Distinguishable promoter elements are involved in transcriptional activation by E1a and cyclic AMP

The sequence motif CGTCA is critical for binding of a group of cellular transcription factors (ATF, CREB, E4F, and EivF) and for activation of certain E1a-inducible and cyclic AMP (cAMP)-inducible promoters. We have tested different promoter elements containing the CGTCA motif (referred to here as ATF-binding sites) for the ability to function as E1a or cAMP response elements. The adenovirus E4 promoter and the cellular vasoactive intestinal peptide (VIP) promoter responded differently to E1a and cAMP, demonstrating that the activating potential of ATF-binding sites within these promoters is not equivalent. While particular ATF-binding sites were sufficient for the activity of both the E4 (E1a inducibility) and VIP (cAMP inducibility) enhancers, these two enhancers had contrasting effects on E1a- and cAMP-inducible transcription. Thus, the relationship between E1a- and cAMP-inducible transcription is not simply explained by the action of these two inducers through the same promoter elements.

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Myocyte nuclear factor, a novel winged-helix transcription factor under both developmental and neural regulation in striated myocytes

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4596-4605.1994 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4596-4605

Author(s):

R Bassel-Duby ◽

M D Hernandez ◽

Q Yang ◽

J M Rochelle ◽

M F Seldin ◽

...

Keyword(s):

Transcription Factors ◽

Transcriptional Activation ◽

Myogenic Differentiation ◽

Specific Binding ◽

Motor Nerve ◽

Sequence Motif ◽

Nuclear Factor ◽

Winged Helix ◽

Amino Terminal ◽

Myocyte Nuclear Factor

A sequence motif (CCAC box) within an upstream enhancer region of the human myoglobin gene is essential for transcriptional activity in both cardiac and skeletal muscle. A cDNA clone, myocyte nuclear factor (MNF), was isolated from a murine expression library on the basis of sequence-specific binding to the myoglobin CCAC box motif and was found to encode a novel member of the winged-helix or HNF-3/fork head family of transcription factors. Probes based on this sequence identify two mRNA species that are upregulated during myocyte differentiation, and antibodies raised against recombinant MNF identify proteins of approximately 90, 68, and 65 kDa whose expression is regulated following differentiation of myogenic cells in culture. In addition, the 90-kDa form of MNF is phosphorylated and is upregulated in intact muscles subjected to chronic motor nerve stimulation, a potent stimulus to myoglobin gene regulation. Amino acid residues 280 to 389 of MNF demonstrate 35 to 89% sequence identity to the winged-helix domain from other known members of this family, but MNF is otherwise divergent. A proline-rich amino-terminal region (residues 1 to 206) of MNF functions as a transcriptional activation domain. These studies provide the first evidence that members of the winged-helix family of transcription factors have a role in myogenic differentiation and in remodeling processes of adult muscles that occur in response to physiological stimuli.

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Covalent labelling of ligand binding sites of human placental S-adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP

Biochemical Journal ◽

10.1042/bj2320643 ◽

1985 ◽

Vol 232 (3) ◽

pp. 643-650 ◽

Cited By ~ 10

Author(s):

V N Aiyar ◽

M S Hershfield

Keyword(s):

