Biomedical subjects

F Schaufele

Publications and source records attributed to F Schaufele.

27 records · Page 2Linked to original sources

PubMed1993

Distance-dependent interactions between basal, cyclic AMP, and thyroid hormone response elements in the rat growth hormone promoter.

Developmental stage- and tissue-specific expression of the rat growth hormone (rGH) gene is conferred by DNA sequences within 237 base pairs of the transcription start site. Although binding of a number of transcription factors including Pit-1, Sp1, GHF3, and thyroid hormone receptor (T3R) stimulates rGH expression, several studies have suggested that interactions between these factors are important in determining cell specificity and responsiveness to extracellular signals. We have directly tested this hypothesis by creating a set of nested insertional mutations at two positions in the rGH promoter. Sequences were inserted at either position -148, separating GHF-3 and T3R binding sites from the downstream Pit-1 and Sp 1 binding sites, or at -51, separating the above elements from the TATA box. All insertions were made in the context of the rGH gene -237/+8 5'-flanking DNA, linked to a chloramphenicol acetyltransferase reporter gene and tested for activity by transient transfection in GC pituitary tumor cells. Insertions at both -148 and -51 caused sharp distance-dependent reductions in serum-stimulated expression such that insertions of 23 base pairs at -51 or 44 base pairs at -148 were sufficient to isolate the effects of sequences upstream of the insertion point. Insertions at -148 reduced T3 responsiveness severalfold but had little or no effect on stimulation by forskolin, whereas insertions at -51 reduced both T3 and forskolin responsiveness. Our results are consistent with the hypothesis that expression and regulation of the rGH gene is dependent on short-range protein-protein interactions, which are more critically dependent on spacing than the relative orientation of the transcription factor binding sites.

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PubMed1993

Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site.

We have characterized the putative AP1 site in the backbone of pUC plasmids and found unique regulatory effects. The site, which mapped to a 19-bp region around nucleotide 37, conferred transcriptional activation by Jun or Jun/Fos that was boosted up to fivefold by unliganded thyroid hormone receptor (TR). Thyroid hormone changed potentiation of the Jun response by TR into repression. Although the plasmid sequence is a near-perfect consensus AP1 site, the perfect consensus AP1 site from the human collagenase promoter did not show the same effects. Deletion of the ligand binding domain of the TR eliminated the ability of the receptor to boost Jun activity, and deletion, mutation, or changes in specificity of the DNA binding domain eliminated both its ability to potentiate Jun activity and repress with hormone. In vitro Jun/Fos complexes bound the operative plasmid fragment, and the presence of TR interfered very little with Jun/Fos binding activity. Protein interaction studies in the absence of DNA showed that TR bound Jun protein in solution either in the presence or in the absence of hormone. These observations suggest a mechanism for synergy and repression by TR through modulation of Jun activity: positive when TR is unliganded, and negative when hormone is bound. They also suggest that the presence of the plasmid element can confound studies of the regulation of linked promoters.

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PubMed1992

Synergistic activation of the rat growth hormone promoter by Pit-1 and the thyroid hormone receptor.

The rat GH (rGH) gene is expressed in the pituitary in a highly tissue-specific manner. A pituitary-specific transcription factor, Pit-1 (or GHF-1), and other, more tissue-general factors, including the thyroid hormone receptor (T3R), are important for regulating rGH promoter activity. The relative roles of Pit-1, T3R, and protein kinases in the activation of the rGH promoter were studied. Each component was supplied individually or in combination with the others to human monocyte U937 cells. The transfected rGH promoter was inactive in these cells even when it was cotransfected with either Pit-1 or T3R expression vectors. The rGH promoter carried in a truncated pUC vector could be activated by expression of the T3R if the cells were cultured with inducers of protein kinase-A (forskolin) and protein kinase-C [phorbol 12-myristate 13-acetate (PMA)] activity. By contrast, the PMA- and forskolin-dependent activation of the rGH promoter by Pit-1 expression was comparatively insignificant unless 1) the sequences deleted from the pUC vector (including a putative site for the transcription factor AP1) were restored to the plasmid carrying the rGH promoter; or 2) the T3R was coexpressed, which led to a marked synergistic response. These results indicate the relative inactivity of Pit-1 in isolation from other factors. Activation by forskolin and PMA did not require de novo protein synthesis. The synergistic activation by Pit-1 and the T3R was enhanced, but was not dependent upon, thyroid hormone (T3). The T3-dependent effect operated predominately through a thyroid hormone response element located up-stream of the two Pit-1-binding sites within the rGH promoter, whereas the T3-independent effect did not require any of the known T3R-binding sites on the rGH promoter. These results suggest a role for the more tissue-general T3R and protein kinases in the activation of the rGH promoter. They demonstrate the synergistic interplay between the T3R and Pit-1, underscore the dependence of Pit-1 action on other transcription factors, and implicate Pit-1 as a cofactor, rather than the dominant factor, influencing the tissue-specific expression of the rGH promoter.

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PubMed1990

Overlapping Pit-1 and Sp1 binding sites are both essential to full rat growth hormone gene promoter activity despite mutually exclusive Pit-1 and Sp1 binding.

