Generalized schemes for high-throughput manipulation of the Desulfovibrio vulgaris genome.

The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.

Automating fruit fly Drosophila embryo injection for high throughput transgenic studies.

To decipher and manipulate the 14 000 identified Drosophila genes, there is a need to inject a large number of embryos with transgenes. We have developed an automated instrument for high throughput injection of Drosophila embryos. It was built on an inverted microscope, equipped with a motorized xy stage, autofocus, a charge coupled device camera, and an injection needle mounted on a high speed vertical stage. A novel, micromachined embryo alignment device was developed to facilitate the arrangement of a large number of eggs. The control system included intelligent and dynamic imaging and analysis software and an embryo injection algorithm imitating a human operator. Once the injection needle and embryo slide are loaded, the software automatically images and characterizes each embryo and subsequently injects DNA into all suitable embryos. The ability to program needle flushing and monitor needle status after each injection ensures reliable delivery of biomaterials. Using this instrument, we performed a set of transformation injection experiments. The robot achieved injection speeds and transformation efficiencies comparable to those of a skilled human injector. Because it can be programed to allow injection at various locations in the embryo, such as the anterior pole or along the dorsal or ventral axes, this system is also suitable for injection of general biochemicals, including drugs and RNAi.

To bind or not to bind.

Gene expression is regulated by transcription factors binding selectively to particular portions of the genome. To what extent are these protein-DNA interactions influenced by the intrinsic sequence-specific recognition properties at each protein, and to what extent are they affected by other factors, such as chromatin structure or cooperative interactions with other proteins. Genome-wide surveys of DNA binding by transcription factors in vivo are beginning to provide some answers.

Transcriptional regulation in Drosophila: the post-genome challenge.

Drosophila melanogaster has long been at the forefront of studies of transcriptional regulation in animals. Many fundamental ideas--such as cis control elements that act over long distances, the regulation of development by hierarchical cascades of transcription factors, dosage compensation, and position effect variegation--originated from studies of the fruit fly. The recent completion of the euchromatic DNA sequence of Drosophila is another breakthrough. The sequence data highlight important unanswered questions. For example, only one-fifth of the 124 Mb of Drosophila euchromatic DNA codes for protein. The function of the remaining 100 Mb of mostly unique DNA is largely unknown. Some proportion of this non-reading frame DNA must encode the functional recognition sites targeted by the approximately 700 sequence-specific DNA binding proteins that regulate transcription in Drosophila, but what proportion? Most or very little? Promoter sequences by definition contain all of the cis information that specifies how gene transcription is regulated. However, it has been difficult to decipher this information and predict the patterns of RNA expression. How do we break this "transcriptional code"? Mechanistic studies, using simple model promoters, indicate that transcription is controlled by the coordinate action of sequence-specific DNA binding proteins interacting with the general transcriptional machinery via intermediary adapters and chromatin remodeling activities. How can we integrate this biochemical information with data from genome-wide studies to describe the generation of highly complex patterns of transcription? Here, we discuss recent studies that may point the way ahead. We also highlight difficulties that the field faces in dissecting transcriptional control in the post-genome era.

Accessibility of transcriptionally inactive genes is specifically reduced at homeoprotein-DNA binding sites in Drosophila.

We showed previously that homeoproteins bind to multiple DNA sites throughout the length of most genes in Drosophila embryos. Based on a comparison of in vivo and in vitro DNA binding specificities, we suggested that homeoprotein binding sites on actively transcribed genes are largely accessible, whereas the binding of homeoproteins to inactive and poorly transcribed genes may be significantly inhibited at most sites, perhaps by chromatin structure. To test this model, we have measured the accessibility of restriction enzyme sites in a number of genes in isolated nuclei. Surprisingly, our data indicate that there is no difference in the overall accessibility of sites for several restriction enzymes on active versus inactive genes. However, consistent with our model, restriction enzyme recognition sequences that overlap with homeoprotein binding sites are less accessible on inactive genes than they are on active genes. We propose that transcriptional activation in all animals may involve a localized increase in accessibility at the AT-rich regions bound by homeo-proteins and perhaps at a few other regions, rather than a generalized effect on all sites throughout a gene.

The specificity of protein-DNA crosslinking by formaldehyde: in vitro and in drosophila embryos.