Cyclic Amp ◽

Binding Sites ◽

Catalytic Mechanism ◽

Periodate Oxidation ◽

Regulatory Subunit ◽

Adenosylhomocysteine Hydrolase ◽

Dependent Protein ◽

Covalently Linked ◽

The Relationship ◽

Derivatives Of

S-Adenosylhomocysteine hydrolase (AdoHcyase) has previously been identified as a cytoplasmic adenosine and cyclic AMP binding protein. In order to examine the relationship between the adenosine and cyclic AMP binding sites on this enzyme we have explored the use of 8-azido analogues of adenosine and cyclic AMP as photoaffinity reagents for covalently labelling AdoHcyase purified from human placenta. 8-Azidoadenosine (8-N3-Ado), like adenosine, inactivated AdoHcyase, and the rate of inactivation was greatly increased by periodate oxidation. In addition, 8-N3-Ado was found to participate in the first step in the catalytic mechanism for AdoHcyase, resulting in conversion of enzyme-bound NAD+ to NADH, although it was not a substrate for the full enzyme-catalysed reaction. Radioactively labelled 8-N3-Ado, its periodate-oxidized derivative and 8-azidoadenosine 3′, 5′-phosphate (8-N3-cAMP) bound specifically to adenosine binding sites on AdoHcyase and, after irradiation, became covalently linked to the enzyme. Photoaffinity-labelled enzyme could be precipitated by monoclonal antibody to human AdoHcyase. Two observations suggested that cyclic AMP and adenosine bind to the same sites on AdoHcyase. First cyclic AMP and adenosine each blocked binding of both radioactively labelled 8-N3-Ado and 8-N3-cAMP, and second, digestion with V8 proteinase generated identical patterns of peptides from AdoHcyase that had been photolabelled with [32P]8-N3-cAMP and [3H]8-N3-Ado. Binding sites for cyclic AMP on AdoHcyase were found to differ functionally and structurally from cyclic AMP binding sites on the R1 regulatory subunit of cyclic AMP-dependent protein kinase.

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Characterization of an E1A-CBP Interaction Defines a Novel Transcriptional Adapter Motif (TRAM) in CBP/p300

Journal of Virology ◽

10.1128/jvi.73.5.3574-3581.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 3574-3581 ◽

Cited By ~ 54

Author(s):

Mark J. O’Connor ◽

Holger Zimmermann ◽

Søren Nielsen ◽

Hans-Ulrich Bernard ◽

Tony Kouzarides

Keyword(s):

Transcription Factors ◽

Cell Fate ◽

Transcriptional Activation ◽

Molecular Model ◽

P53 Protein ◽

Small Region ◽

Adenovirus E1a ◽

Cellular Transcription

ABSTRACT The adenovirus E1A protein subverts cellular processes to induce mitotic activity in quiescent cells. Important targets of E1A include members of the transcriptional adapter family containing CBP/p300. Competition for CBP/p300 binding by various cellular transcription factors has been suggested as a means of integrating different signalling pathways and may also represent a potential mechanism by which E1A manipulates cell fate. Here we describe the characterization of the interaction between E1A and the C/H3 region of CBP. We define a novel conserved 12-residue transcriptional adapter motif (TRAM) within CBP/p300 that represents the binding site for both E1A and numerous cellular transcription factors. We also identify a sequence (FPESLIL) within adenovirus E1A that is required to bind the CBP TRAM. Furthermore, an E1A peptide containing the FPESLIL sequence is capable of preventing the interaction between CBP and TRAM-binding transcription factors, such as p53, E2F, and TFIIB, thus providing a molecular model for E1A action. As an in vivo demonstration of this model, we used a small region of CBP containing a functional TRAM that can bind to the p53 protein. The CBP TRAM binds p53 sequences targeted by the cellular regulator MDM2, and we demonstrate that an MDM2-p53 interaction can be disrupted by the CBP TRAM, leading to stabilization of cellular p53 levels and the activation of p53-dependent transcription. Transcriptional activation of p53 by the CBP TRAM is abolished by wild-type E1A but not by a CBP-binding-deficient E1A mutant.

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The replication activation potential of selected RNA polymerase II promoter elements at the simian virus 40 origin

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3087-3093.1992 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3087-3093

Author(s):

A T Hoang ◽

W Wang ◽

J D Gralla

Keyword(s):

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Binding Sites ◽

Simian Virus 40 ◽

Simian Virus ◽

Cos Cells ◽

Activation Potential ◽

Multiple Copies ◽

Cellular Transcription ◽

Transcriptional Stimulation

Binding sites for cellular transcription factors were placed near the simian virus 40 origin of replication, and their effect on replication and TATA-dependent transcription was measured in COS cells. The hierarchy of transcriptional stimulation changed when the plasmids replicated. Only one of seven inserted sequences, a moderately weak transcription element, stimulated replication detectably. However, when two nonstimulatory sites were present in multiple copies they did activate replication. Multiple sites for the chimeric activator GAL4-VP16 did not stimulate replication even though transcription was stimulated strongly. The results indicate that the ability of a binding site to stimulate replication from the simian virus 40 ori is not based on its transcriptional activation potential but is instead related to a separate replication activation potential that can be increased by having multiple sites.

