Biology subjects

Spicuglia, S.

Publications and source records attributed to Spicuglia, S..

10 recordsLinked to original sources

bioRxiv2026

Epromoter 3D interaction-associated regulation in T-acute Lymphoblastic Leukemia

Background: Promoters have been traditionally seen as contiguous gene-adjacent cis-regulatory elements. Yet, substantial studies corroborate that Epromoters (promoters with enhancer activity) engage in distal forms of gene regulation. Although in the three-dimensional (3D) space enhancer-promoter networks have been well studied, the contribution of the circuits of promoter-promoter (P-P) interactions is poorly understood. Furthermore, whether the regulatory aspects of P-P interactions in cancer may be controlled by physical 3D-mediated Epromoter interactions remains elusive. Results: We show that Epromoter-mediated 3D interactions regulate target genes and participate in cluster co-regulation, playing a critical role in T-cell acute Lymphoblastic Leukemia (T-ALL). To achieve this, we first leveraged survival CRISPR screenings in T-ALL model cells (Jurkat) to identify potential Epromoters. By integrating these findings with an H3K27ac HiChIP dataset from T-ALL cells, we characterized a set of Epromoters that establish 3D genome interactions with other promoters. We observed that promoters organize into dense, promoter-rich genomic clusters, and that among them, the clusters enriched with Epromoters actively regulate complex gene expression networks. To investigate gene coregulation, we integrated transcriptomic data from T-ALL patients and found that promoter-promoter (P-P) pairs exhibit positive correlation at multiple levels, and that several Jurkat Epromoter candidate clusters are significantly co-regulated in the patient cohort. To experimentally validate these candidates, we utilized CRISPRi to inhibit Epromoters, which revealed direct transcriptional regulation of multiple target genes within each hub. Finally, we performed cell competition assays to confirm that these Epromoters are vital for T-ALL cell survival. Conclusions: Our analysis provides support for the role of Epromoters in the regulation of 3D P-P interactions and co-regulation of promoter hubs, and how these interactions play a critical part in T-ALL cell survival.

cancer biology↗

bioRxiv2026

An enhancer-centric approach applied to human immune system epigenomes revealed the association of macrophage enhancers with cardiovascular disease

Complex diseases are influenced by both genetic and environmental factors. Immune cells are key mediating interactions with the environment, but the impact of genetic variation on the immune system and how it influences complex diseases is not fully understood. Moreover, most genome-wide analyses (GWAS) variants associated with complex diseases are non-coding and difficult to interpret. Here, we investigated the association of non-coding variants with immune cell enhancers. As part of BLUEPRINT and the International Human Epigenome (IHEC) consortia, we generated and analysed a comprehensive set of epigenomes for human primary immune cells, including 107 epigenomes derived from 749 ChIP-Seq experiments across 24 cell types. We identified multicell enhancer activity patterns across the genome and examined their links with non-coding variants from 518 GWAS traits. This analysis revealed 117 significant associations, including novel links between cardiovascular disease variants and macrophage-specific enhancers that regulate genes involved in lipid metabolism and immunity, such as the gene encoding for the nuclear receptor LXR-alpha (NR1H3) and many of its known target genes. Together, these data will help to better understand the influence of genetic variability in immune function and related diseases.

