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R S Mann

Publications and source records attributed to R S Mann.

35 records · Page 2Linked to original sources
PubMed1997

Switching the in vivo specificity of a minimal Hox-responsive element.

The homeodomain proteins encoded by the Hox complex genes do not bind DNA with high specificity. In vitro, Hox specificity can be increased by binding to DNA cooperatively with the homeodomain protein extradenticle or its vertebrate homologs, the pbx proteins (together, the PBC family). Here we show that a two basepair change in a Hox-PBC binding site switches the Hox-dependent expression pattern generated in vivo, from labial to Deformed. The change in vivo correlates with an altered Hox binding specificity in vitro. Further, we identify similar Deformed-PBC binding sites in the Deformed and Hoxb-4 genes and show that they generate Deformed or Hoxb-4 expression patterns in Drosophila and mouse embryos, respectively. These results suggest a model in which Hox-PBC binding sites play an instructive role in Hox specificity by promoting the formation of different Hox-PBC heterodimers in vivo. Thus, the choice of Hox partner, and therefore Hox target genes, depends on subtle differences between Hox-PBC binding sites.

Animals↗
PubMed1996

Nuclear import of the homeodomain protein extradenticle in response to Wg and Dpp signalling.

In Drosophila, Decapentaplegic (Dpp) and Wingless (Wg) are two secreted signalling proteins of the transforming growth factor (TGF)-beta and Wnt families, respectively. Although both are often required during development, only a few downstream components of these signalling pathways have been described. Here we present evidence that in the embryonic midgut both signalling pathways control the subcellular localization of the homeodomain protein encoded by the extradenticle (exd) gene. Exd protein is predominantly nuclear in endoderm cells close to Dpp-and Wg-secreting cells of the visceral mesoderm, but is in the cytoplasm in more distant endoderm cells. Both dpp and wg are required for the nuclear localization of Exd in the endoderm, whereas ectopic expression of dpp and wg expands the domain of nuclear Exd. Furthermore, the nuclear import of Exd correlates with the transcription of an exd-dependent reporter gene in the endoderm. Thus one mechanism by which extracellular signals might control pattern is by directing the graded nuclear localization of homeodomain proteins such as Exd that directly control the expression of target genes.

Animals↗
PubMed1996

A structural model for a homeotic protein-extradenticle-DNA complex accounts for the choice of HOX protein in the heterodimer.

The genes of the homeotic complex (HOX) encode DNA binding homeodomain proteins that control developmental fates by differentially regulating the transcription of downstream target genes. Despite their unique in vivo functions, disparate HOX proteins often bind to very similar DNA sequences in vitro. Thus, a critical question is how HOX proteins select the correct sets of target genes in vivo. The homeodomain proteins encoded by the Drosophila extradenticle gene and its mammalian homologues, the pbx genes, contribute to HOX specificity by cooperatively binding to DNA with HOX proteins. For example, the HOX protein labial cooperatively binds with extradenticle protein to a 20-bp oligonucleotide that is sufficient to direct a labial-like expression pattern in Drosophila embryos. Here we have analyzed the protein-DNA interactions that are important for forming the labial-extradenticle-DNA complex. The data suggest a model in which labial and extradenticle, separated by only 4 bp, bind this DNA as a heterodimer in a head-to-tail orientation. We have confirmed several aspects of this model by characterizing extradenticle-HOX binding to mutant oligonucleotides. Most importantly, mutations in base pairs predicted to contact the HOX N-terminal arm resulted in a change in HOX preference in the heterodimer, from labial to Ultrabithorax. These results demonstrate that extradenticle prefers to bind cooperatively with different HOX proteins depending on subtle differences in the heterodimer binding site.

Amino Acid Sequence↗
PubMed1996

An extradenticle-induced conformational change in a HOX protein overcomes an inhibitory function of the conserved hexapeptide motif.

HOX homeoproteins control cell identities during animal development by differentially regulating target genes. The homeoprotein encoded by the extradenticle (exd) gene can selectively modify HOX DNA binding, suggesting that it contributes to HOX specificity in vivo. HOX-EXD interactions are in part mediated by a conserved stretch of amino acids termed the hexapeptide found in many HOX proteins. Here, we demonstrate that a 20 bp oligonucleotide from the 5' region of the mouse Hoxb-1 gene, a homolog of Drosophila labial (lab), is sufficient to direct an expression pattern in Drosophila that is very similar to endogenous lab. In vivo, this expression requires lab and exd and, in vitro, LAB requires EXD to bind this oligonucleotide. In contrast, LAB proteins with mutations in the hexapeptide bind DNA even in the absence of EXD. Moreover, a hexapeptide mutant of LAB has an increased ability to activate transcription in vivo. Partial proteolysis experiments suggest that EXD can induce a conformational change in LAB. These data are consistent with a mechanism whereby the LAB hexapeptide inhibits LAB function by inhibiting DNA binding and that an EXD-induced conformational change in LAB relieves this inhibition, promoting highly specific interactions with biologically relevant binding sites.

