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, Dawn L.; Jean, Annie; Gutierrez‐Hartmann, Arthur

doi:10.1074/jbc.m302433200

Cited by 20 publications

(19 citation statements)

References 43 publications

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“…No effect on the synergistic activity of these proteins at the rat prolactin promoter was observed when Ets-1 proteins carrying mutations on their Pit-1 interaction surface, which significantly reduced binding of the two factors in vitro, were used in in situ assays (6). Ets-1 and Pit-1, however, both require specific DNA binding sites at the promoter.…”

Section: Discussionmentioning

confidence: 99%

Promoter Activation by the Varicella-Zoster Virus Major Transactivator IE62 and the Cellular Transcription Factor USF

Yang

Hua

Hay

et al. 2006

J Virol

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The varicella-zoster virus major transactivator, IE62, can activate expression from homologous and heterologous promoters. High levels of IE62-mediated activation appear to involve synergy with cellular transcription factors. The work presented here focuses on functional interactions of IE62 with the ubiquitously expressed cellular factor USF. We have found that USF can synergize with IE62 to a similar extent on model minimal promoters and the complex native ORF28/29 regulatory element, neither of which contains a consensus IE62 binding site. Using Gal4 fusion constructs, we have found that the activation domain of USF1 is necessary and sufficient for synergistic activation with IE62. We have mapped the regions of USF and IE62 required for direct physical interaction. Deletion of the required region within IE62 does not ablate synergistic activation but does influence its efficiency depending on promoter architecture. Both proteins stabilize/increase binding of TATA binding protein/TFIID to promoter elements. These findings suggest a novel mechanism for the observed synergistic activation which requires neither site-specific IE62 binding to the promoter nor a direct physical interaction with USF.The varicella-zoster virus (VZV) major transcriptional activator, commonly designated the immediate-early 62 protein or IE62, is a potent and promiscuous transactivator of both homologous and heterologous promoters (reviewed in reference 38). The IE62 protein contains 1,310 amino acids (aa), and the major functional domains within the protein with respect to transactivation are an N-terminal acidic activation domain (AD) and a DNA binding domain. The IE62 activation domain (aa 1 to 86) is compositionally similar to other acidic activation domains found in herpes simplex virus VP16 and the pseudorabiesvirus major transactivator but shows little homology to those domains at the individual amino acid level (4, 34).The DNA binding domain of IE62 is contained within aa 468 to 640 of the IE62 sequence. Previous studies showed that bacterially expressed fragments of IE62 containing this region are capable of binding to a variety of VZV and non-VZV promoter elements (2,50,51,53). The ability of IE62 to interact directly with DNA has been shown to be an important aspect of its mechanism of activation since mutations in IE62 which ablate DNA binding also abrogate transactivation (49). However, it is unclear if IE62 is required to bind to a specific sequence. DNase protection studies by Wu and Wilcox (53) identified a consensus sequence (-ATCGT-) to which a recombinant fusion protein containing the IE62 DNA binding domain bound tightly. Other work, however, indicated that IE62 is capable of binding numerous sequences within promoters (2, 22, 50). Finally, an extensive analysis by Perera (32) showed that IE62 is capable of transactivation of minimal promoters containing only a TATA box and lacking all known or permuted IE62 binding sites.The mechanism(s) of IE62 activation is largely unknown. Perera (32) showed that IE62 was able to...

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Section: Discussionmentioning

confidence: 99%

Promoter Activation by the Varicella-Zoster Virus Major Transactivator IE62 and the Cellular Transcription Factor USF

Yang

Hua

Hay

et al. 2006

J Virol

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“…The cooperation between PIT1 and ETS family transcription factor in the PRL promoter is also determined by the spacing between these two transcription factors (Duval et al 2003). To examine the effect of the spacing between PIT1-US and GATA-REs on the transactivation of the Tshb gene, we generated TSHb-2G-CAT and THb-4G-CAT, which have an additional 2 or 4 bp insertion of guanines between PIT1-US and GATA-REs respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Secondly, the synergy of PIT1 with ETS family transcription factor including ETS1 is known to be necessary for the full activation of the Prl gene (Gutierrez-Hartmann et al 2007). Finally, Duval et al (2003) reported that this synergy does not require the physical interaction of PIT1 with ETS1 on DNA but is influenced by the spacing between the binding sites for these transcription factors (8 bp in the wild-type promoter). It should be noted, however, that the cooperation of PIT1 with GATA2 is not conventional synergism but rather derepression against SRBP on SR (Figs 6, 7 and 8).…”

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confidence: 99%

Functions of PIT1 in GATA2-dependent transactivation of the thyrotropin β promoter

Kashiwabara¹,

Sasaki²,

Matsushita³

et al. 2008

Journal of Molecular Endocrinology

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Thyrotropin (TSH) is a heterodimer consisting of a and b chains, and the b chain (TSHb) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHb expression. Using kidneyderived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHb promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHb promoter without PIT1. The deleted region (nt K82/K52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHb promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.

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“…The -domain insertionpoint lies at the boundary between the two TAD exons, R1 and R2, which regulate Pit-1-ETS-1 synergy and Ras response respectively (Duval et al 2003). The lack of requirement for physical interaction between Ets-1 and Pit-1 for either Ras responsiveness or Pit-1-ETS-1 synergy suggests that the -domain may interfere with coactivator recruitment by the Pit-1 and Ets-1 complex.…”

Section: Discussionmentioning

confidence: 99%

Repression of the prolactin promoter: a functional consequence of the heterodimerization between Pit-1 and Pit-1 β

Sporici

Hodskins²,

Locasto³

et al. 2005

Journal of Molecular Endocrinology

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The POU-homeodomain transcription factor Pit-1 is required for the differentiation of the anterior pituitary cells and the expression of their hormone products. Pit-1 , an alternate splicing isoform, has diametrically different outcomes when it is expressed in different cell types. Pit-1 acts as a transcriptional repressor of prolactin (PRL) and growth hormone genes in pituitary cells, and as a transcriptional activator in non-pituitary cells. In order to explore these differences, we: (1) identified the transcriptional cofactors necessary for reconstitution of repression in non-pituitary cells; (2) tested the effect of the -domain on heterodimerization with Pit-1 and physical interaction with the co-activator CREB binding protein (CBP); and (3) determined the -domain sidechain chemistry requirements for repression. Co-expression of both Pit-1 isoforms reconstituted the repression of the PRL promoter in non-pituitary cells. The -domain allowed heterodimerization with Pit-1 but blocked physical interaction with CBP, and specific chemical properties of the -domain beyond hydrophobicity were dispensable. These data strongly suggest that Pit-1 represses hormone gene expression by heterodimerizing with Pit-1 and interfering with the assembly of the Pit-1-CBP complex required for PRL promoter activity in pituitary cells.

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

Cited by 20 publications

References 43 publications

Promoter Activation by the Varicella-Zoster Virus Major Transactivator IE62 and the Cellular Transcription Factor USF

Promoter Activation by the Varicella-Zoster Virus Major Transactivator IE62 and the Cellular Transcription Factor USF

Functions of PIT1 in GATA2-dependent transactivation of the thyrotropin β promoter

Repression of the prolactin promoter: a functional consequence of the heterodimerization between Pit-1 and Pit-1 β

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