Compact Genome Research Articles

Three-Dimensional Genomic Structure and Cohesin Occupancy Correlate with Transcriptional Activity during Spermatogenesis

SummaryMammalian gametogenesis involves dramatic and tightly regulated chromatin remodeling, whose regulatory pathways remain largely unexplored. Here, we generate a comprehensive high-resolution structural and functional atlas of mouse spermatogenesis by combining in situ chromosome conformation capture sequencing (Hi-C), RNA sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) of CCCTC-binding factor (CTCF) and meiotic cohesins, coupled with confocal and super-resolution microscopy. Spermatogonia presents well-defined compartment patterns and topological domains. However, chromosome occupancy and compartmentalization are highly re-arranged during prophase I, with cohesins bound to active promoters in DNA loops out of the chromosomal axes. Compartment patterns re-emerge in round spermatids, where cohesin occupancy correlates with transcriptional activity of key developmental genes. The compact sperm genome contains compartments with actively transcribed genes but no fine-scale topological domains, concomitant with the presence of protamines. Overall, we demonstrate how genome-wide cohesin occupancy and transcriptional activity is associated with three-dimensional (3D) remodeling during spermatogenesis, ultimately reprogramming the genome for the next generation.

Chromosome conformation capture that detects novel cis- and trans-interactions in budding yeast.

Chromosome Conformation Capture (3C) has emerged as a powerful approach for revealing the conformation and features of three-dimensional (3D) genomic organization. Yet attainment of higher resolution in organisms with compact genomes presents a challenge. Here, we describe modifications in the 3C technique that substantially enhance its resolution and sensitivity when applied to the 3D genome of budding yeast. Keys to our approach include use of a 4 bp cutter, Taq I, for cleaving the genome and quantitative PCR for measuring the frequency of ligation. Most importantly, we normalize the percent digestion at each restriction site to account for variation in accessibility of local chromatin structure under a given physiological condition. This strategy has led to the detection of physical interactions between regulatory elements and gene coding regions as well as intricate, stimulus-specific interchromosomal interactions between activated genes. We provide an algorithm that incorporates these and other modifications and allows quantitative determination of chromatin interaction frequencies in yeast under any physiological condition.

The Known, the New, and a Possible Surprise: A Re-Evaluation of the Nucleomorph-Encoded Proteome of Cryptophytes.

Nucleomorphs are small nuclei that evolved from the nucleus of former eukaryotic endosymbionts of cryptophytes and chlorarachniophytes. These enigmatic organelles reside in their complex plastids and harbor the smallest and most compacted eukaryotic genomes investigated so far. Although the coding capacity of the nucleomorph genomes is small, a significant percentage of the encoded proteins (predicted nucleomorph-encoded proteins, pNMPs) is still not functionally annotated. We have analyzed pNMPs with unknown functions via Phyre2, a bioinformatic tool for prediction and modeling of protein structure, resulting in a functional annotation of 215 pNMPs out of 826 uncharacterized open reading frames of cryptophytes. The newly annotated proteins are predicted to participate in nucleomorph-specific functions such as chromosome organization and expression, as well as in modification and degradation of nucleomorph-encoded proteins. Additionally, we have functionally assigned nucleomorph-encoded, putatively plastid-targeted proteins among the reinvestigated pNMPs. Hints for a putative function in the periplastid compartment, the cytoplasm surrounding the nucleomorphs, emerge from the identification of pNMPs that might be homologs of endomembrane system-related proteins. These proteins are discussed in respect to their putative functions.

The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection.

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

Autoantibodies in Systemic Lupus Erythematosus Target Mitochondrial RNA.

