Comparative 3D genome organization in apicomplexan parasites

Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.

Ankylosing Spondylitis (AS) is a chronic inflammatory arthritis of the spine exhibiting a strong genetic background. The mechanistic and functional understanding of the AS-associated genomic loci, identified with Genome Wide Association Studies (GWAS), remains challenging. Chromosome conformation capture (3C) and derivatives are recent techniques which are of great help in elucidating the spatial genome organization and of enormous support in uncover a mechanistic explanation for disease-associated genetic variants. The perturbation of three-dimensional (3D) genome hierarchy may lead to a plethora of human diseases, including rheumatological disorders. Here we illustrate the latest approaches and related findings on the field of genome organization, highlighting how the instability of 3D genome conformation may be among the causes of rheumatological disease phenotypes. We suggest a new perspective on the inclusive potential of a 3C approach to inform GWAS results in rheumatic diseases. 3D genome organization may ultimately lead to a more precise and comprehensive functional interpretation of AS association, which is the starting point for emerging and more specific therapies.

The organization of mammalian genomes within the nucleus features a complex, multiscale three-dimensional (3D) architecture. The functional significance of these 3D genome features, however, remains largely elusive due to limited single-cell technologies that can concurrently profile genome organization and transcriptional activities. Here, we report GAGE-seq, a highly scalable, robust single-cell co-assay that simultaneously measures 3D genome structure and transcriptome within the same cell. Employing GAGE-seq on mouse brain cortex and human bone marrow CD34+ cells, we comprehensively characterized the intricate relationships between 3D genome and gene expression. We found that these multiscale 3D genome features collectively inform cell type-specific gene expressions, hence contributing to defining cell identity at the single-cell level. Integration of GAGE-seq data with spatial transcriptomic data revealed in situ variations of the 3D genome in mouse cortex. Moreover, our observations of lineage commitment in normal human hematopoiesis unveiled notable discordant changes between 3D genome organization and gene expression, underscoring a complex, temporal interplay at the single-cell level that is more nuanced than previously appreciated. Together, GAGE-seq provides a powerful, cost-effective approach for interrogating genome structure and gene expression relationships at the single-cell level across diverse biological contexts.

A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient’s blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a “fast PCR enzyme”. In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the “fast PCR enzyme”, with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses.

To investigate three-dimensional (3D) genome organization in prokaryotic and eukaryotic cells, three main strategies are employed, namely nuclear proximity ligation-based methods, imaging tools (such as fluorescence in situ hybridization (FISH) and its derivatives), and computational/visualization methods. Proximity ligation-based methods are based on digestion and re-ligation of physically proximal cross-linked chromatin fragments accompanied by massively parallel DNA sequencing to measure the relative spatial proximity between genomic loci. Imaging tools enable direct visualization and quantification of spatial distances between genomic loci, and advanced implementation of (super-resolution) microscopy helps to significantly improve the resolution of images. Computational methods are used to map global 3D genome structures at various scales driven by experimental data, and visualization methods are used to visualize genome 3D structures in virtual 3D space-based on algorithms. In this review, we focus on the introduction of novel imaging and visualization methods to study 3D genomes. First, we introduce the progress made recently in 3D genome imaging in both fixed cell and live cells based on long-probe labeling, short-probe labeling, RNA FISH, and the CRISPR system. As the fluorescence-capturing capability of a particular microscope is very important for the sensitivity of bioimaging experiments, we also introduce two novel super-resolution microscopy methods, SDOM and low-power super-resolution STED, which have potential for time-lapse super-resolution live-cell imaging of chromatin. Finally, we review some software tools developed recently to visualize proximity ligation-based data. The imaging and visualization methods are complementary to each other, and all three strategies are not mutually exclusive. These methods provide powerful tools to explore the mechanisms of gene regulation and transcription in cell nuclei.