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Mechanisms of Transcriptional Regulation and Transformation by HOXA9

Blood ◽

10.1182/blood.v120.21.sci-30.sci-30 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-30-SCI-30

Author(s):

Jay L. Hess ◽

Cailin Collins ◽

Joel Bronstein ◽

Yuqing Sun ◽

Surya Nagaraja

Keyword(s):

Transcriptional Regulation ◽

Transcription Factors ◽

Transcriptional Activation ◽

Binding Sites ◽

Target Genes ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

A Genome ◽

The Impact

Abstract Abstract SCI-30 HOXA9 plays important roles in both development and hematopoiesis and is overexpressed in more than 50 percent of acute myeloid leukemias (AML). Nearly all cases of AML with mixed lineage leukemia (MLL) translocations show increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. In most cases, upregulation of HOXA9 is accompanied by upregulation of its homeodomain-containing cofactor MEIS1, which directly interacts with HOXA9. While HOXA9 alone is sufficient for transformation of hematopoietic stem cells in culture, the addition of MEIS1 increases the transformation efficiency and results in rapidly fatal leukemias in transplanted animals. Despite the crucial role that HOXA9 plays in development, hematopoiesis, and leukemia, its transcriptional targets and mechanisms of action are poorly understood. We have used ChIP-seq to identify Hoxa9 and Meis1 binding sites on a genome-wide level in myeloblastic cells, profiled their associated epigenetic modifications, identified the target genes regulated by HOXA9 and identified HOXA9 interacting proteins. HOXA9 and MEIS1 cobind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/P300 binding. These include many proleukemogenic gene loci, such as Erg, Flt3, Myb, Lmo2, and Sox4. In addition, HOXA9 binding sites overlap a subset of enhancers previously implicated in myeloid differentiation and inflammation. HOXA9 binding at enhancers stabilizes association of MEIS1 and lineage-restricted transcription factors, including C/EBPα, PU.1, and STAT5A/B thereby promoting CBP/p300 recruitment, histone acetylation, and transcriptional activation. Current efforts are focused on using both biochemical and genetic approaches to assess the role of HOXA9 “enhanceosome” components C/EBPα, PU.1, and STAT5A/B in transcriptional regulation and leukemogenesis. Studies to date suggest that C/EBPα and PU.1 binding can occur in the absence of HOXA9/MEIS1, supporting a model in which these proteins act as pioneer transcription factors for establishment of poised, but not activated, HOXA9-regulated enhancers. Work is under way to assess the impact of high-level HOXA9 and MEIS1 on enhanceosome assembly and the role of recruitment of transcriptional coactivators involved in target gene up- or downregulation, including histone acetyltransferases and chromatin remodeling complexes. Collectively, our findings suggest that HOXA9-regulated enhancers are a fundamental mechanism of HOX-mediated transcription in normal development that is deregulated in leukemia. Disclosures: No relevant conflicts of interest to declare.

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Nuclear microenvironments modulate transcription from low-affinity enhancers

eLife ◽

10.7554/elife.28975 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 45

Author(s):

Albert Tsai ◽

Anand K Muthusamy ◽

Mariana RP Alves ◽

Luke D Lavis ◽

Robert H Singer ◽

...

Keyword(s):

Transcription Factor ◽

Drosophila Melanogaster ◽

Transcription Factors ◽

Dna Sequences ◽

Transcriptional Activation ◽

Binding Sites ◽

General Feature ◽

Inverse Relationship ◽

Chromosomal Location ◽

Affinity Binding

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here, we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.