The mitochondrion supplies energy to the cell and regulates apoptosis. Unlike other mammalian organelles, mitochondria are formed by binary fission and cannot be directly produced by the cell. They contain numerous copies of a compact circular genome that encodes RNA molecules and proteins involved in mitochondrial oxidative phosphorylation. Whereas, mitochondrial DNA (mtDNA) activates the innate immune system if present in the cytosol or the extracellular milieu, it is also the target of circulating autoantibodies in systemic lupus erythematosus (SLE). However, it is not known whether mitochondrial RNA is also recognized by autoantibodies in SLE. In the present study, we evaluated the presence of autoantibodies targeting mitochondrial RNA (AmtRNA) in SLE. We quantified AmtRNA in an inducible model of murine SLE. The AmtRNA were also determined in SLE patients and healthy volunteers. AmtRNA titers were measured in both our induced model of murine SLE and in human SLE, and biostatistical analyses were performed to determine whether the presence and/or levels of AmtRNA were associated with clinical features expressed by SLE patients. Both IgG and IgM classes of AmtRNA were increased in SLE patients (n = 86) compared to healthy controls (n = 30) (p < 0.0001 and p = 0.0493, respectively). AmtRNA IgG levels correlated with anti-mtDNA-IgG titers (rs = 0.54, p < 0.0001) as well as with both IgG and IgM against β-2-glycoprotein I (anti-β2GPI; rs = 0.22, p = 0.05), and AmtRNA-IgG antibodies were present at higher levels when patients were positive for autoantibodies to double-stranded-genomic DNA (p < 0.0001). AmtRNA-IgG were able to specifically discriminate SLE patients from healthy controls, and were negatively associated with plaque formation (p = 0.04) and lupus nephritis (p = 0.03). Conversely, AmtRNA-IgM titers correlated with those of anti-β2GPI-IgM (rs = 0.48, p < 0.0001). AmtRNA-IgM were higher when patients were positive for anticardiolipin antibodies (aCL-IgG: p = 0.01; aCL-IgM: p = 0.002), but AmtRNA-IgM were not associated with any of the clinical manifestations assessed. These findings identify mtRNA as a novel mitochondrial antigen target in SLE, and support the concept that mitochondria may provide an important source of circulating autoantigens in SLE.

Pervasive and dynamic transcription initiation in Saccharomyces cerevisiae.

Transcription initiation is finely regulated to ensure proper expression and function of genes. The regulated transcription initiation in response to various environmental stimuli in a classic model organism Saccharomyces cerevisiae has not been systematically investigated. In this study, we generated quantitative maps of transcription start sites (TSSs) at a single-nucleotide resolution for S. cerevisiae grown in nine different conditions using no-amplification nontagging Cap analysis of gene expression (nAnT-iCAGE) sequencing. We mapped ∼1 million well-supported TSSs, suggesting highly pervasive transcription initiation in the compact genome of the budding yeast. The comprehensive TSS maps allowed us to identify core promoters for ∼96% verified protein-coding genes. We corrected misannotation of translation start codon for 122 genes and suggested an alternative start codon for 57 genes. We found that 56% of yeast genes are controlled by multiple core promoters, and alternative core promoter usage by a gene is widespread in response to changing environments. Most core promoter shifts are coupled with altered gene expression, indicating that alternative core promoter usage might play an important role in controlling gene transcriptional activities. Based on their activities in responding to environmental cues, we divided core promoters into constitutive class (55%) and inducible class (45%). The two classes of core promoters display distinctive patterns in transcriptional abundance, chromatin structure, promoter shape, and sequence context. In summary, our study improved the annotation of the yeast genome and demonstrated a much more pervasive and dynamic nature of transcription initiation in yeast than previously recognized.

Chemosynthetic symbiont with a drastically reduced genome serves as primary energy storage in the marine flatworm Paracatenula