Amino Acid Sequence↗
PubMed1996

Extra specificity from extradenticle: the partnership between HOX and PBX/EXD homeodomain proteins.

For many DNA-binding transcription factors it is often difficult to reconcile their highly specific in vivo functions with their less specific in vitro DNA-binding properties. Cooperative DNA binding with cofactors often provides part of the answer to this paradox and recent studies have demonstrated this to be the case for the homeotic complex (HOX) family of transcription factors. However, the unique problem posed by these highly related and developmentally important transcription factors requires additional twists to the standard solution, which are beginning to become apparent from the characterization of the HOX cofactors encoded by the extradenticle and PBX genes.

Animals↗
PubMed1995

Segmental expression of Hoxb-1 is controlled by a highly conserved autoregulatory loop dependent upon exd/pbx.

Comparison of Hoxb-1 regulatory regions from different vertebrates identified three related sequence motifs critical for rhombomere 4 (r4) expression in the hindbrain. Functional analysis in transgenic mice and Drosophila embryos demonstrated that the conserved elements are involved in a positive autoregulatory loop dependent on labial (lab) family members. Binding of Hoxb-1 to these elements in vitro requires cofactors, and the motifs closely resemble the consensus binding site for pbx1, a homolog of the Drosophila extradenticle (exd) homoedomain protein. In vitro exd/pbx serves as a Hoxb-1 cofactor in cooperative binding and in Drosophila expression mediated by the r4 enhancer is dependent on both lab and exd. This provides in vivo and in vitro evidence that r4 expression involves direct autoregulation dependent on cooperative interactions of Hoxb-1 with exd/pbx proteins as cofactors.

Animals↗
PubMed1995

The specificity of homeotic gene function.

How transcription factors achieve their in vivo specificities is a fundamental question in biology. For the Homeotic Complex (HOM/Hox) family of homeoproteins, specificity in vivo is likely to be in part determined by subtle differences in the DNA binding properties inherent in these proteins. Some of these differences in DNA binding are due to sequence differences in the N-terminal arms of HOM/Hox homeodomains. Evidence also exists to suggest that cofactors can modify HOM/Hox function by cooperative DNA binding interactions. The Drosophila homeoprotein extradenticle (exd) is likely to be one such cofactor. In HOM/Hox proteins, both the conserved 'YPWM' peptide motif and the homeodomain are important for interacting with exd. Although exd provides part of the answer as to how specificity is achieved, there may be additional cofactors and mechanisms that have yet to be identified.

Amino Acid Sequence↗
PubMed1995

Identification of homeotic target genes in Drosophila melanogaster including nervy, a proto-oncogene homologue.

In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes.

Amino Acid Sequence↗
PubMed1994

The DNA binding specificity of Ultrabithorax is modulated by cooperative interactions with extradenticle, another homeoprotein.

The Ultrabithorax (Ubx) and Antennapedia (Antp) genes of Drosophila encode homeodomain proteins that have very similar DNA binding specificities in vitro but specify the development of different segmental patterns in vivo. We describe cooperative interactions between Ubx protein and a divergent homeodomain protein, extradenticle (exd), that selectively increases the affinity of Ubx, but not Antp, for a particular DNA target. We also provide evidence that Ubx and exd bind to neighboring sites on this DNA and interact directly to stabilize the DNA-bound form of Ubx. Thus, the ability of different homeotic genes to specify distinct segmental patterns may depend on cooperative interactions with proteins such as exd that selectively modulate their otherwise similar DNA binding specificities.

Amino Acids↗
PubMed1994

Engrailed-mediated repression of Ultrabithorax is necessary for the parasegment 6 identity in Drosophila.

The homeotic genes of Drosophila are expressed in overlapping domains along the anterior-to-posterior axis and specify the distinct morphological patterns of each parasegment. Within single parasegments, the levels of homeotic gene expression are often modulated, in part because of cross-regulation by other homeotic gene products. However, the functional significance of different levels of homeotic gene expression is unclear. Here modulations in Ultrabithorax (Ubx) expression within parasegment 6 are examined. Specifically, Ubx is shown to be down-regulated in the posterior compartment of this parasegment by engrailed (en). The significance of Ubx repression by en was demonstrated by characterizing the expression of the Ubx target gene, Distal-less (Dll). In the posterior compartment of parasegment 6, Dll is normally expressed in a small cluster of cells. If Ubx is expressed uniformly via a heat-shock promoter, Dll is inappropriately repressed in these posterior compartment cells. In the anterior compartment of parasegment 6, Dll is normally repressed by high levels of Ubx. However, if en is expressed uniformly via a heat-shock promoter, Ubx is repressed and Dll is derepressed. Because Dll is required for the development of larval sensory structures, these results demonstrate that en-mediated repression of Ubx in the posterior compartment is necessary for the morphology of parasegment 6. Thus, different levels of homeotic gene expression can be important for their segmental patterning functions.

