Daniela Bezdan

Evidence for the biogenesis of more than 1,000 novel human microRNAs

BackgroundMicroRNAs (miRNAs) are established regulators of development, cell identity and disease. Although nearly two thousand human miRNA genes are known and new ones are continuously discovered, no attempt has been made to gauge the total miRNA content of the human genome.ResultsEmploying an innovative computational method on massively pooled small RNA sequencing data, we report 2,469 novel human miRNA candidates of which 1,098 are validated by in-house and published experiments. Almost 300 candidates are robustly expressed in a neuronal cell system and are regulated during differentiation or when biogenesis factors Dicer, Drosha, DGCR8 or Ago2 are silenced. To improve expression profiling, we devised a quantitative miRNA capture system. In a kidney cell system, 400 candidates interact with DGCR8 at transcript positions that suggest miRNA hairpin recognition, and 1,000 of the new miRNA candidates interact with Ago1 or Ago2, indicating that they are directly bound by miRNA effector proteins. From kidney cell CLASH experiments, in which miRNA-target pairs are ligated and sequenced, we observe hundreds of interactions between novel miRNAs and mRNA targets. The novel miRNA candidates are specifically but lowly expressed, raising the possibility that not all may be functional. Interestingly, the majority are evolutionarily young and overrepresented in the human brain.ConclusionsIn summary, we present evidence that the complement of human miRNA genes is substantially larger than anticipated, and that more are likely to be discovered in the future as more tissues and experimental conditions are sequenced to greater depth.

Comparative analysis of Hox downstream genes in Drosophila

Functional diversification of body parts is dependent on the formation of specialized structures along the various body axes. In animals, region-specific morphogenesis along the anteroposterior axis is controlled by a group of conserved transcription factors encoded by the Hox genes. Although it has long been assumed that Hox proteins carry out their function by regulating distinct sets of downstream genes, only a small number of such genes have been found, with very few having direct roles in controlling cellular behavior. We have quantitatively identified hundreds of Hox downstream genes in Drosophila by microarray analysis, and validated many of them by in situ hybridizations on loss- and gain-of-function mutants. One important finding is that Hox proteins, despite their similar DNA-binding properties in vitro, have highly specific effects on the transcriptome in vivo, because expression of many downstream genes respond primarily to a single Hox protein. In addition, a large fraction of downstream genes encodes realizator functions, which directly affect morphogenetic processes, such as orientation and rate of cell divisions, cell-cell adhesion and communication, cell shape and migration, or cell death. Focusing on these realizators, we provide a framework for the morphogenesis of the maxillary segment. As the genomic organization of Hox genes and the interaction of Hox proteins with specific co-factors are conserved in vertebrates and invertebrates, and similar classes of downstream genes are regulated by Hox proteins across the metazoan phylogeny, our findings represent a first step toward a mechanistic understanding of morphological diversification within a species as well as between species.

Antagonistic regulation of apoptosis and differentiation by the Cut transcription factor represents a tumor-suppressing mechanism in Drosophila

Apoptosis is essential to prevent oncogenic transformation by triggering self-destruction of harmful cells, including those unable to differentiate. However, the mechanisms linking impaired cell differentiation and apoptosis during development and disease are not well understood. Here we report that the Drosophila transcription factor Cut coordinately controls differentiation and repression of apoptosis via direct regulation of the pro-apoptotic gene reaper. We also demonstrate that this regulatory circuit acts in diverse cell lineages to remove uncommitted precursor cells in status nascendi and thereby interferes with their potential to develop into cancer cells. Consistent with the role of Cut homologues in controlling cell death in vertebrates, we find repression of apoptosis regulators by Cux1 in human cancer cells. Finally, we present evidence that suggests that other lineage-restricted specification factors employ a similar mechanism to put the brakes on the oncogenic process.

Multifactorial Regulation of a Hox Target Gene

Hox proteins play fundamental roles in controlling morphogenetic diversity along the anterior–posterior body axis of animals by regulating distinct sets of target genes. Within their rather broad expression domains, individual Hox proteins control cell diversification and pattern formation and consequently target gene expression in a highly localized manner, sometimes even only in a single cell. To achieve this high-regulatory specificity, it has been postulated that Hox proteins co-operate with other transcription factors to activate or repress their target genes in a highly context-specific manner in vivo. However, only a few of these factors have been identified. Here, we analyze the regulation of the cell death gene reaper (rpr) by the Hox protein Deformed (Dfd) and suggest that local activation of rpr expression in the anterior part of the maxillary segment is achieved through a combinatorial interaction of Dfd with at least eight functionally diverse transcriptional regulators on a minimal enhancer. It follows that context-dependent combinations of Hox proteins and other transcription factors on small, modular Hox response elements (HREs) could be responsible for the proper spatio-temporal expression of Hox targets. Thus, a large number of transcription factors are likely to be directly involved in Hox target gene regulation in vivo.