Formaldehyde crosslinking has been widely used to study binding of specific proteins to DNA elements in intact cells. However, previous studies have not determined if this crosslinker preserves the bona fide pattern of DNA binding. Here we show that formaldehyde crosslinking of Drosophila embryos maps an interaction of the transcription factor Zeste to a known target element in the Ultrabithorax promoter. This data agrees broadly with previous mapping of the same Zeste binding sites by in vivo UV crosslinking, though the formaldehyde method does give a low, possibly artifactual signal on other DNA fragments that is not detected by the UV method. We also demonstrate, using an in vitro assay, that formaldehyde crosslinking accurately reflects the DNA binding specificities of both Zeste and a second transcription factor, Eve. The crosslinking reagent methylene blue is shown to preserve DNA binding specificity in vitro as well. Our results suggest that crosslinking by formaldehyde, and possibly also by methylene blue, provide an accurate guide to the interaction of proteins with their high affinity target sites in cells.

A comparison of in vivo and in vitro DNA-binding specificities suggests a new model for homeoprotein DNA binding in Drosophila embryos.

Little is known about the range of DNA sequences bound by transcription factors in vivo. Using a sensitive UV cross-linking technique, we show that three classes of homeoprotein bind at significant levels to the majority of genes in Drosophila embryos. The three classes bind with specificities different from each other; however, their levels of binding on any single DNA fragment differ by no more than 5- to 10-fold. On actively transcribed genes, there is a good correlation between the in vivo DNA-binding specificity of each class and its in vitro DNA-binding specificity. In contrast, no such correlation is seen on inactive or weakly transcribed genes. These genes are bound poorly in vivo, even though they contain many high affinity homeoprotein-binding sites. Based on these results, we suggest how the in vivo pattern of homeoprotein DNA binding is determined.

Eve and ftz regulate a wide array of genes in blastoderm embryos: the selector homeoproteins directly or indirectly regulate most genes in Drosophila.

The selector homeoproteins are a highly conserved group of transcription factors found throughout the Eumetazoa. Previously, the Drosophila selector homeoproteins Eve and Ftz were shown to bind with similar specificities to all genes tested, including four genes chosen because they were thought to be unlikely targets of Eve and Ftz. Here, we demonstrate that the expression of these four unexpected targets is controlled by Eve and probably by the other selector homeoproteins as well. A correlation is observed between the level of DNA binding and the degree to which gene expression is regulated by Eve. Suspecting that the selector homeoproteins may affect many more genes than previously thought, we have characterized the expression of randomly selected genes at different stages of embryogenesis. At cellular blastoderm, 25-50% of genes whose transcription can be monitored are regulated by both Eve and Ftz. In late embryogenesis, 87% of genes are directly or indirectly controlled by most or all selector homeoproteins. We argue that this broad control of gene expression is essential to coordinate morphogenesis. Our results raise the possibility that each selector homeoprotein may directly regulate the expression of most genes.

Regulation of segmentation and segmental identity by Drosophila homeoproteins: the role of DNA binding in functional activity and specificity.

Recent advances have shed new light on how the Q50 homeoproteins act in Drosophila. These transcription factors have remarkably similar and promiscuous DNA-binding specificities in vitro; yet they each specify distinct developmental fates in vivo. One current model suggests that, because the Q50 homeoproteins have distinct biological functions, they must each regulate different target genes. According to this 'co-selective binding' model, significant binding of Q50 homeoproteins to functional DNA elements in vivo would be dependent upon cooperative interactions with other transcription factors (cofactors). If the Q50 homeoproteins each interact differently with cofactors, they could be selectively targeted to unique, limited subsets of their in vitro recognition sites and thus control different genes. However, a variety of experiments question this model. Molecular and genetic experiments suggest that the Q50 homeoproteins do not regulate very distinct sets of genes. Instead, they mostly control the expression of a large number of shared targets. The distinct morphogenic properties of the various Q50 homeoproteins may principally result from the different manners in which they either activate or repress these common targets. Further, in vivo binding studies indicate that at least two Q50 homeoproteins have very broad and similar DNA-binding specificities in embryos, a result that is inconsistent with the 'co-selective binding' model. Based on these and other data, we suggest that Q50 homeoproteins bind many of their recognition sites without the aid of cofactors. In this 'widespread binding' model, cofactors act mainly by helping to distinguish the way in which homeoproteins regulate targets to which they are already bound.