Three-dimensional (3D) genome organization becomes altered during development, aging and disease, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multi-scale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screens, a cellular barcode readout method (BARC-FISH) and chromatin tracing. We performed a loss-of-function screen in human cells, and visualized alterations to their 3D chromatin folding conformations, alongside perturbation-paired barcode readout in the same single cells. We discovered tens of new regulators of chromatin folding at different length scales, ranging from chromatin domains and compartments to chromosome territory. A subset of the regulators exhibited 3D genome effects associated with loop extrusion and A–B compartmentalization mechanisms, while others were largely unrelated to these known 3D genome mechanisms. Finally, we identified new regulators of nuclear architectures and found a functional link between chromatin compaction and nuclear shape. Altogether, our method enables scalable, high-content identification of chromatin and nuclear topology regulators that will stimulate new insights into the 3D genome.

The nuclear genome is spatially organized into a three-dimensional (3D) architecture by physical association of large chromosomal domains with subnuclear compartments including the nuclear lamina at the radial periphery and nuclear speckles within the nucleoplasm1–5. However, how spatial genome architecture regulates human brain development has been overlooked owing to technical limitations. Here, we generate high-resolution maps of genomic interactions with the lamina and speckles in cells of the neurogenic lineage isolated from midgestational human cortex, uncovering an intimate association between subnuclear genome compartmentalization, chromatin state and transcription. During cortical neurogenesis, spatial genome organization is extensively remodeled, relocating hundreds of neuronal genes from the lamina to speckles including key neurodevelopmental genes bivalent for H3K27me3 and H3K4me3. At the lamina, bivalent genes have exceptionally low expression, and relocation to speckles enhances resolution of bivalent chromatin to H3K4me3 and increases transcription >7-fold. We further demonstrate that proximity to the nuclear periphery – not the presence of H3K27me3 – is the dominant factor in maintaining the lowly expressed, poised state of bivalent genes embedded in the lamina. In addition to uncovering a critical role of subnuclear genome compartmentalization in neurogenic transcriptional regulation, our results establish a new paradigm in which knowing the spatial location of a gene is necessary to understanding its epigenomic regulation.

BackgroundSeafood is not considered the natural habitat of Salmonella except the river fish, but still, the incidence of Salmonella in seafood is in a steady rise. By extending our understanding of Salmonella growth dynamics and pathogenomics in seafood, we may able to improve seafood safety and offer better strategies to protect the public health. The current study was thus aimed to assess the growth and multiplication of non-typhoidal and typhoidal Salmonella serovars on seafood and further sought to evaluate their virulence and stress genes expression while in contact with seafood at varying temperature exposure.ResultsSalmonella enterica Weltevreden and Salmonella enterica Typhi were left to grow on fish fillets at −20, 4, room temperature (RT) and 45 °C for a period of one week. Total RNA from both Salmonella serovars were extracted and qRT-PCR based relative gene expression approach was used to detect the expression of rpoE, invA, stn and fimA genes at four different temperature conditions studied on incubation days 0, 1, 3, 5 and 7. Salmonella Weltevreden growth on seafood was increased ~4 log10 at RT and 45 °C, nevertheless, nearly 2 and >4 log 10 reduction was observed in cell count stored at 4 and −20 °C on seafood, respectively. Growth pattern of Salmonella Typhi in seafood has shown identical pattern at RT and 45 °C, however, growth was sharply reduced at 4 and −20 °C as compared to the Salmonella Weltevreden. Total RNA of Salmonella Weltevreden was in the range from 1.3 to 17.6 μg/μl and maximum concentration was obtained at 45 °C on day 3. Similarly, RNA concentration of Salmonella Typhi was ranged from 1.2 to 11.8 μg/μl and maximum concentration was obtained at 45 °C on day 3. The study highlighted that expression of invA and stn genes of Salmonella Weltevreden was >8-fold upregulated at RT, whereas, fimA gene was increasingly down regulated at room temperature. Storage of Salmonella Weltevreden at 45 °C on seafood resulted in an increased expression (>13 -fold) of stn genes on day 1 followed by down regulation on days 3, 5, and 7. Nevertheless, other genes i.e. fimA, invA and rpo remained downregulated throughout the storage period. More intense upregulation was observed for invA and stn genes of Salmonella Typhi at RT and 45 °C. Further, incubating Salmonella Weltevreden at 4 °C resulted in down regulation in the expression of rpoE, invA and stn genes. Regarding Salmonella Typhi, fimA and stn genes were upregulated on day one, in addition, an increased expression of fimA was noted on day 3. At −20 °C, there was no obvious expression of target genes of Salmonella Weltevreden and Salmonella Typhi when stored along with seafood.ConclusionHere we demonstrate that nutritional constituents and water content available in seafood has become useful growth ingredients for the proliferation of Salmonella in a temperature dependent manner. Although, it was absence of serovar specific growth pattern of non-typhoidal and typhoidal Salmonella in seafood, there was observation of diverse expression profile of stress and virulent genes in non-typhoidal and typhoidal Salmonella serovars. In presence of seafood, the induced expression of Salmonella virulent genes at ambient temperature is most likely to be impacted by increased risk of seafood borne illness associated with Salmonella.