The cis-regulatory code of Hox function in Drosophila

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis‐regulatory DNA elements. While much is known about TF function in general, our understanding of their cell type‐specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, we have identified binding regions for the Hox TF Deformed (Dfd) in the Drosophila genome. Our analysis of architectural features within Hox cis‐regulatory response elements (HREs) shows that HRE structure is essential for cell type‐specific gene expression. We also find that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non‐overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif‐pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, our results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo.

Analysis of a long-term outbreak of XDR Pseudomonas aeruginosa: a molecular epidemiological study

OBJECTIVES Here we report on a long-term outbreak from 2009 to 2012 with an XDR Pseudomonas aeruginosa on two wards at a university hospital in southern Germany. METHODS Whole-genome sequencing was performed on the outbreak isolates and a core genome was constructed for molecular epidemiological analysis. We applied a time-place-sequence algorithm to improve estimation of transmission probabilities. RESULTS By using conventional infection control methods we identified 49 P. aeruginosa strains, including eight environmental isolates that belonged to ST308 (by MLST) and carried the metallo-β-lactamase IMP-8. Phylogenetic analysis on the basis of a non-recombinant core genome that contained 22 outbreak-specific SNPs revealed a pattern of four dominant clades with a strong phylogeographic structure and allowed us to determine the potential temporal origin of the outbreak to July 2008, 1 year before the index case was diagnosed. Superspreaders at the root of clades exhibited a high number of probable and predicted transmissions, indicating their exceptional position in the outbreak. CONCLUSIONS Our results suggest that the initial expansion of dominant sublineages was driven by a few superspreaders, while environmental contamination seemed to sustain the outbreak for a long period despite regular environmental control measures.

Chromosome-level assembly of Arabidopsis thaliana Ler reveals the extent of translocation and inversion polymorphisms.

Significance Despite widespread reports on deciphering the sequences of all kinds of genomes, most of these reconstructed genomes rely on a comparison of short DNA sequencing reads to a reference sequence, rather than being independently reconstructed. This method limits the insights on genomic differences to local, mostly small-scale variation, because large rearrangements are likely overlooked by current methods. We have de novo assembled the genome of a common strain of Arabidopsis thaliana Landsberg erecta and revealed hundreds of rearranged regions. Some of these differences suppress meiotic recombination, impacting the haplotypes of a worldwide population of A. thaliana. In addition to sequence changes, this work, which, to our knowledge is the first comparison of an independent, chromosome-level assembled A. thaliana genome, revealed hundreds of unknown, accession-specific genes. Resequencing or reference-based assemblies reveal large parts of the small-scale sequence variation. However, they typically fail to separate such local variation into colinear and rearranged variation, because they usually do not recover the complement of large-scale rearrangements, including transpositions and inversions. Besides the availability of hundreds of genomes of diverse Arabidopsis thaliana accessions, there is so far only one full-length assembled genome: the reference sequence. We have assembled 117 Mb of the A. thaliana Landsberg erecta (Ler) genome into five chromosome-equivalent sequences using a combination of short Illumina reads, long PacBio reads, and linkage information. Whole-genome comparison against the reference sequence revealed 564 transpositions and 47 inversions comprising ∼3.6 Mb, in addition to 4.1 Mb of nonreference sequence, mostly originating from duplications. Although rearranged regions are not different in local divergence from colinear regions, they are drastically depleted for meiotic recombination in heterozygotes. Using a 1.2-Mb inversion as an example, we show that such rearrangement-mediated reduction of meiotic recombination can lead to genetically isolated haplotypes in the worldwide population of A. thaliana. Moreover, we found 105 single-copy genes, which were only present in the reference sequence or the Ler assembly, and 334 single-copy orthologs, which showed an additional copy in only one of the genomes. To our knowledge, this work gives first insights into the degree and type of variation, which will be revealed once complete assemblies will replace resequencing or other reference-dependent methods.

