Alexander Kuzin

Drosophila homeodomain protein Nkx6 coordinates motoneuron subtype identity and axonogenesis

The regulatory networks acting in individual neurons to control their stereotyped differentiation, connectivity, and function are not well understood. Here, we demonstrate that homeodomain protein Nkx6 is a key member of the genetic network of transcription factors that specifies neuronal fates in Drosophila. Nkx6 collaborates with the homeodomain protein Hb9 to specify ventrally projecting motoneuron fate and to repress dorsally projecting motoneuron fate. While Nkx6 acts in parallel with hb9 to regulate motoneuron fate, we find that Nkx6 plays a distinct role to promote axonogenesis, as axon growth of Nkx6-positive motoneurons is severely compromised in Nkx6 mutant embryos. Furthermore, Nkx6 is necessary for the expression of the neural adhesion molecule Fasciclin III in Nkx6-positive motoneurons. Thus, this work demonstrates that Nkx6 acts in a specific neuronal population to link neuronal subtype identity to neuronal morphology and connectivity.

Horizontal Gene Transfers Link a Human MRSA Pathogen to Contagious Bovine Mastitis Bacteria

Background Acquisition of virulence factors and antibiotic resistance by many clinically important bacteria can be traced to horizontal gene transfer (HGT) between related or evolutionarily distant microflora. Comparative genomic analysis has become an important tool for identifying HGT DNA in emerging pathogens. We have adapted the multi-genome alignment tool EvoPrinter to facilitate discovery of HGT DNA sequences within bacterial genomes and within their mobile genetic elements. Principal Findings EvoPrinter analysis of 13 different Staphylococcus aureus genomes revealed that one of the human isolates, the hospital epidemic methicillin-resistant MRSA252 strain, uniquely shares multiple putative HGT DNA sequences with different causative agents of bovine mastitis that are not found in the other human S. aureus isolates. MRSA252 shares over 14 different DNA sequence blocks with the bovine mastitis ET3 S. aureus strain RF122, and many of the HGT DNAs encode virulence factors. EvoPrinter analysis of the MRSA252 chromosome also uncovered virulence-factor encoding HGT events with the genome of Listeria monocytogenes and a Staphylococcus saprophyticus associated plasmid. Both bacteria are also causal agents of contagious bovine mastitis. Conclusions EvoPrinter analysis reveals that the human MRSA252 strain uniquely shares multiple DNA sequence blocks with different causative agents of bovine mastitis, suggesting that HGT events may be occurring between these pathogens. These findings have important implications with regard to animal husbandry practices that inadvertently enhance the contact of human and livestock bacterial pathogens.

Nerfin-1 and -2, novel Drosophila Zn-finger transcription factor genes expressed in the developing nervous system

To gain insight into the regulatory networks controlling Drosophila neural-identity decisions, we have identified new neuronal precursor genes by performing an in situ hybridization screen of differentially selected embryonic head cDNAs. Here, we describe the molecular characteristics and expression profile of nerfin-1, a novel pan-neural precursor gene. This paper also documents the embryonic expression of another structurally related gene, nerfin-2. During early CNS development, nerfin-1 gene expression is activated in neuroblasts (NBs) prior to lineage formation. However, after early sublineage development, nerfin-1 expression shifts from NBs to ganglion mother cells (GMCs) but is not expressed in neurons or glia. Differing from nerfin-1, nerfin-2 is expressed only in a subset of brain neurons. Possessing a conserved putative DNA-binding domain, the predicted Nerfin-1 and -2 proteins belong to a subfamily of Zn-finger transcription factors with cognates identified in nematode, mouse and man.

cis-Decoder discovers constellations of conserved DNA sequences shared among tissue-specific enhancers

A systematic approach is described for analysis of evolutionarily conserved cis-regulatory DNA using cis-Decoder, a tool for discovery of conserved sequence elements that are shared between similarly regulated enhancers. Analysis of 2,086 conserved sequence blocks (CSBs), identified from 135 characterized enhancers, reveals most CSBs consist of shorter overlapping/adjacent elements that are either enhancer type-specific or common to enhancers with divergent regulatory behaviors. Our findings suggest that enhancers employ overlapping repertoires of highly conserved core elements.

Making Drosophila lineage–restricted drivers via patterned recombination in neuroblasts

The Drosophila cerebrum originates from about 100 neuroblasts per hemisphere, with each neuroblast producing a characteristic set of neurons. Neurons from a neuroblast are often so diverse that many neuron types remain unexplored. We developed new genetic tools that target neuroblasts and their diverse descendants, increasing our ability to study fly brain structure and development. Common enhancer-based drivers label neurons on the basis of terminal identities rather than origins, which provides limited labeling in the heterogeneous neuronal lineages. We successfully converted conventional drivers that are temporarily expressed in neuroblasts, into drivers expressed in all subsequent neuroblast progeny. One technique involves immortalizing GAL4 expression in neuroblasts and their descendants. Another depends on loss of the GAL4 repressor, GAL80, from neuroblasts during early neurogenesis. Furthermore, we expanded the diversity of MARCM-based reagents and established another site-specific mitotic recombination system. Our transgenic tools can be combined to map individual neurons in specific lineages of various genotypes.

Use of a Drosophila genome‐wide conserved sequence database to identify functionally related cis‐regulatory enhancers

Background: Phylogenetic footprinting has revealed that cis‐regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full‐advantage of the sequence information within enhancer CSCs. Results: We have generated a Drosophila genome‐wide database of conserved DNA consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis‐Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis‐regulatory function. To demonstrate the utility of these tools, a temporally‐restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. Conclusions: cis‐Decoder reveals that co‐regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process. Developmental Dynamics 241:169–189, 2012.