Hosts of chemoautotrophic bacteria typically have much higher biomass than their symbionts and consume symbiont cells for nutrition. In contrast to this, chemoautotrophic Candidatus Riegeria symbionts in mouthless Paracatenula flatworms comprise up to half of the biomass of the consortium. Each species of Paracatenula harbors a specific Ca Riegeria, and the endosymbionts have been vertically transmitted for at least 500 million years. Such prolonged strict vertical transmission leads to streamlining of symbiont genomes, and the retained physiological capacities reveal the functions the symbionts provide to their hosts. Here, we studied a species of Paracatenula from Sant'Andrea, Elba, Italy, using genomics, gene expression, imaging analyses, as well as targeted and untargeted MS. We show that its symbiont, Ca R. santandreae has a drastically smaller genome (1.34 Mb) than the symbiont´s free-living relatives (4.29-4.97 Mb) but retains a versatile and energy-efficient metabolism. It encodes and expresses a complete intermediary carbon metabolism and enhanced carbon fixation through anaplerosis and accumulates massive intracellular inclusions such as sulfur, polyhydroxyalkanoates, and carbohydrates. Compared with symbiotic and free-living chemoautotrophs, Ca R. santandreae's versatility in energy storage is unparalleled in chemoautotrophs with such compact genomes. Transmission EM as well as host and symbiont expression data suggest that Ca R. santandreae largely provisions its host via outer-membrane vesicle secretion. With its high share of biomass in the symbiosis and large standing stocks of carbon and energy reserves, it has a unique role for bacterial symbionts-serving as the primary energy storage for its animal host.

Dynamical modelling of secondary metabolism and metabolic switches in Streptomyces xiamenensis 318.

The production of secondary metabolites, while important for bioengineering purposes, presents a paradox in itself. Though widely existing in plants and bacteria, they have no definite physiological roles. Yet in both native habitats and laboratories, their production appears robust and follows apparent metabolic switches. We show in this work that the enzyme-catalysed process may improve the metabolic stability of the cells. The latter can be responsible for the overall metabolic behaviours such as dynamic metabolic landscape, metabolic switches and robustness, which can in turn affect the genetic formation of the organism in question. Mangrove-derived Streptomyces xiamenensis 318, with a relatively compact genome for secondary metabolism, is used as a model organism in our investigation. Integrated studies via kinetic metabolic modelling, transcriptase measurements and metabolic profiling were performed on this strain. Our results demonstrate that the secondary metabolites increase the metabolic fitness of the organism via stabilizing the underlying metabolic network. And the fluxes directing to NADH, NADPH, acetyl-CoA and glutamate provide the key switches for the overall and secondary metabolism. The information may be helpful for improving the xiamenmycin production on the strain.

Replication timing in Drosophila and its peculiarities in polytene chromosomes

Drosophila melanogaster is one of the popular model organisms in DNA replication studies. Since the 1960s, DNA replication of polytene chromosomes has been extensively studied by cytological methods. In the recent two decades, the progress in our understanding of DNA replication was associated with new techniques. Use of fluorescent dyes increased the resolution of cytological methods significantly. High-throughput methods allowed analysis of DNA replication on a genome scale, as well as its correlation with chromatin structure and gene activi ty. Precise mapping of the cytological structures of polytene chromosomes to the genome assembly allowed comparison of replication between polytene chromosomes and chromosomes of diploid cells. New features of replication characteristic for D. melanogaster were described for both diploid and polytene chromosomes. Comparison of genomic replication profiles revealed a significant similarity between Drosophila and other well-studi ed eukaryotic species, such as human. Early replication is often confined to intensely transcribed gene-dense regions characterized by multiple replication initiation sites. Features of DNA replication in Drosophila might be explained by a compact genome. The organization of replication in polytene chromosomes has much in common with the organization of replication in chromosomes in diploid cells. The most important feature of replication in polytene chromosomes is its low rate and the dependence of S-phase duration on many factors: external and internal, local and global. The speed of replication forks in D. melanogaster polytene chromosomes is affected by SUUR and Rif1 proteins. It is not known yet how universal the mechanisms associated with these factors are, but their study is very promising.

