A DNA element that regulates expression of an endogenous retrovirus during F9 cell differentiation is E1A dependent

The retinoic acid-induced differentiation of F9 cells into parietal endoderm-like cells activates transcription of the endogenous mouse retrovirus, the intracisternal A-particle (IAP). To investigate the elements that control IAP gene differentiation-specific expression, we used methylation interference, Southwestern (DNA-protein), and transient-transfection assays and identified the IAP-proximal enhancer (IPE) element that directs differentiation-specific expression. We find that the IPE is inactive in undifferentiated F9 cells and active in differentiated parietal endoderm-like PYS-2 cells. Three proteins of 40, 60, and 68 kDa bind to the sequence GAGTAGAC located between nucleotides -53 and -47 within the IPE. The 40- and 68-kDa proteins from both the undifferentiated and differentiated cells exhibit similar DNA-binding activities. However, the 60-kDa protein from differentiated cells has greater binding activity than that from undifferentiated cells, suggesting a role for this protein in F9 differentiation-specific expression of the IAP gene. The IAP gene is negatively regulated by the adenovirus E1A proteins, and the E1A sequence responsible for repression is located at the N terminus, between amino acids 2 and 67. The DNA sequence that is the target of E1A repression also maps to the IPE element. Colocalization of the differentiation-specific and E1A-sensitive elements to the same protein-binding site within the IPE suggests that the E1A-like activity functions in F9 cells to repress IAP gene expression. Activation of the IAP gene may result when the E1A-like activity is lost or inactivated during F9 cell differentiation, followed by binding of the 60-kDa positive regulatory protein to the enhancer element.

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Cellular promoters incorporated into the adenovirus genome: effects of viral regulatory elements on transcription rates and cell specificity of albumin and beta-globin promoters

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3798-3806.1986 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3798-3806

Author(s):

L E Babiss ◽

J M Friedman ◽

J E Darnell

Keyword(s):

Cultured Cells ◽

Regulatory Elements ◽

Beta Globin ◽

Specific Expression ◽

Adenovirus E1a ◽

Differentiated Cells ◽

Cell Specificity ◽

293 Cells ◽

Specific Factors ◽

Globin Promoter

In the accompanying paper (Friedman et al., Mol. Cell. Biol. 6:3791-3797, 1986), hepatoma-specific expression of the rat albumin promoter within the adenovirus genome was demonstrated. However, the rate of transcription was very low compared with that of the endogenous chromosomal albumin gene. Here we show that in hepatoma cells the adenovirus E1A enhancer, especially in the presence of E1A protein, greatly stimulates transcription from the albumin promoter but not the mouse beta-globin promoter. This enhancer-dependent stimulation did not occur in myeloma cells in which a virus containing a immunoglobulin promoter and enhancer did function. These experiments suggest a limited distribution in cultured differentiated cells of cell-specific transcription factors. However, either the regulation of such cell-specific factors breaks down in other cultured cells, or strictly cell-specific factors are not at play in controlling cell-specific transcription, because HeLa cells could transcribe the albumin promoter from the same start site about 10% as well as hepatomas could and 293 cells could transcribe both albumin and globin promoters.

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Interaction of several related GC-box- and GT-box-binding proteins with the intronic enhancer is required for differential expression of the gb110 gene in embryonal carcinoma cells

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.5786-5793.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 5786-5793

Author(s):

L Hamann ◽

K U Bayer ◽

K Jensen ◽

K Harbers

Keyword(s):

Transcription Factor ◽

Binding Proteins ◽

Embryonal Carcinoma ◽

Regulatory Elements ◽

Carcinoma Cells ◽

Minor Importance ◽

Embryonal Carcinoma Cells ◽

Enhancer Element ◽

F9 Cells ◽

Parietal Endoderm

The molecular mechanisms by which expression of a gene is down-regulated after differentiation of F9 embryonal carcinoma cells into parietal endoderm-like cells was studied by characterizing the cis- and trans-regulatory elements of the gb110 gene. This gene encodes a putative RNA helicase, and its expression is down-regulated when F9 cells are differentiated with retinoic acid and cyclic AMP. The 5'-flanking region of the gene has all of the features of a GC-rich island promoter and seems to play only a minor role, if any, in the regulated expression. A 133-bp enhancer in the first intron was identified by transient chloramphenicol acetyltransferase assays that activated expression in undifferentiated F9 cells about 50- to 100-fold. As this enhancer was not active in differentiated F9 cells, it seems to be the prime mediator of the differentiation-specific down-regulation of the gb110 gene. Four different protein-binding sites, three of which contain GC- and GT-box motifs, were identified in the enhancer element. The fourth site, interacting with previously described transcription factor FTZ-F1/ELP, seems to be of minor importance for the activity of the enhancer. Mutational analysis showed that the cooperative interaction of several most likely related proteins with the three GC- and GT-box motifs was required for full enhancer activity. On the basis of their binding properties, at least two of these proteins seem to be identical or closely related to ubiquitous transcription factor Sp1. One of the GT-box-binding proteins was present in undifferentiated F9 cells but not, however, in its differentiated derivatives. The cell specificity of this transcription factor explains why the gb110 gene is not expressed or expressed only at low levels in parietal endoderm-like cells.

