The dyad symmetry element is the molecular target for c-fos induction and inhibition during K 562 differentiation along mutually exclusive lineages

Abstract The c-fos proto-oncogene seems to play an important role during differentiation and activation of cells from the hematopoietic lineage. Therefore, it is of interest to investigate the mechanism underlying its transcriptional activation in these cells. To delineate the sequences and factors involved in c-fos transcriptional activation during the course of myeloid cell differentiation, we have used the K 562 chronic leukemic cell line as a model. K 562 cells were transfected with chloramphenicol transacetylase (CAT) reporter constructs, including various regions of the human c-fos promoter, and induced to differentiate by two distinct agents: 12-O-tetradecanoyl phorbol-13- acetate (TPA), which activates a differentiation program along the megakaryoblastic pathway; and hemin, which induces erythroid differentiation. We show here that TPA treatment of K 562 cells induces fos CAT reporter constructs activation, whereas treatment with hemin does not. Furthermore, predifferentiation of the cells with hemin blocks a subsequent induction by TPA, in correlation with the inhibition by hemin of megakaryoblastic differentiation markers appearance. Both the induction by TPA and the inhibition by hemin are mediated by a dyad symmetry element (DSE) located in the upstream regulatory region, between -318 and -296. These results suggest that the protein complex binding to the DSE regulatory element is the target for c-fos activation by TPA and inhibition by hemin in K 562 cells. However, no modulation of protein affinity for the DSE sequence was detected by gel shift assay during the course of induction or inhibition, suggesting that the structural change responsible for the transcriptional modulation is too unstable or too subtle to be detected by this method.

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The dyad symmetry element is the molecular target for c-fos induction and inhibition during K 562 differentiation along mutually exclusive lineages

Blood ◽

10.1182/blood.v77.1.55.bloodjournal77155 ◽

1991 ◽

Vol 77 (1) ◽

pp. 55-63

Author(s):

D Trouche ◽

P Robin ◽

P Sassone-Corsi ◽

WL Farrar ◽

A Harel-Bellan

Keyword(s):

Transcriptional Activation ◽

Regulatory Element ◽

Regulatory Region ◽

Myeloid Cell ◽

Leukemic Cell ◽

Erythroid Differentiation ◽

Symmetry Element ◽

Differentiation Markers ◽

K 562 Cells ◽

Dyad Symmetry

The c-fos proto-oncogene seems to play an important role during differentiation and activation of cells from the hematopoietic lineage. Therefore, it is of interest to investigate the mechanism underlying its transcriptional activation in these cells. To delineate the sequences and factors involved in c-fos transcriptional activation during the course of myeloid cell differentiation, we have used the K 562 chronic leukemic cell line as a model. K 562 cells were transfected with chloramphenicol transacetylase (CAT) reporter constructs, including various regions of the human c-fos promoter, and induced to differentiate by two distinct agents: 12-O-tetradecanoyl phorbol-13- acetate (TPA), which activates a differentiation program along the megakaryoblastic pathway; and hemin, which induces erythroid differentiation. We show here that TPA treatment of K 562 cells induces fos CAT reporter constructs activation, whereas treatment with hemin does not. Furthermore, predifferentiation of the cells with hemin blocks a subsequent induction by TPA, in correlation with the inhibition by hemin of megakaryoblastic differentiation markers appearance. Both the induction by TPA and the inhibition by hemin are mediated by a dyad symmetry element (DSE) located in the upstream regulatory region, between -318 and -296. These results suggest that the protein complex binding to the DSE regulatory element is the target for c-fos activation by TPA and inhibition by hemin in K 562 cells. However, no modulation of protein affinity for the DSE sequence was detected by gel shift assay during the course of induction or inhibition, suggesting that the structural change responsible for the transcriptional modulation is too unstable or too subtle to be detected by this method.

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Forced GATA-1 Expression in the Murine Myeloid Cell Line M1: Induction of c-Mpl Expression and Megakaryocytic/Erythroid Differentiation

Blood ◽

10.1182/blood.v91.2.450.450_450_457 ◽

1998 ◽

Vol 91 (2) ◽

pp. 450-457 ◽

Cited By ~ 6

Author(s):

Yuji Yamaguchi ◽

Leonard I. Zon ◽

Steven J. Ackerman ◽

Masayuki Yamamoto ◽

Toshio Suda

Keyword(s):