Massive Changes of Genome Size Driven by Expansions of Non-autonomous Transposable Elements

In eukaryotes, genome size correlates little with the number of coding genes or the level of organismal complexity (C-value paradox). The underlying causes of variations in genome size, whether adaptive or neutral, remain unclear, although several biological traits often covary with it [1-5]. Rapid increases in genome size occur mainly through whole-genome duplications or bursts in the activity of transposable elements (TEs) [6]. The very small and compact genome of Oikopleura dioica, a tunicate of the larvacean class, lacks elements of most ancient families of animal retrotransposons [7, 8]. Here, we sequenced the genomes of six other larvaceans, all of which are larger than that of Oikopleura (up to 12 times) and which increase in size with greater body length. Although no evidence was found for whole-genome duplications within the group of species, the global amount of TEs strongly correlated with genome size. Compared to other metazoans, however, the TE diversity was reduced in all species, as observed previously in O.dioica, suggesting a common ancestor with a compacted genome. Strikingly, non-autonomous elements, particularly short interspersed nuclear elements (SINEs), massively contributed to genome size variation through species-specific independent amplifications, ranging from 3% in the smallest genome up to 49% in the largest. Variations in SINE abundance explain as much as 83% of interspecific genome size variation. These data support an indirect influence of autonomous TEs on genome size via their ability to mobilize non-autonomous elements.

Long-Read Sequencing - A Powerful Tool in Viral Transcriptome Research.

Long-read sequencing (LRS) has become increasingly popular due to its strengths in de novo assembly and in resolving complex DNA regions as well as in determining full-length RNA molecules. Two important LRS technologies have been developed during the past few years, including single-molecule, real-time sequencing by Pacific Biosciences, and nanopore sequencing by Oxford Nanopore Technologies. Although current LRS methods produce lower coverage, and are more error prone than short-read sequencing, these methods continue to be superior in identifying transcript isoforms including multispliced RNAs and transcript-length variants as well as overlapping transcripts and alternative polycistronic RNA molecules. Viruses have small, compact genomes and therefore these organisms are ideal subjects for transcriptome analysis with the relatively low-throughput LRS techniques. Recent LRS studies have multiplied the number of previously known transcripts and have revealed complex networks of transcriptional overlaps in the examined viruses.

Draft Genome Sequences of Staphylococcus Podophages JBug18, Pike, Pontiff, and Pabna.

We report here the draft genome sequences of Staphylococcus bacteriophages JBug18, Pike, Pontiff, and Pabna, which infect and lyse S. epidermidis and S. aureus strains. All bacteriophages belong to the morphological family Podoviridae and constitute attractive candidates for use as whole-phage therapeutics due to their compact genomes and lytic lifestyles.

Identification of functional long non-coding RNAs in C. elegans

BackgroundFunctional characterisation of the compact genome of the model organism Caenorhabditis elegans remains incomplete despite its sequencing 20 years ago. The last decade of research has seen a tremendous increase in the number of non-coding RNAs identified in various organisms. While we have mechanistic understandings of small non-coding RNA pathways, long non-coding RNAs represent a diverse class of active transcripts whose function remains less well characterised.ResultsBy analysing hundreds of published transcriptome datasets, we annotated 3392 potential lncRNAs including 143 multi-exonic loci that showed increased nucleotide conservation and GC content relative to other non-coding regions. Using CRISPR/Cas9 genome editing, we generated deletion mutants for ten long non-coding RNA loci. Using automated microscopy for in-depth phenotyping, we show that six of the long non-coding RNA loci are required for normal development and fertility. Using RNA interference-mediated gene knock-down, we provide evidence that for two of the long non-coding RNA loci, the observed phenotypes are dependent on the corresponding RNA transcripts.ConclusionsOur results highlight that a large section of the non-coding regions of the C. elegans genome remains unexplored. Based on our in vivo analysis of a selection of high-confidence lncRNA loci, we expect that a significant proportion of these high-confidence regions is likely to have a biological function at either the genomic or the transcript level.

Stage-specific testes proteomics of Drosophila melanogaster identifies essential proteins for male fertility

