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T Hoey

Publications and source records attributed to T Hoey.

35 records · Page 2Linked to original sources
PubMed1993

Molecular cloning and functional analysis of Drosophila TAF110 reveal properties expected of coactivators.

The general transcription factor TFIID is a multiprotein complex containing the TATA-binding protein and several associated factors (TAFs), some of which may function as coactivators that are essential for activated, but not basal, transcription. Here we describe the isolation and characterization of the first gene encoding a TAF protein. The deduced amino acid sequence of TAF110 revealed the presence of several glutamine- and serine/threonine-rich regions reminiscent of the protein-protein interaction domains of the regulatory transcription factor Sp1 that are involved in transcription activation and multimerization. In both Drosophila cells and yeast, TAF110 specifically interacts with the glutamine-rich activation domains of Sp1. Moreover, purified Sp1 selectively binds recombinant TAF110 in vitro. These findings taken together suggest that TAF110 may function as a coactivator by serving as a site of protein-protein contact between activators like Sp1 and the TFIID complex.

Amino Acid Sequence↗
PubMed1991

Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation.

A key step in the regulation of transcription involves interactions between promoter-selective factors and various components of the transcriptional apparatus. Here we report the requirements for transcriptional activation directed by NTF-1, a developmentally regulated transcription factor in Drosophila. Reconstituted transcription with fractionated Drosophila basal factors reveals that activation by NTF-1 requires factors present in the endogenous TFIID fraction that are distinct from the purified TATA-binding protein (TBP). Glycerol gradient sedimentation and immunoprecipitation analyses indicate that TFIID is a multiprotein complex containing TBP and at least six tightly bound TBP-associated factors (TAFs). Preparations of TBP lacking TAFs after fractionation with denaturants no longer support activation by NTF-1 but retain basal level activity. Addition of immunopurified and renatured TAFs to free TBP restores the ability of NTF-1 to activate transcription without influencing basal transcription. These results suggest that one or more of the TAF polypeptides confer coactivator function.

Animals↗
PubMed1991

Autoregulation of a segmentation gene in Drosophila: combinatorial interaction of the even-skipped homeo box protein with a distal enhancer element.

Autoregulation has been implicated in the expression of many patterning genes in Drosophila, but the molecular details of this process are largely unknown. In the case of the segmentation gene even-skipped (eve), autoregulation is important for the specification of sharp stripes of gene expression at the onset of gastrulation. Here, we use a combination of DNA binding and P-transformation assays to characterize the cis- and trans-acting factors responsible for autoregulation. We show that eve autoregulation is mediated, at least in part, by a 100-bp minimal autoregulatory sequence (MAS) located approximately 5 kb upstream from the eve transcription start site. Multimerization of a 200-bp DNA fragment that encompasses the MAS drives optimal autoregulatory activity, comparable to that obtained with the native distal enhancer element located between -5.9 and -5.2 kb. The MAS contains two eve protein-binding sites, as well as binding sites for two nuclear factors present in early embryos. Directed mutagenesis of these binding sites suggests that both the eve protein and nuclear factors are essential for autoregulation. These results provide evidence that the eve protein acts combinatorially with other transcription factors to enhance its own expression.

Animals↗
PubMed1991

Transcriptional regulation of a pair-rule stripe in Drosophila.

The periodic, seven-stripe pattern of the primary pair-rule gene even-skipped (eve) is initiated by crude, overlapping gradients of maternal and gap gene proteins in the early Drosophila embryo. Previous genetic studies suggest that one of the stripes, stripe 2, is initiated by the maternal morphogen bicoid (bcd) and the gap protein hunchback (hb), while the borders of the stripe are formed by selective repression, involving the gap protein giant (gt) in anterior regions and the Krüppel (Kr) protein in posterior regions. Here, we present several lines of evidence that are consistent with this model for stripe 2 expression, including in vitro DNA-binding experiments and transient cotransfection assays in cultured cells. These experiments suggest that repression involves a competition or short-range quenching mechanism, whereby the binding of gt and Kr interferes with the binding or activity of bcd and hb activators at overlapping or neighboring sites within the eve stripe 2 promoter element. Such short-range repression could reflect a general property of promoters composed of multiple, but autonomous regulatory elements.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of the Drosophila gene encoding the TATA box binding protein, TFIID.

To investigate the biochemical mechanisms involved in interactions between regulatory factors and the general transcription complex, we have cloned, expressed, and characterized the Drosophila gene encoding the TATA binding protein, dTFIID. Comparison of the protein sequences of the Drosophila and yeast TATA binding proteins reveals a bipartite organization consisting of a highly conserved, basic carboxy-terminal domain and a nonconserved amino-terminal region rich in Gln, Gly, Ser, and Met residues. Purified dTFIID protein binds specifically to the TATA sequence and activates basal-level transcription, and the conserved carboxy-terminal half of the molecule is sufficient for both activities. Partially purified TFIID from Drosophila cells mediates activation by the transcription factor Sp1. In contrast, purified dTFIID expressed from the cloned gene is unable to support Sp1-dependent activation, suggesting that other factors may be required to mediate interactions between upstream activators like Sp1 and the TATA binding protein.

