Biology subjects

Cantu, C.

Publications and source records attributed to Cantu, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Enhancer pioneering activity of Wnt/β-catenin signaling

Wnt/{beta}-catenin signaling is widely understood as a molecular switch that activates preprogrammed regulatory states. Whether it also actively reshapes chromatin to establish those states has remained unclear. To address this, we performed time-resolved ATAC-seq and CUT&RUN analyses in HEK293T cells and monitored {beta}-catenin-dependent changes in chromatin accessibility and complex assembly following pathway activation. In addition to conventional Wnt-responsive elements (WREs), which are constitutively accessible and pre-bound by TCF/LEF transcription factors (TFs), we identified a second class of WREs, that are initially inaccessible but become activated upon stimulus. At these latent enhancers, {beta}-catenin functions as a pioneer-like factor together with HDAC1 and CBP, mediating chromatin remodeling and TCF/LEF occupancy. Together, these results extend the current view of Wnt/{beta}-catenin signaling by showing that it can establish regulatory elements de novo. Here, {beta}-catenin emerges as the central regulator of transcriptional control and remodeling, providing a framework for the dynamic and context-dependent nature of Wnt-driven gene regulation in development and disease.

genomics↗

bioRxiv2026

GATA transcription factors act as gatekeepers between the Wnt/β-catenin-mediated proliferation and differentiation in the intestinal epithelium

Wnt/{beta}-catenin signalling drives gene expression in a plethora of processes during development and tissue homeostasis. Yet, little is known about how {beta}-catenin, the transducer of the pathway, gives rise to context-specific transcriptional outcomes. For example, in the intestinal epithelium, stem cells and secretory cells lie adjacent to each other, are both exposed to and dependent on Wnt signals, but display divergent identities. We find that, in this tissue, GATA transcription factors functionally interact with Wnt/{beta}-catenin signaling to drive differential cell identity. This occurs because GATA selectively repress Wnt target secretory-lineage genes in stem cells. Mechanistically, we found that GATA factors physically engage with the {beta}-catenin transcriptional complex, and that this association is mediated by the bridge protein BCL9/9L. Our work implicates GATA factors as context dependent modulators of the Wnt/{beta}-catenin transcription, shows that the GATA/BCL9 module acts as a switch at the crossroads of cell identity decision, and uncovers how this epithelium maintains the fine balance between self-renewal and differentiation.

developmental biology↗

bioRxiv2025

CUT&ID for simultaneous profiling of protein-DNA and protein-protein interactions

We developed CUT&ID to simultaneously profile the native genomics and proteomics associations of any endogenous target, in a single streamlined workflow with no need of transgenesis. CUT&ID is enabled by a fusion protein that drives sequential target recognition, proximal protein biotinylation and DNA cleavage in living cells. CUT&ID is broadly applicable and yields high signal-to-noise genomics and proteomics identification across all types of gene regulators tested.

molecular biology↗

bioRxiv2025

Differential Wnt/β-Catenin Signaling via TCF7L2/LEF1 Binding Specificity Shapes Cellular and Tumor Phenotypes

The mechanisms by which Wnt/{beta}-catenin signaling regulates gene expression in a tissue- and context-specific manner remain poorly understood, limiting our ability to target the aberrant cell growth typical of many Wnt-driven cancers. Here we focus on malignant liver tumors driven by activating CTNNB1 ({beta}-catenin) mutations that nevertheless display distinct phenotypic states and Wnt outputs. By profiling patient-derived organoids via single-cell transcriptomics and chromatin dynamics, we identify subtype-specific transcriptional and epigenetic profiles. Using CUT&RUN, we show that {beta}-catenin engages distinct genomic regions, dictated by differential association with TCF/LEF family transcription factors. Specifically, we define a novel sequence-specific regulatory element engaged by {beta}-catenin only upon interaction with TCF7L2, revealing that partner choice, independent of CTNNB1 mutational status, ultimately determines cell fate. Our findings, validated across multiple tumor models and patient tissues, offer a framework for understanding how differential {beta}-catenin-TCF/LEF interaction orchestrates context-specific Wnt signaling outcomes. SignificanceWnt/{beta}-catenin signaling is crucial for development and cancer, yet how it drives different gene programs across tissues is unclear. Using patient-derived liver tumor organoids, we show that {beta}-catenins transcriptional output depends on its binding partner: LEF1 or TCF7L2. These factors guide {beta}-catenin to distinct genomic regions, activating either stemness or differentiation genes. We identify a novel helper motif that directs {beta}-catenin-TCF7L2 binding and target selection. By linking partner choice and motif specificity to context-dependent gene regulation, our work provides a unifying mechanism explaining how Wnt/{beta}-catenin signaling produces diverse cellular outcomes.