Minerva: An Alignment and Reference Free Approach to Deconvolve Linked-Reads for Metagenomics

Emerging Linked-Read technologies (aka Read-Cloud or barcoded short-reads) have revived interest in standard short-read technology as a viable way to understand large-scale structure in genomes and metagenomes. Linked-Read technologies, such as the 10X Chromium system, use a microfluidic system and a set of specially designed 3’ barcodes (aka UIDs) to tag short DNA reads which were originally sourced from the same long fragment of DNA; subsequently, these specially barcoded reads are sequenced on standard short read platforms. This approach results in interesting compromises. Each long fragment of DNA is covered only sparsely by short reads, no information about the relative ordering of reads from the same fragment is preserved, and typically each 3’ barcode matches reads from 2-20 long fragments of DNA. However, compared to long read platforms like those produced by Pacific Biosciences and Oxford Nanopore the cost per base to sequence is far lower, far less input DNA is required, and the per base error rate is that of Illumina short-reads. The use of Linked-Reads presents a new set of algorithmic challenges. In this paper, we formally describe one particular issue common to all applications of Linked-Read technology: the deconvolution of reads with a single 3’ barcode into clusters that correspond to a single long fragment of DNA. We introduce Minerva, A graph-based algorithm that approximately solves the barcode deconvolution problem for metagenomic data (where reference genomes may be incomplete or unavailable). Additionally, we develop two demonstrations where the deconvolution of barcoded reads improves downstream results: improving the specificity of taxonomic assignments, and by improving clustering of related sequences. To the best of our knowledge, we are the first to address the problem of barcode deconvolution in metagenomics.

Multi-omics approach identifies novel pathogen-derived prognostic biomarkers in patients with Pseudomonas aeruginosa bloodstream infection

Pseudomonas aeruginosa is a human pathogen that causes health-care associated blood stream infections (BSI). Although P. aeruginosa BSI are associated with high mortality rates, the clinical relevance of pathogen-derived prognostic biomarker to identify patients at risk for unfavorable outcome remains largely unexplored. We found novel pathogen-derived prognostic biomarker candidates by applying a multi-omics approach on a multicenter sepsis patient cohort. Multi-level Cox regression was used to investigate the relation between patient characteristics and pathogen features (2298 accessory genes, 1078 core protein levels, 107 parsimony-informative variations in reported virulence factors) with 30-day mortality. Our analysis revealed that presence of the helP gene encoding a putative DEAD-box helicase was independently associated with a fatal outcome (hazard ratio 2.01, p = 0.05). helP is located within a region related to the pathogenicity island PAPI-1 in close proximity to a pil gene cluster, which has been associated with horizontal gene transfer. Besides helP, elevated protein levels of the bacterial flagellum protein FliL (hazard ratio 3.44, p < 0.001) and of a bacterioferritin-like protein (hazard ratio 1.74, p = 0.003) increased the risk of death, while high protein levels of a putative aminotransferase were associated with an improved outcome (hazard ratio 0.12, p < 0.001). The prognostic potential of biomarker candidates and clinical factors was confirmed with different machine learning approaches using training and hold-out datasets. The helP genotype appeared the most attractive biomarker for clinical risk stratification due to its relevant predictive power and ease of detection.

Whole-genome sequencing enabling the detection of a colistin-resistant hypermutating Citrobacter werkmanii strain harbouring a novel metallo-β-lactamase VIM-48

Citrobacter spp. harbouring metallo-β-lactamases (MBLs) have been reported from various countries and different sources, but their isolation from clinical specimens remains a rare event in Europe. MBL-harbouring Enterobacteriaceae are considered a major threat in infection control as therapeutic options are often limited to colistin. In this study, whole-genome sequencing was applied to characterise five clinical isolates of multidrug-resistant Citrobacter werkmanii obtained from rectal swabs. Four strains possessed a class 1 integron with a novel blaVIM-48 MBL resistance gene and the aminoglycoside acetyltransferase gene aacA4, whilst one isolate harboured a blaIMP-8 MBL. Resistance to colistin evolved in one strain isolated from a patient who had received colistin orally for 8 days. Genomic comparison of this strain with a colistin-susceptible pre-treatment isolate from the same patient revealed 66 single nucleotide polymorphisms (SNPs) and 26 indels, indicating the presence of a mutator phenotype. This was confirmed by the finding of a SNP in the mutL gene that led to a significantly truncated protein. Additionally, an amino acid change from glycine to serine at position 53 was observed in PmrA. Mutations in the pmrA gene have been previously described as mediating colistin resistance in different bacterial species and are the most likely reason for the susceptibility change observed. To the best of our knowledge, this is the first description of a colistin-resistant Citrobacter spp. isolated from a human sample. This study demonstrates the power of applying next-generation sequencing in a hospital setting to trace and understand evolving resistance at the level of individual patients.