Amino Acid Sequence↗
PubMed1989

Sequence-specific DNA-binding activities of the gap proteins encoded by hunchback and Krüppel in Drosophila.

The segmentation of the Drosophila body plan depends on a hierarchy of interactions among approximately 20-25 regulatory genes that are active in the early embryo (refs 1-4; for a review see ref. 5). The gap genes have a key role in this process and are responsible for the periodic expression of certain pair-rule genes and the localized expression of several homoeotic genes. The two best characterized gap genes, hunchback (hb) and Krüppel (Kr), contain homologies with the zinc-finger DNA-binding motif, although their mode of action in the early embryo is unknown. Here we report that both of the proteins encoded by these genes possess sequence-specific DNA-binding activities, which indicates that they might regulate gene expression at the level of transcription. The binding sites of the hb gene product are related by a 10-base pair (bp) consensus sequence, G/A C/C ATAAAAAA, whereas the binding sites of the Kr gene product share a distinct 10-bp motif, AACGGGTTAA. It is possible that the hb and Kr proteins cooperatively regulate gene expression, because they are expressed in broad, overlapping gradients in the early embryo. We also provide evidence that the on/off periodicity of the pair-rule gene even-skipped (eve) involves the interaction of the hb and Kr proteins with defined eve promoter elements.

Animals↗
PubMed1989

Autoregulatory and gap gene response elements of the even-skipped promoter of Drosophila.

The pair-rule gene even-skipped (eve) plays a key role in the regulatory hierarchy governing segmentation in Drosophila. Here we describe the use of P-transformation and eve promoter fusions to identify cis elements that regulate the periodic seven-stripe eve pattern. A distal region of the eve promoter, located between -5.9 and -5.2 kb, controls autoregulation. Sequences from this region will induce striped expression of a heterologous hsp70 basal promoter in the presence, but not absence, of endogenous eve+ products. Autoregulatory activity was localized to a 200-bp region of the distal eve promoter. We also provide evidence that individual eve expression stripes are regulated by separate cis sequences. eve promoter sequences located between -4.7 and -3 kb upstream of the transcription start site are important for the initiation of stripe 3, whereas sequences between -1.7 and -0.4 kb are needed for stripes 2 and 7. It is possible that these latter regions are directly regulated by the products of gap genes.

Animals↗
PubMed1988

Divergent homeo box proteins recognize similar DNA sequences in Drosophila.

A member of a small group of genes in Drosophila that define the segmentation pattern of the early embryo even-skipped (eve), which plays a key role in a network of interactions among segmentation genes. It appears to control morphogenesis by regulating the expression of the segmentation gene engrailed (en), and by autoregulating its own expression (M. Frasch and M.L., in preparation). Here we show that these regulatory interactions could occur at the level of transcription as a full-length eve protein binds with high affinity to specific sequences located near the 5' ends of the eve and en genes. The en binding sites contain at least one copy of a 10-base pair consensus sequence: T-C-A-A-T-T-A-A-A-T. In contrast, the 5' eve binding sites are relatively G-C rich and do not share obvious similarities with the 10-base pair consensus sequence associated with en. Other homeo box proteins can recognize both classes of eve binding sites, lending support to the proposal that regulatory interactions among homeo box genes involve a competition of different homeo box proteins for similar cis regulatory sequences.

Animals↗
PubMed1988

Purification and properties of the Drosophila zen protein.

The zen protein is encoded by the zerknullt gene required for normal early development in Drosophila. Like many regulatory proteins of this type, zen contains a 60 amino acid homeobox sequence. We have purified the zen protein and studied its solution behavior and its interaction with DNA. The zen protein exists as a monomer in solution with a molecular weight of about 40,000. It binds specifically to a site about 900 bases upstream from the zen gene. Within this binding site DNase protection experiments indicate that binding is confined to two regions approximately 11 and 14 bases in length that are separated by about 30 base pairs. The protein concentration dependence of the binding curve suggests that protein binding is non cooperative.

Animals↗
PubMed1988

DNA-binding activities of the Drosophila melanogaster even-skipped protein are mediated by its homeo domain and influenced by protein context.

The homeo box gene even-skipped (eve) encodes a 376-amino-acid protein that binds with high affinity to sequences located near the 5' termini of the eve and en genes. The 5' en sites are A + T rich and contain copies of the 10-base-pair (bp) consensus sequence T-C-A-A-T-T-A-A-A-T. In contrast, the 5' eve sites are G + C rich and contain the 9-bp sequence T-C-A-G-C-A-C-C-G. Among the five different homeo box proteins that have been tested for binding, eve is unique in that it shows virtually equal preference for the A + T-rich 5' en binding sites and the G + C-rich 5' eve sites. Most of the other proteins bind with a relatively higher affinity to the en sites than to the eve sites. In an effort to identify the regions of the eve protein that are responsible for its efficient binding to both classes of recognition sequences, we analyzed the DNA-binding properties of various mutant eve proteins. These studies suggest that the homeo domain of the eve protein is responsible for both binding activities. However, mutations in distant regions of the protein influenced the binding behavior of the eve homeo domain and caused a reduction in binding to the G + C class of recognition sites. We propose that the protein context of the homeo domain can influence its DNA-binding properties.

