Biology subjects

Siegel, T. N.

Publications and source records attributed to Siegel, T. N..

8 recordsLinked to original sources

bioRxiv2026

Super-resolution imaging flow cytometry

Organelle architecture is a key determinant of cellular state. However, its heterogeneity remains difficult to quantify at population scale because conventional imaging flow cytometry is diffraction-limited. Here we present super-resolution imaging flow cytometry (SR-IFC), a high-throughput structured-illumination platform combining stroboscopic excitation, microfluidic hydrodynamic focusing and AI-assisted reconstruction. From three phase-shifted frames acquired along the flow axis, SR-IFC reconstructs images with approximately 118-nm isotropic in-plane resolution, about two-fold beyond the diffraction limit, at up to approximately 720 cells per second. In microbiology, SR-IFC resolves individual lipid droplets in nitrogen-starved Euglena gracilis and reveals spatial reorganization during lipid biogenesis missed by diffraction-limited imaging. In immunology, it maps activation-associated mitochondrial remodeling across primary human CD8+ T-cell differentiation, including a transient networked state. SR-IFC thus provides a broadly applicable platform for population-scale super-resolution phenotyping across diverse areas of biology.

bioengineering↗

bioRxiv2025

First Genome-Wide Centromere Map of Trypanosoma cruzi Reveals Linear and 3D Compartment Boundaries and Spatial Clustering

Background: Trypanosoma cruzi, the etiological agent of Chagas disease, possesses a highly repetitive genome that has historically hindered high-quality assembly and structural characterization. Despite significant advances in assembling T. cruzi genomes, major gaps remain. Among these, the complete repertoire of centromeric sequences has remained elusive, representing a critical missing piece in our understanding of chromosome structure and inheritance. Results: Here, we generated high-coverage Hi-C (genome-wide chromosome conformation capture) data for the widely used T. cruzi Dm28c strain improving its genome assembly, reducing the number of scaffolds and producing a more contiguous and accurate genome. To investigate centromere organization, we performed ChIP-seq using the mNeonGreen-myc-tagged kinetochore proteins KKT2 and KKT3, resulting in the identification of 40 KKT-enriched peaks across 29 scaffolds. These peaks were located in regions enriched in retrotransposable elements, particularly L1Tc and VIPER, near strand switch regions, areas of high GC content, and at the boundaries between conserved genes and virulence-factor multigene families. Conclusion: Notably, Hi-C analysis revealed that centromeres may act as structural boundaries contributing to genome compartmentalization and frequently engage in 3D spatial clustering, suggesting a role in higher-order nuclear architecture. Overall, our study provides a high-quality reference genome for the Dm28c strain, presents the first genome-wide centromere map in T. cruzi, and offers novel insights into centromere-mediated 3D genome organization

genomics↗

bioRxiv2024

SLAM-seq reveals independent contributions of RNA processing and stability to gene expression in African trypanosomes

Gene expression is a multi-step process that converts DNA-encoded information into proteins, involving RNA transcription, maturation, degradation, and translation. While transcriptional control is a major regulator of protein levels, the role of post-transcriptional processes such as RNA processing and degradation is less well understood due to the challenge of measuring their contributions individually. To address this challenge, we investigated the control of gene expression in Trypanosoma brucei, a unicellular parasite assumed to lack transcriptional control. Instead, mRNA levels in T. brucei are controlled by post-transcriptional processes, which enabled us to disentangle the contribution of both processes to total mRNA levels. In this study, we developed an efficient metabolic RNA labeling approach and combined ultra-short metabolic labeling with transient transcriptome sequencing (TT-seq) to confirm the long-standing assumption that RNA polymerase II transcription is unregulated in T. brucei. In addition, we established thiol (SH)-linked alkylation for metabolic sequencing of RNA (SLAM-seq) to globally quantify RNA processing rates and half-lives. Our data, combined with scRNA-seq data, indicate that RNA processing and stability independently affect total mRNA levels and contribute to the variability seen between individual cells in African trypanosomes.

molecular biology↗

bioRxiv2024

High-resolution scRNA-seq reveals genomic determinants of antigen expression hierarchy in African Trypanosomes