Zeste-mediated activation by an enhancer is independent of cooperative DNA binding in vivo.

It is not clear how transcription factors bound at distal enhancer and proximal promoter sequences cooperate to stimulate transcription in vivo. To distinguish between different models for the action of enhancer elements, we have directly measured DNA binding of the Drosophila activator zeste by in vivo UV crosslinking. Experiments in Drosophila embryos show that binding of zeste protein to either the proximal promoter of the Ultrabithorax (Ubx) gene or to a Ubx enhancer element does not require the presence of the other element. However, significant transcription is observed only when both elements are present and bound by zeste. The results indicate that stimulation by an enhancer can occur by a mechanism other than increasing the occupancy of an activator to binding sites near the start site of transcription.

Measurement of in vivo DNA binding by sequence-specific transcription factors using UV cross-linking.

This paper describes an in vivo UV cross-linking protocol that is sensitive enough to detect DNA binding by sequence-specific transcription factors in Drosophila embryos and tissue culture cells. The strength of this approach is that it provides a quantitative measure of DNA binding in vivo with unambiguous identification of the factor involved in the binding. This assay often detects DNA binding properties of proteins that were not predicted from previous experiments, and it can be used to directly test diverse models of gene regulation in the context of a living organism.

Redundant control of Ultrabithorax by zeste involves functional levels of zeste protein binding at the Ultrabithorax promoter.

Many biological processes appear to be controlled by functionally redundant genes or pathways, but it has proven difficult to understand the nature of this redundancy. Here, we have analyzed a redundant regulatory interaction between the Drosophila transcription factor zeste and the homeotic gene Ultrabithorax. Mutations in zeste do not affect the cis-regulation of the endogenous Ultrabithorax gene; however, the expression of small Ultrabithorax promoter constructs is strongly dependent upon zeste. We show that this difference is due to redundant cis-regulatory elements in the Ultrabithorax gene, which presumably contain binding sites for factors that share the function of zeste. We also provide evidence suggesting that zeste and the gene encoding the GAGA factor have an overlapping function in regulating Ultrabithorax. Furthermore, we show that the zeste protein is bound at equal levels in vivo to a Ultrabithorax promoter construct, which zeste strongly activates, and to the identical promoter region in the endogenous Ultrabithorax gene, which zeste redundantly regulates. These results suggest that zeste is significantly active in the wild-type animal and not simply a factor that is induced as a back-up when other activators fail.

Purification of the Drosophila RNA polymerase II general transcription factors.

We describe a fractionation and purification scheme for the Drosophila RNA polymerase II general transcription factors. Drosophila TFIIE, TFIIF, TFIIH, and RNA polymerase II have been purified to greater than 50% homogeneity from Drosophila embryo nuclear extracts. TFIID has been purified 80-fold and is not significantly contaminated with any of the other general factors. This is the first reported identification and purification of Drosophila TFIIH and TFIIE. Further analysis shows that, similar to their mammalian counterparts, Drosophila TFIIH is composed of eight polypeptides sized between 30 and 100 kDa, and Drosophila TFIIE is composed of two polypeptides sized at 34 and 60 kDa. When all of these fractions are combined with recombinant Drosophila TFlIB, a highly purified in vitro transcription system is generated that has not previously been available in Drosophila. The TFIID fraction can be replaced with recombinant Drosophila TBP to give a transcription system that is nearly free of contaminating proteins.

DNA binding specificity of two homeodomain proteins in vitro and in Drosophila embryos.

In previous experiments, the homeodomain proteins even-skipped and fushi-tarazu were found to UV cross-link to a surprisingly wide array of DNA sites in living Drosophila embryos. We now show that UV cross-linking gives a highly accurate measure of DNA binding by these proteins. In addition, the binding of even-skipped and fushi-tarazu proteins has been measured in vitro to the same DNA fragments that were examined in vivo. This analysis shows that these proteins have broad DNA recognition properties in vitro that are likely to be important determinants of their distribution on DNA in vivo, but it also shows that in vitro DNA binding specificity alone is not sufficient to explain the distribution of these proteins in embryos.

Bending DNA can repress a eukaryotic basal promoter and inhibit TFIID binding.

