Biology subjects

Barozzi, I.

Publications and source records attributed to Barozzi, I..

At least 19 recordsLinked to original sources

bioRxiv2026

Systemic antibody repertoires target specific microbial antigens in patients with colorectal cancer

Colorectal cancer (CRC) represents the third leading cause of cancer related deaths worldwide and is influenced by multiple risk factors. Both exposure to microbiota as well as adaptive immune responses have been implicated in CRC development and suggested as candidate biomarkers. However, the extent to which CRC and adenomas associate with systemic immune signatures against microbes remains unclear. Here, we apply Phage-display ImmunoPrecipitation Sequencing (PhIP-Seq) on 321 individuals of an Austrian CORSA cohort (Colorectal Cancer Study of Austria) including controls, adenomas, and CRC patients. Although antibody repertoires showed global similarity across the different disease status and limited predictive performance, prevalence-based analyses revealed specific signals, such as Helicobacter pylori CagY as candidate marker of CRC and viral antigens as candidate for non-cancer stages. Taken together, this large-scale analysis of antibody reactivity in CRC highlights the potential of serological markers to reflect disease-associated immune signatures.

immunology↗

bioRxiv2026

Multi-scale electrophysiological characterization of human cardioids reveals progressive maturation

Background: Human induced pluripotent stem cell-derived cardiomyocytes are valuable for cardiac disease modelling and drug testing but remain limited by immature structural and electrophysiological properties in conventional two-dimensional cultures. Three-dimensional cardiac models, including cardioids, provide a more physiological environment through multicellular organization and enable tissue-level functional analysis. Their electrophysiological properties and maturation, however, remain incompletely characterized. Methods: We performed an integrated electrophysiological characterization of human cardioids over the first three weeks of maturation using sharp microelectrode recordings, calcium transient measurements, whole-cell patch clamp recordings, immunofluorescence, transcriptomic analysis, and pharmacological interventions. Results: During maturation, cardioids exhibited progressive electrophysiological maturation, characterized by more physiological resting membrane potentials (-54.0{+/-}3.1mV to -68.1{+/-}1.2mV), increased action potential amplitude (73.5{+/-}4.8mV to 101.7{+/-}2.1mV) and upstroke velocity (44.3{+/-}5mV/ms to 86.6{+/-}8.4mV/ms), and shorter action potential duration (178.9{+/-}8.7ms to 144.0{+/-}4.8ms at 1Hz stimulation). These changes were associated with progressively increased INa density and enhanced SCN5A mRNA and Nav1.5 protein expression, consistent with progressive maturation of cardiac excitability. IKr current and KCNH2/hERG expression were already present at early maturation stages and remained stable, as evidenced by preserved sensitivity to dofetilide-blockade. Pharmacological inhibition of barium- and HMR-sensitive currents altered resting membrane and/or action potential characteristics in intact cardioids, whereas in isolated cardiomyocytes classical IK1 remained undetectable and IKs was small, highlighting a certain degree of persisting immaturity of the model. Conclusions: These findings demonstrate that human cardioids undergo progressive electrophysiological maturation and provide complementary tissue- and cellular-level insights into cardiac function, establishing their suitability for investigating INa and IKr-related cardiac pathologies.

physiology↗

bioRxiv2026

Tumor Heterogeneity Induces Pro- and Anti-metastatic Myeloid Cell Phenotypes in Breast Cancer Lung Metastasis

Metastasis is a major cause of cancer-related mortality, yet targeting metastatic cells directly has been largely unsuccessful due to their plasticity and heterogeneity. Myeloid cells play diverse pro- and anti-metastatic functions and are an attractive alternative target for treating metastasis, but how tumor heterogeneity influences myeloid cell phenotypes during metastasis remains poorly understood. Here, we profiled myeloid cells from primary tumors and matched metastatic lungs of 12 heterogeneous and differentially metastatic patient-derived xenograft models of breast cancer. Comparative analysis of cell type abundances revealed distinct myeloid remodeling specific to primary tumors or metastatic lungs. Beyond compositional differences, we identified gene expression programs that were associated with metastatic burden, such as number or size of metastatic nodules, indicating distinct microenvironmental requirements for metastatic seeding and outgrowth. Examining these metastasis-associated programs using time-course datasets, we discovered an evolution from anti- to pro-metastatic monocyte phenotypes during metastatic progression. We further showed that this phenotypic shift was driven by an increase in two distinct myeloid-derived suppressor cell signatures, and a transcriptionally regulated impairment of monocyte differentiation leading to the depletion of non-classical monocytes. Our results comprehensively dissect the heterogeneity of myeloid cell phenotypes across primary tumor and metastatic sites, opening novel avenues for myeloid-targeting therapies specific to metastasis.

