Biology subjects

Zinzen, R. P.

Publications and source records attributed to Zinzen, R. P..

9 recordsLinked to original sources

bioRxiv2026

Evolution of CTCF underlies the establishment of loop extrusion as a predominant chromatin folding mechanism in vertebrates

CTCF is a DNA-binding protein that emerged in the last bilaterian ancestor and that plays a central role in 3D genome organization. Yet, despite its relevance, genomic studies have revealed notable differences in CTCF function across animal clades. In vertebrates, CTCF is essential for TAD organization through cohesin-mediated loop extrusion, whereas its contribution to chromatin organization outside this clade appears to be more limited. Here we investigate the evolution of CTCF as an architectural factor through a cross-species functional replacement approach, in which orthologs from divergent bilaterian lineages are expressed in either mouse embryonic stem cells or Drosophila embryos. Chromosome conformation analyses demonstrated that the ability of CTCF to mediate loop extrusion-dependent TAD formation is encoded within the vertebrate protein. Yet, this property alone is insufficient to establish vertebrate-like genome architecture in an invertebrate background, suggesting the existence of additional lineage-specific factors. ChIP-seq analyses further revealed that CTCF DNA-binding specificity has been progressively refined during bilaterian evolution, with the vertebrate lineage exhibiting a preferential shift towards distal genomic regions that is consistent with an expansion of regulatory domains. Notably, despite failing to form TADs, the ortholog from a non-vertebrate chordate partially rescued the lethality associated with the loss of the vertebrate CTCF protein, indicating that its architectural and regulatory functions are evolutionary independent. Further Hi-C analyses in tunicate and cyclostome species suggest that the ability of CTCF to act as a loop extrusion barrier was already present in the last common ancestor of all vertebrates. Altogether, our findings establish the diversification of CTCF regulation and function as key drivers for the specialization of bilaterian 3D genome organization.

genomics↗

bioRxiv2025

Evolutionarily conserved transcriptional regulators control monoaminergic neuron development

To what extent conserved developmental programs specify homologous cell types is a central question in biology. Here, we address this by focusing on reconstructing monoaminergic neuron development in Drosophila melanogaster embryo using time- resolved single-cell genomics, spatial transcript mapping with hybridisation chain reaction, and targeted metabolomics. We uncover a regulatory landscape in which specific transcription factors are activated before biosynthetic enzymes, establishing a prospective temporal architecture for monoaminergic fate specification. Comparative analyses of developmental single-cell atlases from zebrafish and sea urchin indicate that components of this machinery are conserved across [~]550 million years of bilaterian evolution with orthologous transcription factors showing similar temporal dynamics. Together, these findings point to a putatively conserved regulatory core that interfaces with other context-dependent transcription factors; this interplay accommodates monoaminergic multifunction and subtype diversity across distinct neuroanatomies.

evolutionary biology↗

bioRxiv2025

Dissecting the role of RNA-binding proteins in early herpes simplex virus 1 transcription using acute protein depletion

Herpes simplex virus 1 (HSV-1) infects about 50-80% of the entire human population and persists in the neurons of affected individuals. A fraction of affected individuals suffer from recurrent cold sores caused by reactivating virus, in rare but severe cases the virus can cause encephalitis. During lytic infection, the virus relies on host factors such as RNA polymerase II and accessory proteins involved in transcription to express its genes and ensure successful replication. In general, RNA molecules in cells are bound by RNA-binding proteins (RBPs) during their entire lifecycle. Importantly, RBPs are increasingly described to also regulate transcription, an aspect long time outside the scope of investigations, especially during viral infections. Here, we examined the impact of five nuclear proteins (FUBP1, SLBP, SFPQ, SPT5 and SAF-B) with known RNA-binding activities on HSV-1 transcription. Additionally, we evaluated their importance for human adenovirus C5 (HAdV) growth to assess whether these host factors are specific to HSV-1 infections or might have broader relevance for the general transcription of dsDNA viruses. We show that the transcriptional elongation factor SPT5 coded by SUPT5H accumulates on HSV-1 genomes early during the infection and is required for the transcription of the immediate-early gene UL54. Its depletion affects also HAdV replication, indicating a general role in transcription of viruses that depend on the host transcriptional machinery. In contrast, depletion of the transcriptional repressor and paraspeckle protein SFPQ reduces UL54 RNA levels in HSV-1 infection, but does not cause significant changes in HAdV growth. Since SFPQ does not co-localize with HSV-1 genomes, this suggests a function not directly associated to viral DNA.

molecular biology↗

bioRxiv2023

Respective contribution of Appl to mushroom body axon growth and long-term memory in Drosophila

The Amyloid Precursor Protein (APP) is associated with Alzheimers disease. Appl is the single Drosophila APP ortholog and is expressed in all neurons throughout development. Appl was previously shown to cell-autonomously modulate axon outgrowth in the mushroom bodies (MBs), the fly olfactory memory center. However, we found that Appld, the only reported null allele, affects the normal function of vnd, the gene just proximal to Appl. To decipher developmental defects specifically due to a loss of only Appl function, we generated a precise Appl null allele (ApplC2.1) by CRISPR/Cas9 genomic engineering. With ApplC2.1, we confirmed the partial requirement for Appl in MB axon outgrowth. We also produced new CRISPR vnd alleles removing either vnd-B or vnd-A function. We report here that vnd is also required for MB {beta}-branch axon outgrowth and to a much greater extent than Appl itself. Moreover, vnd is expressed in neurons close to, but not within, the MB during development and is required non-cell-autonomously for MB axon outgrowth

neuroscience↗

bioRxiv2023

Differential regulation of the proteome and phosphosproteome along the dorso-ventral axis of the early Drosophila embryo