cancer biology↗

bioRxiv2025

Mechanistic dissection of GRHL2 and PR transcriptional co-regulation in breast cells

Gene expression is controlled by complex transcriptional networks in which transcription factors and their cognate enhancer elements integrate developmental and environmental cues. The progesterone receptor (PR), a hormone-activated transcription factor, is essential for breast development and physiology, yet how it engages with the chromatin and lineage-specific cofactors remains unclear. Using an unbiased approach, we identify the epithelial transcription factor grainyhead-like 2 (GRHL2) as a key co-regulator of PR activity in hormone responsive breast cancer cells. We show that GRHL2 interacts with PR in a progesterone-independent manner. Upon progesterone stimulation, GRHL2 and PR are both recruited to distal enhancer elements of target genes. Furthermore, GRHL2- and PR-bound elements connect spatially through chromatin looping to regulate shared targets. These findings uncover a previously unrecognized mechanism by which GRHL2 and PR coordinate gene regulation through both chromatin binding and 3D genome architecture modification, positioning GRHL2 as a crucial modulator of steroid hormone receptor function.

genomics↗

bioRxiv2024

The Transcription Factor Binding Landscape of Mouse Development

Gene regulators physically associate to the genome, in a combinatorial fashion, to drive tissue-specific gene expression programs. Uncovering the genome-wide activity of all gene regulators across tissues is therefore needed to understand how the genome is regulated during development. Here, we take a first step forward towards achieving this goal. Using CUT&RUN, we systematically measured the genome-wide binding profiles of key transcription factors and cofactors that mediate the activity of ontogenetically relevant signaling pathways in select mouse tissues at two developmental stages. Computation of the numerous genome-wide binding datasets unveiled a large degree of tissue and time-specific activity for each gene regulator, and several factor-specific idiosyncrasies. Moreover, we identified "popular" regulatory regions that are bound by a multitude of pathway regulators. Popular regions tend to be more evolutionarily conserved, implying their essentiality. Consistently, they lie in the proximity of genes whose dysregulation causes early embryonic lethality in the mouse. Moreover, the human homologs of these regions are also bound by many gene regulators and are highly conserved in human populations, indicating that they retain functional relevance for human development. This work constitutes a decisive step towards understanding how the genome is simultaneously read and used by gene regulators in a holistic fashion and unveils multiple genomic mechanisms that drive embryonic development.

genomics↗

bioRxiv2024

Wnt signaling alters CTCF binding patterns and global chromatin structure

Wnt signaling plays a pivotal role during development, stem cell maintenance, and tissue homeostasis. Upon Wnt pathway activation, {beta}-catenin translocates to the nucleus where it binds the TCF/LEF transcription factors to drive the context-specific expression of Wnt target genes. Coordinating gene expression programs in vertebrates requires a complex interplay between the regulatory and the 3D organization of the genome. However, the impact of Wnt signaling on genome structure has been poorly explored. Here we investigated how Wnt signaling activation influences the binding patterns of CTCF, one of the core architectural proteins that helps establish the 3D genome organization be demarcating topologically associated domains (TAD). This study uncovered a series of CTCF rearrangements under Wnt, that we termed RUW. Notably, RUW sites that were gained upon Wnt activation were typically dependent on {beta}-catenin and were characterized by both CTCF and TCF/LEF binding. Accordingly, many CTCF RUWs aligned with {beta}-catenin binding patterns, and {beta}-catenin and CTCF co-localized in vivo in discreet nuclear puncta only upon pathway activation. Genome-wide investigation of CTCF-mediated 3D genomic interactions upon Wnt pathway stimulation supported the role of the identified RUWs in mediating Wnt-dependent chromatin loops. Lastly, targeted disruption of selected CTCF binding sites demonstrated their functional contribution to Wnt target gene regulation, implicating regulation of the 3D genomic structure in the execution of transcriptional programs orchestrated by developmental pathways.

developmental biology↗

bioRxiv2023

SOX2 and NR2F1 coordinate the gene expression program of the early postnatal visual thalamus

