Biology subjects

Ciofani, M.

Publications and source records attributed to Ciofani, M..

9 recordsLinked to original sources

bioRxiv2026

Enhancer hubs govern chromatin topology and Th17 identity

Between one and two million noncoding regulatory elements have been described across mammalian genomes, but determining their functional role remains a challenge. To address this gap, we measure the regulatory potential of open chromatin regions (OCRs) in five closely related mouse CD4+ T cell subsets with ATAC-STARR-seq, then map endogenous enhancer function and three-dimensional contacts in Th17 cells with pooled CRISPR-based noncoding screens and high-resolution Micro-C. Of all CD4+ T cell OCRs, approximately 25% demonstrate largely subset-shared regulatory activity, though we identify subset-restricted active elements distinguishable by their sequence features. In Th17 cells, we reveal a set of core regulatory OCRs essential for subset polarization at the Batf, Rorc(t) and Il17a/f loci, and confirm their requirement for cell identity in vivo. At these three loci, we resolve nested yet selective enhancer-to-enhancer and enhancer-to-promoter physical interactions that converge into multi-enhancer hubs. Importantly, these hubs contain many of the strongest functional elements. Perturbation of a single enhancer within the Batf locus selectively disrupts hub contacts and Batf expression in Th17 cells, recapitulating the genome-wide transcriptional and chromatin accessibility signatures of a germline knockout. Together, this work assigns regulatory activity to accessible chromatin across CD4+ T cell subsets and couples the function of a set of essential elements to selective three-dimensional contacts in Th17 cells.

genomics↗

bioRxiv2024

Restriction of innate Tγδ17 cell plasticity by an AP-1 regulatory axis

IL-17-producing {gamma}{delta} T (T{gamma}{delta}17) cells are innate-like mediators of intestinal barrier immunity. While Th17 cell and ILC3 plasticity have been extensively studied, the mechanisms governing T{gamma}{delta}17 cell effector flexibility remain undefined. Here, we combined type 3 fate-mapping with single cell ATAC/RNA-seq multiome profiling to define the cellular features and regulatory networks underlying T{gamma}{delta}17 cell plasticity. During homeostasis, T{gamma}{delta}17 cell effector identity was stable across tissues, including for intestinal T-bet+ T{gamma}{delta}17 cells that restrained IFN{gamma} production. However, S. typhimurium infection induced intestinal V{gamma}6+ T{gamma}{delta}17 cell conversion into type 1 effectors, with loss of IL-17A production and partial ROR{gamma}t downregulation. Multiome analysis revealed a trajectory along V{gamma}6+ T{gamma}{delta}17 effector conversion, with TIM-3 marking ex-T{gamma}{delta}17 cells with enhanced type 1 functionality. Lastly, we characterized and validated a critical AP-1 regulatory axis centered around JunB and Fosl2 that controls V{gamma}6+ T{gamma}{delta}17 cell plasticity by stabilizing type 3 identity and restricting type 1 effector conversion.

immunology↗

bioRxiv2024

Structure-guided design, synthesis, and characterization of C22- and C32-modified FK520 analogs with enhanced activity against human pathogenic fungi

Invasive fungal infections are a leading cause of death worldwide. Translating molecular insights into clinical benefits is challenging because fungal pathogens and their hosts share similar eukaryotic physiology. Consequently, current antifungal treatments have limited efficacy, may be poorly fungicidal in the host, can exhibit toxicity, and are increasingly compromised by emerging resistance. We have established that the phosphatase calcineurin (CaN) is required for invasive fungal disease and an attractive target for antifungal drug development. CaN is a druggable target, and there is vast clinical experience with the CaN inhibitors FK506 and cyclosporin A (CsA). However, while FK506 and its natural analog FK520 exhibit antifungal activity, they are also immunosuppressive in the host and thus not fungal-selective. We leverage our pathogenic fungal CaN-FK506-FKBP12 complex X-ray structures and biophysical data to support structure-based ligand design as well as structure-activity relationship analyses of broad-spectrum FK506/FK520 derivatives with potent antifungal activity and reduced immunosuppressive activity. Here we apply molecular docking studies to develop antifungal C22- or C32-modified FK520 derivatives with improved therapeutic index scores. Among them, the C32-modified FK520 derivative JH-FK-44 (7) demonstrates a significantly improved therapeutic index compared to JH-FK-08, our lead compound to date. NMR binding studies with C32-derivatives are consistent with our hypothesis that C32 modifications disrupt the hydrogen bonding network in the human complex while introducing favorable electrostatic and cation-{pi} interactions with the fungal FKBP12 R86 residue. These findings further reinforce calcineurin inhibition as a promising strategy for antifungal therapy. SignificanceInvasive fungal infections cause significant mortality worldwide, and current antifungal treatments are often ineffective, toxic, or face growing resistance. This research identifies calcineurin (CaN), a critical protein for fungal survival, as a potential target for developing new antifungal drugs. Although existing CaN inhibitors such as FK506 (tacrolimus) and FK520 (ascomycin) possess antifungal properties, their immunosuppressive effects limit their clinical utility. By studying the structure of human and fungal FKBP12-FK506 or FK520 complexes with CaN, we have designed and synthesized modified FK520 derivatives with strong antifungal activity and reduced immunosuppressive effects. These new derivatives are expected to have significantly improved therapeutic profiles, offering hope for more effective and safer antifungal treatments.

