GHF-1/Pit-1 Functions as a Cell-specific Integrator of Ras Signaling by Targeting the Ras Pathway to a Composite Ets-1/GHF-1 Response Element

Bradford, Andrew P.; Conrad, Kerry E.; Tran, Phat H.; Ostrowski, Michael C.; Gutierrez‐Hartmann, Arthur

doi:10.1074/jbc.271.40.24639

Cited by 59 publications

(95 citation statements)

References 68 publications

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“…Future studies will define the synergistic domains of Pit-1 with GATA-2 on the TSH␤ promoter, as well as the specific sites of protein-protein interaction. Such factor interactions with Pit-1 may represent a more general mechanism whereby a tissue-restricted homeodomain factor can recruit widely expressed factors to a particular composite element and integrate high levels of tissue-specific gene expression, as has been proposed by Gutierrez-Hartmann's group (64,65).…”

Section: Discussionmentioning

confidence: 99%

Pit-1 and GATA-2 Interact and Functionally Cooperate to Activate the Thyrotropin β-Subunit Promoter

Gordon

Lewis

Haugen

et al. 1997

Journal of Biological Chemistry

128

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The molecular determinants governing cell-specific expression of the thyrotropin (TSH) ␤-subunit gene in pituitary thyrotropes are not well understood. The P1 region of the mouse TSH␤ promoter (؊133 to ؊88) region interacts with Pit-1 and an additional 50-kDa factor at an adjacent site that resembles a consensus GATA binding site. Northern and Western blot assays demonstrated the presence of GATA-2 transcripts and protein in TtT-97 thyrotropic tumors. In electrophoretic mobility shift assays, a comigrating complex was observed with both TtT-97 nuclear extracts and GATA-2 expressed in COS cells. The complex demonstrated binding specificity to the P1 region DNA probe and could be disrupted by a GATA-2 antibody. When both Pit-1 and GATA-2 were combined, a slower migrating complex, indicative of a ternary protein-DNA interaction was observed. Cotransfection of both Pit-1 and GATA-2 into CV-1 cells synergistically stimulated mouse TSH␤ promoter activity 8.5-fold, while each factor alone had a minimal effect. Mutations that abrogated this functional stimulatory effect mapped to the P1 region. Finally, we show that GATA-2 directly interacts with Pit-1 in solution. In summary, these data demonstrate functional synergy and physical interaction between homeobox and zinc finger factors and provide insights into the transcriptional mechanisms of thyrotrope-specific gene expression.Cell-specific expression of eukaryotic genes involves the functional interaction of sets of transcription factors that interact with essential cis-acting promoter regions to initiate RNA transcription. The repertoires of transcription factors that are involved in the expression of genes in highly differentiated cells often consist of those ubiquitously expressed in many cell types in cooperation with others whose expression are cell type-restricted (1, 2). Expression of the thyrotropin (TSH) 1

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Section: Discussionmentioning

confidence: 99%

Pit-1 and GATA-2 Interact and Functionally Cooperate to Activate the Thyrotropin β-Subunit Promoter

Gordon

Lewis

Haugen

et al. 1997

Journal of Biological Chemistry

128

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“…It is also probable that the activation of specific genes by p42Ets-1 is due to the interaction of the protein with a different set of transcriptional coregulators than p51Ets-1; p51Ets-1 activity is regulated by cofactors such as CBP/p300 (Yang et al, 1998;Jayaraman et al, 1999), pit-1 (Howard andMaurer, 1995;Bradford et al, 1996Bradford et al, , 1997 or pax-5 (Fitzsimmons et al, 1996;Garvie et al, 2001), which interact with different regions of Ets-1. Thus, p51Ets-1 and p42Ets-1 activities could be regulated by common cofactors such as Pit-1, because it interacts with a region different from the exon VIIcoded fragment (Bradford et al, 1995;Bradford et al, 1997) or more specifically by cofactors which interact with the exon VII-coded region, such as by CBFa2 (Goetz et al, 2000), which would not affect p42Ets-1 function.…”

Section: Discussionmentioning

confidence: 99%

Characterization and functional analysis of the p42Ets-1 variant of the mouse Ets-1 transcription factor

et al. 2003

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We have identified the mouse exon VII splice variant of the Ets-1 transcription factor. The variant is expressed in all cell lines which express ets-1, at lower levels, it is also expressed in the mouse embryo in vivo. The corresponding protein, p42Ets-1, is a transcription factor as it is able to bind to specific DNA sequences and to transactivate a bona fide ETS reporter vector. A comparison of optimal DNA-binding sites shows that p42Ets-1 binds to more various DNA sequences than p51Ets-1; p42Ets-1 recognizes the same optimal consensus sequence as p51Ets-1, but also many variations of it, mainly at base À1, which is located just prior to the GGAA/T core sequence. The binding differences were quantified by surface plasmon resonance analyses and the protein region responsible for the differences in DNA sequence recognition located in the Val 280 -Glu 302 fragment, which is encoded by exon VII. The specific DNA-binding properties of each isoform translates into clear differences in activity, p42Ets-1 transactivates the natural VE-cadherin gene promoter through both ETS-binding site (EBS)2 and EBS4 whereas p51Ets-1 is mainly active on EBS4. Altogether, our data suggest that p42Ets-1 acts as a distinct transcription factor from p51Ets-1. Oncogene (2003) 22, 9156-9164.

