Cilia as Wnt signaling organelles.

Cilia and Wnt signaling have a complex relationship, wherein Wnt regulates cilia and, conversely, cilia may affect Wnt signaling. Recently, it was shown that Wnt receptors are present in flagella, primary cilia, and multicilia, where they transmit an intraciliary signal that is independent of ß-catenin. Intraciliary Wnt signaling promotes ciliogenesis, affecting male fertility, adipogenesis, and mucociliary clearance. Wnt also stimulates the beating of motile cilia, highlighting that these nanomotors, too, are chemosensory. Intraciliary Wnt signaling employs a Wnt-protein phosphatase 1 (PP1) signaling axis, involving the canonical Wnt pathway's inhibition of glycogen synthase kinase 3 (GSK3) to repress PP1 activity. Collectively, these findings support that cilia are Wnt signaling organelles, with implications for ciliopathies and cancer.

R-Spondin 2 governs Xenopus left-right body axis formation by establishing an FGF signaling gradient.

Establishment of the left-right (LR, sinistral, dextral) body axis in many vertebrate embryos relies on cilia-driven leftward fluid flow within an LR organizer (LRO). A cardinal question is how leftward flow triggers symmetry breakage. The chemosensation model posits that ciliary flow enriches a signaling molecule on the left side of the LRO that promotes sinistral cell fate. However, the nature of this sinistralizing signal has remained elusive. In the Xenopus LRO, we identified the stem cell growth factor R-Spondin 2 (Rspo2) as a symmetrically expressed, sinistralizing signal. As predicted for a flow-mediated signal, Rspo2 operates downstream of leftward flow but upstream of the asymmetrically expressed gene dand5. Unexpectedly, in LR patterning, Rspo2 acts as an FGF receptor antagonist: Rspo2 via its TSP1 domain binds Fgfr4 and promotes its membrane clearance by Znrf3-mediated endocytosis. Concordantly, we find that at flow-stage, FGF signaling is dextralizing and forms a gradient across the LRO, high on the dextral- and low on the sinistral side. Rspo2 gain- and loss-of function equalize this FGF signaling gradient and sinistralize and dextralize development, respectively. We propose that leftward flow of Rspo2 produces an FGF signaling gradient that governs LR-symmetry breakage.

DNA methylation clocks for clawed frogs reveal evolutionary conservation of epigenetic aging.

To address how conserved DNA methylation-based epigenetic aging is in diverse branches of the tree of life, we generated DNA methylation data from African clawed frogs (Xenopus laevis) and Western clawed frogs (Xenopus tropicalis) and built multiple epigenetic clocks. Dual species clocks were developed that apply to both humans and frogs (human-clawed frog clocks), supporting that epigenetic aging processes are evolutionary conserved outside mammals. Highly conserved positively age-related CpGs are located in neural-developmental genes such as uncx, tfap2d as well as nr4a2 implicated in age-associated disease. We conclude that signatures of epigenetic aging are evolutionary conserved between frogs and mammals and that the associated genes relate to neural processes, altogether opening opportunities to employ Xenopus as a model organism to study aging.

ROTACs leverage signaling-incompetent R-spondin for targeted protein degradation.

Proteolysis-targeting chimeras (PROTACs) are an emerging technology for therapeutic intervention, but options to target cell surface proteins and receptors remain limited. Here we introduce ROTACs, bispecific WNT- and BMP-signaling-disabled R-spondin (RSPO) chimeras, which leverage the specificity of these stem cell growth factors for ZNRF3/RNF43 E3 transmembrane ligases, to target degradation of transmembrane proteins. As a proof-of-concept, we targeted the immune checkpoint protein, programmed death ligand 1 (PD-L1), a prominent cancer therapeutic target, with a bispecific RSPO2 chimera, R2PD1. The R2PD1 chimeric protein binds to PD-L1 and at picomolar concentration induces its lysosomal degradation. In three melanoma cell lines, R2PD1 induced between 50 and 90% PD-L1 protein degradation. PD-L1 degradation was strictly dependent on ZNRF3/RNF43. Moreover, R2PD1 reactivates cytotoxic T cells and inhibits tumor cell proliferation more potently than Atezolizumab. We suggest that signaling-disabled ROTACs represent a paradigm to target cell surface proteins for degradation in a range of applications.