cancer biology↗

bioRxiv2025

Functional architecture of cardiac TF regulatory landscapes in control of mammalian heart development

Congenital heart disease (CHD), the most common human birth defect, often results from disruptions in gene regulatory networks (GRNs) that control cardiac lineage specification and cell type identity during heart development. A conserved core set of cardiac transcription factors (TFs) orchestrates these processes through combinatorial interactions that are cell type-specific and tightly regulated across space and time. However, the genomic enhancer architecture that integrates upstream effectors to establish precise cardiac TF dosage and downstream transcriptional output remains largely unresolved. Here, we assessed the functional necessity of five developmental heart enhancer modules previously linked to the regulation of Gata4 and Hand2, core cardiac TFs exhibiting overlapping roles in myocardial and endocardial development. While individual enhancer deletions in mouse embryonic hearts revealed a surprising degree of transcriptional resilience, a subset of Gata4 enhancers proved indispensable for embryonic progression in a genetically compromised background. To achieve higher precision in cardiac cell type-specific enhancer prediction, we applied single-nucleus multiome profiling, enabling the delineation of cardiac cistromes underlying heart morphogenesis. By integrating this resource with deep learning applications, site-directed transgenesis, and chromatin conformation modeling, we mapped the cardiac enhancer repertoire and regulatory signatures that orchestrate Hand2 dynamics across distinct cardiac compartments and lineages. Genome editing further revealed an essential role for the Hand2-upstream regulatory interval (H2-URI) in transcriptional control of endocardial lineage effectors and, consequently, trabecular network formation and cardiac cushion patterning. Together our findings highlight substantial resilience in the cis-regulatory architectures governing cardiac TF dynamics and demonstrate that combinatorial integration of upstream lineage identities across modular enhancer landscapes establishes the cardiac cell type-specific programs driving heart morphogenesis. These results advance the reconstruction of cardiac GRNs and enhance the functional interpretation of CHD-associated variants.

developmental biology↗

bioRxiv2023

Cell Type- and Tissue-specific Enhancers in Craniofacial Development

The genetic basis of craniofacial birth defects and general variation in human facial shape remains poorly understood. Distant-acting transcriptional enhancers are a major category of non-coding genome function and have been shown to control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development1-3. However, a lack of accurate maps of the genomic location and cell type-specific in vivo activities of all craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combined histone modification and chromatin accessibility profiling from different stages of human craniofacial development with single-cell analyses of the developing mouse face to create a comprehensive catalogue of the regulatory landscape of facial development at tissue- and single cell-resolution. In total, we identified approximately 14,000 enhancers across seven developmental stages from weeks 4 through 8 of human embryonic face development. We used transgenic mouse reporter assays to determine the in vivo activity patterns of human face enhancers predicted from these data. Across 16 in vivo validated human enhancers, we observed a rich diversity of craniofacial subregions in which these enhancers are active in vivo. To annotate the cell type specificities of human-mouse conserved enhancers, we performed single-cell RNA-seq and single-nucleus ATAC-seq of mouse craniofacial tissues from embryonic days e11.5 to e15.5. By integrating these data across species, we find that the majority (56%) of human craniofacial enhancers are functionally conserved in mice, providing cell type- and embryonic stage-resolved predictions of their in vivo activity profiles. Using retrospective analysis of known craniofacial enhancers in combination with single cell-resolved transgenic reporter assays, we demonstrate the utility of these data for predicting the in vivo cell type specificity of enhancers. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=138 HEIGHT=200 SRC="FIGDIR/small/546603v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@7595a9org.highwire.dtl.DTLVardef@1b48b81org.highwire.dtl.DTLVardef@32fe86org.highwire.dtl.DTLVardef@1e41fed_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2022

Coexisting morpho-biotypes unveil the regulatory bases of phenotypic plasticity in pancreatic ductal adenocarcinoma