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Cell differentiation induces TIF1β association with centromeric heterochromatin via an HP1 interaction

Journal of Cell Science ◽

10.1242/jcs.115.17.3439 ◽

2002 ◽

Vol 115 (17) ◽

pp. 3439-3448 ◽

Cited By ~ 4

Author(s):

Florence Cammas ◽

Mustapha Oulad-Abdelghani ◽

Jean-Luc Vonesch ◽

Yolande Huss-Garcia ◽

Pierre Chambon ◽

...

Keyword(s):

Cell Differentiation ◽

Critical Role ◽

Centromeric Heterochromatin ◽

Heterochromatin Protein 1 ◽

F9 Cells ◽

Differentiated Cells ◽

Confocal Immunofluorescence Microscopy ◽

Confocal Immunofluorescence ◽

Family Protein ◽

Undifferentiated Cells

The transcriptional intermediary factor 1 (TIF1) family protein TIF1βis a corepressor for Krüppel-associated box (KRAB)-domain-containing zinc finger proteins and plays a critical role in early embryogenesis. Here, we examined TIF1β distribution in the nucleus of mouse embryonic carcinoma F9 cells during retinoic-acid-induced primitive endodermal differentiation. Using confocal immunofluorescence microscopy, we show that, although TIF1β is diffusely distributed throughout the nucleoplasm of undifferentiated cells, it relocates and concentrates into distinct foci of centromeric heterochromatin in differentiated cells characterized by a low proliferation rate and a well developed cytokeratin network. This relocation was not observed in isoleucine-deprived cells, which are growth arrested, or in compound RXRα-/-/RARγ-/- null mutant cells, which are resistant to RA-induced differentiation. Amino-acid substitutions in the PxVxL motif of TIF1β, which abolish interaction with members of the heterochromatin protein 1 (HP1) family, prevent its centromeric localization in differentiated cells. Collectively, these data provide compelling evidence for a dynamic nuclear compartmentalization of TIF1βthat is regulated during cell differentiation through a mechanism that requires HP1 interaction.

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Cellular promoters incorporated into the adenovirus genome: effects of viral regulatory elements on transcription rates and cell specificity of albumin and beta-globin promoters.

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3798 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3798-3806 ◽

Cited By ~ 35

Author(s):

L E Babiss ◽

J M Friedman ◽

J E Darnell

Keyword(s):

Cultured Cells ◽

Regulatory Elements ◽

Beta Globin ◽

Specific Expression ◽

Adenovirus E1a ◽

Differentiated Cells ◽

Cell Specificity ◽

293 Cells ◽

Specific Factors ◽

Globin Promoter

In the accompanying paper (Friedman et al., Mol. Cell. Biol. 6:3791-3797, 1986), hepatoma-specific expression of the rat albumin promoter within the adenovirus genome was demonstrated. However, the rate of transcription was very low compared with that of the endogenous chromosomal albumin gene. Here we show that in hepatoma cells the adenovirus E1A enhancer, especially in the presence of E1A protein, greatly stimulates transcription from the albumin promoter but not the mouse beta-globin promoter. This enhancer-dependent stimulation did not occur in myeloma cells in which a virus containing a immunoglobulin promoter and enhancer did function. These experiments suggest a limited distribution in cultured differentiated cells of cell-specific transcription factors. However, either the regulation of such cell-specific factors breaks down in other cultured cells, or strictly cell-specific factors are not at play in controlling cell-specific transcription, because HeLa cells could transcribe the albumin promoter from the same start site about 10% as well as hepatomas could and 293 cells could transcribe both albumin and globin promoters.

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Interaction of several related GC-box- and GT-box-binding proteins with the intronic enhancer is required for differential expression of the gb110 gene in embryonal carcinoma cells.