Antigenic variation is an immune evasion strategy used by many different pathogens. It involves the periodic, non-random switch in the expression of different antigens throughout an infection. How the observed hierarchy in antigen expression is achieved has remained a mystery. A key challenge in uncovering this process has been the inability to track transcriptome changes and potential genomic rearrangements in individual cells during a switch event. Here, we report the establishment of a highly sensitive single-cell RNA-seq (scRNA-seq) approach for the model protozoan parasite Trypanosoma brucei. This approach has revealed genomic rearrangements that occur in individual cells during a switch event. Our data show that following a double-strand break (DSB) in the transcribed antigen-coding gene - an important trigger for antigen switching - the type of repair mechanism and the resultant antigen expression depend on the availability of a homologous repair template in the genome. When such a template was available, repair proceeded through segmental gene conversion, creating new, mosaic antigen-coding genes. Conversely, in the absence of a suitable template, a telomere-adjacent antigen-coding gene from a different part of the genome was activated by break-induced replication. Our results reveal the critical role of available repair sequence in the antigen selection mechanism. Additionally, our study demonstrates the power of highly sensitive scRNA-seq methods in detecting genomic rearrangements that drive transcriptional changes at the single-cell level.

microbiology↗

bioRxiv2021

{-}{-} Histone H2B.V demarcates strategic regions in the Trypanosoma cruzi genome, associates with a bromodomain factor and affects parasite differentiation and host cell invasion

Histone variants play a crucial role in chromatin structure organization and gene expression. Trypanosomatids have an unusual H2B variant (H2B.V) that is known to dimerize with the variant H2A.Z generating unstable nucleosomes. Previously, we found that H2B.V protein is enriched in nonreplicative life forms of Trypanosoma cruzi, suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among parasite life forms. Here, we performed the first genome-wide profiling of histone localization in T. cruzi using replicative and nonreplicative life forms, and we found that H2B.V was preferentially located at the edges of divergent switch regions, which encompass putative transcriptional start regions; at some tDNA loci; and between the conserved and disrupted genome compartments, mainly at trans-sialidase, mucin and MASP genes. Remarkably, the chromatin of nonreplicative forms was depleted of H2B.V-enriched peaks in comparison to replicative forms. Interactome assays indicated that H2B.V associated specifically with H2A.Z, bromodomain factor 2, nucleolar proteins and a histone chaperone, among others. Parasites expressing reduced H2B.V levels were associated with higher rates of parasite differentiation and mammalian cell infectivity. Taken together, H2B.V demarcates critical genomic regions and associates with regulatory chromatin proteins, suggesting a scenario wherein local chromatin structures associated with parasite differentiation and invasion are regulated during the parasite life cycle. Author SummaryTrypanosomatids have to adapt to different environmental conditions, changing their morphology, gene expression and metabolism. These organisms have many unique features in terms of gene expression regulation. The genomic organization includes polycistronic regions with the absence of well-defined transcription start sites. In T. brucei, histone variants mark the start and ending sites of transcription; however, little is known about whether these proteins change their genome location, expression levels and interactors along life forms and what the impact is of these changes on parasite differentiation and infection. In T. cruzi, the causative agent of Chagas disease, we previously found that the histone variant of H2B is enriched in nonreplicative and infective forms, suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among these life forms. Here, we aimed to go one step further and performed the first histone ChIP-seq analysis in T. cruzi, in which we found that H2B.V was enriched at divergent strand switch regions, some tDNA loci and other critical genomic regions associated with T. cruzi genome compartments. We found that H2B.V interacts with a bromodomain factor, suggesting an intricate network involving chromatin acetylation around H2B.V enriched sites. Moreover, parasites expressing reduced H2B.V levels were associated with higher rates of differentiation and mammalian cell infectivity.