Cell Line ◽

Expression Vector ◽

Myeloid Cell ◽

Leukemic Cell ◽

Erythroid Differentiation ◽

Acetylcholinesterase Activity ◽

Globin Mrna ◽

Platelet Factor ◽

Hematopoietic Stem ◽

In Cells

The “zinc-finger” transcription factor GATA-1 was first shown in cells of erythroid lineage. It is also expressed in cells of other hematopoietic lineages including megakaryocytes, mast cells, and eosinophils. GATA-1 is now considered to be one of the central regulators in hematopoietic cell differentiation. To further analyze the role of GATA-1 in controlling differentiation from hematopoietic stem cells, we investigated the phenotypic changes induced by the overexpression of murine GATA-1 in the murine myeloid leukemic cell line, M1. Forced expression of GATA-1 induced the appearance of erythroid cells and megakaryocytes as assessed by cellular morphology, acetylcholinesterase activity, and expression of platelet factor 4 and β-globin mRNA synthesis. Because the c-mpl ligand, thrombopoietin, plays an important role in megakaryopoiesis, the expression of c-mpl and c-mpl ligand (thrombopoietin) mRNA was analyzed by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in M1 cells overexpressing GATA-1. The c-mpl ligand mRNA was equally expressed both in parental M1 cells and in those transfected with the GATA-1 expression vector. In contrast, the mRNA expression of c-mpl was increased only in GATA-1 expressing M1 cells differentiated towards erythroid and megakaryocyte lineages. The increased expression of c-mpl mRNA induced by GATA-1 raised the question as to whether or not GATA-1 transactivated the c-mpl promoter. The activity of the c-mpl promoter in the presence of cotransfected GATA-1 was significantly increased compared with that of the control. A plasmid with the mutated GATA-binding site did not show transactivation ability in the cotransfection with a GATA expression vector. These findings suggest that the upregulation of c-mpl induced by GATA-1 expression in M1 cells is closely associated with erythroid and megakaryocytic differentiation.

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A novel Sp1-relatedciselement involved in intestinal alkaline phosphatase gene transcription

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.276.4.g800 ◽

1999 ◽

Vol 276 (4) ◽

pp. G800-G807 ◽

Cited By ~ 5

Author(s):

Jeong H. Kim ◽

Shufen Meng ◽

Amy Shei ◽

Richard A. Hodin

Keyword(s):

Alkaline Phosphatase ◽

Transcriptional Activation ◽

Molecular Mechanisms ◽

Regulatory Element ◽

Gene Activation ◽

Regulatory Region ◽

Intestinal Alkaline Phosphatase ◽

Mobility Shift ◽

Sv40 Promoter

We have used sodium butyrate-treated HT-29 cells as an in vitro model system to study the molecular mechanisms underlying intestinal alkaline phosphatase (IAP) gene activation. Transient transfection assays using human IAP-CAT reporter genes along with DNase I footprinting were used to localize a critical cis element (IF-III) corresponding to the sequence 5′-GACTGGGCGGGGTCAAGATGGA-3′. Deletion of the IF-III element resulted in a dramatic reduction in reporter gene activity, and IF-III was shown to function in the context of a heterologous (SV40) promoter in a cell type-specific manner, further supporting its functional role in IAP transactivation. Electrophoretic mobility shift assays revealed that IF-III binds Sp1 and Sp3, but these factors comprise only a portion of the total nuclear binding and appear to mediate only a small portion of its transcriptional activity. IF-III does not correspond to any previously characterized regulatory region from other intestine-specific genes. We have thus identified a novel, Sp1-related cis-regulatory element in the human IAP gene that appears to play a role in its transcriptional activation during differentiation in vitro.

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Transcriptional activation by TGFβ1 mediated by the dyad symmetry element (DSE) and the TPA responsive element (TRE)

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(90)91139-j ◽

1990 ◽

Vol 168 (3) ◽

pp. 1074-1081 ◽

Cited By ~ 31

Author(s):

Rolf P. de Groot ◽

Wiebe Kruijer

Keyword(s):

Transcriptional Activation ◽

Symmetry Element ◽

Responsive Element ◽

Dyad Symmetry

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Specific Methylation Patterns in Two Control Regions of Epstein-Barr Virus Latency: the LMP-1-Coding Upstream Regulatory Region and an Origin of DNA Replication (oriP)

Journal of Virology ◽

10.1128/jvi.72.4.2969-2974.1998 ◽

1998 ◽

Vol 72 (4) ◽

pp. 2969-2974 ◽

Cited By ~ 30

Author(s):

Kerstin I. Falk ◽

Laszlo Szekely ◽

Anna Aleman ◽

Ingemar Ernberg

Keyword(s):