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.5786 ◽

1994 ◽

Vol 14 (9) ◽

pp. 5786-5793 ◽

Cited By ~ 8

Author(s):

L Hamann ◽

K U Bayer ◽

K Jensen ◽

K Harbers

Keyword(s):

Transcription Factor ◽

Binding Proteins ◽

Embryonal Carcinoma ◽

Regulatory Elements ◽

Carcinoma Cells ◽

Minor Importance ◽

Embryonal Carcinoma Cells ◽

Enhancer Element ◽

F9 Cells ◽

Parietal Endoderm

The molecular mechanisms by which expression of a gene is down-regulated after differentiation of F9 embryonal carcinoma cells into parietal endoderm-like cells was studied by characterizing the cis- and trans-regulatory elements of the gb110 gene. This gene encodes a putative RNA helicase, and its expression is down-regulated when F9 cells are differentiated with retinoic acid and cyclic AMP. The 5'-flanking region of the gene has all of the features of a GC-rich island promoter and seems to play only a minor role, if any, in the regulated expression. A 133-bp enhancer in the first intron was identified by transient chloramphenicol acetyltransferase assays that activated expression in undifferentiated F9 cells about 50- to 100-fold. As this enhancer was not active in differentiated F9 cells, it seems to be the prime mediator of the differentiation-specific down-regulation of the gb110 gene. Four different protein-binding sites, three of which contain GC- and GT-box motifs, were identified in the enhancer element. The fourth site, interacting with previously described transcription factor FTZ-F1/ELP, seems to be of minor importance for the activity of the enhancer. Mutational analysis showed that the cooperative interaction of several most likely related proteins with the three GC- and GT-box motifs was required for full enhancer activity. On the basis of their binding properties, at least two of these proteins seem to be identical or closely related to ubiquitous transcription factor Sp1. One of the GT-box-binding proteins was present in undifferentiated F9 cells but not, however, in its differentiated derivatives. The cell specificity of this transcription factor explains why the gb110 gene is not expressed or expressed only at low levels in parietal endoderm-like cells.

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Binding of a factor to an enhancer element responsible for the tissue-specific expression of the chicken alpha A-crystallin gene

Development ◽

10.1242/dev.113.2.539 ◽

1991 ◽

Vol 113 (2) ◽

pp. 539-550 ◽

Cited By ~ 2

Author(s):

I. Matsuo ◽

M. Kitamura ◽

K. Okazaki ◽

K. Yasuda

Keyword(s):

Gene Expression ◽

Dna Sequences ◽

Nuclear Protein ◽

Fusion Gene ◽

Regulatory Region ◽

Binding Activity ◽

Flanking Sequence ◽

Specific Expression ◽

Enhancer Element

We have characterized a regulatory region of the chicken alpha A-crystallin gene using transfection assays, which revealed that a 84 base pair element (−162 to −79) in the 5′ flanking sequence is necessary and sufficient for lens-specific expression. A multimer of this element functions as lens-specific enhancer and synergistically activates transcription from chicken alpha A-crystallin or beta-actin basal promoters fused to the CAT gene. In vivo competition experiments demonstrated that DNA sequences containing the 84 bp element reduced alpha A-crystallin-CAT fusion gene expression. A nuclear factor present exclusively in lens cells binds to the 84 bp element in the region between positions −165 and −140. Southwestern blot analysis showed that 61,000 Mr (61 × 10(3) Mr) lens nuclear protein exhibited DNA-binding activity specific to the 84 bp element. Our data suggested that the 61 × 10(3) Mr nuclear protein, and the 84 bp element that it interacts with, may be involved in regulating the alpha A-crystallin gene expression in vivo.

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An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element

Blood ◽

10.1182/blood.v98.8.2555 ◽

2001 ◽

Vol 98 (8) ◽

pp. 2555-2562 ◽

Cited By ~ 26

Author(s):

Mark Loyevsky ◽

Timothy LaVaute ◽

Charles R. Allerson ◽

Robert Stearman ◽

Olakunle O. Kassim ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Regulatory Protein ◽

Protein S ◽

Fold Increase ◽

Binding Activity ◽

Mobility Shift ◽

Iron Responsive Element ◽

Element Binding Protein ◽

Responsive Element ◽

Electrophoretic Mobility Shift Assays

Abstract This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)–like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex fromP falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.

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Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

Molecular and Cellular Biology ◽

10.1128/mcb.7.12.4400 ◽

1987 ◽

Vol 7 (12) ◽

pp. 4400-4406 ◽

Cited By ~ 50

Author(s):

K D Breunig ◽

P Kuger

Keyword(s):

Protein Binding ◽

Binding Site ◽

Transcriptional Activation ◽

Kluyveromyces Lactis ◽

Regulatory Protein ◽

Binding Activity ◽

Protein Binding Site ◽

Regulatory Sequence ◽

Functional Homology ◽

Beta Galactosidase

As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription.

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