Early ◽

Mrna Levels ◽

3T3 Cells ◽

Weakly Bound ◽

Murine Embryonic Fibroblast ◽

Early Protein ◽

Nih 3T3 ◽

Nih 3T3 Cells

ABSTRACT The NF-κB/Rel family has been implicated in control of transcription of the Bcl-xL gene, a target which mediates cell survival signals. The cytomegalovirus (CMV) immediate-early protein 1 (IE1) was previously shown to induce NF-κB activity. Here, we report that in both vascular smooth muscle cells (SMCs) and NIH 3T3 cells, surprisingly, IE1 failed to induce Bcl-xL promoter activity, although it induced activity of E8-CAT, a reporter construct driven by two copies of the NF-κB element upstream of the c-myc promoter (upstream regulatory element [URE]). Thus, the subunit nature of the NF-κB/Rel factors induced by IE1 was examined using immunofluorescence and immunoblotting. IE1 was found to selectively induce nuclear RelB and p50 in SMCs and NIH 3T3 cells. An increase in RelB protein mediated by IE1 could, in part, be related to an increase in steady-state relB mRNA levels. Consistent with this subunit identification, IE1 was unable to induce E8-CAT activity in relB −/− murine embryonic fibroblast cells. In cotransfection analysis of SMCs and NIH 3T3 cells, RelB and p50 proteins failed to induce Bcl-xL promoter activity while inducing E8-CAT. Furthermore, the NF-κB element of the Bcl-xL promoter only weakly bound RelB-p50 complexes compared to the URE NF-κB element. Overall, these findings demonstrate in SMCs and NIH 3T3 cells that the CMV IE1 protein selectively induces RelB and p50, which fail to activate the Bcl-xL promoter, indicating a strong specificity of binding and activity for the RelB member of the NF-κB family. Furthermore, our results implicate RelB in CMV infection of cells such as vascular SMCs.

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Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1334-1343.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1334-1343

Author(s):

R Paciucci ◽

A Pellicer

Keyword(s):

Transcription Initiation ◽

Regulatory Element ◽

Transcription Unit ◽

Regulatory Sequences ◽

Hypersensitive Site ◽

Ras Gene ◽

Rnase Protection ◽

Negative Regulatory Element ◽

Flanking Region

The 5' flanking region of the mouse N-ras gene was investigated to determine the elements governing transcriptional activity of the gene. The promoter did not contain typical TATA or CCAAT boxes, and according to primer extension and RNase protection analyses, transcription started at several sites. These assays also confirmed the short nucleotide distance interposed between the N-ras transcription unit and the previously described upstream unr gene. Chromatin studies performed by digestion of nuclei with DNase I revealed the presence of four hypersensitive sites: a, b, c, and d. Deletion mutagenesis of the 5' flanking region revealed sequences responsible for both promotion and inhibition of transcription. These sequences resided within 230 bp upstream of the transcription initiation site. Hypersensitive site b colocalized with the 76-bp segment with promoter activity. The negative regulatory element at position -180 colocalized with hypersensitive site a, was active on the N-ras promoter in stable as well as transient assays, and down-regulated the heterologous herpes simplex virus thymidine kinase promoter. Footprint analysis and in vivo transfection-competition experiments indicated that a trans-acting factor is responsible for the negative effect on transcription. The interaction between the cis-acting negative regulatory element and the promoter region may play a role in the tissue- and developmental-stage-specific patterns of expression of the N-ras gene.

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Human erythropoietin gene expression in transgenic mice: multiple transcription initiation sites and cis-acting regulatory elements.

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.930 ◽

1990 ◽

Vol 10 (3) ◽

pp. 930-938 ◽

Cited By ~ 78

Author(s):

G L Semenza ◽

R C Dureza ◽

M D Traystman ◽

J D Gearhart ◽

S E Antonarakis

Keyword(s):

Transgenic Mice ◽

Dna Sequences ◽

Transcription Initiation ◽

Cobalt Chloride ◽

Fetal Liver ◽

Regulatory Element ◽

Regulatory Elements ◽

Flanking Sequence ◽

Rnase Protection ◽

Cis Acting

Erythropoietin (EPO) is the primary humoral regulator of mammalian erythropoiesis. The single-copy EPO gene is normally expressed in liver and kidney, and increased transcription is induced by anemia or cobalt chloride administration. To identify cis-acting DNA sequences responsible for regulated expression, transgenic mice were generated by microinjection of a 4-kilobase-pair (kb) (tgEPO4) or 10-kb (tgEPO10) cloned DNA fragment containing the human EPO gene, 0.7 kb of 3'-flanking sequence, and either 0.4 or 6 kb of 5'-flanking sequence, respectively. tgEPO4 mice expressed the transgene in liver, where expression was inducible by anemia or cobalt chloride, kidney, where expression was not inducible, and other tissues that do not normally express EPO. Human EPO RNA in tgEPO10 mice was detected only in liver of anemic or cobalt-treated mice. Both tgEPO4 and tgEPO10 mice were polycythemic, demonstrating that the human EPO RNA transcribed in liver is functional. These results suggest that (i) a liver inducibility element maps within 4 kb encompassing the gene, 0.4 kb of 5'-flanking sequence, and 0.7 kb of 3'-flanking sequence; (ii) a negative regulatory element is located between 0.4 and 6 kb 5' to the gene; and (iii) sequences required for inducible kidney expression are located greater than 6 kb 5' or 0.7 kb 3' to the gene. RNase protection analysis revealed that human EPO RNA in anemic transgenic mouse liver and hypoxic human hepatoma cells is initiated from several sites, only a subset of which is utilized in nonanemic transgenic liver and human fetal liver.

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Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1334 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1334-1343 ◽

Cited By ~ 14

Author(s):

R Paciucci ◽

A Pellicer

Keyword(s):

Transcription Initiation ◽

Regulatory Element ◽

Transcription Unit ◽

Regulatory Sequences ◽

Hypersensitive Site ◽

Ras Gene ◽

Rnase Protection ◽

Negative Regulatory Element ◽

Flanking Region

The 5' flanking region of the mouse N-ras gene was investigated to determine the elements governing transcriptional activity of the gene. The promoter did not contain typical TATA or CCAAT boxes, and according to primer extension and RNase protection analyses, transcription started at several sites. These assays also confirmed the short nucleotide distance interposed between the N-ras transcription unit and the previously described upstream unr gene. Chromatin studies performed by digestion of nuclei with DNase I revealed the presence of four hypersensitive sites: a, b, c, and d. Deletion mutagenesis of the 5' flanking region revealed sequences responsible for both promotion and inhibition of transcription. These sequences resided within 230 bp upstream of the transcription initiation site. Hypersensitive site b colocalized with the 76-bp segment with promoter activity. The negative regulatory element at position -180 colocalized with hypersensitive site a, was active on the N-ras promoter in stable as well as transient assays, and down-regulated the heterologous herpes simplex virus thymidine kinase promoter. Footprint analysis and in vivo transfection-competition experiments indicated that a trans-acting factor is responsible for the negative effect on transcription. The interaction between the cis-acting negative regulatory element and the promoter region may play a role in the tissue- and developmental-stage-specific patterns of expression of the N-ras gene.

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