scholarly journals Determination of the consensus DNA-binding sequence and a transcriptional activation domain for ESE-2

2006 ◽  
Vol 398 (3) ◽  
pp. 497-507 ◽  
Author(s):  
Yeon Sook Choi ◽  
Satrajit Sinha
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Regulatory Elements ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Ets Proteins ◽  
Flanking Sequences ◽  
Binding Sequence

The ESE (epithelium-specific Ets) subfamily of Ets transcription factors plays an important role in regulating gene expression in a variety of epithelial cell types. Although ESE proteins have been shown to bind to regulatory elements of some epithelial genes, the optimal DNA-binding sequence has not been experimentally ascertained for any member of the ESE subfamily of transcription factors. This has made the identification and validation of their targets difficult. We are studying ESE-2 (Elf5), which is highly expressed in epithelial cells of many tissues including skin keratinocytes. Here, we identify the preferred DNA-binding site of ESE-2 by performing CASTing (cyclic amplification and selection of targets) experiments. Our analysis shows that the optimal ESE-2 consensus motif consists of a GGA core and an AT-rich 5′- and 3′-flanking sequences. Mutational and competition experiments demonstrate that the flanking sequences that confer high DNA-binding affinity for ESE-2 show considerable differences from the known consensus DNA-binding sites of other Ets proteins, thus reinforcing the idea that the flanking sequences may impart recognition specificity for Ets proteins. In addition, we have identified a novel isoform of murine ESE-2, ESE-2L, that is generated by use of a hitherto unreported new exon and an alternate promoter. Interestingly, transient transfection assays with an optimal ESE-2 responsive reporter show that both ESE-2 and ESE-2L are weak transactivators. However, similar studies utilizing GAL4 chimaeras of ESE-2 demonstrate that while the DNA-binding ETS (E twenty-six) domain functions as a repressor, the PNT (pointed domain) of ESE-2 can act as a potent transcriptional activation domain. This novel transactivating property of PNT is also shared by ESE-3, another ESE family member. Identification of the ESE-2 consensus site and characterization of the transcriptional activation properties of ESE-2 shed new light on its potential as a regulator of target genes.

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

scholarly journals Stress-induced binding of the transcriptional factor CHOP to a novel DNA control element.

1996 ◽  
Vol 16 (4) ◽  
pp. 1479-1489 ◽  
Author(s):  
M Ubeda ◽  
X Z Wang ◽  
H Zinszner ◽  
I Wu ◽  
J F Habener ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Activity ◽  
Control Element ◽  
Core Element ◽  
Transcriptional Activation Domain ◽  
Dna Binding Activity ◽  
Basic Region

CHOP (GADD153) is a mammalian nuclear protein that dimerizes with members of the C/EBP family of transcriptional factors. Absent under normal conditions, CHOP is induced by the stress encountered during nutrient deprivation, the acute-phase response, and treatment of cells with certain toxins. The basic region of CHOP deviates considerably in sequence from that of other C/EBP proteins, and CHOP-C/EBP heterodimers are incapable of binding to a common class of C/EBP sites. With respect to such sites, CHOP serves as an inhibitor of the activity of C/EBP proteins. However, recent studies indicate that certain functions of CHOP, such as the induction of growth arrest by overexpression of the wild-type protein and oncogenic transformation by the TLS-CHOP fusion protein, require an intact basic region, suggesting that DNA binding by CHOP may be implicated in these activities. In this study an in vitro PCR-based selection assay was used to identify sequences bound by CHOP-C/EBP dimers. These sequences were found to contain a unique core element PuPuPuTGCAAT(A/C)CCC. Competition in DNA-binding assays, DNase 1 footprint analysis, and methylation interference demonstrate that the binding is sequence specific. Deletions in the basic region of CHOP lead to a loss of DNA binding, suggesting that CHOP participates in this process. Stress induction in NIH 3T3 cells leads to the appearance of CHOP-containing DNA-binding activity. CHOP is found to contain a transcriptional activation domain which is inducible by cellular stress, lending further support to the notion that the protein can function as a positively acting transcription factor. We conclude that CHOP may serve a dual role both as an inhibitor of the ability of C/EBP proteins to activate some target genes and as a direct activator of others.