The thalamic dorsolateral geniculate nucleus, (dLGN) receives visual input from the retina via the optic nerve, and projects to the cortical visual area, where eye-derived signals are elaborated. The transcription factors SOX2 and NR2F1 are directly involved in the differentiation of dLGN neurons, based on mouse work and patient mutations leading to vision defects. However, whether they regulate each other, or control common targets is still unclear. By RNA-seq analysis of neonatal dLGN from thalamo-specific Sox2 and Nr2f1 mouse mutants, we found a striking overlap of deregulated genes. Among them, VGF, a cytokine transported along thalamic-cortical axons is strongly downregulated in both mutants. CUT&RUN analysis of SOX2 binding in dLGN chromatin identified a binding pattern characteristic of the dLGN. Collectively, the SOX2 and NR2F1- coregulated genes, and cognate SOX2 binding sites, contribute as a basis to understand the gene regulatory network driving the differentiation and connectivity of thalamic neurons.

developmental biology↗

bioRxiv2023

Exhaustive identification of genome-wide binding events of transcriptional regulators with ICEBERG

Genome-wide protein interaction assays aspire to map the complete binding pattern of gene regulators. How-ever, common practice relies on replication and high stringency statistics which favor false negatives over false positives, thereby excluding portions of signal which may represent biologically relevant events. Here, we present ICEBERG (Increased Capture of Enrichment By Exhaustive Replicate aGgregation), an experimental and analytical pipeline that harnesses large numbers of CUT&RUN replicates to discover the full set of binding events and chart the line between false positives and false negatives. We employed ICEBERG to map the full set of H3K4me3-marked regulatory regions and {beta}-catenin targets in human colorectal cancer cells. The ICE-BERG datasets allow benchmarking of individual replicates, comparison of the performance of peak calling and replication approaches and expose the arbitrary nature of other strategies to identify reproducible peaks. Instead of a static view of genomic targets, ICEBERG established a spectrum of detection probabilities across the genome for a given factor, underlying the intrinsic dynamicity of its mechanism of action, and permitting to distinguish frequent from rare regulation events. Finally, ICEBERG discovered instances, undetectable with other approaches, that might underlie novel mechanisms of colorectal cancer progression.

genomics↗

bioRxiv2023

Single-cell response to Wnt activation in human embryonic stem cells reveals uncoupling of Wnt target gene expression

Wnt signaling drives nuclear translocation of {beta}-catenin and its subsequent association with the DNA-bound TCF/LEF transcription factors, which dictate target gene specificity by recognizing Wnt responsive elements across the genome. {beta}-catenin target genes are therefore thought to be collectively activated upon Wnt pathway stimulation. However, this appears in contrast with the non-overlapping patterns of Wnt target gene expression in several contexts, including early mammalian embryogenesis. Here we followed Wnt target gene expression in human embryonic stem cells after Wnt pathway stimulation at a single-cell resolution. Cells changed gene expression program over time consistent with three key developmental events: i) loss of pluripotency, ii) induction of Wnt target genes, and iii) mesoderm specification. Contrary to our expectation, not all cells displayed equal amplitude of Wnt target gene activation; rather, they distributed in a continuum from strong to weak responders when ranked based on the expression of the target AXIN2. Moreover, high AXIN2 did not always correspond to elevated expression of other Wnt targets, which were activated in different proportions in individual cells. This uncoupling of Wnt target gene expression, which was also identified in single colorectal cancer cells with hyper-activated Wnt signaling, underlines the necessity to identify additional mechanisms that explain the heterogeneity of the Wnt/{beta}-catenin-mediated transcriptional outputs in single cells.

developmental biology↗

bioRxiv2022

The CUT&RUN Blacklist of Problematic Regions of the Genome

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an increasingly popular technique to map genome-wide binding profiles of histone modifications, transcription factors and co-factors. The ENCODE project and others have compiled blacklists for ChIP-seq which have been widely adopted: these lists contain regions of high and unstructured signal, regardless of cell type or protein target. While CUT&RUN obtains similar results to ChIP-seq, its biochemistry and subsequent data analyses are different. We found that this results in a CUT&RUN-specific set of undesired high-signal regions. For this reason, we have compiled blacklists based on CUT&RUN data for the human and mouse genomes, identifying regions consistently called as peaks in negative controls by the CUT&RUN peak caller SEACR. Using published CUT&RUN data from our and other labs, we show that the CUT&RUN blacklist regions can persist even when peak calling is performed with SEACR against a negative control, and after ENCODE blacklist removal. Moreover, we experimentally validated the CUT&RUN Blacklists by performing reiterative negative control experiments in which no specific protein is targeted, showing that they capture >80% of the peaks identified. We propose that removing these problematic regions prior to peak calling can substantially improve the performance of SEACR-based peak calling in CUT&RUN experiments, resulting in more reliable peak datasets.