In eukaryotic cells, 3D genome plays an important role in the regulation of gene spatiotemporal expression, which is essential for the biological and developmental processes in a life cycle. In the past decade, the development of high-throughput technologies greatly enhances our ability to map the 3D genome organization, identifies multiple 3D genome structures, and investigates the functional role of 3D genome organization in gene regulation, which facilitates our understandings of cis-regulatory landscape and biological development. Comparing with the comprehensive analyses of 3D genome in mammals and model plants, the progress in soybean is much less. Future development and application of tools to precisely manipulate 3D genome structure at different levels will significantly strengthen the functional genome study and molecular breeding in soybean. Here, we review the recent progresses in 3D genome study and discuss future directions, which may help to improve soybean 3D functional genome study and molecular breeding.

CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription

The 3D folding of the genome is tightly linked to its epigenetic state which maintains gene expression programmes. Although the relationship between gene expression and genome organisation is highly context dependent, 3D genome organisation is emerging as a novel epigenetic layer to reinforce and stabilise transcriptional states. Whether regulatory information carried in genome folding could be transmitted through mitosis is an area of active investigation. In this review, we discuss the relationship between epigenetic state and nuclear organisation, as well as the interplay between transcriptional regulation and epigenetic genome folding. We also consider the architectural remodelling of nuclei as cells enter and exit mitosis, and evaluate the potential of the 3D genome to contribute to cellular memory.

BackgroundMalaria is a vector-borne parasitic disease which is prevalent in many developing countries. Recently, it has been found that Plasmodium knowlesi, a simian malaria parasite can be life-threatening to humans. Long-tailed macaques, which are widely distributed in Malaysia, are the natural hosts for simian malaria, including P. knowlesi. The aim of the present study was to determine the prevalence of simian malaria parasites in long-tailed macaques in the district of Hulu Selangor, Selangor, Malaysia.MethodsA total of 70 blood samples were collected from Macaca fascicularis dwelling in the forest of Hulu Selangor by the Department of Wildlife and National Parks Peninsular Malaysia, Kuala Lumpur, Malaysia. DNA was extracted using PureLink™ Genomic DNA Kits. Conventional and nested PCR were used to detect the genus and species of Plasmodium parasites respectively. In addition, phylogenetic analysis was carried out to confirm the species of Plasmodium parasites.ResultsThirty-five (50 %) of the 70 samples were positive for Plasmodium using genus-specific primers. These positive samples were then subjected to nested PCR targeting the 18S ribosomal RNA genes to detect all five simian malaria parasites: namely, P. knowlesi, Plasmodium inui, Plasmodium cynomolgi, Plasmodium fieldi, and Plasmodium coatneyi. All five species of simian malaria parasites were detected. Of these, P. inui was the predominant (65.7 %), followed by P. knowlesi (60 %), P. cynomolgi (51.4 %) P. coatneyi (45.7 %) and P.fieldi (2.9 %). A total of nine macaques had mono-infection with P. knowlesi (four), P. cynomolgi (two), P. coatneyi (two) and P. fieldi (one). Eleven of the macaques had dual infections while 12 had triple infections. Three macaques were infected with four species of Plasmodium. Molecular and phylogenetic analysis confirmed the five species of Plasmodium parasites.ConclusionThis study has provided evidence to elucidate the presence of transmission of malaria parasites among the local macaques in Hulu Selangor. Since malaria is a zoonosis, it is important to determine the new control strategies for the control of malaria.

