CONDOR: a database resource of developmentally associated conserved non-coding elements

<p>Abstract</p> <p>Background</p> <p>Comparative genomics is currently one of the most popular approaches to study the regulatory architecture of vertebrate genomes. Fish-mammal genomic comparisons have proved powerful in identifying conserved non-coding elements likely to be distal <it>cis-</it>regulatory modules such as enhancers, silencers or insulators that control the expression of genes involved in the regulation of early development. The scientific community is showing increasing interest in characterizing the function, evolution and language of these sequences. Despite this, there remains little in the way of user-friendly access to a large dataset of such elements in conjunction with the analysis and the visualization tools needed to study them.</p> <p>Description</p> <p>Here we present CONDOR (COnserved Non-coDing Orthologous Regions) available at: <url>http://condor.fugu.biology.qmul.ac.uk</url>. In an interactive and intuitive way the website displays data on > 6800 non-coding elements associated with over 120 early developmental genes and conserved across vertebrates. The database regularly incorporates results of ongoing <it>in vivo </it>zebrafish enhancer assays of the CNEs carried out in-house, which currently number ~100. Included and highlighted within this set are elements derived from duplication events both at the origin of vertebrates and more recently in the teleost lineage, thus providing valuable data for studying the divergence of regulatory roles between paralogs. CONDOR therefore provides a number of tools and facilities to allow scientists to progress in their own studies on the function and evolution of developmental <it>cis</it>-regulation.</p> <p>Conclusion</p> <p>By providing access to data with an approachable graphics interface, the CONDOR database presents a rich resource for further studies into the regulation and evolution of genes involved in early development.</p

Effect of Citrus Byproducts on Survival of O157:H7 and Non-O157 \u3ci\u3eEscherichia coli\u3c/i\u3e Serogroups within \u3ci\u3eIn Vitro\u3c/i\u3e Bovine Ruminal Microbial Fermentations

Citrus byproducts (CBPs) are utilized as a low cost nutritional supplement to the diets of cattle and have been suggested to inhibit the growth of both Escherichia coli O157:H7 and Salmonella. The objective of this study was to examine the effects in vitro that varying concentrations of CBP in the powdered or pelleted variety have on the survival of Shiga-toxin Escherichia coli (STEC) serotypes O26:H11, O103:H8, O111:H8, O145:H28, and O157:H7 in bovine ruminal microorganism media. The O26:H11, O111:H8, O145:H28, and O157:H7 serotypes did not exhibit a change in populations in media supplemented with CBP with either variety. The O103:H8 serotype displayed a general trend for an approximate 1 log10 reduction in 5% powdered CBP and 20% pelleted CBP over 6 h. There was a trend for reductions in populations of a variant form of O157:H7 mutated in the stx1 and stx2 genes in higher concentrations of CBP. These results suggest that variations exist in the survival of these serotypes of STEC within mixed ruminal microorganism fluid media when supplemented with CBP. Further research is needed to determine why CBPs affect STEC serotypes differently

Examination and reconstruction of three ancient endogenous parvovirus capsid protein gene remnants found in rodent genomes

Parvovirus-derived endogenous viral elements (EVEs) have been found in the genomes of many different animal species, resulting from integration events that may have occurred from more than 50 million years ago to much more recently. Here, we further investigate the properties of autonomous parvovirus EVEs and describe their relationships to contemporary viruses. While we did not find any intact capsid protein open reading frames in the integrated viral sequences, we examined three EVEs that were repaired to form full-length sequences with relatively few changes. These sequences were found in the genomes of Rattus norvegicus (brown rat), Mus spretus (Algerian mouse), and Apodemus sylvaticus (wood mouse). The R. norvegicus sequence was not present in the genomes of the closely related species R. rattus, R. tanezumi, R. exulans, and R. everetti, indicating that it was less than 2 million years old, and the M. spretus and A. sylvaticus sequences were not found in the published genomes of other mouse species, also indicating relatively recent insertions. The M. spretus VP2 sequence assembled into capsids, which had high thermal stability, bound the sialic acid N-acetylneuraminic acid, and entered murine L cells. The 3.89-Å structure of the M. spretus virus-like particles (VLPs), determined using cryo-electron microscopy, showed similarities to rodent and porcine parvovirus capsids. The repaired VP2 sequences from R. norvegicus and A. sylvaticus did not assemble as first prepared, but chimeras combining capsid surface loops from R. norvegicus with canine parvovirus assembled, allowing some of that capsid’s structures and functions to be examined

Functional Analysis of Conserved Non-Coding Regions Around the Short Stature hox Gene (shox) in Whole Zebrafish Embryos

