A Cellular Repressor of E1A-Stimulated Genes That Inhibits Activation by E2F

ABSTRACT The adenovirus E1A protein both activates and represses gene expression to promote cellular proliferation and inhibit differentiation. Here we report the identification and characterization of a cellular protein that antagonizes transcriptional activation and cellular transformation by E1A. This protein, termed CREG for cellular repressor of E1A-stimulated genes, shares limited sequence similarity with E1A and binds both the general transcription factor TBP and the tumor suppressor pRb in vitro. In transfection assays, CREG represses transcription and antagonizes 12SE1A-mediated activation of both the adenovirus E2 and cellular hsp70 promoters. CREG also antagonizes E1A-mediated transformation, as expression of CREG reduces the efficiency with which E1A and the oncogeneras cooperate to transform primary cells. Binding sites for E2F, a key transcriptional regulator of cell cycle progression, were found to be required for repression of the adenovirus E2 promoter by CREG, and CREG was shown to inhibit activation by E2F. Since both the adenovirus E1A protein and transcriptional activation by E2F function to promote cellular proliferation, the results presented here suggest that CREG activity may contribute to the transcriptional control of cell growth and differentiation.

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The hsp70 gene CCAAT-binding factor mediates transcriptional activation by the adenovirus E1a protein.

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2599 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2599-2605 ◽

Cited By ~ 30

Author(s):

L S Lum ◽

S Hsu ◽

M Vaewhongs ◽

B Wu

Keyword(s):

Transcriptional Activation ◽

Dependent Manner ◽

Hsp70 Gene ◽

Hsp70 Promoter ◽

Adenovirus E1a ◽

Cell Biol ◽

Binding Factor ◽

E1a Protein

Expression of the human hsp70 gene is cell cycle regulated and is inducible by both serum and the adenovirus E1a protein (K. Milarski and R. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986; M. C. Simon, K. Kitchener, H.-T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987; B. Wu, H. Hurst, N. Jones, and R. Morimoto, Mol. Cell. Biol. 6:2994-2999, 1986; B. Wu and R. Morimoto, Proc. Natl. Acad. Sci. USA 82:6070-6074, 1985). This regulated expression is predominantly controlled by the CCAAT element at position -70 relative to the transcriptional initiation site (G. Williams, T. McClanahan, and R. Morimoto, Mol. Cell. Biol. 9:2574-2587, 1989; B. Wu, H. Hurst, N. Jones, and R. Morimoto, Mol. Cell. Biol. 6:2994-2999, 1986). A corresponding CCAAT-binding factor (CBF) of 999 amino acids has recently been cloned and shown to stimulate transcription selectively from the hsp70 promoter in a CCAAT element-dependent manner (L. Lum, L. Sultzman, R. Kaufman, D. Linzer, and B. Wu, Mol. Cell. Biol. 10:6709-6717, 1990). We report here that the first 192 residues of CBF, when fused to the DNA-binding domain of the heterologous activator GAL-4, are necessary and sufficient to mediate E1a-dependent transcriptional activation. E1a and CBF exhibit complex formation in vitro, suggesting that an in vivo interaction between these proteins may be relevant to the well-characterized E1a-induced transcriptional activation of the hsp70 promoter.

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AMF-1/Gps2 Binds p300 and Enhances Its Interaction with Papillomavirus E2 Proteins

Journal of Virology ◽

10.1128/jvi.74.13.5872-5879.2000 ◽

2000 ◽

Vol 74 (13) ◽

pp. 5872-5879 ◽

Cited By ~ 60

Author(s):