Epstein Barr Virus ◽

Methylation Status ◽

Regulatory Region ◽

Cell Types ◽

Symmetry Element ◽

Regulatory Sequence ◽

Type I ◽

Barr Virus ◽

Epstein Barr ◽

Dyad Symmetry

ABSTRACT The Epstein-Barr virus (EBV) can establish at least four different forms of latent infection. Previously, we have shown that the level of methylation of the EBV genome varies, depending on the form of latency. The methylation status of CpGs was analyzed by the bisulfite genomic sequencing technique in four different cell types representing different forms of latency. The dyad symmetry element of the origin of replication (oriP) region and the latent membrane protein 1 (LMP-1) regulatory sequence (LRS) were studied. The dyad symmetry element has four binding sites for EBNA-1. In a cell with type I latency, a region upstream of the dyad symmetry element was highly methylated, whereas the dyad symmetry element was unmethylated in the EBNA-1-binding region. The LRS was extensively methylated in the LMP-1-negative cell line Rael, in contrast to a LMP-1-expressing nasopharyngeal carcinoma tumor (NPC C15), which was almost completely unmethylated. The methylation pattern of LRS in type I and type III Burkitt lymphoma cells of similar parental origins confirmed that demethylation of some regions takes place upon phenotypic drift.

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A novel retinoic acid-response element requires an enhancer element mediator for transcriptional activation

Biochemical Journal ◽

10.1042/bj20040715 ◽

2004 ◽

Vol 383 (1) ◽

pp. 37-43 ◽

Cited By ~ 7

Author(s):

Laura R. HARRIS ◽

Olli-Pekka KAMARAINEN ◽

Minna SEVAKIVI ◽

Gwen C. MILLER ◽

James W. CLARKE ◽

...

Keyword(s):

Retinoic Acid ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Regulatory Element ◽

Regulatory Region ◽

Mobility Shift ◽

Experimental Conditions ◽

Response Element ◽

Enhancer Element ◽

Retinoic Acid Response Element

The Col11a2 gene codes for α2(XI), a subunit of type XI collagen that is a critical component of the cartilage extracellular matrix. The 5′ regulatory region of Col11a2 was subjected to deletional analysis to detect any regulatory element in addition to the two known chondrocyte-specific enhancer elements B/C and D/E. Deletion of the region from −342 to −242 bp reduced transcriptional activity to less than 50% of wild-type, but the sequence showed no independent ability to increase transcription from a minimal promoter. When cloned downstream of the D/E enhancer, however, a subsection of the sequence nearly doubled transcriptional activity and produced an additional 3-fold activation in response to RA (retinoic acid). A 6-bp direct repeat, separated by 4 bp (a DR-4 element) near the 5′-end of this region, was found to be essential for its activity, and was further shown to bind the RA X receptor β in electrophoretic mobility-shift assays. The present study has revealed a novel RA-response element in Col11a2 that does not interact directly with the promoter, but instead requires the D/E enhancer to mediate transcriptional activation. Proteins bound at the enhancer, therefore, would be expected to affect the transcriptional response to RA. Such a system of regulation, particularly if found to be operating in other cartilage genes, could explain the conflicting responses RA produces in chondrocytes under different experimental conditions.

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Functional role of a novel cis-acting element (GAGA box) in human type-1 angiotensin II receptor gene transcription

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0250097 ◽

2000 ◽

Vol 25 (1) ◽

pp. 97-108 ◽

Cited By ~ 21

Author(s):

BD Wyse ◽

SL Linas ◽

TJ Thekkumkara

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Transcriptional Activation ◽

De Novo ◽

Regulatory Element ◽

Regulatory Region ◽

Receptor Expression ◽

Cis Acting ◽

Human Type

GH/growth factors have been shown to increase angiotensin type 1 receptor expression. In the present study we determined the cis-acting regulatory region controlling GH-induced transcription of the human type-1 angiotensin receptor (hAT(1)). In human proximal tubule cells transfected with a chloramphenicol acetyl transferase (CAT) reporter plasmid under the control of the hAT(1) promoter, GH induced CAT activity. Serial deletions of the hAT(1) promoter region indicated that an area between -314 bp and -70 bp upstream of the 5'-end of the cDNA sequence was essential for this activation to occur. Although sequence analysis identified putative multiple nuclear protein binding sites in this region, we determined that a 12 bp sequence (5'-GAGAGGGAGGAG-3', GAGA box) located between -161 bp and -149 bp was important for GH-mediated activation. Using mobility shift assays we demonstrated increased DNA binding activity to the labeled GAGA box in nuclear extracts treated with GH, suggesting this sequence is a GH response element. Southwestern analysis identified an 18 kDa GAGA box-binding protein (GAGA-BP). GH-induced activity of the GAGA-BP occurred within 2.5 min and reached a maximum at 5 min. Activation did not require de novo protein synthesis. Removal of the GAGA box abolished GH-induced transcription as well as basal transcription of the hAT(1) gene. Additional studies demonstrated that epidermal growth factor, platelet-derived growth factor and insulin activate the GAGA-BP, suggesting these growth factors can also regulate the transcription of the hAT(1) gene through the GAGA box. Our data show that the GAGA-BP acts as a trans-acting factor binding to the cis-acting regulatory element in the hAT(1) promoter, which is necessary for the basal and growth factor(s)-mediated transcriptional activation of the hAT(1) gene.