BackgroundImmunoassay platforms that simultaneously detect malaria antigens including histidine-rich protein 2 (HRP2)/HRP3 and Plasmodium lactate dehydrogenase (pLDH), are useful epidemiological tools for rapid diagnostic test evaluation. This study presents the comparative evaluation of two multiplex platforms in identifying Plasmodium falciparum with presence or absence of HRP2/HRP3 expression as being indicative of hrp2/hrp3 deletions and other Plasmodium species. Moreover, correlation between the malaria antigen measurements performed at these platforms is assessed after calibrating with either assay standards or international standards and the cross-reactivity among Plasmodium species is examined.MethodsA 77-member panel of specimens composed of the World Health Organization (WHO) international Plasmodium antigen standards, cultured parasites for P. falciparum and Plasmodium knowlesi, and clinical specimens with mono-infections for P. falciparum, Plasmodium vivax, and Plasmodium malariae was generated as both whole blood and dried blood spot (DBS) specimens. Assays for HRP2, P. falciparum–specific pLDH (PfLDH), P. vivax–specific pLDH (PvLDH), and all human Plasmodium species Pan malaria pLDH (PanLDH) on the Human Malaria Array Q-Plex and the xMAP platforms were evaluated with these panels.ResultsThe xMAP showed a higher percent positive agreement for identification of hrp2-deleted P. falciparum and Plasmodium species in whole blood and DBS than the Q-Plex. For whole blood samples, there was a highly positive correlation between the two platforms for PfLDH (Pearson r = 0.9926) and PvLDH (r = 0. 9792), moderate positive correlation for HRP2 (r = 0.7432), and poor correlation for PanLDH (r = 0.6139). In Pearson correlation analysis between the two platforms on the DBS, the same assays were r = 0.9828, r = 0.7679, r = 0.6432, and r = 0.8957, respectively. The xMAP HRP2 assay appeared to cross-react with HRP3, while the Q-Plex did not. The Q-Plex PfLDH assay cross-reacted with P. malariae, while the xMAP did not. For both platforms, P. knowlesi was detected on the PvLDH assay. The WHO international standards allowed normalization across both platforms on their HRP2, PfLDH, and PvLDH assays in whole blood and DBS.ConclusionsQ-Plex and xMAP show good agreement for identification of P. falciparum mutants with hrp2/hrp3 deletions, and other Plasmodium species. Quantitative results from both platforms, normalized into international units for HRP2, PfLDH, and PvLDH, showed good agreement and should allow comparison and analysis of results generated by either platform.

Malaria is one of the most common infectious diseases that affect millions of people globally. It is caused by protozoan parasites in the genus Plasmodium. Existing antimalarials have lost efficacy thanks to the emergence of resistant parasite populations and the incidence of the disease is on the rise. It is believed that the completion of the genome sequence of the human malaria parasite Plasmodium falciparum and its sibling species will lead to a better understanding of the molecular mechanisms underlying parasite biology and complex parasite-host interactions. In this study we focus on the investigation of genome evolution and genome plasticity in six malaria parasite species with various mammalian hosts, ranging from humans to rodents. We identify core genome components that are commonly present in these species. These components play important roles in fundamental functions as well as in parasite-specific activities.

Genome-wide Mapping of DNA Methylation in the Human Malaria Parasite Plasmodium falciparum