The initially homogeneous epithelium of the early Drosophila embryo differentiates into regional subpopulations with different behaviours and physical properties that are needed for morphogenesis. The factors at top of the genetic hierarchy that control these behaviours are known, but many of their targets are not. To understand how proteins work together to mediate differential cellular activities, we studied in an unbiased manner the proteomes and phosphoproteomes of the three main cell populations along the dorso-ventral axis during gastrulation using mutant embryos that represent the different populations. We detected 6111 protein groups and 6259 phosphosites of which 3399 and 3433 respectively, were differentially regulated. The changes in phosphosite abundance did not correlate with changes in host protein abundance, showing phosphorylation to be a regulatory step during gastrulation. Hierarchical clustering of protein groups and phosphosites identified clusters that contain known fate determinants such as Doc1, Sog, Snail and Twist. The recovery of the appropriate known marker proteins in each of the different mutants we used validated the approach, but also revealed that two mutations that both interfere with the dorsal fate pathway, Toll10B and serpin27aex do this in very different manners. Diffused network analyses within each cluster point to microtubule components as one of the main groups of regulated proteins. Functional studies on the role of microtubules provide the proof of principle that microtubules have different functions in different domains along the DV axis of the embryo.

developmental biology↗

bioRxiv2021

Optogenetic perturbations of RNA expression in tissue space

Organoids derived from stem cells become increasingly important to study human development and to model disease. However, methods are needed to control and study spatio-temporal patterns of gene expression in organoids. To this aim, we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes, at single-cell resolution and in programmable spatio-temporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. High-resolution spatial transcriptomic and single-cell analyses showed that this local induction was sufficient to generate stereotypically patterned organoids in three dimensions and revealed new insights into SHHs contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.

bioengineering↗

bioRxiv2018

A gene expression atlas of embryonic neurogenesis in Drosophila reveals complex spatiotemporal regulation of lncRNAs.

Cell type specification during early nervous system development in Drosophila melanogaster requires precise regulation of gene expression in time and space. Resolving the programs driving neurogenesis has been a major challenge owing to the complexity and rapidity with which distinct cell populations arise. To resolve the cell type-specific gene expression dynamics in early nervous system development, we have sequenced the transcriptomes of purified neurogenic cell types across consecutive time points covering critical events in neurogenesis. The resulting gene expression atlas comprises a detailed resource of global transcriptome dynamics that permits systematic analysis of how cells in the nervous system acquire distinct fates. We resolve known gene expression dynamics and uncover novel expression signatures for hundreds of genes among diverse neurogenic cell types, most of which remain unstudied. We also identified a set of conserved and processed long-noncoding RNAs (lncRNAs) that exhibit spatiotemporal expression during neurogenesis with exquisite specificity. LncRNA expression is highly dynamic and demarcates specific subpopulations within neurogenic cell types. Our spatiotemporal transcriptome atlas provides a comprehensive resource to investigate the function of coding genes and noncoding RNAs during critical stages of early neurogenesis.\n\nSummary statementWe present a spatiotemporal transcriptome during early Drosophila embryonic nervous system development, revealing a complex cell type-specific network of mRNAs and IncRNAs.

developmental biology↗

bioRxiv2017

Determinants of Transcription Initiation Directionality in Metazoans

Divergent transcription from promoters and enhancers is pervasive in many species, but it remains unclear if it is a general and passive feature of all eukaryotic cis regulatory elements. To address this, we define promoters and enhancers in C. elegans, D. melanogaster and H. sapiens using ATAC-Seq and investigate the determinants of their transcription initiation directionalities by analyzing genome-wide nascent, cap-selected, polymerase run-on assays. All three species initiate divergent transcription from separate core promoter sequences. Sequence asymmetry downstream of forward and reverse initiation sites, known to be important for termination and stability in H. sapiens, is unique in each species. Chromatin states of divergent promoters are not entirely conserved, but in all three species, the levels of histone modifications on the +1 nucleosome are independent from those on the -1 nucleosome, arguing for independent initiation events. This is supported by an integrative model of H3K4me3 levels and core promoter sequence that is highly predictive of promoter directionality and of two types of promoters: those with balanced initiation directionality and those with skewed directionality. Lastly, D. melanogaster enhancers display variation in chromatin architecture depending on enhancer location, and D. melanogaster promoter regions with dual enhancer/promoter potential are enriched for divergent transcription. Our results point to a high degree of variation in regulatory element transcription initiation directionality within and between metazoans, and to non-passive regulatory mechanisms of transcription initiation directionality in those species.

molecular biology↗

bioRxiv2017

The Drosophila Embryo at Single Cell Transcriptome Resolution

Drosophila is a premier model system for understanding the molecular mechanisms of development. By the onset of morphogenesis, ~6000 cells express distinct gene combinations according to embryonic position. Despite extensive mRNA in situ screens, combinatorial gene expression within individual cells is largely unknown. Therefore, it is difficult to comprehensively identify the coding and non-coding transcripts that drive patterning and to decipher the molecular basis of cellular identity. Here, we single-cell sequence precisely staged embryos, measuring >3100 genes per cell. We produce a transcriptomic blueprint of development - a virtual embryo where 3D locations of sequenced cells are confidently identified. Our \"Drosophila-Virtual-Expression-eXplorer\" performs virtual in situ hybridizations and computes expression gradients. Using DVEX, we predict spatial expression and discover patterned lncRNAs. DEVX is sensitive enough to detect subtle evolutionary changes in expression patterns between Drosophila species. We believe DVEX is a prototype for powerful single cell studies in complex tissues.

genomics↗