Spermiogenesis in Drosophila melanogaster is a highly conserved process and essential for male fertility. In this haploid phase of spermatogenesis, motile sperm are assembled from round cells, and flagella and needle-shaped nuclei with highly compacted genomes are formed. As transcription takes place mainly in spermatocytes and transcripts relevant for post-meiotic sperm development are translationally repressed for days, we comparatively analysed the proteome of larval testes (only germ cell stages before meiotic divisions), testes of 1–2-day-old pupae (germ cell stages before meiotic divisions, meiotic and early spermatid stages) and adult flies (germ cell stages before meiotic divisions, meiotic and early spermatid stages, late spermatids and sperm). We identified 6,171 proteins; 61 proteins were detected solely in one stage and are thus enriched, namely 34 in larval testes, 77 in pupal testes and 214 in adult testes. To substantiate our mass spectrometric data, we analysed the stage-specific synthesis and importance for male fertility of a number of uncharacterized proteins. For example, Mst84B (gene CG1988), a very basic cysteine- and lysine-rich nuclear protein and was present in the transition phase from a histone-based to a protamine-based chromatin structure. CG6332 encodes d-Theg, which is related to the mouse tHEG and human THEG proteins. Mutants of d-Theg were sterile due to the lack of sperm in the seminal vesicles. Our catalogue of proteins of the different stages of testis development in D. melanogaster will pave the road for future analyses of spermatogenesis.

Drosophila as a Model for Developmental Toxicology: Using and Extending the Drosophotoxicology Model.

Fruit flies, Drosophila melanogaster, have been traditionally valued as a simple model system due to their easy and inexpensive culture, their relatively compact genome, and the variety of available genetic tools. However, due to similarities of their neurological and developmental pathways with those of vertebrates, Drosophila also offers advantages for developmental toxicity assays. The ability to distinguish the effects of a toxicant on adult females, males, and the developing offspring adds to the usefulness of this model. Here we describe key techniques to screen chemicals and other potential emerging toxicants such as nanoparticles on adult Drosophila female and male reproductive success. In addition, assessments of relative toxicity can be revealed by viability assays at each developmental stage from the embryo to the pharate, or preemergent, adult.

A Dynamic &lt;i&gt;E. coli&lt;/i&gt; Genome: Widespread DNA Contacts Revealed by Monitoring Mu Transposition

The problem of compacting genomes while still performing cellular processes is common to all life forms. The current view for E. coli is a compacted genome with well-organized domains where replication initiates and terminates. Some studies have suggested that these and other regions are sequestered or compartmentalized such that there is little to no interaction between them. In this work, we have exploited the high efficiency and promiscuity of phage Mu transposition to directly measure the in vivo rates of interactions between genomic loci, and have developed new tools for analyzing the proximity of loci across the genome. We observe widespread contacts between all regions of the E. coli chromosome, revealing a dynamic, effectively un-compartmentalized genome. We detect long-range interactions between several genes in different gene families such as dna and rrna, implicating spatial proximity of many distantly co-regulated genes for the first time in a prokaryote. We also see a higher interaction between the two halves of the chromosome during replication, consistent with the deduced proximity and higher mobility of the chromosomal arms as they segregate during replication. Our work advances a new view of genome organization in E. coli.

Automated morphometry toolbox for analysis of microscopic model organisms using simple bright-field imaging.

ABSTRACTModel organisms with compact genomes, such as yeast and Caenorhabditis elegans, are particularly useful for understanding organism growth and life/cell cycle. Organism morphology is a critical parameter to measure in monitoring growth and stage in the life cycle. However, manual measurements are both time consuming and potentially inaccurate, due to variations among users and user fatigue. In this paper we present an automated method to segment bright-field images of fission yeast, budding yeast, and C. elegans roundworm, reporting a wide range of morphometric parameters, such as length, width, eccentricity, and others. Comparisons between automated and manual methods on fission yeast reveal good correlation in size values, with the 95% confidence interval lying between −0.8 and +0.6 μm in cell length, similar to the 95% confidence interval between two manual users. In a head-to-head comparison with other published algorithms on multiple datasets, our method achieves more accurate and robust results with substantially less computation time. We demonstrate the method's versatility on several model organisms, and demonstrate its utility through automated analysis of changes in fission yeast growth due to single kinase deletions. The algorithm has additionally been implemented as a stand-alone executable program to aid dissemination to other researchers.