genomics↗

bioRxiv2026

PREMATURE BIRTH AND CESAREAN SECTION AFFECT NEONATAL CD4+ T CELL GENE EXPRESSION AND CELLULAR FUNCTION

Premature birth and cesarean section are associated with increased morbidity and inflammatory diseases. However, their impact on neonatal immunity remains incompletely defined. To explore how gestational age and mode of delivery contribute to early immune programming, we analyzed CD4+ T cells, central regulators of adaptive responses, from preterm neonates and full-term neonates born by cesarean section or vaginal delivery. We performed transcriptomic profiling (mRNA-seq) and functional assessment of T cell activation, proliferation, and cytokine production following stimulation. The mode of delivery emerged as a key factor for CD4+ T cell transcriptome and function. CD4+ T cells from full-term neonates born by vaginal delivery exhibited an immune activation signature, produced higher levels of multiple cytokines, and showed reduced proliferative capacity. In contrast, prematurity was associated with modest changes in basal gene expression relative to full-term cesarean section neonates. CD4+ T cells from preterm neonates displayed enhanced proliferation and increased secretion of inflammatory cytokines (IL-13, TNF, IL-6, and IL-17F) upon stimulation, consistent with heightened responsiveness. Collectively, our findings show that CD4+ T cells from preterm neonates exhibit augmented inflammatory potential, which becomes more regulated at term. Mode of delivery further contributes to this developmental trajectory: cesarean section is associated with a restrained functional profile, whereas vaginal delivery is associated with a mild immune activation signature and increased responsiveness. These results support a model in which neonatal CD4+ T cell trajectories are established during fetal life and further modulated at birth, highlighting the layered influence of perinatal factors on immune development. Summary sentenceNeonatal CD4+ T cell trajectories are established during fetal life and further shaped at birth by the mode of delivery, influencing early immune responsiveness.

immunology↗

bioRxiv2025

Exonic enhancers are a widespread class of dual-function regulatory elements

Exonic enhancers (EEs) occupy an under-appreciated niche in gene regulation. By integrating transcription factor binding, chromatin accessibility, and high-throughput enhancer-reporter assays, we demonstrate that many protein-coding exons possess enhancer activity across species. These EEs exhibit characteristic epigenomic signatures, form long-range interactions with gene promoters, and can be altered by both nonsynonymous and synonymous variants. CRISPR-mediated inactivation demonstrated the involvement of EEs in the cis-regulation of host and distal gene expression. Through large-scale cancer genome analyses, we reveal that EE mutations correlate with dysregulated target-gene expression and clinical outcomes, highlighting their potential relevance in disease. Evolutionary comparisons show that EEs exhibit both strong sequence constraint and lineage-specific plasticity, suggesting that they serve ancient regulatory functions while also contributing to species divergence. Our findings redefine the landscape of functional elements by establishing EEs as a component of gene regulation, while revealing how coding regions can simultaneously fulfil both protein-coding and cis-regulatory roles.

genomics↗

bioRxiv2025

Harnessing ALDH1A2 vulnerability in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with limited therapeutic options, particularly in the relapsed/refractory (R/R) setting. Unlike other hematological malignancies, which benefit from targeted immunotherapies, T-ALL remains reliant on chemotherapy, leading to poor outcomes in R/R cases. Identifying novel therapeutic vulnerabilities is crucial to improving the outcomes of patients. Herein, we identify aldehyde dehydrogenase 1A2 (ALDH1A2) as a T-ALL-specific enzyme essential for leukemic cell survival. Transcriptomic and epigenetic analyses reveal its selective expression, regulated by the TAL1 oncogene. Pharmacological inhibition of ALDH1A2 using Dimate demonstrates potent anti-leukemic activity across diverse T-ALL subtypes, including primary samples of relapsed disease. These findings establish ALDH1A2 as a therapeutic target in T-ALL and support ALDH inhibition as a promising strategy to overcome drug resistance and improve treatment outcomes in R/R T-ALL.

cancer biology↗

bioRxiv2025

Identification and Functional Characterization of Regulatory Variants in DPP9 Associated with COVID-19 Severity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to a wide-range of clinical outcomes, which have been extensively studied through genome-wide association studies (GWAS). Starting from lead genetic variants associated with COVID-19 infection and severity, we identified a subset of non-coding candidate variants with potential regulatory functions. Using bioinformatics analysis and functional screening in three cell lines, we prioritized two DPP9 variants within a haplotype that increases the risk of severe COVID-19. This haplotype exhibited increased regulatory activity and altered transcription factor binding, suggesting its role in influencing COVID-19 severity through modulation of DPP9 expression in immune and lung cell types. The interest of our study lies in the functional characterization of regulatory variants responsible for the higher levels of DPP9 and lung damage observed in patients with severe COVID-19. These findings advance our understanding of genetic risk factors for COVID-19 and highlight functional SNPs that may guide future therapeutic research.

