A Toolkit and Robust Pipeline for the Generation of Fosmid-Based Reporter Genes in C. elegans

Engineering fluorescent proteins into large genomic clones, contained within BACs or fosmid vectors, is a tool to visualize and study spatiotemporal gene expression patterns in transgenic animals. Because these reporters cover large genomic regions, they most likely capture all cis-regulatory information and can therefore be expected to recapitulate all aspects of endogenous gene expression. Inserting tags at the target gene locus contained within genomic clones by homologous recombination (“recombineering”) represents the most straightforward method to generate these reporters. In this methodology paper, we describe a simple and robust pipeline for recombineering of fosmids, which we apply to generate reporter constructs in the nematode C. elegans, whose genome is almost entirely covered in an available fosmid library. We have generated a toolkit that allows for insertion of fluorescent proteins (GFP, YFP, CFP, VENUS, mCherry) and affinity tags at specific target sites within fosmid clones in a virtually seamless manner. Our new pipeline is less complex and, in our hands, works more robustly than previously described recombineering strategies to generate reporter fusions for C. elegans expression studies. Furthermore, our toolkit provides a novel recombineering cassette which inserts a SL2-spliced intercistronic region between the gene of interest and the fluorescent protein, thus creating a reporter controlled by all 5′ and 3′ cis-acting regulatory elements of the examined gene without the direct translational fusion between the two. With this configuration, the onset of expression and tissue specificity of secreted, sub-cellular compartmentalized or short-lived gene products can be easily detected. We describe other applications of fosmid recombineering as well. The simplicity, speed and robustness of the recombineering pipeline described here should prompt the routine use of this strategy for expression studies in C. elegans

Molecular cloning and expression of the biodegradative threonine dehydratase gene ( tdc ) of Escherichia coli K12

The biodegradative threonine dehydratase gene ( tdc ) of Escherichia coli was cloned by isolating a dehydratase-negative mutant after Tn5 mutagenesis, cloning the tdc ::Tn5 DNA into pBR322 and then replacing the Tn5 element on the plasmid in vivo. Subcloning and nucleotide sequence data revealed two distinct procaryotic promoterlike elements each containing a potential CAP-binding site and AT-rich regions, and a Shine-Dalgarno sequence. One of these putative promoters, P 2 , was located immediately upstream from the tdc coding region, and a second, P 1 , was approximately 1 kilobase upstream from P 2 . Deletion of the potential CAP-binding site from P 1 prevented tdc gene expression. However, removal of P 2 and a large segment of the upstream DNA had no discernible effect on dehydratase synthesis. A 936-base pair open reading frame was found between P 1 and the tdc coding region, which produced a polypeptide of about 32 kilodaltons. The data suggest that P 1 , and not P 2 , is necessary for tdc gene expression, and that the DNA sequences coding for the 32 KD polypeptide and threonine dehydratase are part of a single transcriptional unit.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47562/1/438_2004_Article_BF00425676.pd

Influence of DNA topology on expression of the tdc operon in Escherichia coli K-12

TdcB activity expressed from the chromosomal gene and LacZ expression from single-copy tdc-lacZ transcriptional and translational fusions were measured in Escherichia coli strains harboring mutations in the genes encoding DNA gyrase, topoisomerase I and the HU protein. The pattern of tdc operon expression in these mutants suggests that relaxation of supercoiled DNA enhances tdc transcription in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47585/1/438_2004_Article_BF00290409.pd

DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of migratory defects in both pathways in Pax6(Sey/Sey) mice

T-cell identity and epigenetic memory

T-cell development endows cells with a flexible range of effector differentiation options, superimposed on a stable core of lineage-specific gene expression that is maintained while access to alternative hematopoietic lineages is permanently renounced. This combination of features could be explained by environmentally responsive transcription factor mobilization overlaying an epigenetically stabilized base gene expression state. For example, "poising" of promoters could offer preferential access to T-cell genes, while repressive histone modifications and DNA methylation of non-T regulatory genes could be responsible for keeping non-T developmental options closed. Here, we critically review the evidence for the actual deployment of epigenetic marking to support the stable aspects of T-cell identity. Much of epigenetic marking is dynamically maintained or subject to rapid modification by local action of transcription factors. Repressive histone marks are used in gene-specific ways that do not fit a simple, developmental lineage-exclusion hierarchy. We argue that epigenetic analysis may achieve its greatest impact for illuminating regulatory biology when it is used to locate cis-regulatory elements by catching them in the act of mediating regulatory change