Misexpression of dHAND induces ectopic digits in the developing limb bud in the absence of direct DNA binding

Development ◽  
2002 ◽  
Vol 129 (13) ◽  
pp. 3077-3088 ◽  
Author(s):  
David G. McFadden ◽  
John McAnally ◽  
James A. Richardson ◽  
Jeroen Charité ◽  
Eric N. Olson
Keyword(s):  
Dna Binding ◽  
Sonic Hedgehog ◽  
Transcriptional Activation ◽  
Function Analysis ◽  
Limb Bud ◽  
Activation Domain ◽  
Limb Patterning ◽  
Transcriptional Activation Domain ◽  
Wide Range ◽  
E Box

Basic helix-loop-helix (bHLH) transcription factors control developmental decisions in a wide range of embryonic cell types. The HLH motif mediates homo- and heterodimerization, which juxtaposes the basic regions within the dimeric complex to form a bipartite DNA binding domain that recognizes a DNA consensus sequence known as an E-box. eHAND and dHAND (also known as HAND1 and HAND2) are closely related bHLH proteins that control cardiac, craniofacial and limb development. Within the developing limb, dHAND expression encompasses the zone of polarizing activity in the posterior region, where it has been shown to be necessary and sufficient to induce the expression of the morphogen sonic hedgehog. Misexpression of dHAND in the anterior compartment of the limb bud induces ectopic expression of sonic hedgehog, with resulting preaxial polydactyly and mirror image duplications of posterior digits. To investigate the potential transcriptional mechanisms involved in limb patterning by dHAND, we have performed a structure-function analysis of the protein in cultured cells and ectopically expressed dHAND mutant proteins in the developing limbs of transgenic mice. We show that an N-terminal transcriptional activation domain, and the bHLH region, are required for E-box-dependent transcription in vitro. Remarkably, however, digit duplication by dHAND requires neither the transcriptional activation domain nor the basic region, but only the HLH motif. eHAND has a similar limb patterning activity to dHAND in these misexpression experiments, indicating a conserved function of the HLH regions of these proteins. These findings suggest that dHAND may act via novel transcriptional mechanisms mediated by protein-protein interactions independent of direct DNA binding.

scholarly journals Cooperative interactions between HOX and PBX proteins mediated by a conserved peptide motif.

1995 ◽  
Vol 15 (8) ◽  
pp. 3989-3997 ◽  
Author(s):  
M L Phelan ◽  
I Rambaldi ◽  
M S Featherstone
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Activity ◽  
Cooperative Interaction ◽  
Transcriptional Activation Domain ◽  
Conserved Sequence ◽  
Dna Binding Activity ◽  
Group Protein

Homeoprotein products of the Hox/HOM gene family pattern the animal embryo through the transcriptional regulation of target genes. We have previously shown that the labial group protein HOXA-1 has intrinsically weak DNA-binding activity due to residues in the N-terminal arm of its homeodomain (M. L. Phelan, R. Sadoul, and M. S. Featherstone, Mol. Cell. Biol. 14:5066-5075, 1994). This observation, among others, suggests that HOX and HOM proteins require cofactors for stable interactions with DNA. We have demonstrated that a putative HOX cofactor, PBX1A, participates in cooperative DNA binding with HOXA-1 and the Deformed group protein HOXD-4. Three Abdominal-B class HOX proteins failed to cooperate with PBX1A. We mapped the interacting domain of HOXD-4 to the YPWMK pentapeptide motif, a conserved sequence found N terminal to the homeodomain of HOXA-1 and many other homeoproteins but absent from the Abdominal-B class. The naturally occurring fusion of the transcriptional activation domain of E2A with PBX1 creates an oncoprotein implicated in human pre-B-cell leukemias (M. P. Kamps, C. Murre, X.-H. Sun, and D. Baltimore, Cell 60:547-555, 1990; J. Nourse, J. D. Mellentin, N. Galili, J. Wilkinson, E. Starbridge, S. D. Smith, and M. L. Cleary, Cell 60:535-545, 1990). A pentapeptide mutation that abolished cooperative interaction with PBX1A in vitro also abrogated synergistic transcriptional activation with the E2A/PBX oncoprotein. The direct contact of PBX family members by the HOX pentapeptide is likely to play an important role in developmental and oncogenic processes.