Intratumor morphological heterogeneity predicts clinical outcomes of pancreatic ductal adenocarcinoma (PDAC). However, it is only partially understood at the molecular level and devoid of clinical actionability. In this study we set out to determine the gene regulatory networks and expression programs underpinning intra-tumor morphological variation in PDAC. To this aim, we identified and deconvoluted at single cell level the molecular profiles characteristic of morphologically distinguishable clusters of PDAC cells that coexisted in individual tumors. We identified three major morpho-biotypes that co-occurred in various proportions in most PDACs: a glandular biotype with classical epithelial ductal features; a biotype with abortive ductal structures and expressing a partial epithelial-to-mesenchymal transition program; and a poorly differentiated biotype showing partial neuronal lineage priming and absence of both ductal features and basement membrane. The identification of PDAC morpho-biotypes may help improve patient stratification and therapeutic schemes taking into account the spectrum of actionable targets expressed by coexisting tumor components.

cancer biology↗

bioRxiv2022

Widespread Increase in Enhancer-Promoter Interactions during Developmental Enhancer Activation in Mammals

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here, we investigate the 3D conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. 61% of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general significance of enhancer- promoter physical proximity for developmental gene activation in mammals.

genomics↗

bioRxiv2022

Logical design of synthetic cis-regulatory DNA for genetic tracing of cell identities and state changes

Descriptive data are rapidly expanding in biomedical research. Instead, functional validation methods with sufficient complexity remain underdeveloped. Transcriptional reporters allow experimental characterization and manipulation of developmental and disease cell states, but their design lacks flexibility. Here, we report logical design of synthetic cis-regulatory DNA(LSD), a computational framework leveraging phenotypic biomarkers and trans-regulatory networks as input to design reporters marking the activity of selected cellular states and pathways. LSD uses bulk or single-cell biomarkers and a reference genome or custom cis-regulatory DNA datasets with user-defined boundary regions. By benchmarking validated reporters, we integrated LSD with a computational classifier to rank phenotypic specificity of putative cis-regulatory DNA. Experimentally, LSD-designed reporters targeting a wide range of cell states are functional without minimal promoters. In silico, an LSD-unsupervised mesenchymal glioblastoma reporter outperformed previously validated ones. In genome-scale CRISPRa screens, it discovered known and novel bona fide cell-state-drivers. Thus, LSD captures core principles of cis-regulation and is broadly applicable to studying complex cell states and mechanisms of transcriptional regulation.

bioengineering↗

bioRxiv2022

A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2

During development cell fates are specified by tightly controlled gene expression programs. PBX TALE transcription factors control gene regulatory networks (GRN) that direct vertebrate tissue patterning and organ morphogenesis. How PBX1/2 proteins acquire context-specific functions, despite widespread embryonic expression of Pbx1/2, remains elusive. In mouse limb buds, mesenchymal-specific loss of PBX1/2 or of the transcriptional regulator HAND2 results in similar phenotypes, suggesting that PBX1/2- and HAND2-dependent programs converge to control limb development. To investigate this scenario, we combined tissue-specific and temporally-controlled mutagenesis with multi-omics approaches using the murine hindlimb bud as a model. We reconstructed a GRN collaboratively directed by PBX1/2 and HAND2, demonstrating that Pbx1-Hand2 genetically interact in vivo during hindlimb patterning, with PBX1 concomitantly acting as an upstream regulator of Hand2. At organismal-level resolution the GRN is active within restricted subsets of posterior-proximal hindlimb mesenchymal cells, wherein Pbx1/2 and Hand2 are co-expressed with their target genes. Genome-wide profiling of PBX1 binding across multiple tissues further revealed that HAND2 selects a subset of PBX-bound regions to impart limb patterning functionality. This research elucidates mechanisms underlying limb bud-specific functions by PBX1/2, while informing general principles by which promiscuous transcription factors cooperate with select cofactors to instruct distinct developmental programs.

developmental biology↗

bioRxiv2022

Genetic and epigenetic driven variation in regulatory regions activitycontribute to adaptation and evolution under endocrine treatment

Comprehensive profiling of hormone-dependent breast cancer (HDBC) has identified hundreds of protein-coding alterations contributing to cancer initiation1, 2, but only a handful have been linked to endocrine therapy resistance, potentially contributing to 40% of relapses1, 3-9. If other mechanisms underlie the evolution of HDBC under adjuvant therapy is currently unknown. In this work, we employ integrative functional genomics to dissect the contribution of cis-regulatory elements (CREs) to cancer evolution by focusing on 12 megabases of non-coding DNA, including clonal enhancers10, gene promoters, and boundaries of topologically associating domains11. Massive parallel perturbation in vitro reveals context-dependent roles for many of these CREs, with a specific impact on dormancy entrance12, 13 and endocrine therapy resistance9. Profiling of CRE somatic alterations in a unique, longitudinal cohort of patients treated with endocrine therapies identifies non-coding changes involved in therapy resistance. Overall, our data uncover actionable transient transcriptional programs critical for dormant persister cells and unveil new regulatory nodes driving evolutionary trajectories towards disease progression.

