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H G Stunnenberg

Publications and source records attributed to H G Stunnenberg.

9 recordsLinked to original sources
PubMed1992

Cooperativity in transactivation between retinoic acid receptor and TFIID requires an activity analogous to E1A.

In embryonal carcinoma (EC) cells retinoic acid (RA) strongly induces transcription from the RA receptor beta 2 (RAR beta 2) promoter through an RA response element (RARE) located in close proximity to the TATA box. Here we demonstrate that recombinant human TATA box-binding protein, hTFIID, and RAR functionally cooperate in transactivation of the RAR beta 2 promoter in EC cells in a strictly RA-dependent manner. We demonstrate that the core domain of hTFIID is sufficient to mediate RAR-dependent transcription and that Drosophila, but not yeast, TFIID can substitute for hTFIID. In COS cells ectopic expression of the E1A protein is a prerequisite for hTFIID and RAR to cooperate in transactivation. We propose a model for transcriptional regulation of the RAR beta 2 promoter in EC cells in which RAR, following activation by RA, functionally interacts with hTFIID via an E1A-like activity present in EC cells.

Adenovirus Early Proteins↗
PubMed1992

Identification of multiple SRF N-terminal phosphorylation sites affecting DNA binding properties.

Human serum response factor (SRF) bearing a histidine tag was expressed using vaccinia virus. The recombinant protein was purified and shown to be phosphorylated mainly in its N-terminal part. The corresponding phosphorylation sites were mapped by microsequencing and also appear to be phosphorylated in endogenous serum response factor. Four phosphorylation sites are located on serines within amino acids 77-85, while another phosphorylation site has been identified at Ser103. Mutations that considerably reduced or abolished phosphorylation at amino acids 77-85 caused a decrease in binding to the c-fos serum response element accompanied by markedly reduced association and dissociation rates. In contrast, replacing Ser103 by alanine decreased DNA binding activity without drastically affecting the on/off rates. The combination of abolishing phosphorylation at amino acids 77-85 and 103 displayed greatly reduced on/off rates of DNA binding, but the reduction of DNA binding activity was partially alleviated. None of these mutations affect either the ability to interact with p62TCF or stimulation of transcription in vitro. These findings imply possible roles for SRF phosphorylation in the regulation of c-fos transcription.

Amino Acid Sequence↗
PubMed1992

RXR alpha, a promiscuous partner of retinoic acid and thyroid hormone receptors.

Retinoic acid receptor (RAR), thyroid hormone receptor (T3R) and vitamin D3 receptor (VD3R) differ from steroid hormone receptors in that they bind and transactivate through responsive elements organized as direct rather than inverted repeats. We now show that recombinant RAR and T3R are monomers in solution and cannot form stable homodimeric complexes on their responsive elements. Stable binding of the receptors to their responsive elements requires heterodimerization with a nuclear factor. This auxiliary factor is tightly associated with RAR and T3R in the absence of DNA and co-purifies with both receptors. As demonstrated by extensive purification, the same auxiliary factor is required for stable DNA binding of RAR as for that of T3R; the factor also facilitates the formation of a stable VD3R-DNA complex. The auxiliary factor is identical to the retinoid X receptor alpha (RXR alpha) by biochemical and functional criteria. The identification of RXR alpha as a dimerization partner for the RARs, T3Rs and VD3R has important implications as to the function of these receptors and their ligands in development, homeostasis and neoplasia.

Animals↗
PubMed1992

The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation.

UBF is a DNA binding protein which interacts with both the promoter and the enhancer of various vertebrate ribosomal RNA genes and functions as a transcription initiation factor for RNA polymerase I (pol I). We have purified murine UBF to apparent molecular homogeneity and demonstrate that its transactivating potential, but not its DNA binding activity, is modulated in response to cell growth. In vivo labelling experiments demonstrate that UBF is a phosphoprotein and that the phosphorylation state is different in growing and quiescent cells. We show that UBF is phosphorylated in vitro by a cellular protein kinase which by several criteria closely resembles casein kinase II (CKII). A major modification involves serine phosphoesterifications in the carboxy terminal hyperacidic tail of UBF. Deletions of this C-terminal domain severely decreases the UBF directed activation of transcription. The data suggest that phosphorylation of UBF by CKII may play an important role in growth dependent control of rRNA synthesis.

