Mohamed-Ali Hakimi

Regeneration requires injured tissues to selectively reactivate certain developmental programs while triggering regeneration-specific mechanisms and responding to the immediate consequences of tissue damage. How chromatin states are reorganized during this transition, and whether the underlying sequence of regulatory events is conserved across regenerating animals, remain poorly understood. We address these questions in the two-spotted cricket Gryllus bimaculatus , in which limb regeneration progressively declines during nymphal development and is absent in adults, and extend our analysis to the crustacean Parhyale hawaiensis . We first establish a chromosome-scale genomic and epigenomic framework for the cricket and then follow chromatin and transcriptional changes after limb amputation. Amputation triggers a striking genome-wide shutdown of enhancer-associated chromatin, marked by loss of accessible regulatory regions and depletion of H3K27ac and H3K4me1. This is followed, in regeneration-competent tissue, by selective reopening of a restricted population of regulatory elements and strong induction of the pioneer transcription factor Zelda (GbZelda). The GbZelda locus is unusually marked by both H3K27me3 and H3K9me3 in intact leg tissue; after injury, H3K9me3 is reduced broadly, whereas H3K27me3 is selectively relieved in regenerating nymphs. Finally, analysis of Parhyale reveals the same sequence of chromatin events during appendage regeneration: an initial enhancer shutdown followed by selective regulatory reopening concomitant with Zelda upregulation. Thus, rather than restoring the pre-injury regulatory landscape wholesale, appendage regeneration in these two pancrustacean models proceeds through a conserved sequence of genome-wide chromatin resetting, selective regulatory reopening and activation of a pioneer transcription factor.

Histone acetylation governs Toxoplasma gondii gene expression, developmental plasticity and virulence, yet the organization and deployment of its acetyltransferase machinery remain poorly understood. Here, we show that T. gondii rewired this process using a plant-like system built around the acetyltransferase Tg GCN5b, which differs from the typical SAGA complex found in other eukaryotes. Interactome and structural analyses reveal a modular assembly that integrates multiple acetyltransferase (GNAT) enzymes and chromatin-reader proteins carrying PHD and PZP domains and Apetala-related transcriptional regulators, an organization unique to apicomplexan parasites. Tg GCN5b catalyzes a selective tri-site acetylation pattern on histone H3 at lysines 9, 14, and 18 that maintains open chromatin and sustains transcription of core metabolic, invasion, and virulence genes. Its conditional depletion disrupts these post-translational modifications, silencing key promoters and decoupling transcription from histone methylation. Genome-wide analyses further show that Tg GCN5b functions independently of the MORC/HDAC3 repressive pathway, defining a distinct regulatory circuit that connects chromatin acetylation with gene expression and developmental transitions. These findings reveal that the SAGA complex has been evolutionarily reconfigured in T. gondii from a plant origin, yet featuring divergent and apicomplexan-specific feature, positioning Tg GCN5b as a central regulator that links epigenetic control to parasite growth, adaptation, and virulence.

Toxoplasma gondii and Cryptosporidium species are apicomplexan parasites of significant medical and veterinary importance. Although current therapeutic options for toxoplasmosis and cryptosporidiosis demonstrate notable efficacy, their clinical efficacy is often limited by suboptimal efficacy and frequent adverse effects. Moreover, therapeutic alternatives remain limited or nonexistent, particularly for cryptosporidiosis, for which nitazoxanide is currently the only approved medication to treat diarrhea in adults and children older than 1 year of age. To identify alternative therapeutic options for addressing these health challenges, we performed a phenotypic screening of an FDA-approved drug repurposing library against Toxoplasma . This screening identifies LY2090314 as a potent inhibitor of T. gondii and Cryptosporidium growth in mammalian cells. Through a target deconvolution strategy combining forward genetics, transcriptome sequencing, and computational mutation analysis, we elucidate the parasiticidal mechanism of LY2090314 and demonstrate that Tg GSK3 kinase is its primary molecular target. We also report the first X-ray crystal structure of LY2090314 bound to Tg GSK3, resolved at 2.1 Å, which reveals an interaction mode characteristic of type I ATP-competitive inhibitors. Furthermore, interactome analysis uncovers functional connections between Tg GSK3 and key cytoskeletal and signaling regulators, providing insights into compound’s effects. Collectively, these findings validate Tg GSK3 as a promising therapeutic target for toxoplasmosis and offer mechanistic insights into apicomplexan GSK3 biology.

ISWI chromatin remodelers are conserved regulators of nucleosome positioning and chromatin accessibility across eukaryotes, yet their evolutionary diversification is poorly understood. In the apicomplexan parasite Toxoplasma gondii , we identify Hydra, a previously unrecognized globular domain embedded within Tg SNF2L, one of two ISWI paralogues. Hydra is structurally unique, lacking homology to any known protein fold, and represents a lineage-specific insertion in an otherwise structurally conserved protein family. Biochemical analyses reveal that the isolated Hydra domain self-assembles into stable oligomers, undergoing reversible equilibrium with its monomeric form. Cryo-electron microscopy analysis reveals discrete globular assemblies, though little consistency could be obtained, suggesting a highly dynamic complex. Deletion of Hydra from full-length Tg SNF2L disrupts its intrinsic ability to form higher order oligomers in solution, yielding predominantly monomeric and dimeric species. Functionally, the Hydra-driven multimerization of Tg SNF2L modulates its availability for chromatin engagement in response to cell-cycle cues. Hydra thus represents the first reported structural innovation within the ISWI family.