microbiology↗

bioRxiv2021

A phased genome assembly for allele-specific analysis in Trypanosoma brucei

Many eukaryotic organisms are diploid or even polyploid, i.e. they harbour two or more independent copies of each chromosome. Yet, to date most reference genome assemblies represent a mosaic consensus sequence in which the homologous chromosomes have been collapsed into one sequence. This procedure generates sequence artefacts and impedes analyses of allele-specific mechanisms. Here, we report the allele-specific genome assembly of the diploid unicellular protozoan parasite Trypanosoma brucei. As a first step, we called variants on the allele-collapsed assembly of the T. brucei Lister 427 isolate using short-read error-corrected PacBio reads. We identified 96 thousand heterozygote variants across the genome (average of 4.2 variants / kb), and observed that the variant density along the chromosomes was highly uneven. Several long (>100 kb) regions of loss-of-heterozigosity (LOH) were identified, suggesting recent recombination events between the alleles. By analysing available genomic sequencing data of multiple Lister 427 derived clones, we found that most LOH regions were conserved, except for some that were specific to clones adapted to the insect lifecycle stage. Surprisingly, we also found that some Lister 427 clones were aneuploid. We found evidence of trisomy in chromosome five (chr 5), chr 2, chr 6 and chr 7. Moreover, by analysing RNA-seq data, we showed that the transcript level is proportional to the ploidy, evidencing the lack of a general expression control at the transcript level in T. brucei. As a second step, to generate an allele-specific genome assembly, we used two powerful datatypes for haplotype reconstruction: raw long reads (PacBio) and chromosome conformation (Hi-C) data. With this approach, we were able to assign 99.5% of all heterozygote variants to a specific homologous chromosome, building a 66 Mb long T. brucei Lister 427 allele-specific genome assembly. Hereby, we identified genes with allele-specific premature termination codons and showed that differences in allele-specific expression at the level of transcription and translation can be accurately monitored with the fully phased genome assembly. The obtained reference-grade allele-specific genome assembly of T. brucei will enable the analysis of allele-specific phenomena, as well as the better understanding of recombination and evolutionary processes. Furthermore, it will serve as a standard to benchmark much needed automatic genome assembly pipelines for highly heterozygous wild species isolates.

genomics↗

bioRxiv2020

Antigenic variation by switching inter-chromosomal interactions with an RNA splicing locus in trypanosomes

Highly selective gene expression is a key requirement for antigenic variation in several pathogens, allowing evasion of host immune responses and maintenance of persistent infections. African trypanosomes, parasites that cause lethal diseases in humans and livestock, employ an antigenic variation mechanism that involves monogenic antigen expression from a pool of >2500 antigen coding genes. In other eukaryotes, the expression of individual genes can be enhanced by mechanisms involving the juxtaposition of otherwise distal chromosomal loci in the three-dimensional nuclear space. However, trypanosomes lack classical enhancer sequences or regulated transcription initiation and the monogenic expression mechanism has remained enigmatic. Here, we show that the single expressed antigen coding gene displays a specific inter-chromosomal interaction with a major mRNA splicing locus. Chromosome conformation capture (Hi-C), revealed a dynamic reconfiguration of this inter-chromosomal interaction upon activation of another antigen. Super-resolution microscopy showed the interaction to be heritable and splicing dependent. We find that the two genomic loci are connected by the antigen exclusion complex, whereby VEX1 associated with the splicing locus and VEX2 with the antigen coding locus. Following VEX2 depletion, loss of monogenic antigen expression was accompanied by increased interactions between previously silent antigen genes and the splicing locus. Our results reveal a novel mechanism to ensure monogenic expression, requiring the spatial integration of antigen transcription and mRNA splicing in a dedicated compartment. These findings suggest a new means of post-transcriptional gene regulation.

molecular biology↗

bioRxiv2019

Efficient and specific oligo-based depletion of rRNA

In most organisms, ribosomal RNA (rRNA) contributes to >85% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA or other highly abundant transcripts is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNAs and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, their efficiency relies on a high degree of sequence homology between oligonucleotide probes and the target RNA. This restricts the use of such kits to a limited number of organisms with conserved rRNA sequences.\n\nIn this study we describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. Our approach reduces the levels of the most abundant rRNA transcripts to less than 5% with minimal off-target effects.\n\nBy adjusting the sequence of the oligonucleotide probes, our approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, our protocol provides a useful alternative for rRNA removal where enrichment of polyadenylated transcripts is not an option and commercial kits for rRNA are not available.

molecular biology↗