Amino Acid Sequence↗
PubMed1991

Functional characterization of a natural retinoic acid responsive element.

Retinoic acid receptor (RAR) and thyroid hormone receptor (T3R) are thought to bind as dimers to a T3 responsive element (T3REpal) comprised of inverted repeats of the half-site motif GGTCA. However, a RA responsive element (beta RARE) was previously identified in the promoter of the RAR beta 2 gene which contains two direct repeats of the motif GTTCA spaced by a six nucleotide gap. We now demonstrate the ability of RAR alpha, beta and gamma to bind to and transactivate through this element and that the two direct repeats comprise the beta RARE. Surprisingly, the GTTCA motifs rearranged to form a palindrome do not confer RA responsiveness to a heterologous promoter. Furthermore, no significant level of transactivation is detected by ligand-activated RAR through the Moloney murine leukaemia virus T3RE, which comprises two direct repeats of the sequence GGTCA/C spaced by a five nucleotide gap. Similarly, T3R does not induce gene expression through the beta RARE. This study establishes the preference of T3R to transactivate through direct repeats spaced by a five nucleotide gap as opposed to a six nucleotide gap. In contrast, RAR appears to be more flexible with respect to spacing requirements between repeats, although higher levels of transactivation are obtained through direct repeats spaced by a six nucleotide gap. Interestingly, although some elements mediate either RA or T3 induction, changing a single nucleotide in the MoMLV T3RE with a five nucleotide spacing creates a promiscuous RA/T3 responsive element.

Animals↗
PubMed1991

Vaccinia virus capping enzyme is a transcription initiation factor.

It has previously been demonstrated that vaccinia virus capping enzyme is involved both in the formation of a 5' cap structure and in termination of early transcription. Here we show that capping enzyme has an additional activity which is required for transcription of intermediate genes. VITF-A and VITF-B have been defined as two activities which together with RNA polymerase are necessary and sufficient to transcribe intermediate genes in vitro. VITF-A and the viral capping enzyme are shown to copurify to near homogeneity. Direct evidence that capping enzyme is VITF-A was obtained by complementation of a reconstituted transcription system with viral capping enzyme expressed in Escherichia coli. Although capping enzyme is a cofactor in early transcription termination, intermediate transcription is not terminated in response to the early termination signal. Capping enzyme is shown to form a complex with RNA polymerase in the absence of VITF-B. This appears to be a prerequisite for the formation of a stable initiation complex.

DNA-Directed RNA Polymerases↗
PubMed1979

RNA polymerase from the fungus, Aspergillus nidulans. Large-scale purification of DNA-dependent RNA polymerase I (or A).

The DNA-dependent RNA polymerase I (or A) from the lower eukaryote Aspergillus nidulans has been purified on a large scale to apparent homogeneity by homogenizing the fungal hyphae in liquid nitrogen, extraction of the enzyme at high salt concentration, precipitation of RNA polymerase activity with polymin P (a polyethylene imine), elution of the RNA polymerase from the polymin P precipitate, ammonium sulphate precipitation, molecular sieving on Bio-Gel A-1.5m, binding to ion-exchangers and DNA-cellulose affinity chromatography. By this procedure 1.6 mg of RNA polymerase I can be purified over 2000-fold from 500 g wet weight of starting material with a yield of 30--35%. The isolated RNA polymerase I is stable for several months at -20 degrees C. The subunit compostion has been resolved by polyacrylamide gel electrophoresis on two-dimensional gels, using either non-denaturing of 8 M urea (pH 8.7) cylindrical gels in the first dimension and sodium dodecyl sulphate slab gels in the second dimension. The putative subunits have molecular weights of 190,000, 135,000, 63,000, 62,000, 43,000, 29,000, (28,000), 16,000 and probably 13,000 and 12,000. Two distinct forms of RNA polymerase I (Ia and Ib) have been resolved by DEAE-Sephadex A-25 chromatography showing ample differences in enzymatic properties and subunit pattern. Additional information is given on RNA polymerase II (or B) which appears to be highly insensitive to alpha-amanitin at concentrations up to 400 micrograms/ml.

Aspergillus nidulans↗
PubMed1979

Protoplasts from Aspergillus nidulans.

A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.

Aspergillus nidulans↗