Structure-Function Analysis of the Rat Prolactin Promoter: Phasing Requirements of Proximal Cell-Specific Elements

Harvey, C; Jackson, Sara L.; Siddiqui, Shahid K.; Gutierrez‐Hartmann, Arthur

doi:10.1210/mend-5-6-836

Cited by 30 publications

(12 citation statements)

References 28 publications

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“…Stimulation of the reporter plasmids showed a 10 bp periodicity and changes of only 2 or 3 bp could drastically reduce the degree of co-operativity [46]. DNA-turn-dependent reductions in rat prolactin promoter activity have also been reported for the two proximal elements [47]. In addition, the region of spacing divergence between the human and rat insulin 1 promoters begins only 2 bp upstream of the 6 bp E1 consensus sequence and would, therefore, be within the binding footprint of E47/β2.…”

Section: Discussionmentioning

confidence: 83%

Relative contribution of PDX-1, MafA and E47/β2 to the regulation of the human insulin promoter

Docherty

Hay

Ferguson

et al. 2005

Biochemical Journal

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The insulin promoter binds a number of tissue-specific and ubiquitous transcription factors. Of these, the homoeodomain protein PDX-1 (pancreatic duodenal homeobox factor-1), the basic leucine zipper protein MafA and the basic helix-loop-helix heterodimer E47/BETA2 (beta-cell E box transactivator 2; referred to here as beta2) bind to important regulatory sites. Previous studies have shown that PDX-1 can interact synergistically with E47 and beta2 to activate the rat insulin 1 promoter. The aim of the present study was to determine the relative contribution of PDX-1, MafA and E47/beta2 in regulating the human insulin promoter, and whether these factors could interact synergistically in the context of the human promoter. Mutagenesis of the PDX-1, MafA and E47/beta2 binding sites reduced promoter activity by 60, 74 and 94% respectively, in INS-1 beta-cells. In the islet glucagonoma cell line alphaTC1.6, overexpression of PDX-1 and MafA separately increased promoter activity approx. 2.5-3-fold, and in combination approx. 6-fold, indicating that their overall effect was additive. Overexpression of E47 and beta2 had no effect. In HeLa cells, PDX-1 stimulated the basal promoter by approx. 40-fold, whereas MafA, E47 and beta2 each increased activity by less than 2-fold. There was no indication of any synergistic effects on the human insulin promoter. On the other hand, the rat insulin 1 promoter and a mutated version of the human insulin promoter, in which the relevant regulatory elements were separated by the same distances as in the rat insulin 1 promoter, did exhibit synergy. PDX-1 was shown further to activate the endogenous insulin 1 gene in alphaTC1.6 cells, whereas MafA activated the insulin 2 gene. In combination, PDX-1 and MafA activated both insulin genes. Chromatin immunoprecipitation assays confirmed that PDX-1 increased the association of acetylated histones H3 and H4 with the insulin 1 gene and MafA increased the association of acetylated histone H3 with the insulin 2 gene.

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Section: Discussionmentioning

confidence: 83%

Relative contribution of PDX-1, MafA and E47/β2 to the regulation of the human insulin promoter

Docherty

Hay

Ferguson

et al. 2005

Biochemical Journal

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“…To examine the possibility of an Ets‐DNA‐Ets complex formed on the DYT1 Ets direct repeat motif, we tested the effects of increasing the distance between the two Ets cores. Changing the distance between adjacent binding sites in promoters affects the relative position of a core sequence on the DNA turn as well as their relative distance along the DNA helix, resulting in decreased promoter activity in the majority of genes (Harvey et al. 1991; Baillat et al.…”

