Biology subjects

Doniger, T.

Publications and source records attributed to Doniger, T..

6 recordsLinked to original sources

bioRxiv2023

A single DPE core promoter motif contributes to in vivo transcriptional regulation and affects cardiac function

Transcription is initiated at the core promoter, which confers specific functions depending on the unique combination of core promoter elements. The downstream core promoter element (DPE) is found in many genes related to heart and mesodermal development. However, the function of these core promoter elements has thus far been studied primarily in isolated, in vitro or reporter gene settings. tinman (tin) encodes a key transcription factor that regulates the formation of the dorsal musculature and heart. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, we show that a substitution mutation of the functional tin DPE motif within the natural context of the core promoter results in a massive perturbation of Tinmans regulatory network orchestrating dorsal musculature and heart formation. Mutation of endogenous tin DPE reduced the expression of tin and distinct target genes, resulting in significantly reduced viability and an overall decrease in adult heart function. We demonstrate the feasibility and importance of characterizing DNA sequence elements in vivo in their natural context, and accentuate the critical impact a single DPE motif has during Drosophila embryogenesis and functional heart formation.

developmental biology↗

bioRxiv2023

Uncovering the function of the evolutionary conserved Clock gene in the sea anemone Nematostella vectensis

The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCKs functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis, pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant (NvClk{Delta}), revealing circadian behavior loss in constant light conditions (LL and DD) while a 24-hour rhythm was maintained under light-dark condition (LD). Transcriptomics showed distinct rhythmic genes in wild-type (WT) genes in LD compared to DD. The LD NvClk{Delta}/{Delta} showed comparable numbers of rhythmic genes, but were greatly reduced in DD. Furthermore, the LD NvClk{Delta}/{Delta} showed alterations in temporal pacemaker gene expression, affecting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior to the diel rhythm of the earths biosphere.

evolutionary biology↗

bioRxiv2021

Genome instability drives epistatic adaptation in the human pathogen Leishmania

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches we provide first evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover post-transcriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in snoRNA gene dosage with changes in rRNA 2-O-methylation and pseudouridylation, suggesting translational control is an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression, and genome-independent, compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery, and may be relevant to other fast evolving, eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer. One Sentence SummaryEpistatic interactions harness genome instability for Leishmania fitness gain.

genomics↗

bioRxiv2021

Post-transcriptional regulation of Leishmania fitness gain

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network driving parasite fitness, with genome instability causing highly reproducible, gene dosage-dependent changes in protein abundance linked to post-transcriptional regulation. These in turn were associated with a gene dosage-independent reduction in abundance of flagellar transcripts and a coordinated increase in abundance of coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of fitness-adapted ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

microbiology↗

bioRxiv2020

Characterization of novel pollen-expressed transcripts reveals their potential roles in pollen heat stress response in Arabidopsis thaliana.

The male gametophyte is the most heat-sensitive of all plant tissues. In recent years, long noncoding RNAs (lncRNAs) have emerged as important components of cellular regulatory networks involved in most biological processes, including response to stress. While examining RNAseq datasets of developing and germinating Arabidopsis thaliana pollen exposed to heat stress (HS), we identified 66 novel and 246 recently-annotated intergenic expressed loci (XLOCs) of unknown function, with the majority encoding lncRNAs. Comparison to HS in cauline leaves and other RNAseq experiments, indicated 74% of the 312 XLOCs are pollen-specific, and at least 42% are HS-responsive. Phylogenetic analysis revealed 96% of the genes evolved recently in Brassicaceae. We found that 50 genes are putative targets of microRNAs, and that 30% of the XLOCs contain small open reading frames (ORFs) with homology to protein sequences. Finally, RNAseq of ribosome-protected RNA fragments together with predictions of periodic footprint of the ribosome P-sites indicated that 23 of these ORFs are likely to be translated. Our findings indicate that many of the 312 unknown genes might be functional, and play significant role in pollen biology, including the HS response.

plant biology↗

bioRxiv2020

The transcription factor TRF2 has a unique function in regulating cell cycle and apoptosis

TRF2 (TATA-box-binding protein-related factor 2) is an evolutionarily conserved general transcription factor that is essential for embryonic development of Drosophila melanogaster, C. elegans, zebrafish and Xenopus. Nevertheless, the cellular processes that are regulated by TRF2 are largely underexplored. Here, using Drosophila Schneider cells as a model, we discovered that TRF2 regulates cell cycle progression. Using flow cytometry, high-throughput microscopy and advanced imaging-flow cytometry, we demonstrate that TRF2 knockdown regulates cell cycle progression and exerts distinct effects on G1 and specific mitotic phases. RNA-seq analysis revealed that TRF2 regulates the expression of Cyclin E and the mitotic cyclins, Cyclin A, Cyclin B and Cyclin B3, but not Cyclin D or Cyclin C. To identify proteins that could account for the observed regulation of these cyclin genes, we searched for TRF2-interacting proteins. Interestingly, mass spectrometry analysis of TRF2-containing complexes identified GFZF, a nuclear glutathione S-transferase implicated in cell cycle regulation, and Motif 1 binding protein (M1BP). Furthermore, available ChIP-exo data revealed that TRF2, GFZF and M1BP co-occupy the promoters of TRF2-regulated genes. Using RNAi to knockdown the expression of either M1BP, GFZF, TRF2 or their combinations, we demonstrate that although GFZF and M1BP interact with TRF2, it is TRF2, rather than GFZF or M1BP, that is the main factor regulating the expression of Cyclin E and the mitotic cyclins. Taken together, our findings uncover a critical and unanticipated role of a general transcription factor as a key regulator of cell cycle.

molecular biology↗