cancer biology↗

bioRxiv2021

SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning

SMAD4 regulates gene expression in response to BMP and TGF{beta} signal transduction and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we use an epitope-tagged Smad4 allele for ChIP-seq analysis together with transcriptome analysis of wild-type and mouse forelimb buds lacking Smad4 in the mesenchyme. This analysis identifies the SMAD4 target genes during establishment of the feedback signaling system and establishes that SMAD4 predominantly mediates BMP signal-transduction during early limb bud development. Unexpectedly, the initial analysis reveals that the expression of cholesterol biosynthesis enzymes is precociously down-regulated and intracellular cholesterol levels reduced in Smad4-deficient limb bud mesenchymal progenitors. The SMAD4 target GRNs includes genes, whose expression in the anterior limb bud is up-regulated by interactions of SMAD4 complexes with enhancers active in the anterior mesenchyme. This reveals a predominant function of SMAD4 in up-regulating target gene expression in the anterior limb bud mesenchyme. Analysis of differentially expressed genes that are shared between Smad4- and Shh-deficient limb buds corroborates the positive role of SMAD4 in transcriptional regulation of anterior genes and reveals a repressive effect on posterior genes that are positively regulated by SHH signaling. This analysis uncovers the overall opposing effects of SMAD4-mediated BMP and SHH signalling on transcriptional regulation during early limb bud development. In summary, this analysis indicates that during early digit patterning and limb bud outgrowth, the anterior/proximal and proximo/distal expression dynamics of co-regulated genes are controlled by distinct and contrasting trans-regulatory inputs from SHH and SMAD4-mediated BMP signal transduction.

developmental biology↗

bioRxiv2021

The co-evolution of the genome and epigenome in colorectal cancer

Colorectal malignancies are a leading cause of cancer death. Despite large-scale genomic efforts, DNA mutations do not fully explain malignant evolution. Here we study the co-evolution of the genome and epigenome of colorectal tumours at single-clone resolution using spatial multi-omic profiling of individual glands. We collected 1,373 samples from 30 primary cancers and 9 concomitant adenomas and generated 1,212 chromatin accessibility profiles, 527 whole-genomes and 297 whole-transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent chromatin changes in regulatory regions of cancer drivers with otherwise no mutation. Genome-wide alterations in transcription factor binding accessibility involved CTCF, downregulation of interferon, and increased accessibility for SOX and HOX, indicating developmental genes reactivation. Epigenetic aberrations were heritable, distinguishing adenomas from cancers. Mutational signature analysis showed the epigenome influencing DNA mutation accumulation. This study provides a map of (epi)genetic tumour heterogeneity, with fundamental implications for understanding colorectal cancer biology.

cancer biology↗

bioRxiv2021

Topologically Associating Domain Boundaries are Commonly Required for Normal Genome Function

Topologically associating domain (TAD) boundaries are thought to partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotype1-3, but the overall extent to which this occurs remains unknown. Here we show that TAD boundary deletions commonly disrupt normal genome function in vivo. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11-80kb in size) from the genome in mice. All deletions examined resulted in at least one detectable molecular or organismal phenotype, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. For 5 of 8 (62%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and suggest that noncoding deletions affecting TAD boundaries should be carefully considered for potential pathogenicity in clinical genetics screening.

genomics↗

bioRxiv2020

A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival

Gene deserts are defined as genomic regions devoid of protein coding genes and spanning more than 500 kilobases, collectively encompassing about 25% of the human genome. Approximately 30% of all gene deserts are enriched for conserved elements with cis-regulatory signatures. These are located predominantly near developmental transcription factors (TFs) but despite predicted critical functions, the transcriptional contributions and biological necessity of most gene deserts remain elusive. Here, we explore the cis-regulatory impact of a gene desert flanking the Shox2 gene, a TF indispensable for proximal limb, craniofacial and cardiac pacemaker development. Using a functional genomics approach in mouse embryos we identify the gene desert as a hub for numerous Shox2-overlapping enhancers arranged in a globular chromatin domain with tissue-specific features. In accordance, using endogenous CRISPR deletion, we demonstrate that the gene desert interval is essential for Shox2 transcriptional control in developing limbs, craniofacial compartments, and the heart. Phenotypically, gene desert ablation leads to pacemaker-related embryonic lethality due to Shox2 depletion in the cardiac sinus venosus. We show that this role is partially mediated through a distal gene desert enhancer, providing evidence for intra-gene desert regulatory robustness. Finally, we uncover a multi-layered functional role of the gene desert by revealing an additional requirement for stylopod morphogenesis, mediated through an array of proximal limb enhancers (PLEs). In summary, our study establishes the Shox2 gene desert as a fundamental genomic unit that controls pleiotropic gene expression through modular arrangement and coordinated dynamics of tissue-specific enhancers.