genetics↗

bioRxiv2024

Epromoters bind key stress-related transcription factors to regulate clusters of stress response genes

Stress insults trigger the rapid and global reprogramming of gene transcription by coordinated recruitment of a limited number of key inducible transcription factors to cis-regulatory elements. Here, we performed a comprehensive analysis of different stress models and observed that co-induced genes are generally located in close genomic proximity. By integrating gene expression and transcription factor binding resources in different stress models, we found an enrichment for clusters whereby only one of the promoters of the cluster recruits the key transcription factors, reminiscent of Epromoters, a type of cis-regulatory elements displaying both promoter and enhancer function. Epromoter-regulated clusters were frequently found irrespectively of the stress or inflammatory response. Predicted Epromoters displayed enhancer activity and regulated clusters of stress-response genes independently of their genomic location. These findings have significant implications for understanding complex gene regulation following the response to acute perturbations. TeaserWhen cells face stress, they undergo rapid changes in gene expression, orchestrated by a handful of key transcription factors. But how do these factors coordinate such a complex response? Our study reveals that "Epromoters"--cis-regulatory elements that combine the functions of both promoters and enhancers help organize stress-response genes into tightly regulated clusters. This discovery not only deepens our understanding of gene regulation in the face of stress but also offers exciting implications for studying inflammation and other acute cellular responses.

genomics↗

bioRxiv2024

Unraveling a novel dual-function regulatory element showing epistatic interaction with a variant that escapes genome-wide association studies.

Regulation of gene expression has recently been complexified by the identification of Epromoters, a subset of promoters with enhancer function. Here, we uncovered the first dual cis-regulatory element, "ESpromoter," exhibiting both enhancer and silencer function, as a regulator of the nearby genes ATP2B4 and LAX1 in single human T cells. Through integrative approach, we pinpointed functional rs11240391, a severe malaria risk variant that escapes detection in genome-wide association studies, challenging conventional strategies for identifying causal variants. CRISPR-modified cells demonstrated the regulatory effect of ESpromoter and rs11240391 on LAX1 expression and T cell activation. Furthermore, our findings revealed an epistatic interaction between ESpromoter SNPs and rs11240391, impacting severe malaria susceptibility by further reducing LAX1 expression. This groundbreaking discovery challenges the conventional enhancer-silencer dichotomy. It highlights the sophistication of transcriptional regulation and argues for an integrated approach combining genetics, epigenetics, and genomics to identify new therapeutic targets for complex diseases. HIGHLIGHTSO_LINovel dual enhancer-silencer element (ESpromoter) in a single human cell type C_LIO_LIFunctional SNP for severe malaria risk that escapes genome-wide association studies C_LIO_LIGenome editing at the SNP demonstrates a regulatory effect on LAX1 and T cell activation C_LIO_LIEpistatic interaction between SNPs increases the risk of severe malaria C_LI In briefEpistatic interaction between common variants within a novel dual enhancer-silencer regulatory element and the LAX1 promoter variant is responsible for severe malaria susceptibility through T-cell activation.

genomics↗

bioRxiv2023

Architecture and evolutionary conservation of Xenopus tropicalis osteoblast-specific regulatory regions shed light on bone diseases and early skeletal evolution