microbiology↗

bioRxiv2024

E protein control of NKgammadeltaT cell development through both generation and function of the stereotypic Vgamma1Vdelta6.3 TCR

T cell receptor (TCR) signals regulate important developmental transitions through induction of the E protein antagonist, Id3; however, Id3-deficiency produces paradoxical effects on {gamma}{delta} T cell subsets. Here, we show here that Id3-deficiency attenuates the development of V{gamma}3-expressing {gamma}{delta} T cells, while markedly enhancing the development of V{gamma}1V{delta}6.3-expressing NK{gamma}{delta}T cells. Id3-deficiency does so by regulating both the generation of the stereotypic V{gamma}1V{delta}6.3 TCR expressed by NK{gamma}{delta}T cells and its capacity to support development. Indeed, we determined that the Trav15 segment, which encodes the V{delta}6.3 TCR subunit, is directly bound by E proteins that control its expression. Moreover, once expressed, the resulting V{gamma}1V{delta}6.3 TCR in capable of specifying the innate-like NK{gamma}{delta}T cell fate in a cell-autonomous and developmentally-unrestricted manner that is restrained by the Id3/E axis. Together, these data indicate that the paradoxical behavior of NK{gamma}{delta}T cells in the Id3-deficient setting is entirely determined by its stereotypic V{gamma}1V{delta}6.3 TCR complex.

immunology↗

bioRxiv2023

Enhanced fungal specificity and in vivo therapeutic efficacy of a C-22 modified FK520 analog against C. neoformans

Fungal infections are of mounting global concern, and the current limited treatment arsenal poses challenges when treating such infections. In particular, infections by Cryptococcus neoformans are associated with high mortality, emphasizing the need for novel therapeutic options. Calcineurin is a protein phosphatase that mediates fungal stress responses, and calcineurin inhibition by the natural product FK506 blocks C. neoformans growth at 37{degrees}C. Calcineurin is also required for pathogenesis. However, because calcineurin is conserved in humans, and inhibition with FK506 results in immunosuppression, the use of FK506 as an anti-infective agent is precluded. We previously elucidated the structures of multiple fungal calcineurin-FK506-FKBP12 complexes and implicated the C-22 position on FK506 as a key point for differential modification of ligand inhibition of the mammalian versus fungal target proteins. Through in vitro antifungal and immunosuppressive testing of FK520 (a natural analog of FK506) derivatives, we identified JH-FK-08 as a lead candidate for further antifungal development. JH-FK-08 exhibited significantly reduced immunosuppressive activity and both reduced fungal burden and prolonged survival of infected animals. JH-FK-08 exhibited additive activity in combination with fluconazole in vivo. These findings further advance calcineurin inhibition as an antifungal therapeutic approach. ImportanceFungal infections cause significant morbidity and mortality globally. The therapeutic armamentarium against these infections is limited and development of antifungal drugs has been hindered by the evolutionary conservation between fungi and the human host. With rising resistance to the current antifungal arsenal and an increasing at-risk population, there is an urgent need for the development of new antifungal compounds. The FK520 analogs described in this study display potent antifungal activity as a novel class of antifungals centered on modifying an existing orally-active FDA approved therapy. This research advances the development of much needed newer antifungal treatment options with novel mechanisms of action.

microbiology↗

bioRxiv2022

Structure-guided synthesis of FK506 and FK520 analogs with increased selectivity exhibit in vivo therapeutic efficacy against C. neoformans