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The replication activation potential of selected RNA polymerase II promoter elements at the simian virus 40 origin.

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3087 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3087-3093 ◽

Cited By ~ 8

Author(s):

A T Hoang ◽

W Wang ◽

J D Gralla

Keyword(s):

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Binding Sites ◽

Simian Virus 40 ◽

Simian Virus ◽

Cos Cells ◽

Activation Potential ◽

Multiple Copies ◽

Cellular Transcription ◽

Transcriptional Stimulation

Binding sites for cellular transcription factors were placed near the simian virus 40 origin of replication, and their effect on replication and TATA-dependent transcription was measured in COS cells. The hierarchy of transcriptional stimulation changed when the plasmids replicated. Only one of seven inserted sequences, a moderately weak transcription element, stimulated replication detectably. However, when two nonstimulatory sites were present in multiple copies they did activate replication. Multiple sites for the chimeric activator GAL4-VP16 did not stimulate replication even though transcription was stimulated strongly. The results indicate that the ability of a binding site to stimulate replication from the simian virus 40 ori is not based on its transcriptional activation potential but is instead related to a separate replication activation potential that can be increased by having multiple sites.

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Regulation of cyclin E gene expression by the human papillomavirus type 16 E7 oncoprotein

Journal of General Virology ◽

10.1099/0022-1317-80-8-2103 ◽

1999 ◽

Vol 80 (8) ◽

pp. 2103-2113 ◽

Cited By ~ 9

Author(s):

Beate Vogt ◽

Karin Zerfaß-Thome ◽

Almut Schulze ◽

Jürgen W. Botz ◽

Werner Zwerschke ◽

...

Keyword(s):

Human Papillomavirus ◽

Transcriptional Activation ◽

Binding Sites ◽

Cyclin E ◽

Regulatory Region ◽

Regulatory Elements ◽

E Gene ◽

Human Papillomavirus Type 16 ◽

Activation Of Transcription ◽

Cellular Transcription

In this study, we characterized the 5′ regulatory region of the murine cyclin E gene and analysed activation of the gene by the E7 oncogene of human papillomavirus type 16 in transfection experiments. We found that the murine cyclin E promoter is composed of multiple regulatory elements, and we present evidence for at least two independent transcription units, designated P1 and P2. Overlapping binding sites for the cellular transcription factors Sp1 and E2F were identified in both promoters, and we found that E2F-mediated activation of transcription is inhibited by Sp1 in cotransfection experiments. The E2F/Sp1 binding sites contribute to transcriptional activation by E7, and the data suggest that the cyclin E gene is rendered E7-inducible through the combination of several cis-acting elements which display only weak intrinsic responsiveness to E7.

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Nuclear microenvironments modulate transcription from low-affinity enhancers

10.1101/128280 ◽

2017 ◽

Cited By ~ 3

Author(s):

Justin Crocker ◽

Albert Tsai ◽

Anand K. Muthusamy ◽

Luke D. Lavish ◽

Robert H. Singer ◽

...

Keyword(s):

Transcription Factors ◽

Dna Sequences ◽

Transcriptional Activation ◽

Binding Sites ◽

General Feature ◽

Inverse Relationship ◽

Affinity Binding ◽

Regulate Gene Expression ◽

High Concentrations ◽

Regulate Gene

AbstractTranscription factors regulate gene expression by binding to DNA for short durations and by often binding to low-affinity DNA sequences. It is not clear how such temporally brief, low-affinity interactions can drive efficient transcription. Here we report that the transcription factor Ultrabithorax (Ubx) functionally utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus in nuclear microenvironments of relatively high Ubx concentration. By manipulating the affinity of svb enhancers, we revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. A Ubx cofactor, Homothorax (Hth), was enriched together with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. These results suggest that low affinity sites overcome their kinetic inefficiency by utilizing microenvironments with high concentrations of transcription factors and cofactors. Mechanisms that generate these microenvironments are likely to be a general feature of eukaryotic transcriptional regulation.

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