Previous studies indicated that repression by eve involves cooperative DNA binding and leads to the formation of a DNA loop which encompasses the DNA sequences normally bound by the RNA polymerase II general transcription factors. To test the general principle of whether bending of a basal promoter sequence can contribute directly to repression of transcription, a minicircle template of 245 bp was used. In a purified transcription system, transcription from the minicircular DNA is greatly reduced compared with that from the identical DNA fragment in linear form. Transcription is also reduced when the minicircle contains a single-stranded nick, indicating that transcription is reduced because of DNA bending, rather than any constraint on supercoiling. We show that the reduced transcription from the minicircle in these experiments is not due to a reduced rate of elongation by RNA polymerase II. Rather, repression occurs, at least in part, because binding of the general transcription factor TFIID to the minicircle is strongly inhibited compared with binding to the linear DNA. We suggest that bending DNA may be a mechanism by which eukaryotic transcription may be regulated, by modulating the activity of the general transcription factors.

A domain of the even-skipped protein represses transcription by preventing TFIID binding to a promoter: repression by cooperative blocking.

We examined the mechanism by which the C-terminal 236 amino acids of the even-skipped protein (region CD) repress transcription. A fusion protein, CDGB, was created that contains region CD fused to the glucocorticoid receptor DNA binding domain. This protein repressed transcription in an in vitro system containing purified fractions of the RNA polymerase II general transcription factors, and repression was dependent upon the presence of high-affinity glucocorticoid receptor binding sites in the promoter. Repression by CDGB was prevented when the promoter DNA was preincubated with TFIID or TBP, whereas preincubation of the template DNA with CDGB prevented TFIID binding. Together, these results strongly imply that CDGB represses transcription by inhibiting TFIID binding, and further experiments suggested a mechanism by which this may occur. Region CD can mediate cooperative interactions between repressor molecules such that molecules bound at the glucocorticoid receptor binding sites stabilize binding of additional CDGB molecules to low-affinity binding sites throughout the basal promoter. Binding to some of these low-affinity sites was shown to contribute to repression. Further experiments suggested that the full-length eve protein also represses transcription by the same mechanism. We speculate that occupancy of secondary sites within the basal promoter by CDGB or the eve protein inhibits subsequent TFIID binding to repress transcription, a mechanism we term cooperative blocking.

Two homeo domain proteins bind with similar specificity to a wide range of DNA sites in Drosophila embryos.

We have used in vivo UV cross-linking to directly measure DNA binding by the homeo domain proteins even-skipped (eve) and fushi tarazu (ftz) in Drosophila embryos. Strikingly, these two proteins bind at uniformly high levels throughout the length of their genetically identified target genes and at lower, but significant, levels to genes that they are not expected to regulate. The data also suggest that these two proteins have very similar DNA-binding specificities in vivo. In contrast, a non-homeo domain transcription factor, zeste, is only detected on short DNA elements within a target promoter and not on other genes. These results are consistent with the in vitro properties of these various proteins, their respective concentrations in the nucleus, and with earlier predictions of how transcription factors bind DNA in vivo. We propose that these data favor the model that eve, ftz, and closely related homeo domain proteins act by directly regulating mostly the same target genes.

Cooperative binding at a distance by even-skipped protein correlates with repression and suggests a mechanism of silencing.

In this study, we examined how the Drosophila developmental control gene even-skipped (eve) represses transcription. Tissue culture cells were used to show that eve contains domains which inhibit transcriptional activators present at the Ultrabithorax (Ubx) proximal promoter when bound up to 1.5 kb away from these activators. Different portions of eve were fused to a heterologous DNA binding domain to show that three adjacent regions of eve contribute to silencing. There appear to be two mechanisms by which eve protein represses transcription. In this study, we used in vitro transcription and DNA binding experiments to provide evidence for one of these mechanisms. Repression in vitro correlates with binding of eve protein to two low-affinity sites in the Ubx proximal promoter. Occupancy of these low-affinity sites is dependent upon cooperative binding of other eve molecules to a separate high-affinity site. Some of these sites are separated by over 150 bp of DNA, and the data suggest that this intervening DNA is bent to form a looped structure similar to those caused by prokaryotic repressors. One of the low-affinity sites overlaps an activator element bound by the zeste transcription factor. Binding of eve protein is shown to exclude binding by zeste protein. These data suggest a mechanism for silencing whereby a repressor protein would be targeted to DNA by a high-affinity element, which itself does not overlap activator elements. Cooperative binding of further repressor molecules to distant low-affinity sites, and competition with activators bound at these sites lead to repression at a distance.