RNA stability controlled by m6A methylation contributes to X-to-autosome dosage compensation in mammals.

In mammals, X-chromosomal genes are expressed from a single copy since males (XY) possess a single X chromosome, while females (XX) undergo X inactivation. To compensate for this reduction in dosage compared with two active copies of autosomes, it has been proposed that genes from the active X chromosome exhibit dosage compensation. However, the existence and mechanisms of X-to-autosome dosage compensation are still under debate. Here we show that X-chromosomal transcripts have fewer m6A modifications and are more stable than their autosomal counterparts. Acute depletion of m6A selectively stabilizes autosomal transcripts, resulting in perturbed dosage compensation in mouse embryonic stem cells. We propose that higher stability of X-chromosomal transcripts is directed by lower levels of m6A, indicating that mammalian dosage compensation is partly regulated by epitranscriptomic RNA modifications.

Multimodal Wnt signalling in the mouse neocortex.

The neocortex is the site of higher cognitive functions and its development is tightly regulated by cell signalling pathways. Wnt signalling is inexorably linked with neocortex development but its precise role remains unclear. Most studies demonstrate that Wnt/ß-catenin regulates neural progenitor self-renewal but others suggest it can also promote differentiation. Wnt/STOP signalling is a novel branch of the Wnt pathway that stabilizes proteins during G2/M by inhibiting glycogen synthase kinase 3 (GSK3)-mediated protein degradation. Recent data from our work in Da Silva et al. (2021) demonstrate that Wnt/STOP is involved in neocortex development where, by stabilizing the neurogenic transcription factors Sox4 and Sox11, it promotes neural progenitor differentiation. We also show that Wnt/STOP regulates asymmetric cell division and cell cycle dynamics in apical and basal progenitors, respectively. Our study reveals a division of labour in the Wnt signalling pathway by suggesting that Wnt/STOP is the primary driver of cortical neurogenesis while Wnt/ß-catenin is mainly responsible for self-renewal. These results resolve a decades-old question on the role of Wnt signalling in cortical neural progenitors.

DEAD box RNA helicases act as nucleotide exchange factors for casein kinase 2.

DDX RNA helicases promote RNA processing, but DDX3X also activates casein kinase 1 (CK1ε). We show that other DDX proteins also stimulate the protein kinase activity of CK1ε and that this extends to casein kinase 2 (CK2). CK2 enzymatic activity was stimulated by various DDX proteins at high substrate concentrations. DDX1, DDX24, DDX41, and DDX54 were required for full kinase activity in vitro and in Xenopus embryos. Mutational analysis of DDX3X indicated that CK1 and CK2 kinase stimulation engages its RNA binding but not catalytic motifs. Mathematical modeling of enzyme kinetics and stopped-flow spectroscopy showed that DDX proteins function as nucleotide exchange factors toward CK2 and reduce unproductive reaction intermediates and substrate inhibition. Our study reveals protein kinase stimulation by nucleotide exchange as important for kinase regulation and as a generic function of DDX proteins.

Mucociliary Wnt signaling promotes cilia biogenesis and beating.

It is widely thought that Wnt/Lrp6 signaling proceeds through the cytoplasm and that motile cilia are signaling-inert nanomotors. Contrasting both views, we here show in the mucociliary epidermis of X. tropicalis embryos that motile cilia transduce a ciliary Wnt signal that is distinct from canonical ß-catenin signaling. Instead, it engages a Wnt-Gsk3-Ppp1r11-Pp1 signaling axis. Mucociliary Wnt signaling is essential for ciliogenesis and it engages Lrp6 co-receptors that localize to cilia via a VxP ciliary targeting sequence. Live-cell imaging using a ciliary Gsk3 biosensor reveals an immediate response of motile cilia to Wnt ligand. Wnt treatment stimulates ciliary beating in X. tropicalis embryos and primary human airway mucociliary epithelia. Moreover, Wnt treatment improves ciliary function in X. tropicalis ciliopathy models of male infertility and primary ciliary dyskinesia (ccdc108, gas2l2). We conclude that X. tropicalis motile cilia are Wnt signaling organelles that transduce a distinct Wnt-Pp1 response.