Understanding the genetic mechanisms underpinning the differentiation of osteoblasts, the bone producing cells, has far reaching implications for skeletal diseases and evolution. To this end, it is crucial to characterize osteoblastic regulatory landscape in a diverse array of distantly-related vertebrate species. By comparing of the ATAC-seq profile of Xenopus tropicalis (Xt) osteoblasts to liver, heart and lung control tissues, we identified 524 promoters and 6,750 distal regions whose chromatin is specifically open in osteoblasts. Nucleotide composition, Gene Ontology, and RNA-Seq confirmed that the identified elements correspond to bona fide osteogenic transcriptional enhancers, and TFBS enrichment revealed a well-conserved regulatory logic with mammals. Amongst the 357 Xt osteoblast-specific enhancers aligning to homologous human loci, 127 map to regions annotated as enhancers. Phenotype predictions based on the genes neighbouring these conserved enhancers are tightly related to impaired skeletal development. In addition, six conserved enhancers are located at loci associated to craniosynostosis (mx2, tcf12), osteopoikilosis (lemd3), osteopenia (gorab), skeletal dysplasia (flnb) and craniofacial abnormalities (gpc4). From an evolutionary perspective, the elephant shark genome aligns to 53 Xt osteoblast-specific enhancers that are also conserved and annotated as enhancers in humans, revealing an ancestral osteogenic role for the ATOH8, IRX3, NFAT, NFIB and MEF2C transcription factors, as well as for the FGF, IHH and BMP/TGFb signalling pathways. As the absence of bone in sharks is a derived feature, we propose that, in this lineage, the osteogenic regulatory network has been maintained for its function in odontoblasts. Our data argues in favour of a common origin for dentine and bone, and provides a glimpse into the key regulatory elements and upstream activators that drove the formation of an ancient type of mineralized tissue in the vertebrates that inhabited the oceans more than 460 million years ago. Author SummaryDuring animal embryogenesis, distinct type of tissues are formed and assembled, resulting in an integrated, functional organism. During this process, cells must make important decisions, which largely rely on an accurate use of their genetic material. Here, we have studied how the genome "knows" that it must participate to the formation of the bone tissue in a frog animal model. We therefore identified important genomic regions that are involved in driving the expression of genes involved in the formation of a mineralized skeleton. On the one hand, we show that some of these regions are also present in humans, and, therefore, skeletal pathologies could be studied in the frog model at a genetic level. On the other hand, we also identify regions that are present in the genome of a shark, which allows us to propose an evolutionary framework for the early evolutionary origin of the vertebrate skeleton.

genomics↗

bioRxiv2023

A workflow combining single-cell CRISPRi screening and a supervised autoencoder neural network to detect subtle transcriptomic perturbations induced by lncRNA Knock-Down

Single-cell CRISPR-based transcriptome screens are potent genetic tools for concomitantly assessing the expression profiles of cells targeted by a set of guides RNA (gRNA), and inferring target gene functions from the observed perturbations. However, due to various limitations, this approach lacks sensitivity in detecting weak perturbations and is essentially reliable when studying master regulators such as transcription factors. To overcome the challenge of detecting subtle gRNA induced transcriptomic perturbations and classifying the most responsive cells, we developed a new supervised autoencoder neural network method. Our Sparse supervised autoencoder (SSAE) neural network provides selection of both relevant features (genes) and actual perturbed cells. We applied this method on an in-house single-cell CRISPR-interference-based (CRISPRi) transcriptome screening (CROP-Seq) focusing on a subset of long non-coding RNAs (lncRNAs) regulated by hypoxia, a condition that promote tumor aggressiveness and drug resistance, in the context of lung adenocarcinoma (LUAD). The CROP-seq library of validated gRNA against a subset of lncRNAs and, as positive controls, HIF1A and HIF2A, the 2 main transcription factors of the hypoxic response, was transduced in A549 LUAD cells cultured in normoxia or exposed to hypoxic conditions during 3, 6 or 24 hours. We first validated the SSAE approach on HIF1A and HIF2 by confirming the specific effect of their knock-down during the temporal switch of the hypoxic response. Next, the SSAE method was able to detect stable short hypoxia-dependent transcriptomic signatures induced by the knock-down of some lncRNAs candidates, outperforming previously published machine learning approaches. This proof of concept demonstrates the relevance of the SSAE approach for deciphering weak perturbations in single-cell transcriptomic data readout as part of CRISPR-based screening.

bioinformatics↗