Biology subjects

Nordin, A.

Publications and source records attributed to Nordin, A..

8 recordsLinked to original sources

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

The Developmental Transcription Factor TBX3 Physically Engages with the Wnt/β-catenin Transcriptional Complex in Human Colorectal Cancer Cells to Regulate Metastasis Genes

Wnt signaling orchestrates gene expression in a plethora of processes during development and adult cell homeostasis via the action of nuclear {beta}-catenin. Furthermore, neoplasia of the colorectal epithelium begins with aberrant Wnt/{beta}-catenin signaling. Yet, little is known about how {beta}-catenin generates context-specific transcriptional outcomes. We have previously identified the developmental transcription factor TBX3 as a tissue-specific component of the Wnt/{beta}-catenin nuclear complex during mouse forelimb development. In this study, we show that TBX3 is present and functionally active in human colorectal cancers. TBX3s genomic binding pattern suggests a regulatory role that broadly coincides with that of Wnt/{beta}-catenin. Moreover, proteomics proximity labelling indicated that, during Wnt pathway activation, TBX3 is vicinal to several protein partners, including the transcription factors TCF/LEF and chromatin remodeling complexes which are usually found at Wnt responsive elements. Sequence and structure analysis revealed that TBX3 possesses an exposed Asp-Pro-Phe (NPF) motif predicted by AlphaFold2 Multimer to mediate direct interactions with several Wnt-activated TBX3 partners. Deletion of NPF abrogates TBX3 proximity to these partners and its ability to modulate Wnt-dependent transcription. TBX3 emerges as a key modulator of the oncogenic activity of Wnt/{beta}-catenin in colorectal cancer, and its mechanism of action exposes a novel druggable protein-interaction surface.

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↗