Yu-Cai Peng ◽

David E. Breiding ◽

Francis Sverdrup ◽

James Richard ◽

Elliot J. Androphy

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Functional Significance ◽

Cellular Protein ◽

Transcriptional Coactivator ◽

Adenovirus E1a ◽

Coactivator P300 ◽

P300 Recruitment

ABSTRACT The cellular protein AMF-1 (Gps2) positively modulates gene expression by the papillomavirus E2 protein (D. E. Breiding et al., Mol. Cell. Biol. 17:7208–7219, 1997). We show here that AMF-1 also binds the transcriptional coactivator p300 in vitro and in vivo. E2 interacted weakly with p300. These observations led to a model in which AMF-1 recruits p300 into a complex with E2. Cotransfection of AMF-1 or p300 stimulated levels of E2-dependent transcription, while cotransfection of both AMF-1 and p300 showed an additive effect. The functional significance of p300 recruitment for E2 transactivation was evidenced by repression of E2-activated transcription by adenovirus E1A, which inhibits both coactivator and acetylase activities of p300. Antibodies to AMF-1 or E2 immunoprecipitated histone acetylase activity from cell lysates. Western blotting using antibody against acetyl-lysine failed to detect acetylation of AMF-1 or E2 in complex with p300. These results suggest that AMF-1 facilitates the recruitment of p300 and its histone acetylase activity into complexes with E2 and represents a novel mechanism of transcriptional activation.

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The hsp70 gene CCAAT-binding factor mediates transcriptional activation by the adenovirus E1a protein

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2599-2605.1992 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2599-2605 ◽

Cited By ~ 1

Author(s):

L S Lum ◽

S Hsu ◽

M Vaewhongs ◽

B Wu

Keyword(s):

Transcriptional Activation ◽

Dependent Manner ◽

Hsp70 Gene ◽

Hsp70 Promoter ◽

Adenovirus E1a ◽

Cell Biol ◽

Binding Factor ◽

E1a Protein

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SCL-mediated regulation of the cell-cycle regulator p21 is critical for murine megakaryopoiesis

Blood ◽

10.1182/blood-2011-01-328765 ◽

2011 ◽

Vol 118 (3) ◽

pp. 723-735 ◽

Cited By ~ 28

Author(s):

Hedia Chagraoui ◽

Mira Kassouf ◽

Sreemoti Banerjee ◽

Nicolas Goardon ◽

Kevin Clark ◽

...

Keyword(s):

Cell Cycle ◽

Transcriptional Control ◽

Cell Cycle Progression ◽

Cellular Proliferation ◽

Loss Of Function ◽

Cycle Progression ◽

Cell Cycle Regulator ◽

Nuclear Changes ◽

Cytoplasmic Maturation

Abstract Megakaryopoiesis is a complex process that involves major cellular and nuclear changes and relies on controlled coordination of cellular proliferation and differentiation. These mechanisms are orchestrated in part by transcriptional regulators. The key hematopoietic transcription factor stem cell leukemia (SCL)/TAL1 is required in early hematopoietic progenitors for specification of the megakaryocytic lineage. These early functions have, so far, prevented full investigation of its role in megakaryocyte development in loss-of-function studies. Here, we report that SCL critically controls terminal megakaryocyte maturation. In vivo deletion of Scl specifically in the megakaryocytic lineage affects all key attributes of megakaryocyte progenitors (MkPs), namely, proliferation, ploidization, cytoplasmic maturation, and platelet release. Genome-wide expression analysis reveals increased expression of the cell-cycle regulator p21 in Scl-deleted MkPs. Importantly, p21 knockdown-mediated rescue of Scl-mutant MkPs shows full restoration of cell-cycle progression and partial rescue of the nuclear and cytoplasmic maturation defects. Therefore, SCL-mediated transcriptional control of p21 is essential for terminal maturation of MkPs. Our study provides a mechanistic link between a major hematopoietic transcriptional regulator, cell-cycle progression, and megakaryocytic differentiation.

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Adenovirus E1A specifically blocks SWI/SNF-dependent transcriptional activation.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5737 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5737-5743 ◽

Cited By ~ 18

Author(s):

M E Miller ◽

B R Cairns ◽

R S Levinson ◽

K R Yamamoto ◽

D A Engel ◽

...