Amino Acid Sequence↗
PubMed1987

Characterization and localization of the even-skipped protein of Drosophila.

On the basis of homeo box cross-homology we have isolated the pair-rule gene even-skipped (eve) of Drosophila. The eve transcription unit appears to be less than 1.5 kb in length, and encodes a single mRNA of approximately 1.4 kb. The nucleotide sequence of genomic and cDNA clones indicates that the eve protein is composed of 376 amino acid residues, and that its homeo domain shares only approximately 50% amino acid identity with the homeo domains of previously characterized genes. Using antibodies raised against a beta-galactosidase fusion protein we show that the eve protein is distributed in a series of seven transverse stripes at the cellular blastoderm stage, and is localized primarily within the nuclear regions of those embryonic cells that express the gene. After gastrulation, seven weakly stained stripes of eve expression appear, resulting in a transient pattern that consists of a total of 14 evenly spaced stripes. Both the original and new stripes gradually disappear during germ band elongation. A second expression pattern emerges during neurogenesis, whereby eve protein is detected in discrete subsets of neurons in each of the ventral ganglia.

Amino Acid Sequence↗
PubMed1987

Molecular characterization of the zerknüllt region of the Antennapedia gene complex in Drosophila.

zerknüllt (zen) is unique among the 18 known homeo box genes in Drosophila since it is required for the differentiation of the dorsal-ventral pattern, and does not appear to be involved in the process of segmentation. Here we show that the zen region of the Antennapedia complex (ANT-C) consists of two closely linked homeo box genes, designated z1 and z2. The z1 and z2 transcription units show essentially identical patterns of expression during early development, which are consistent with the timing and sites of zen+ gene activity. The putative proteins encoded by z1 and z2 are highly divergent and are related only by virtue of homeo box homology. We have used P-element-mediated germ line transformation to show that z1 alone can provide zen+ gene function, suggesting that the z2 gene might be dispensable. The occurrence of closely linked homeo box genes that display similar patterns of expression is not unique to the zen locus. Such gene duplications might provide important clues to the evolution of the homeo box gene family in Drosophila and other organisms.

Amino Acid Sequence↗
PubMed1986

Cross-regulatory interactions among pair-rule genes in Drosophila.

The pair-rule genes of Drosophila are required for the subdivision of the developing embryo into a repeating series of homologous body segments. One of the pair-rule genes, even-skipped (eve), appears to be particularly important for the overall segmentation pattern since eve- embryos lack all segmental subdivisions in the middle body region. On the basis of homeo box cross-homology we have isolated a gene, S72, which probably corresponds to eve. In embryo tissue sections S72 transcripts show a periodic distribution pattern. The eve- phenotype appears to involve altered patterns of fushi tarazu and engrailed expression. These and other findings suggest that pair-rule gene expression might involve hierarchical cross-regulatory interactions.

DNA↗
PubMed1986

Homeo box gene expression in anterior and posterior regions of the Drosophila embryo.

The homeo box is a 180-base-pair coding sequence that has been implicated in the control of Drosophila development. A common feature of the nine previously reported homeo box genes is their involvement in the establishment of the segmentation pattern of the embryo. In this report we describe the isolation and properties of two additional homeo box genes, F90-2 and S67. Transcripts encoded by the two genes are detected in embryonic tissues that derive from regions near the anterior and posterior poles of the embryo, which are outside the limits of expression of known homeotic genes. These results suggest that at least some homeo box genes specify positional identity along the anterior-posterior body axis that is independent of the process of segmentation.

Amino Acid Sequence↗
PubMedDate not supplied

Transcripts encoded by a homoeo box gene are restricted to dorsal tissues of Drosophila embryos.

In Drosophila elaboration of positional identity along the anterior-posterior and dorsal-ventral embryonic body axes involves early zygotic gene functions that are expressed in response to maternal cues present in the unfertilized egg. Zygotic loci that are required for the specification of positional identity along the anterior-posterior body axis have been described in detail. Less is known about the zygotic loci responsible for differentiation of the dorsal-ventral pattern; however, several genes that might be involved have been identified. Zerknüllt (zen) is an example of a zygotic gene required for correct differentiation of dorsally derived embryonic tissues. On the basis of homoeo box cross homology, we have now isolated a gene, called S60, that derives from the zen region of the Antennapedia complex (ANT-C). Transcripts encoded by S60 transiently accumulate in the dorsal-most tissues of developing embryos. This pattern of expression suggests that S60 corresponds to zen. Since S60 contains a homoeo box, it is possible that differentiation of the anterior-posterior and dorsal-ventral embryonic patterns involves similar molecular mechanisms.

Amino Acid Sequence↗