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“…Mutations mEBS and mFPIV have been previously described (11). Mutations of pA3-425PRL-Luc including monomer, dimer, 12 bp, 16 bp, and 24 bp were generated by overlap extension PCR amplification from Ϫ425PRLpGEM7 using mutant internal primers and SP6 and T7 primers with dual rounds of amplification.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…For example, transient transfection of pituitary-derived GH4T2 somatolactotrope cells with a dominant-negative Ets factor encoding only the Ets DNA binding domain (DBD), or with Pit-1␤ (an alternatively spliced isoform that functions as a dominant-negative effector in pituitary cells), results in diminished activity of the rPRL promoter in an effector-dependent manner (3). Similarly, transient transfection of rPRL promoter constructs containing site-specific mutations of either the EBS or FPIV sites within the composite element results in a loss of Ras responsiveness (10,11). In non-pituitary HeLa cells, it has been shown that although individual Ets-1 or Pit-1 effectors strongly activate the rPRL promoter, the combination results in a response that is 6-fold greater than the sum of the individual responses (i.e.…”

mentioning

confidence: 99%

Ras Signaling and Transcriptional Synergy at a Flexible Ets-1/Pit-1 Composite DNA Element Is Defined by the Assembly of Selective Activation Domains

Duval

Jean

Gutierrez‐Hartmann

2003

Journal of Biological Chemistry

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Pit-1 and Ets-1 binding to a composite element synergistically activates and targets Ras-mitogen-activated protein kinase signaling to the rat prolactin promoter. These transcriptional responses appear to depend on three molecular features: organization of the Ets-1/Pit-1 composite element, physical interaction of these two factors via the Pit-1 homeodomain (amino acids 199 -291) and the Ets-1 regulatory III domain (amino acids 190 -257), and assembly of their transcriptional activation domains (TADs). Here we show that the organization of the Ets-1/Pit-1 composite element tolerates significant flexibility with regard to Ras stimulation and synergy. Specifically, the putative monomeric Pit-1 binding site can be substituted with bona fide binding sites for either a Pit-1 monomer or dimer, and these sites tolerated a separation of 28 bp. Additionally, we show that the physical interaction of Ets-1 and Pit-1 is not required for Ras responsiveness or synergy because block mutations of the Pit-1 interaction surface in Ets-1, which reduced Ets-1/Pit-1 binding in vitro, did not significantly affect Ets-1 stimulation of Ras responsiveness or synergy. We also show differential use of distinct TAD subtypes and Pit-1 TAD subregions to mediate either synergy or Ras responsiveness. Specifically, TADs from Gal4, VP16, or Ets-2 regulatory III domain linked to Ets-1 DNA binding domain constructs restored synergy to these TAD/Ets-1 DNA binding domain fusions. Conversely, deletion of the defined Pit-1 TAD (amino acids 2-80) retained synergy, but not Ras responsiveness. Consequently, we further defined the Pit-1 amino-terminal TAD into region 1 (R1, amino acids 2-45) and region 2 (R2, amino acids 46 -80). R1 appears to regulate basal and synergistic responses, whereas the Ras response was mapped to R2. In summary, Ras responsiveness and Pit-1/Ets-1 synergy are mediated through the assembly of distinct TADs at a flexible composite element, indicating that different mechanisms underlie these two transcriptional responses and that the Pit-1 R2 subregion represents a novel, tissue-specific Ras-responsive TAD.

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GHF-1/Pit-1 Functions as a Cell-specific Integrator of Ras Signaling by Targeting the Ras Pathway to a Composite Ets-1/GHF-1 Response Element

Cited by 59 publications

References 68 publications

Pit-1 and GATA-2 Interact and Functionally Cooperate to Activate the Thyrotropin β-Subunit Promoter

Pit-1 and GATA-2 Interact and Functionally Cooperate to Activate the Thyrotropin β-Subunit Promoter

Characterization and functional analysis of the p42Ets-1 variant of the mouse Ets-1 transcription factor

Ras Signaling and Transcriptional Synergy at a Flexible Ets-1/Pit-1 Composite DNA Element Is Defined by the Assembly of Selective Activation Domains

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