Keyword(s):

Transcriptional Activation ◽

Cellular Transformation ◽

Transcriptional Coactivator ◽

Gene Encoding ◽

Adenovirus E1a ◽

Transcriptional Regulatory ◽

Genes Encoding ◽

Regulatory Complex ◽

Activation Pathways ◽

Coactivator P300

Expression of the adenovirus E1A243 oncoprotein in Saccharomyces cerevisiae produces a slow-growth phenotype with accumulation of cells in the G1 phase of the cell cycle. This effect is due to the N-terminal and CR1 domains of E1A243, which in rodent cells are involved in triggering cellular transformation and also in binding to the cellular transcriptional coactivator p300. A genetic screen was undertaken to identify genes required for the function of E1A243 in S. cerevisiae. This screen identified SNF12, a gene encoding the 73-kDa subunit of the SWI/SNF transcriptional regulatory complex. Mutation of genes encoding known members of the SWI/SNF complex also led to loss of E1A function, suggesting that the SWI/SNF complex is a target of E1A243. Moreover, expression of E1A in wild-type cells specifically blocked transcriptional activation of the INO1 and SUC2 genes, whose activation pathways are distinct but have a common requirement for the SWI/SNF complex. These data demonstrate a specific functional interaction between E1A and the SWI/SNF complex and suggest that a similar interaction takes place in rodent and human cells.

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Transcriptional Activation of the Telomerase hTERT Gene by Human Papillomavirus Type 16 E6 Oncoprotein

Journal of Virology ◽

10.1128/jvi.75.9.4467-4472.2001 ◽

2001 ◽

Vol 75 (9) ◽

pp. 4467-4472 ◽

Cited By ~ 177

Author(s):

Tim Veldman ◽

Izumi Horikawa ◽

J. Carl Barrett ◽

Richard Schlegel

Keyword(s):

Human Papillomavirus ◽

Transcriptional Activation ◽

Transcriptional Control ◽

Regulatory Region ◽

Rnase Protection ◽

Hpv 16 ◽

Htert Gene ◽

Human Papillomavirus Type 16 ◽

E6 Gene

ABSTRACT The E6 and E7 oncogenes of human papillomavirus type 16 (HPV-16) are sufficient for the immortalization of human genital keratinocytes in vitro. The products of these viral genes associate with p53 and pRb tumor suppressor proteins, respectively, and interfere with their normal growth-regulatory functions. The HPV-16 E6 protein has also been shown to increase the telomerase enzyme activity in primary epithelial cells by an unknown mechanism. We report here that a study using reverse transcription-PCR and RNase protection assays in transduced primary human foreskin keratinocytes (HFKs) shows that the E6 gene (but not the E7 gene) increases telomerase hTERT gene transcription coordinately with E6-induced telomerase activity. In these same cells, the E6 gene induces a 6.5-fold increase in the activity of a 1,165-bp 5′ promoter/regulatory region of the hTERT gene, and this induction is attributable to a minimal 251-bp sequence (−211 to +40). Furthermore, there is a 35-bp region (+5 to +40) within this minimal E6-responsive promoter that is responsible for 60% of E6 activity. Although the minimal hTERT promoter contains Myc-responsive E-box elements and recent studies have suggested a role for Myc protein in hTERT transcriptional control, we found no alterations in the abundance of either c-Myc or c-Mad in E6-transduced HFKs, suggesting that there are other or additional transcription factors critical for regulating hTERT expression.

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The Cold-Shock Domain Protein YB-1 Is Important for Cellular Proliferation and the Prevention of Premature Senescence.

Blood ◽

10.1182/blood.v104.11.2571.2571 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2571-2571

Author(s):