scholarly journals Intramolecular Regulation of MyoD Activation Domain Conformation and Function

1998 ◽  
Vol 18 (9) ◽  
pp. 5478-5484 ◽  
Author(s):  
Jing Huang ◽  
Hal Weintraub ◽  
Larry Kedes
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Helix Loop Helix ◽  
Dna Binding Specificity ◽  
E Box ◽  
And Function ◽  
Bhlh Proteins ◽  
Basic Region

ABSTRACT The MyoD family of basic helix-loop-helix (bHLH) proteins is required for myogenic determination and differentiation. The basic region carries the myogenic code and DNA binding specificity, while the N terminus contains a potent transcriptional activation domain. Myogenic activation is abolished when the basic region, bound to a myogenic E box, carries a mutation of Ala-114. It has been proposed that DNA binding of the MyoD basic region leads to recruitment of a recognition factor that unmasks the activation domain. Here we demonstrate that an A114N mutant exhibits an altered conformation in the basic region and that this local conformational difference can lead to a more global change affecting the conformation of the activation domain. This suggests that the deleterious effects of this class of mutations may result directly from defective conformation. Thus, the activation domain is unmasked only upon DNA binding by the correct basic region. Such a coupled conformational relationship may have evolved to restrict myogenic specificity to a small number of bHLH proteins among many with diverse functions yet with DNA binding specificities known to be similar.

scholarly journals The cis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements in Drosophila

2018 ◽  
Author(s):  
Arya Zandvakili ◽  
Juli Uhl ◽  
Ian Campbell ◽  
Yuntao Charlie Song ◽  
Brian Gebelein
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Target Genes ◽  
Hox Genes ◽  
Regulatory Elements ◽  
Protease Gene ◽  
Precise Integration

AbstractHox genes encode a family of transcription factors that, despite having similar in vitro DNA binding preferences, regulate distinct genetic programs along the metazoan anterior-posterior axis. To better define mechanisms of Hox specificity, we compared and contrasted the ability of abdominal Hox factors to regulate two cis-regulatory elements within the Drosophila embryo. Both the Ultrabithorax (Ubx) and Abdominal-A (Abd-A) Hox factors form cooperative complexes with the Extradenticle (Exd) and Homothorax (Hth) transcription factors to repress the distal-less leg selector gene via the DCRE, whereas only Abd-A interacts with Exd and Hth on the RhoA element to activate a rhomboid serine protease gene that stimulates Epidermal Growth Factor secretion. By swapping binding sites between these elements, we found that the RhoA Exd/Hth/Hox site configuration that mediates Abd-A specific activation can also convey transcriptional repression by both Ubx and Abd-A when placed into the DCRE, but only in one orientation. We further show that the orientation and spacing of Hox sites relative to additional transcription factor binding sites within the RhoA and DCRE elements is critical to mediate appropriate cell- and segment-specific output. These results indicate that the interaction between Hox, Exd, and Hth neither determines activation vs repression specificity nor defines Ubx vs Abd-A specificity. Instead the precise integration of Hox sites with additional TF inputs is required for accurate transcriptional output. Taken together, these studies provide new insight into the mechanisms of Hox target and regulatory specificity as well as the constraints placed on regulatory elements to convey appropriate outputs.Author SummaryThe Hox genes encode a family of transcription factors that give cells within each region along the developing body plan a unique identity in animals from worms to mammals. Surprisingly, however, most of the Hox factors bind the same or highly similar DNA sequences. These findings raise a paradox: How can proteins that have highly similar DNA binding properties perform different functions in the animal by regulating different sets of target genes? In this study, we address this question by studying how two Hox factors regulate the expression of target genes that specify leg development and the making of liver-like cells in the developing fly. By comparing and contrasting how Hox target genes are activated and/or repressed, we found that the same Hox binding sites can mediate either activation or repression in a manner that depends upon context. In addition, we found that a Hox binding site that is normally regulated by only one Hox factor, can also be used by more than one Hox factor swapped into another target gene. These findings indicate that the specificity of a Hox factor to regulate target genes does not rely solely upon DNA binding specificity but also requires regulatory specificity.

scholarly journals Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs

1993 ◽  
Vol 13 (8) ◽  
pp. 4640-4647
Author(s):  
F E Johansen ◽  
R Prywes
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Binding Domain ◽  
Response Factor ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Serum Response

The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos. Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells. To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4. Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity. First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells. Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain. Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition. Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct. The implications of these inhibitory domains for SRF regulation are discussed.