genomics↗

bioRxiv2022

Time-resolved analysis of Wnt-signaling reveals β-catenin temporal genomic repositioning and cell type-specific plastic or elastic chromatin responses

Wnt signaling orchestrates gene expression via its effector {beta}-catenin. Whether {beta}-catenin targets genomic regions simultaneously or in a temporal fashion, and how this impacts the chromatin dynamics to modulate cell behavior, is currently unknown. Here we find that {beta}-catenin binds different loci at each time-point after stimulation, implying that the definition of Wnt-targets is fundamentally temporal. This process is intrinsically cell-type specific. In fact, Wnt/{beta}-catenin progressively shapes the chromatin of human embryonic stem cells consistent with their mesodermal differentiation: we call this genomic response plastic. In embryonic kidney cells, on the other hand, Wnt/{beta}-catenin drives a transient chromatin opening, followed by a re-establishment of the pre-stimulation state: a response that we define elastic. Finally, the Wnt-induced transient chromatin opening requires {beta}-catenin, suggesting a previously unappreciated pioneering role for this molecule. We submit that the plastic-vs-elastic behavior constitutes part of the mechanism explaining how Wnt/{beta}-catenin drives divergent cell-fate decisions during development and homeostasis.

genomics↗

bioRxiv2022

A New CUT&RUN Low Volume-Urea (LoV-U) protocol uncovers Wnt/β-catenin tissue-specific genomic targets

Upon WNT/{beta}-catenin pathway activation, stabilized {beta}-catenin travels to the nucleus where it associates with the TCF/LEF family of transcription factors, which constitutively bind to genomic Wnt Responsive Elements (WREs), to activate transcription of target genes. Discovering the binding profile of {beta}-catenin is therefore required to unambiguously assign direct targets of WNT signaling. Cleavage Under Target and Release Using Nuclease (CUT&RUN) has recently emerged as a prime technique for mapping the binding profile of chromatin interacting proteins. In our attempts to profile different regulators of the WNT/{beta}-catenin transcriptional complex, CUT&RUN performed reliably when targeting transcription factors such as TCF/LEF, but it failed to produce consistent binding patterns of the non-DNA-binding {beta}-catenin. Here, we present a biochemical modification of the CUT&RUN protocol, which we refer to as LoV-U (Low Volume and Urea), that enables the generation of robust and reproducible {beta}-catenin binding profiles. CUT&RUN-LoV-U uncovers direct WNT/{beta}-catenin target genes in human cells, as well as in ex vivo cells isolated from developing mouse tissue. CUT&RUN-LoV-U can profile all classes of chromatin regulators tested and is well suited for simultaneous processing of several samples. We submit that the application of our protocol will allow the detection of the complex system of tissue-specific WNT/{beta}-catenin target genes, together with other non-DNA-binding transcriptional regulators that act downstream of ontogenetically fundamental signaling cascades.

developmental biology↗

bioRxiv2022

The oncogene FOXQ1 is a selective activator of Wnt/β-catenin signalling

The forkhead box transcription factor FOXQ1 is aberrantly induced in various cancers, and contributes to tumour growth and metastasis. It has been suggested that FOXQ1 exacerbates cancer by activating the oncogenic Wnt/{beta}-catenin signalling pathway. However, the mode of action of FOXQ1 in the Wnt pathway remains to be resolved. Here, we report that FOXQ1 is a bimodal transcriptional activator of Wnt target gene expression in normal and cancer cells. Using co-immunoprecipitation, proximity proteomics, and reporter assays, we show that FOXQ1 engages the Wnt transcriptional complex to promote gene expression via TCF/LEF transcription factors. In parallel, FOXQ1 differentially regulates the expression of Wnt target genes independently of {beta}-catenin and TCF/LEFs, which is facilitated by spatially separated activator and repressor domains. Our results suggest that FOXQ1 is a novel component of the Wnt transcriptional complex that reinforces and specifies Wnt signalling in a context-dependent manner.