Zhi Hong Lu ◽

Jason T. Books ◽

Timothy James Ley

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Cycle Progression ◽

Binding Proteins ◽

Cellular Proliferation ◽

Cold Shock ◽

Premature Senescence ◽

Cycle Progression

Abstract Mammalian proteins containing “cold-shock” domains belong to the most evolutionarily conserved family of nucleic acid-binding proteins known in bacteria, plants, and animals. One of these proteins, YB-1, has been implicated in basic cellular functions such as cell proliferation and responses to environmental stresses. In mammalian cells, YB-1 has been shown to shuttle between the nuclear and cytoplasmic compartments. Within the nucleus, YB-1 interacts with several DNA-and pre-mRNA-binding proteins, and has been implicated in nuclear activities, including transcriptional regulation, chromatin remodeling, and pre-mRNA splicing. YB-1 is also abundant in the cytoplasm, where it binds nonspecifically to mRNA, and may act as a general regulator of mRNA stability, cytoplasmic localization, and translation. Thus, YB-1 has been proposed to function as a multifunctional regulator for the control of gene expression in both the nucleus and cytoplasm. YB-1 overexpression has been frequently detected in a variety of human cancers, often associated with unfavorable clinical outcomes. However, it remains unclear whether YB-1 overexpression contributes directly to the malignant phenotype, or whether it is simply a non-causal “marker” associated with rapid cell growth (and poor prognostic outcomes). To further assess the role of this protein in health and disease, we created mice deficient for YB-1. Complete loss of function of this gene results in fully-penetrant late embryonic and perinatal lethality. Morphological and histological analyses revealed that YB-1−/− embryos displayed major developmental and functional defects, including neurological abnormalities, hemorrhage, and respiratory failure, which probably contributed to lethality. Growth retardation occurred in all late-stage embryos, and was the result of hypoplasia in multiple organ systems. Consistent with these in vivo results, fibroblasts isolated from YB-1−/− embryos (MEFs) grew slowly and entered senescence prematurely in vitro; these defects were rescued by ectopic expression of a GFP-tagged human YB-1 cDNA. This data suggests that YB-1 plays an important cell-autonomous role in cell proliferation and prevention of premature senescence. We further showed that loss of YB-1 in early passage MEFs resulted a delay in G0/G1 to S-phase progression, and a defect in a transcriptional mechanism that normally represses the expression of the G1-specific CDK inhibitor gene p16Ink4a, and the p53 target genes p21Cip1 and Mdm2. However, YB-1 does not cause “global” changes in the transcriptome, the proteome, or protein synthesis efficiency. As predicted, p16Ink4a and p21Cip1 double knockdown by siRNA treatment led to an increase in the rate of cell proliferation, and an extension of proliferative capacity during late passages in YB-1−/− cells. Furthermore, YB-1 deficiency reduced the ability of MEFs to proliferate normally in response to c-Myc overexpression. In conclusion, our data has revealed that YB-1 is required for normal mouse development and survival, and that it plays an important role in supporting rapid cellular proliferation both in vivo and in vitro. Our data further suggests that YB-1 is a cell cycle progression regulator that is important for preventing the early onset of senescence in cultured MEF cells. This data raises the possibility that disregulated expression of YB-1 may contribute to malignant phenotypes by supporting rapid cell cycle progression, and by protecting cells from cytotoxic stresses.

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Cyclin A expression is under negative transcriptional control during the cell cycle.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3789 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3789-3798 ◽

Cited By ~ 73

Author(s):

X Huet ◽

J Rech ◽

A Plet ◽

A Vié ◽

J M Blanchard

Keyword(s):

Cell Cycle ◽

Transcriptional Repression ◽

Transcriptional Control ◽

Cell Cycle Progression ◽

Cyclin A ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Proliferating Cells

Transcription of the gene coding for cyclin A, a protein required for S-phase transit, is cell cycle regulated and is restricted to proliferating cells. To further explore transcriptional regulation linked to cell division cycle control, a genomic clone containing 5' flanking sequences of the murine cyclin A gene was isolated. When it was fused to a luciferase reporter gene, it was shown to function as a proliferation-regulated promoter in NIH 3T3 cells. Transcription of the mouse cyclin A gene is negatively regulated by arrest of cell proliferation. A mutation of a GC-rich sequence conserved between mice and humans is sufficient to relieve transcriptional repression, resulting in a promoter with constitutively high activity. In agreement with this result, in vivo footprinting reveals a protection of the cell cycle-responsive element in G0/early G1 cells which is not observed at later stages of the cell cycle. Moreover, the footprint is present in dimethyl sulfoxide-induced differentiating and not in proliferating Friend erythroleukemia cells. Conversely, two other sites, which in vitro bind ATF-1 and NF-Y, respectively, are constitutively occupied throughout cell cycle progression.