The cis- Regulatory Dynamics of the Drosophila CNS Determinant castor are Controlled by Multiple Sub-Pattern Enhancers

In the developing CNS, unique functional identities among neurons and glia are, in part, established as a result of successive transitions in gene expression programs within neural precursor cells. One of the temporal-identity windows within Drosophila CNS neural precursor cells or neuroblasts (NBs) is marked by the expression of a zinc-finger transcription factor (TF) gene, castor (cas). Our analysis of cis-regulatory DNA within a cas loss-of-function rescue fragment has identified seven enhancers that independently activate reporter transgene expression in specific sub-patterns of the wild-type embryonic cas gene expression domain. Most of these enhancers also regulate different aspects of cas expression within the larval and adult CNS. Phylogenetic footprinting reveals that each enhancer is made up of clusters of highly conserved DNA sequence blocks that are flanked by less-conserved inter-cluster spacer sequences. Comparative analysis of the conserved DNA also reveals that cas enhancers share different combinations of sequence elements and many of these shared elements contain core DNA-binding recognition motifs for characterized temporal-identity TFs. Intra-species alignments show that two of the sub-pattern enhancers originated from an inverted duplication and that this repeat is unique to the cas locus in all sequenced Drosophila species. Finally we show that three of the enhancers differentially require cas function for their wild-type regulatory behavior. Cas limits the expression of one enhancer while two others require cas function for full expression. These studies represent a starting point for the further analysis of cas gene expression and the TFs that regulate it.

Conserved sequence block clustering and flanking inter-cluster flexibility delineate enhancers that regulate nerfin-1 expression during Drosophila CNS development

We have identified clusters of conserved sequences constituting discrete modular enhancers within the Drosophilanerfin-1 locus. nerfin-1 encodes a Zn-finger transcription factor that directs pioneer interneuron axon guidance. nerfin-1 mRNA is detected in many early delaminating neuroblasts, ganglion mother cells and transiently in nascent neurons. The comparative genomics analysis program EvoPrinter revealed conserved sequence blocks both upstream and downstream of the transcribed region. By using the aligning regions of different drosophilids as the reference DNA, EvoPrinter detects sequence length flexibility between clusters of conserved sequences and thus facilitates differentiation between closely associated modular enhancers. Expression analysis of enhancer-reporter transgenes identified enhancers that drive expression in different regions of the developing embryonic and adult nervous system, including subsets of embryonic CNS neuroblasts, GMCs, neurons and PNS neurons. In summary, EvoPrinter facilitates the discovery and analysis of enhancers that control crucial aspects of nerfin-1 expression.

Sequence conservation and combinatorial complexity of Drosophila neural precursor cell enhancers

BackgroundThe presence of highly conserved sequences within cis-regulatory regions can serve as a valuable starting point for elucidating the basis of enhancer function. This study focuses on regulation of gene expression during the early events of Drosophila neural development. We describe the use of EvoPrinter and cis-Decoder, a suite of interrelated phylogenetic footprinting and alignment programs, to characterize highly conserved sequences that are shared among co-regulating enhancers.ResultsAnalysis of in vivo characterized enhancers that drive neural precursor gene expression has revealed that they contain clusters of highly conserved sequence blocks (CSBs) made up of shorter shared sequence elements which are present in different combinations and orientations within the different co-regulating enhancers; these elements contain either known consensus transcription factor binding sites or consist of novel sequences that have not been functionally characterized. The CSBs of co-regulated enhancers share a large number of sequence elements, suggesting that a diverse repertoire of transcription factors may interact in a highly combinatorial fashion to coordinately regulate gene expression. We have used information gained from our comparative analysis to discover an enhancer that directs expression of the nervy gene in neural precursor cells of the CNS and PNS.ConclusionThe combined use EvoPrinter and cis-Decoder has yielded important insights into the combinatorial appearance of fundamental sequence elements required for neural enhancer function. Each of the 30 enhancers examined conformed to a pattern of highly conserved blocks of sequences containing shared constituent elements. These data establish a basis for further analysis and understanding of neural enhancer function.

Functional analysis of conserved sequences within a temporally restricted neural precursor cell enhancer

Many of the key regulators of Drosophila CNS neural identity are expressed in defined temporal orders during neuroblast (NB) lineage development. To begin to understand the structural and functional complexity of enhancers that regulate ordered NB gene expression programs, we have undertaken the mutational analysis of the temporally restricted nerfin-1 NB enhancer. Our previous studies have localized the enhancer to a region just proximal to the nerfin-1 transcription start site. Analysis of this enhancer, using the phylogenetic footprint program EvoPrinter, reveals the presence of multiple sequence blocks that are conserved among drosophilids. cis-Decoder alignments of these conserved sequence blocks (CSBs) has identified shorter elements that are conserved in other Drosophila NB enhancers. Mutagenesis of the enhancer reveals that although each CSB is required for wild-type expression, neither position nor orientation of the CSBs within the enhancer is crucial for enhancer function; removal of less-conserved or non-conserved sequences flanking CSB clusters also does not significantly alter enhancer activity. While all three conserved E-box transcription factor (TF) binding sites (CAGCTG) are required for full function, adding an additional site at different locations within non-conserved sequences interferes with enhancer activity. Of particular note, none of the mutations resulted in ectopic reporter expression outside of the early NB expression window, suggesting that the temporally restricted pattern is defined by transcriptional activators and not by direct DNA binding repressors. Our work also points to an unexpectedly large number of TFs required for optimal enhancer function - mutant TF analysis has identified at least four that are required for full enhancer regulation.