A DNA-binding factor present in a wide variety of cell types is shown to bind to a site (GHF2) within the rat growth hormone gene promoter between nucleotides -147 and -129. Biochemical and transcriptional properties identify this factor as Sp1. The degree of glycosylation of one of the forms of Sp1 is observed to vary in a cell type-specific fashion. The GHF2 binding site overlaps with the more distal (dGHF1) of the two binding sites for the pituitary-specific transcription factor Pit-1. DNase I footprinting and gel retardation analysis with partially purified Sp1 and Pit-1 show that Sp1 and Pit-1 binding to these sites is mutually exclusive. Despite the inability of these two transcription factors to be simultaneously bound to their overlapping sites in vitro, the structural integrity of both binding sites appears essential to the maximal expression of the rat growth hormone promoter after transfection into pituitary GC cells. These findings suggest that activation of the rat growth hormone promoter by Sp1 and Pit-1 may occur through a multistage mechanism.

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PubMed1990

Somatotroph- and lactotroph-specific interactions with the homeobox protein binding sites in the rat growth hormone gene promoter.

Nuclear extracts prepared from growth hormone-secreting (GC) and prolactin-secreting (235-1) rat anterior pituitary cell lines were compared for their ability to bind to the DNA sequences conferring tissue-specificity to the expression of the rat growth hormone (rGH) gene promoter. Cell-specific differences in the interaction of Pit-1, a tissue-specific member of the POU-domain transcription factor family, with the pGHF1 binding site were detected by methylation interference experiments; otherwise the Pit-1 proteins present in GC cell and 235-1 cell extracts were similar. Two other protein/DNA complexes, GHF5 and GHF7, were detected by gel mobility shift assays and the binding of both complexes to the rGH promoter depended upon DNA sequences contained within the two binding sites for Pit-1. In contrast to Pit-1 which can bind to either of the two sites independently, a single Pit-1 binding site was insufficient for GHF5 and GHF7 binding; i.e. both Pit-1 binding sites within the rGH promoter were required. Whereas GHF5 was present in nuclear extracts of GC cells and a variety of cells not producing growth hormone, GHF7 binding activity was detected only in the GC cell line (and not in the 235-1 cell line). GHF7 binding activity was therefore more closely correlated with growth hormone gene transcription than was Pit-1. rGH promoters containing mutations which inhibited GHF5, GHF7 and Pit-1 binding were expressed less efficiently than the wild type promoter after transfection into GC cells. One promoter mutation to which the GHF7 complex but not the Pit-1 factor can bind was also transcription deficient demonstrating that Pit-1 binding, independent of GHF7 binding, was nevertheless important to the expression of the rat growth hormone promoter.

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PubMed1990

Resolution by diagonal gel mobility shift assays of multisubunit complexes binding to a functionally important element of the rat growth hormone gene promoter.

DNase I footprinting identifies a tissue-general factor, GHF3, binding to the rat growth hormone promoter between nucleotides -239 and -219. Mutation of the GHF3-binding site reduces promoter activity to 30% of that of the wild-type promoter after transfection into GC cells. Southwestern blotting and protein/DNA cross-linking experiments demonstrate that the GHF3-binding factor migrates as a 43-kDa protein. However, multiple GHF3 factor/DNA complexes with different electrophoretic mobilities are detected by gel retardation analysis. A novel technique, the diagonal gel mobility shift assay, is used to demonstrate that five of the different complexes represent multisubunit structures containing a common DNA-binding subunit. In this method, the multisubunit complexes resolved by one-dimensional gel mobility shift assays are observed to partially dissociate during electrophoresis in a second dimension with the DNA-binding subunit detected as a common signal directly below those signals representing the undissociated complexes which lie on a diagonal line. Two of the five complexes also contain an additional subunit in common whereas two other complexes appear to contain completely different subunits interacting with the common DNA-binding subunit. All five complexes copurify during GHF3-binding site-specific DNA affinity chromatography, and this fraction stimulates in vitro transcription in a GHF3-binding site-dependent fashion. Thus, a functionally important region of the rat growth hormone gene promoter interacts with a DNA-binding transcription factor which in turn acts as a docking site for other proteins.

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PubMed1988

Functional analysis of the sea urchin U7 small nuclear RNA.

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.

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PubMed1987

The inability of the Psammechinus miliaris H3 RNA to be processed in the Xenopus oocyte is associated with sequences distinct from those highly conserved amongst sea urchin histone RNAs.

3' processing of precursors of the H3 RNA of the sea urchin Psammechinus miliaris in Xenopus oocytes is dependent upon sea urchin U7 snRNA. Sequences necessary for this interaction are highly conserved in all sea urchin histone precursor RNAs (including the Psammechinus H3) which, in contrast, are efficiently processed in the Xenopus oocyte without the addition of the homologous U7 snRNA. We resolve this seeming paradox by demonstrating here that the inability of the sea urchin Psammechinus miliaris H3 histone RNA to be processed in the Xenopus oocyte is associated with nucleotides immediately 3' to the conserved downstream sea urchin histone sequence element. Thus, a sequence-specific element (or lack of it) is responsible for the poor recognition of the Psammechinus H3 precursor RNA by the Xenopus processing machinery.

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PubMedDate not supplied

Compensatory mutations suggest that base-pairing with a small nuclear RNA is required to form the 3' end of H3 messenger RNA.

Processing of the 3' end of sea urchin H3 histone pre-mRNA requires conserved sequence elements and the presence of U7 snRNA. A mutation in the conserved CAAGAAGA sequence of the H3 pre-mRNA that renders 3' processing of this precursor defective is shown to be suppressed by a compensatory change in the U7 snRNA, restoring the base-pairing potential of the two RNAs. RNA-RNA contacts between these two molecules appear to be an essential feature of the 3' processing reaction.

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