DNA-binding specificities of the GATA transcription factor family

1993 ◽  
Vol 13 (7) ◽  
pp. 4011-4022
Author(s):  
L J Ko ◽  
J D Engel
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Target Genes ◽  
Polymerase Chain Reaction Amplification ◽  
Consensus Sequence ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Erythroid Cell ◽  
Binding Motif ◽  
Gata Factors

Members of the GATA family of transcription factors, which are related by a high degree of amino acid sequence identity within their zinc finger DNA-binding domains, each show distinct but overlapping patterns of tissue-restricted expression. Although GATA-1, -2, and -3 have been shown to recognize a consensus sequence derived from regulatory elements in erythroid cell-specific genes, WGATAR (in which W indicates A/T and R indicates A/G), the potential for more subtle differences in the binding preferences of each factor has not been previously addressed. By employing a binding selection and polymerase chain reaction amplification scheme with randomized oligonucleotides, we have determined the binding-site specificities of bacterially expressed chicken GATA-1, -2, and -3 transcription factors. Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA-2 and GATA-3 but only poorly by GATA-1. These studies suggest that all three GATA factors are capable of mediating transcriptional effects via a common erythroid consensus DNA-binding motif. Furthermore, GATA-2 and GATA-3, because of their distinct expression patterns and broader DNA recognition properties, may be involved in additional regulatory processes beyond those of GATA-1. The definition of an alternative GATA-2-GATA-3 consensus sequence may facilitate the identification of new target genes in the further elucidation of the roles that these transcription factors play during development.

scholarly journals The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors

2012 ◽  
Vol 70 (5) ◽  
pp. 855-865 ◽  
Author(s):  
Shiv B. Tiwari ◽  
Alemu Belachew ◽  
Siu Fong Ma ◽  
Melinda Young ◽  
Jules Ade ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Transcriptional Activation ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Erf Transcription Factors

Mechanisms regulating target gene selection by the homeodomain-containing protein Fushi tarazu

Development ◽  
2000 ◽  
Vol 127 (13) ◽  
pp. 2965-2976 ◽  
Author(s):  
A. Nasiadka ◽  
A. Grill ◽  
H.M. Krause
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Site ◽  
Target Gene ◽  
Target Genes ◽  
Gene Selection ◽  
Activity Regulation ◽  
Fushi Tarazu ◽  
Activation Domain ◽  
Regulation Model

Homeodomain proteins are DNA-binding transcription factors that control major developmental patterning events. Although DNA binding is mediated by the homeodomain, interactions with other transcription factors play an unusually important role in the selection and regulation of target genes. A major question in the field is whether these cofactor interactions select target genes by modulating DNA binding site specificity (selective binding model), transcriptional activity (activity regulation model) or both. A related issue is whether the number of target genes bound and regulated is a small or large percentage of genes in the genome. In this study, we have addressed these issues using a chimeric protein that contains the strong activation domain of the viral VP16 protein fused to the Drosophila homeodomain-containing protein Fushi tarazu (Ftz). We find that genes previously thought not to be direct targets of Ftz remain unaffected by FtzVP16. Addition of the VP16 activation domain to Ftz does, however, allow it to regulate previously identified target genes at times and in regions that Ftz alone cannot. It also changes Ftz into an activator of two genes that it normally represses. Taken together, the results suggest that Ftz binds and regulates a relatively limited number of target genes, and that cofactors affect target gene specificity primarily by controlling binding site selection. Activity regulation then fine-tunes the temporal and spatial domains of promoter responses, the magnitude of these responses, and whether they are positive or negative.

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