molecular biology↗

bioRxiv2020

TBX3 acts as tissue-specific component of the Wnt/β-catenin enhanceosome

BCL9 and PYGO are {beta}-catenin cofactors that enhance the transcription of Wnt target genes. They have been proposed as therapeutic targets to diminish Wnt signalling output in intestinal malignancies. Here we find that, in colorectal cancer cells and in developing mouse forelimbs, BCL9 proteins sustain the action of {beta}-catenin in a largely PYGO-independent manner. Our genetic analyses implied that BCL9 necessitates other interaction partners in mediating its transcriptional output. We identified the transcription factor TBX3 as a candidate tissue-specific member of the {beta}-catenin transcriptional complex. In developing forelimbs, TBX3 and BCL9 co-occupy a large number of Wnt-responsive regulatory elements, genome-wide. Moreover, mutations in Bcl9 affect the expression of TBX3 targets in vivo, and modulation of TBX3 abundance impacts on Wnt target genes transcription in a {beta}-catenin- and TCF/LEF-dependent manner. Finally, TBX3 overexpression exacerbates the metastatic potential of Wnt-dependent human colorectal cancer cells. Our work implicates TBX3 as a new, context-dependent component of the Wnt/{beta}-catenin-dependent enhanceosome.

developmental biology↗

bioRxiv2018

Gene fitness landscape of group A streptococcus during necrotizing myositis

Necrotizing fasciitis and myositis are devastating infections characterized by high mortality. Group A streptococcus (GAS) is a common cause of these infections, but the molecular pathogenesis is poorly understood. We report a genome-wide analysis using serotype M1 and M28 strains that identified novel GAS genes contributing to necrotizing myositis in nonhuman primates (NHP), a clinically relevant model. Using transposon directed insertion-site sequencing (TraDIS) we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively. For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters. Thirteen GAS transporters contributed to both M1 and M28 strain fitness in NHP myositis, including putative importers for amino acids, carbohydrates, and vitamins, and exporters for toxins, quorum sensing peptides, and uncharacterized molecules. Targeted deletion of genes encoding five transporters confirmed that each isogenic mutant strain was significantly impaired in causing necrotizing myositis in NHPs. qRT-PCR analysis showed that these five genes are expressed in infected NHP and human skeletal muscle. Certain substrate-binding lipoproteins of these transporters, such as Spy0271 and Spy1728, were previously documented to be surface-exposed, suggesting that our findings have translational research implications.

microbiology↗

bioRxiv2018

Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and cell death

CDC25 phosphatases have a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational approaches we defined a minimal common pharmacophore in established CDC25 inhibitors and performed a virtual screening of a proprietary library. Taking advantage of the availability of crystal structures for CDC25A and CDC25B and using a molecular docking strategy, we carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, studies on 3D organoids derived from intestinal crypt stem cells of Apc/K-Ras mice revealed that the compounds caused arrest of proliferation.

cancer biology↗

bioRxiv2018

Mutations in Bcl9 and Pygo genes cause congenital heart defects by tissue-specific perturbation of Wnt/β-catenin signaling

Genetic alterations in human BCL9 genes have repeatedly been found in congenital heart disease (CHD) with as-of-yet unclear causality. BCL9 proteins and their Pygopus (Pygo) co-factors can participate in canonical Wnt signaling via binding to {beta}-catenin. Nonetheless, their contributions to vertebrate heart development remain uncharted. Here, combining zebrafish and mouse genetics, we document tissue-specific functions in canonical Wnt signaling for BCL9 and Pygo proteins during heart development. In a CRISPR-Cas9-based genotype-phenotype association screen, we uncovered that zebrafish mutants for bcl9 and pygo genes largely retain {beta}-catenin activity, yet develop cardiac malformations. In mouse, both systemic and lineage-specific loss of the Pygo-BCL9-{beta}-catenin complex caused heart defects with outflow tract malformations, aberrant cardiac septation and valve formation, and compact myocardium hypoplasia. Mechanistically, these phenotypes coincide with transcriptional deregulation during heart patterning, and Pygo2 associates with {beta}-catenin at cis-regulatory regions of cardiac genes. Taken together, our results establish BCL9 and Pygo as tissue-specific {beta}-catenin co-factors during vertebrate heart development. Our results further implicate alterations in BCL9 and BCL9L in human CHDs as possibly causative.

genetics↗