Toxoplasma gondii , a neurotropic protozoan, causes toxoplasmosis, a prevalent zoonotic and food-borne infection, posing significant risks to immunocompromised individuals and congenital cases. The chronic phase, characterized by dormant, cyst-forming bradyzoites, is central to disease progression but is poorly understood due to the lack of serological tests to detect bradyzoite-specific antigens. This study identifies the bradyzoite serological marker (BSM) and cyst-associated BCLA as effective biomarkers for chronic toxoplasmosis. These markers showed high sensitivity and specificity in detecting cyst-bearing mice and had a positivity rate of 30% in humans with prior immunity. Bradyzoite serology helps to discriminate between recent and past infections, with BCLA improving the accuracy of the diagnosis of congenital infections. Mechanistic analyses show that the chromatin modifiers MORC and HDAC3 epistatically regulate BFD1, a key bradyzoite regulator. While BFD1 controls the expression of bradyzoite genes such as BCLA, a specific subset, including BSM, is regulated independently of BFD1. This multilayered regulation complicates the understanding of parasite persistence in humans, but offers promise for improved serologic diagnosis during pregnancy, but also in individuals with mental illness.

ATP-dependent chromatin remodellers are specialized multiprotein machines that organize the genome in eukaryotic cells and regulate its accessibility by repositioning, ejecting or modifying nucleosomes. However, their role in Toxoplasma gondii is poorly understood. Here we show that T. gondii has evolved two divergent proteins within the imitation switch (ISWI) family: TgSNF2h and TgSNF2L. TgSNF2h specifically forms a core complex with the transcription factor AP2VIII-2 and the scaffold protein TgRFTS. Depletion of TgRFTS phenocopies the knockdown of TgSNF2h, restricting access to chromatin and altering local gene expression. At the genomic level, TgSNF2h insulates highly transcribed genes from silenced neighbours, ensuring stage-specific gene regulation. By modulating chromatin accessibility to transcription factors, TgSNF2h exerts epistatic control over MORC, a key regulator of sexual commitment. Our findings show that a specific ISWI complex orchestrates the partitioning of developmental genes and ensures transcriptional fidelity throughout the parasite life cycle.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described^1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. ¹), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

The study presents a model of sexual differentiation in the malaria parasite, Plasmodium berghei, where male and female gametocytes differentiate from a common sexual precursor. This process relies on a cascade of nucleic acid-binding proteins, co-expressed downstream of AP2-G, which perform distinct functions in a hierarchy of regulatory events. The zinc finger protein GD1 is identified as a top-level factor for female determination, operating upstream of the female-specific transcription factor AP2-FG. The study also explores how sex ratio is determined in P. berghei. The researchers found no evidence of a sex-specific transcriptomic signature preceding the commitment to sexual development. Instead, they suggest that sex may be determined downstream of AP2-G induction, potentially involving non-transcriptional mechanisms like chromatin marks, differential splicing, or phosphorylation states. The study identifies a group of proteins co-expressed downstream of AP2-G, whose deletion affects either the determination of, or differentiation along, a male or female cellular trajectory. The screen revealed an abundance of putative RNA binding proteins, including the maleness inducing factor MD1 and the early response genes MD4, MD5, and FD1. The LOTUS/OST-HTH domain gene md1 suggests the possibility of the function of this domain in gametogenesis being conserved beyond animals. The findings provide a route to establishing these mechanisms in more detail, which will shed light on the precise molecular mechanisms of sex ratio determination and sexual development in malaria parasites.

The 3′‐end processing of mRNA is a co‐transcriptional process that leads to the formation of a poly‐adenosine tail on the mRNA and directly controls termination of the RNA polymerase II juggernaut. This process involves a megadalton complex composed of cleavage and polyadenylation specificity factors (CPSFs) that are able to recognize cis‐sequence elements on nascent mRNA to then carry out cleavage and polyadenylation reactions. Recent structural and biochemical studies have defined the roles played by different subunits of the complex and provided a comprehensive mechanistic understanding of this machinery in yeast or metazoans. More recently, the discovery of small molecule inhibitors of CPSF function in Apicomplexa has stimulated interest in studying the specificities of this ancient eukaryotic machinery in these organisms. Although its function is conserved in Apicomplexa, the CPSF complex integrates a novel reader of the N6‐methyladenosine (m6A). This feature, inherited from the plant kingdom, bridges m6A metabolism directly to 3′‐end processing and by extension, to transcription termination. In this review, we will examine convergence and divergence of CPSF within the apicomplexan parasites and explore the potential of small molecule inhibition of this machinery within these organisms.This article is categorized under: RNA Processing > 3′ End Processing RNA Processing > RNA Editing and Modification