Background: Mutations in the SHOX gene are responsible for Leri-Weill Dyschondrosteosis, a disorder characterised by mesomelic limb shortening. Recent investigations into regulatory elements surrounding SHOX have shown that deletions of conserved non-coding elements (CNEs) downstream of the SHOX gene produce a phenotype indistinguishable from Leri-Weill Dyschondrosteosis. As this gene is not found in rodents, we used zebrafish as a model to characterise the expression pattern of the shox gene across the whole embryo and characterise the enhancer domains of different CNEs associated with this gene. Methodology/Principal Findings: Expression of the shox gene in zebrafish was identified using in situ hybridization, with embryos showing expression in the blood, putative heart, hatching gland, brain pharyngeal arch, olfactory epithelium, and fin bud apical ectodermal ridge. By identifying sequences showing 65% identity over at least 40 nucleotides between Fugu, human, dog and opossum we uncovered 35 CNEs around the shox gene. These CNEs were compared with CNEs previously discovered by Sabherwal et al. ,resulting in the identification of smaller more deeply conserved sub-sequence. Sabherwal et al.’s CNEs were assayed for regulatory function in whole zebrafish embryos resulting in the identification of additional tissues under the regulatory control of these CNEs. Conclusion/Significance: Our results using whole zebrafish embryos have provided a more comprehensive picture of the expression pattern of the shox gene, and a better understanding of its regulation via deeply conserved noncoding elements. In particular, we identify additional tissues under the regulatory control of previously identified SHOX CNEs. We also demonstrate the importance of these CNEs in evolution by identifying duplicated shox CNEs and more deeply conserved sub-sequences within already identified CNEs

Early Evolution of Conserved Regulatory Sequences Associated with Development in Vertebrates

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA

Human GLI3 Intragenic Conserved Non-Coding Sequences Are Tissue-Specific Enhancers

The zinc-finger transcription factor GLI3 is a key regulator of development, acting as a primary transducer of Sonic hedgehog (SHH) signaling in a combinatorial context dependent fashion controlling multiple patterning steps in different tissues/organs. A tight temporal and spatial control of gene expression is indispensable, however, cis-acting sequence elements regulating GLI3 expression have not yet been reported. We show that 11 ancient genomic DNA signatures, conserved from the pufferfish Takifugu (Fugu) rubripes to man, are distributed throughout the introns of human GLI3. They map within larger conserved non-coding elements (CNEs) that are found in the tetrapod lineage. Full length CNEs transiently transfected into human cell cultures acted as cell type specific enhancers of gene transcription. The regulatory potential of these elements is conserved and was exploited to direct tissue specific expression of a reporter gene in zebrafish embryos. Assays of deletion constructs revealed that the human-Fugu conserved sequences within the GLI3 intronic CNEs were essential but not sufficient for full-scale transcriptional activation. The enhancer activity of the CNEs is determined by a combinatorial effect of a core sequence conserved between human and teleosts (Fugu) and flanking tetrapod-specific sequences, suggesting that successive clustering of sequences with regulatory potential around an ancient, highly conserved nucleus might be a possible mechanism for the evolution of cis-acting regulatory elements

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

PARVOVIRUS CAPSID STRUCTURES, LIGAND BINDING INTERACTIONS, AND ENDOGENOUS VIRAL ELEMENTS

Parvoviruses are among the simplest of viruses, with capsids composed of variants of a single structural protein. These capsids mediate many of the processes required for infection and are remarkably stable. However, antibody binding or mutations to the capsid can disrupt these processes and block infection. This dissertation discusses mutations to the canine parvovirus (CPV) capsid that result in loss of infection, the interaction between CPV and the transferrin receptor (TfR), and parvovirus endogenous viral elements (EVEs), which contain ancient parvovirus gene sequences. I found that point mutations to CPV VP2 residues 270, 272, 273, and 299/300 result in loss of viral infectivity. Mutation of residue 270 results in loss of a sub-molar proteolytic cleavage event in VP2 and increases capsid stability, residue 272 mutation causes loss of capsid assembly, and residue 273 mutation results in assembled capsids being trapped in the nucleus. Mutation of VP2 residues 299 and 300, which are associated with TfR binding, to lysine disrupts the interaction between CPV and the TfR, inhibiting infection but still allowing receptor binding and uptake into cells. I also found that CPV has different interactions with TfRs from different host species, binding strongly to some TfRs and very weakly to others, even though each TfR can mediate a successful infection. TfRs from different species also had varying levels of occupancy on CPV capsids, with up to 12 black-backed jackal TfRs, but only 1-2 feline TfRs binding to each capsid, and it is possible that the feline TfR induces a conformational change in CPV that inhibits binding of additional TfRs. Antibody binding could also disrupt the CPV/TfR binding interaction, suggesting a possible mechanism of virus neutralization. To examine the structure and function of ancient parvovirus capsids, I expressed the VP2 gene from three different parvovirus EVEs. VP2 expressed from an EVE in the M. spretus genome assembled into capsids, and I determined that these capsids were highly stable, could bind to N-Acetylneuraminic acid, and were endocytosed into murine cells. The results in this dissertation provide new information about parvovirus infection, receptor binding interactions, and evolution, and further the understanding of how infection occurs and may be disrupted