developmental biology↗

bioRxiv2020

Transcriptional Network Orchestrating Regional Patterning of Cortical Progenitors

We uncovered a transcription factor (TF) network that regulates cortical regional patterning. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1 and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer-gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning.

neuroscience↗

bioRxiv2019

Genome-Wide Fetalization of Enhancer Architecture in Heart Disease

Heart disease is associated with re-expression of key transcription factors normally active only during prenatal development of the heart. However, the impact of this reactivation on the genome-wide regulatory landscape in heart disease has remained obscure. Here we show that pervasive epigenomic changes occur in heart disease, with thousands of regulatory sequences reacquiring fetal-like chromatin signatures. We used RNA-seq and ChIP-seq targeting a histone modification associated with active transcriptional enhancers to generate genome-wide enhancer maps from left ventricle tissue from 18 healthy controls and 18 individuals with idiopathic dilated cardiomyopathy (DCM). Healthy individuals had a highly reproducible epigenomic landscape, consisting of more than 31,000 predicted heart enhancers. In contrast, we observed reproducible disease-associated gains or losses of activity at more than 7,500 predicted heart enhancers. Next, we profiled human fetal heart tissue by ChIP-seq and RNA-seq. Comparison with adult tissues revealed that the heart disease epigenome and transcrip-tome both shift toward a fetal-like state, with 3,400 individual enhancers sharing fetal regulatory properties. Our results demonstrate widespread epigenomic changes in DCM, and we provide a comprehensive data resource (http://heart.lbl.gov) for the mechanistic exploration of heart disease etiology.

genomics↗

bioRxiv2019

Dissection of acute stimulus-inducible nucleosome remodeling in mammalian cells

Accessibility of the genomic regulatory information is largely controlled by the nucleosome-organizing activity of transcription factors (TFs). Whereas stimulus-induced TFs bind to genomic regions that are maintained accessible by lineage-determining TFs, they also increase accessibility of thousands of cis-regulatory elements. Nucleosome remodeling events underlying such changes and their interplay with basal positioning are unknown. Here, we devised a novel quantitative framework discriminating different types of nucleosome remodeling events in micrococcal nuclease ChIP-seq datasets and used it to analyze nucleosome dynamics at stimulus-regulated cis-regulatory elements. At enhancers, remodeling preferentially affected poorly positioned nucleosomes while sparing well-positioned nucleosomes flanking the enhancer core, indicating that inducible TFs do not suffice to overrule basal nucleosomal organization maintained by lineage-determining TFs. Remodeling events appeared to be combinatorially driven by multiple TFs, with distinct TFs showing however different remodeling efficiencies. Overall, these data provide a systematic view of the impact of stimulation on nucleosome organization and genome accessibility in mammalian cells.

genomics↗

bioRxiv2019

Exploiting evolutionary herding to control drug resistance in cancer

Drug resistance mediated by clonal evolution is arguably the biggest problem in cancer therapy today. However, evolving resistance to one drug may come at a cost of decreased growth rate or increased sensitivity to another drug due to evolutionary trade-offs. This weakness can be exploited in the clinic using an approach called evolutionary herding that aims at controlling the tumour cell population to delay or prevent resistance. However, recapitulating cancer evolutionary dynamics experimentally remains challenging. Here we present a novel approach for evolutionary herding based on a combination of single-cell barcoding, very large populations of 108-109 cells grown without re-plating, longitudinal non-destructive monitoring of cancer clones, and mathematical modelling of tumour evolution. We demonstrate evolutionary herding in non-small cell lung cancer, showing that herding allows shifting the clonal composition of a tumour in our favour, leading to collateral drug sensitivity and proliferative fitness costs. Through genomic analysis and single-cell sequencing, we were also able to determine the mechanisms that drive such evolved sensitivity. Our approach allows modelling evolutionary trade-offs experimentally to test patient-specific evolutionary herding strategies that can potentially be translated into the clinic to control treatment resistance.

cancer biology↗