Calcineurin is an essential virulence factor that is conserved across human fungal pathogens including Cryptococcus neoformans, Aspergillus fumigatus, and Candida albicans. Although an excellent target for antifungal drug development, the serine-threonine phosphatase activity of calcineurin is conserved in mammals and inhibition of this activity results in immunosuppression. FK506 (tacrolimus) is a naturally produced macrocyclic compound that inhibits calcineurin by binding to the immunophilin FKBP12. Previously, our fungal calcineurin-FK506-FKBP12 structure-based approaches identified a non-conserved region of FKBP12 that can be exploited for fungal-specific targeting. These studies led to the design of an FK506 analog, APX879, modified at the C22 position that was less immunosuppressive yet maintained antifungal activity. We now report high resolution protein crystal structures of fungal FKBP12 and a human truncated calcineurin-FKBP12 bound to a natural FK506 analog, FK520 (ascomycin). Based on information from these structures and the success of APX879, we synthesized and screened a novel panel of C22-modified compounds derived from both FK506 and FK520. One compound, JH-FK-05 demonstrates broad-spectrum antifungal activity in vitro and is non-immunosuppressive in vivo. In murine models of pulmonary and disseminated C. neoformans infection, JH-FK-05 treatment significantly reduced fungal burden and extended animal survival alone and in combination with fluconazole. Furthermore, molecular dynamic simulations performed with JH-FK-05 binding to fungal and human FKBP12 identified additional residues outside of the C22 and C21 positions that could be modified to generate novel FK506 analogs with improved antifungal activity. SignificanceDue to rising rates of antifungal drug resistance and a limited armamentarium of antifungal treatments, there is a paramount need for novel antifungal drugs to treat systemic fungal infections. Calcineurin has been established as an essential and conserved virulence factor in several fungi, making it an attractive antifungal target. However, due to the immunosuppressive action of calcineurin inhibitors, they have not been successfully utilized clinically for antifungal treatment in humans. Recent availability of crystal structures of fungal calcineurin bound inhibitor complexes have enabled the structure-guided design of FK506 analogs and led to a breakthrough in the development of a compound increased for fungal specificity. The development of a calcineurin inhibitor with reduced immunosuppressive activity and therapeutic antifungal activity would add a significant tool to the treatment options for these invasive fungal infections with exceedingly high rates of mortality.

microbiology↗

bioRxiv2021

Transgenic mice for in vivo epigenome editing with CRISPR-based systems

The discovery, characterization, and adaptation of the RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 system has greatly increased the ease with which genome and epigenome editing can be performed. Fusion of chromatin-modifying domains to the nuclease-deactivated form of Cas9 (dCas9) has enabled targeted gene activation or repression in both cultured cells and in vivo in animal models. However, delivery of the large dCas9 fusion proteins to target cell types and tissues is an obstacle to widespread adoption of these tools for in vivo studies. Here we describe the generation and validation of two conditional transgenic mouse lines for targeted gene regulation, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. Using the dCas9p300 and dCas9KRAB transgenic mice we demonstrate activation or repression of genes in both the brain and liver in vivo, and T cells and fibroblasts ex vivo. We show gene regulation and targeted epigenetic modification with gRNAs targeting either transcriptional start sites (TSS) or distal enhancer elements, as well as corresponding changes to downstream phenotypes. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.

genomics↗

bioRxiv2019

JunB controls tissue-specific effector programs in regulatory T cells

Foxp3-expressing regulatory T (Treg) cells are critical mediators of immunological tolerance to both self and microbial antigens. Tregs activate context-dependent transcriptional programs to adapt effector function to specific tissues; however, the factors controlling tissue-specific gene expression in Tregs remain unclear. Here, we find that the AP-1 transcription factor JunB regulates the intestinal adaptation of Tregs by controlling select gene expression programs in multiple Treg subsets. Treg-specific ablation of JunB results in immune dysregulation characterized by enhanced colonic T helper cell accumulation and cytokine production. However, in contrast to its classical binding-partner BATF, JunB is dispensable for maintenance of effector Tregs as well as most specialized Treg subsets. In the Peyers patches, JunB activates a transcriptional program facilitating the maintenance of CD25- Tregs, leading to the complete loss of T follicular regulatory cells in the absence of JunB. This defect is compounded by loss of a separate effector program found in both major colonic Treg subsets that includes the cytolytic effector molecule granzyme B. Therefore, JunB is an essential regulator of intestinal Treg effector function through pleiotropic effects on gene expression.

immunology↗

bioRxiv2018

Leveraging chromatin accessibility for transcriptional regulatory network inference in T Helper 17 Cells

Transcriptional regulatory networks (TRNs) provide insight into cellular behavior by describing interactions between transcription factors (TFs) and their gene targets. The Assay for Transposase Accessible Chromatin (ATAC)-seq, coupled with transcription-factor motif analysis, provides indirect evidence of chromatin binding for hundreds of TFs genome-wide. Here, we propose methods for TRN inference in a mammalian setting, using ATAC-seq data to influence gene expression modeling. We rigorously test our methods in the context of T Helper Cell Type 17 (Th17) differentiation, generating new ATAC-seq data to complement existing Th17 genomic resources (plentiful gene expression data, TF knock-outs and ChIP-seq experiments). In this resource-rich mammalian setting, our extensive benchmarking provides quantitative, genome-scale evaluation of TRN inference combining ATAC-seq and RNA-seq data. We refine and extend our previous Th17 TRN, using our new TRN inference methods to integrate all Th17 data (gene expression, ATAC-seq, TF KO, ChIP-seq). We highlight new roles for individual TFs and groups of TFs (\"TF-TF modules\") in Th17 gene regulation. Given the popularity of ATAC-seq, which provides high-resolution with low sample input requirements, we anticipate that application of our methods will improve TRN inference in new mammalian systems, especially in vivo, for cells directly from humans and animal models.

systems biology↗