Draft genome of the cotton aphid Aphis gossypii

The cotton aphid Aphis gossypii Glover is a worldwide agricultural pest that feeds on cotton, melon, and other landscape plants, causing a high level of economic loss. In addition to the common characteristics shared with other aphids, Ap. gossypii has evolved multiple biotypes that present substantial differences in host adaption. These intriguing biological features are of interest from both a fundamental and applied perspective. However, the molecular studies of Ap. gossypii have been restrained by the lack of a reference genome. Furthermore, in order to establish a platform for the development of novel and sustainable control methods, it is necessary to generate genomic resources for Ap. gossypii. Here, we present a 294 Mb draft genome sequence of Ap. gossypii, which consists of 4,724 scaffolds with an N50 size of 438 kb. Compared to other aphid species with published genomes, Ap. gossypii presents the most compact genome size. A total of 14,694 protein-coding genes were predicted and annotated in the consensus gene set, 98.03% of CEGMA genes and 93.5% of BUSCO genes were captured respectively. Genome-wide selection analyses revealed that significantly evolving pathways in the genus Aphis are related to biological processes of detoxification, steroid biosynthesis, and ethylbenzene degradation. The acquisition of the genome of Ap. gossypii makes it possible to understand the molecular mechanism of intricate biological traits of this species, and will further facilitate the study of aphid evolution.

Comprehensive Analysis of Hepatitis B Virus Promoter Region Mutations.

Over 250 million people are infected chronically with hepatitis B virus (HBV), the leading cause of liver cancer worldwide. HBV persists, due, in part, to its compact, stable minichromosome, the covalently-closed, circular DNA (cccDNA), which resides in the hepatocytes’ nuclei. Current therapies target downstream replication products, however, a true virological cure will require targeting the cccDNA. Finding targets on such a small, compact genome is challenging. For HBV, to remain replication-competent, it needs to maintain nucleotide fidelity in key regions, such as the promoter regions, to ensure that it can continue to utilize the necessary host proteins. HBVdb (HBV database) is a repository of HBV sequences spanning all genotypes (A–H) amplified from clinical samples, and hence implying an extensive collection of replication-competent viruses. Here, we analyzed the HBV sequences from HBVdb using bioinformatics tools to comprehensively assess the HBV core and X promoter regions amongst the nearly 70,000 HBV sequences for highly-conserved nucleotides and variant frequencies. Notably, there is a high degree of nucleotide conservation within specific segments of these promoter regions highlighting their importance in potential host protein-viral interactions and thus the virus’ viability. Such findings may have key implications for designing antivirals to target these areas.

Spermatogenesis as a tool for staging gonad development in the gonochoric appendicularian Oikopleura dioica Fol 1872

Oikopleura dioica, the only gonochoric species among appendicularians, has a spematozoon with a mid-piece and a conspicuous acrosome that, during fertilisation, undergoes a reaction forming an acrosomal process. To provide more insight into the spermatogenesis of a holoplanktonic tunicate species that completes its life cycle in three to five days, changes in the testis during individual growth have been examined. Spermatogenesis has been subdivided into seven stages based on ultrastructural features during the formation and organisation of the male gonad and the relationships between its macroscopic anatomy and the events of sperm differentiation. Gametes undergo highly synchronised differentiation due to the presence of widespread syncytial structures. Both meiosis and spermiogenesis are brief, and the passage from spermatocytes to spermatids involves a progressive segregation of the germ cells from the syncytial mass with the formation of large cytoplasmic bridges and volume reduction for nucleus compacting and cytoplasmic material changing. The nucleus is small and penetrated anteriorly by a complex acrosome and posteriorly by the distal centriole and part of the flagellum. In spermatids, the single, large mitochondrion appears laterally to the nucleus, and finally, in spermatozoa, it migrates into the mid-piece, wrapping the proximal portion of the axoneme. Because this mitochondrial position is reached only in the late phases of spermatogenesis, it suggests that appendicularians have derived oligopyrenic sperms in which the small nucleus results from adaptation to the assembly of numerous spermatozoa inside the narrow space of the testis compacted in the genital cavity. The formulation of a staging system of gonad development in a model tunicate species known for having the most compacted genome in chordates led to a comparison of histological observations with recent molecular data, improving the characterisation of its biology and life cycle in light of evolutionary implications.