Section: Discussionmentioning

confidence: 99%

Regulation of DYT1 gene expression by the Ets family of transcription factors

Armata¹,

Ananthanarayanan²,

Balasubramaniyan³

et al. 2008

Journal of Neurochemistry

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The DYT1 gene encodes for torsinA, a protein with widespread tissue distribution, involved in early onset dystonia (EOD). Numerous studies have focused on torsinA function but no information is available on its transcriptional regulation. We cloned mouse and human 5′‐upstream DYT1 DNA fragments, exhibiting high transcriptional activity, as well as tissue specificity. We identified a proximal minimal DYT1 promoter within −141 bp for mouse and −191 bp for human with respect to the ATG codon. Primer extension analysis indicated multiple transcription start sites. In silico analysis of approximately 500 bp 5′‐upstream DYT1 fragment demonstrated lack of a classical TATA or CAAT box and the presence of a highly conserved direct repeat of two Ets binding cores within −86 bp to −77 bp and −78 bp to −69 bp of the mouse and human DYT1 gene, respectively. A single or a two base nucleotide alteration within the downstream Ets core resulted in approximately 90% (mouse) or 45–60% (human) drop in activity. Interestingly, a 3‐bp distance increase between the two Ets cores dramatically decreased transcriptional activity which was partially restored when the distance was increased up to 10 bp. Ets‐like dominant negatives confirmed the Ets factors as DYT1 transcriptional activators.

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“…These results suggested that GHF-1 alone does not mediate the Ras/Raf activation of the rPRL promoter. However, examination of the rPRL promoter DNA sequence reveals that the EBS at Ϫ217 to Ϫ209 is immediately adjacent to FPIV, the most distal and lowest-affinity GHF-1-binding site (28) (Fig. 2B), suggesting that deletion at Ϫ212 not only destroys the EBS but, by doing so, also interferes with a composite c-Ets-1/GHF-1 Ras/Raf-responsive element.…”

Section: Ghf-1 But Not Ghf-2 Synergistically Enhances Ras and Raf Actmentioning

confidence: 99%

Functional Interaction of c-Ets-1 and GHF-1/Pit-1 Mediates Ras Activation of Pituitary-Specific Gene Expression: Mapping of the Essential c-Ets-1 Domain

Bradford

Conrad

Wasylyk

et al. 1995

Molecular and Cellular Biology

117

139

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The mechanism by which activation of common signal transduction pathways can elicit cell-specific responses remains an important question in biology. To elucidate the molecular mechanism by which the Ras signaling pathway activates a cell-type-specific gene, we have used the pituitary-specific rat prolactin (rPRL) promoter as a target of oncogenic Ras and Raf in GH4 rat pituitary cells. Here we show that expression of either c-Ets-1 or the POU homeo-domain transcription factor GHF-1/Pit-1 enhance the Ras/Raf activation of the rPRL promoter and that coexpression of the two transcription factors results in an even greater synergistic Ras response. By contrast, the related GHF-1-dependent rat growth hormone promoter fails to respond to Ras or Raf, indicating that GHF-1 alone is insufficient to mediate the Ras/Raf effect. Using amino-terminal truncations of c-Ets-1, we have mapped the c-Ets-1 region required to mediate the optimal Ras response to a 40-amino-acid segment which contains a putative mitogen-activated protein kinase site. Finally, dominant-negative Ets and GHF constructs block Ras activation of the rPRL promoter, and each blocks the synergistic activation mediated by the other partner protein, further corroborating that a functional interaction between c-Ets-1 and GHF-1 is required for an optimal Ras response. Thus, the functional interaction of a pituitary-specific transcription factor, GHF-1, with a widely expressed nuclear proto-oncogene product, c-Ets-1, provides one important molecular mechanism by which the general Ras signaling cascade can be interpreted in a cell-type-specific manner.

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Structure-Function Analysis of the Rat Prolactin Promoter: Phasing Requirements of Proximal Cell-Specific Elements

Cited by 30 publications

References 28 publications

Relative contribution of PDX-1, MafA and E47/β2 to the regulation of the human insulin promoter

Relative contribution of PDX-1, MafA and E47/β2 to the regulation of the human insulin promoter

Regulation of DYT1 gene expression by the Ets family of transcription factors

Functional Interaction of c-Ets-1 and GHF-1/Pit-1 Mediates Ras Activation of Pituitary-Specific Gene Expression: Mapping of the Essential c-Ets-1 Domain

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