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Forced GATA-1 Expression in the Murine Myeloid Cell Line M1: Induction of c-Mpl Expression and Megakaryocytic/Erythroid Differentiation

Blood ◽

10.1182/blood.v91.2.450 ◽

1998 ◽

Vol 91 (2) ◽

pp. 450-457 ◽

Cited By ~ 54

Author(s):

Yuji Yamaguchi ◽

Leonard I. Zon ◽

Steven J. Ackerman ◽

Masayuki Yamamoto ◽

Toshio Suda

Keyword(s):

Cell Line ◽

Expression Vector ◽

Myeloid Cell ◽

Leukemic Cell ◽

Erythroid Differentiation ◽

Acetylcholinesterase Activity ◽

Globin Mrna ◽

Platelet Factor ◽

Hematopoietic Stem ◽

In Cells

Abstract The “zinc-finger” transcription factor GATA-1 was first shown in cells of erythroid lineage. It is also expressed in cells of other hematopoietic lineages including megakaryocytes, mast cells, and eosinophils. GATA-1 is now considered to be one of the central regulators in hematopoietic cell differentiation. To further analyze the role of GATA-1 in controlling differentiation from hematopoietic stem cells, we investigated the phenotypic changes induced by the overexpression of murine GATA-1 in the murine myeloid leukemic cell line, M1. Forced expression of GATA-1 induced the appearance of erythroid cells and megakaryocytes as assessed by cellular morphology, acetylcholinesterase activity, and expression of platelet factor 4 and β-globin mRNA synthesis. Because the c-mpl ligand, thrombopoietin, plays an important role in megakaryopoiesis, the expression of c-mpl and c-mpl ligand (thrombopoietin) mRNA was analyzed by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in M1 cells overexpressing GATA-1. The c-mpl ligand mRNA was equally expressed both in parental M1 cells and in those transfected with the GATA-1 expression vector. In contrast, the mRNA expression of c-mpl was increased only in GATA-1 expressing M1 cells differentiated towards erythroid and megakaryocyte lineages. The increased expression of c-mpl mRNA induced by GATA-1 raised the question as to whether or not GATA-1 transactivated the c-mpl promoter. The activity of the c-mpl promoter in the presence of cotransfected GATA-1 was significantly increased compared with that of the control. A plasmid with the mutated GATA-binding site did not show transactivation ability in the cotransfection with a GATA expression vector. These findings suggest that the upregulation of c-mpl induced by GATA-1 expression in M1 cells is closely associated with erythroid and megakaryocytic differentiation.

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Calcium and growth factor pathways of c-fos transcriptional activation require distinct upstream regulatory sequences

Molecular and Cellular Biology ◽

10.1128/mcb.8.7.2787-2796.1988 ◽

1988 ◽

Vol 8 (7) ◽

pp. 2787-2796 ◽

Cited By ~ 4

Author(s):

M Sheng ◽

S T Dougan ◽

G McFadden ◽

M E Greenberg

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Transcriptional Activation ◽

Serum Response Factor ◽

Symmetry Element ◽

Cis Acting ◽

Polypeptide Growth Factors ◽

Dyad Symmetry ◽

Serum Response

Transcription of the c-fos proto-oncogene is rapidly induced in the rat pheochromocytoma PC12 cell line by a wide variety of stimuli, including polypeptide growth factors, phorbol esters, and calcium ion fluxes. We have mapped the upstream sequence requirements for this activation in PC12 cells by analysis of promoter deletion mutants in a transient expression assay. Two distinct pathways of c-fos induction are defined that differ in their requirement for cis-acting DNA sequences. Calcium activation of c-fos transcription is dependent on a DNA element located approximately 60 base pairs upstream of the transcription start site. This region is highly conserved between human, mouse, and chicken c-fos genes and contains a sequence that resembles the consensus for a cyclic AMP response element. The dyad symmetry element at position -300, which is necessary for serum responsiveness of c-fos, appears to be unimportant for calcium activation of the gene. The dyad symmetry element is, however, an essential cis-acting sequence for c-fos inducibility by nerve growth factor, epidermal growth factor, fibroblast growth factor, and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate. Studies in vivo and in vitro with various mutants of the dyad symmetry element indicate that c-fos activation by polypeptide growth factors and 12-O-tetradecanoyl activation by polypeptide growth factors and 12-O-tetradecanoyl phorbol-13-acetate is mediated by a common transcription factor, and that this factor is identical to the previously described serum response factor. In vitro DNA-binding assays suggest that the quantity of serum response factor-binding activity remains unchanged during c-fos transcriptional activation.

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