Animals↗
PubMed1993

The segment identity functions of Ultrabithorax are contained within its homeo domain and carboxy-terminal sequences.

Using an in vivo assay for segment identity, the structural differences that distinguish two Drosophila homeotic selector proteins, Ultrabithorax (Ubx) and Antennapedia (Antp), have been investigated. There are at least two independent parts of Ubx and Antp that contribute to their functional specificities: (1) their homeo domains and (2) residues carboxy-terminal to their homeo domains (C-tails). In the absence of any C-tail, differences in 5 homeo domain amino acids are sufficient to distinguish between the functions of Ubx and Antp. Two of these are at the amino terminus of the homeo domain and could contact DNA directly. A three dimensional model suggests that the other 3 homeo domain residues and the C-tails are unlikely to contact DNA. In addition, we demonstrate that the assay used to measure the segment identity functions of Ubx and Antp is independent of any homeotic selector gene normally active in thoracic and abdominal segments. Therefore, it is likely that this assay measures the coordinate regulation of many downstream target genes. This expectation is confirmed for at least one Ubx target gene, Distal-less.

Amino Acid Sequence↗
PubMed1993

Negative autoregulation by Ultrabithorax controls the level and pattern of its expression.

The Drosophila homeotic gene Ultrabithorax (Ubx) encodes transcriptional regulatory proteins (UBX) that specify thoracic and abdominal segmental identities. Ubx autoregulation was examined by manipulating UBX levels, both genetically and with an inducible transgene, and monitoring the effect of these manipulations on the expression of Ubx and Ubx-lacZ reporter genes. Positive autoregulation by Ubx is restricted to the visceral mesoderm, while in other tissues Ubx negatively autoregulates. In some cases, negative autoregulation stabilizes UBX levels, while in others it modulates the spatial and temporal patterns of UBX expression. This modulation of UBX expression may enable Ubx to specify distinct identities in different segments. The upstream control region of Ubx contains multiple autoregulatory elements for both positive and negative autoregulation.

Animals↗
PubMed1990

Functional dissection of Ultrabithorax proteins in D. melanogaster.

Expression of Ultrabithorax (UBX) proteins via a heat-inducible promoter generated homeotic transformations of segmental identities in the embryonic cuticle and peripheral nervous system (PNS) of Drosophila and transformed antennae into legs in the adult. The embryonic transformations were used to determine the identity functions of members of the UBX family and UBX mutant forms. Whereas UBX forms I and IV each induced the cuticle transformations, only form I induced the PNS transformations. Analysis of the transformations generated by UBX deletions and by a chimeric Ultrabithorax-Antennapedia protein demonstrated that the majority of the UBX identity information is contained within the C-terminal, homeodomain-containing portion of the protein. Implications of these results for how homeotic proteins select particular metameric identities are discussed.

Amino Acid Sequence↗
PubMed1984

Cross-reaction of BK virus large T antigen with monoclonal antibodies directed against SV40 large T antigen.

Seventy-five percent of the amino acid sequence of simian virus 40 (SV40) large T antigen is identical to that of the large T antigen of the human papovavirus, BK virus (BKV). Cross-reactivity between BKV T antigen and monoclonal antibodies directed against SV40 T antigen was studied by immunofluorescence, an enzyme immunoassay, immunoprecipitation of radiolabeled extracts, and Western blotting. BKV T antigen was found to be recognized by two monoclonal antibodies, PAb 416 and 430, which react with two distinct sites toward the amino terminus of SV40 large T antigen. These two sites may correspond to two hydrophilic regions of shared amino acid sequence which exist toward the amino termini of the T antigens.

Amino Acid Sequence↗
PubMed1978

Suppression of histone deacetylation in vivo and in vitro by sodium butyrate.

In HeLa cells which have been exposed to 5 mM sodium butyrate for 21 h, the level of histone acetylation is greatly increased as compared to control cells (Riggs, M.G., Whittaker, R.G., Neumann, J.R., and Ingram, V.R. (1977) Nature 268, 462-464). Our experiments indicate that the increase in the relative amounts of multiacetylated forms of histones H4 and H3 following butyrate treatment is the result of an inhibition of histone deacetylase activity.

Butyrates↗