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Characterization of the iPSC-derived conditioned medium that promotes the growth of bovine corneal endothelial cells

PeerJ ◽

10.7717/peerj.6734 ◽

2019 ◽

Vol 7 ◽

pp. e6734

Author(s):

Qing Liu ◽

Yonglong Guo ◽

Shiwei Liu ◽

Peiyuan Wang ◽

Yunxia Xue ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Conditioned Medium ◽

Cell Morphology ◽

Cell Cycle Progression ◽

Cellular Proliferation ◽

Activin A ◽

Corneal Endothelial Cells ◽

Induced Pluripotent

Corneal endothelial cells (CECs) maintain corneal transparency and visual acuity. However, the limited proliferative capability of these cells in vitro has prompted researchers to find efficient culturing techniques for them. The aim of our study was to evaluate the use of conditioned medium (CM) obtained from induced pluripotent stem cells (iPSCs) as a source for the effective proliferation of bovine CECs (B-CECs). In our study, the proliferative ability of B-CECs was moderately enhanced when the cells were grown in 25% iPSC conditioned medium (iPSC-CM). Additionally, hexagonal cell morphology was maintained until passage 4, as opposed to the irregular and enlarged shape observed in control corneal endothelial medium (CEM). B-CECs in both the 25% iPSC-CM and CEM groups expressed and Na+-K+-ATPase. The gene expression levels of NIFK, Na+-K+-ATPase, Col4A and Col8A and the percentage of cells entering S and G2 phases were higher in the iPSC-CM group. The number of apoptotic cells also decreased in the iPSC-CM group. In comparison to the control cultures, iPSC-CM facilitated cell migration, and these cells showed better barrier functions after several passages. The mechanism of cell proliferation mediated by iPSC-CM was also investigated, and phosphorylation of Akt was observed in B-CECs after exposure to iPSC-CM and showed sustained phosphorylation induced for up to 180 min in iPSC-CM. Our findings indicate that iPSC-CM may employ PI3-kinase signaling in regulating cell cycle progression, which can lead to enhanced cellular proliferation. Effective component analysis of the CM showed that in the iPSC-CM group, the expression of activin-A was significantly increased. If activin-A is added as a supplement, it could help to maintain the morphology of the cells, similar to that of CM. Hence, we conclude that activin-A is one of the effective components of CM in promoting cell proliferation and maintaining cell morphology.

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The Adrenomedullin Gene Is a Target for Negative Regulation by the Myc Transcription Complex

Molecular Endocrinology ◽

10.1210/mend.13.2.0240 ◽

1999 ◽

Vol 13 (2) ◽

pp. 254-267 ◽

Cited By ~ 5

Author(s):

Xueyan Wang ◽

Mette A. Peters ◽

Fransiscus E. Utama ◽

Yuzhen Wang ◽

Elizabeth J. Taparowsky

Keyword(s):

Gene Expression ◽

Cell Line ◽

Cell Lines ◽

Sequence Similarity ◽

Constitutive Expression ◽

Cellular Transformation ◽

Nucleotide Sequence Analysis ◽

Adenovirus E1a ◽

Major Late Promoter ◽

Ras P21

Abstract The Myc family of transcription factors plays a central role in vertebrate growth and development although relatively few genetic targets of the Myc transcription complex have been identified. In this study, we used mRNA differential display to investigate gene expression changes induced by the overexpression of the MC29 v-Myc oncoprotein in C3H10T1/2 mouse fibroblasts. We identified the transcript of the adrenomedullin gene (AM) as an mRNA that is specifically down-regulated in v-Myc overexpressing C3H10T1/2 cell lines as well as in a Rat 1a cell line inducible for c-Myc. Nucleotide sequence analysis of the mouse AM promoter reveals the presence of consensus CAAT and TATA boxes as well as an initiator element (INR) with significant sequence similarity to the INR responsible for Myc-mediated repression of the adenovirus major late promoter (AdMLP). Reporter gene assays confirm that the region of the AM promoter containing the INR is the target of Myc-mediated repression. Exogenous application of AM peptide to quiescent C3H10T1/2 cultures does not stimulate growth, and constitutive expression of AM mRNA in C3H10T1/2 cells correlates with a reduced potential of the cells to be cotransformed by v-Myc and oncogenic Ras p21. Additional studies showing that AM mRNA is underrepresented in C3H10T1/2 cell lines stably transformed by Ras p21 or adenovirus E1A suggest that AM gene expression is incompatible with deregulated growth in this cell line. We propose a model in which the repression of AM gene expression by Myc is important to the role of this oncoprotein as a potentiator of cellular transformation in C3H10T1/2 and perhaps other cell lines.

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