DNA methylation and cardiovascular disease in humans: a systematic review and database of known CpG methylation sites.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death worldwide and considered one of the most environmentally driven diseases. The role of DNA methylation in response to the individual exposure for the development and progression of CVD is still poorly understood and a synthesis of the evidence is lacking. RESULTS: A systematic review of articles examining measurements of DNA cytosine methylation in CVD was conducted in accordance with PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines. The search yielded 5,563 articles from PubMed and CENTRAL databases. From 99 studies with a total of 87,827 individuals eligible for analysis, a database was created combining all CpG-, gene- and study-related information. It contains 74,580 unique CpG sites, of which 1452 CpG sites were mentioned in ≥ 2, and 441 CpG sites in ≥ 3 publications. Two sites were referenced in ≥ 6 publications: cg01656216 (near ZNF438) related to vascular disease and epigenetic age, and cg03636183 (near F2RL3) related to coronary heart disease, myocardial infarction, smoking and air pollution. Of 19,127 mapped genes, 5,807 were reported in ≥ 2 studies. Most frequently reported were TEAD1 (TEA Domain Transcription Factor 1) and PTPRN2 (Protein Tyrosine Phosphatase Receptor Type N2) in association with outcomes ranging from vascular to cardiac disease. Gene set enrichment analysis of 4,532 overlapping genes revealed enrichment for Gene Ontology molecular function "DNA-binding transcription activator activity" (q = 1.65 × 10-11) and biological processes "skeletal system development" (q = 1.89 × 10-23). Gene enrichment demonstrated that general CVD-related terms are shared, while "heart" and "vasculature" specific genes have more disease-specific terms as PR interval for "heart" or platelet distribution width for "vasculature." STRING analysis revealed significant protein-protein interactions between the products of the differentially methylated genes (p = 0.003) suggesting that dysregulation of the protein interaction network could contribute to CVD. Overlaps with curated gene sets from the Molecular Signatures Database showed enrichment of genes in hemostasis (p = 2.9 × 10-6) and atherosclerosis (p = 4.9 × 10-4). CONCLUSION: This review highlights the current state of knowledge on significant relationship between DNA methylation and CVD in humans. An open-access database has been compiled of reported CpG methylation sites, genes and pathways that may play an important role in this relationship.

Primary cilia are WNT-transducing organelles whose biogenesis is controlled by a WNT-PP1 axis.

WNT signaling is important in development, stem cell maintenance, and disease. WNT ligands typically signal via receptor activation across the plasma membrane to induce ß-catenin-dependent gene activation. Here, we show that in mammalian primary cilia, WNT receptors relay a WNT/GSK3 signal that ß-catenin-independently promotes ciliogenesis. Characterization of a LRP6 ciliary targeting sequence and monitoring of acute WNT co-receptor activation (phospho-LRP6) support this conclusion. Ciliary WNT signaling inhibits protein phosphatase 1 (PP1) activity, a negative regulator of ciliogenesis, by preventing GSK3-mediated phosphorylation of the PP1 regulatory inhibitor subunit PPP1R2. Concordantly, deficiency of WNT/GSK3 signaling by depletion of cyclin Y and cyclin-Y-like protein 1 induces primary cilia defects in mouse embryonic neuronal precursors, kidney proximal tubules, and adult mice preadipocytes.

Human SLFN5 and its Xenopus Laevis ortholog regulate entry into mitosis and oocyte meiotic resumption.

The Schlafen gene family was first described in mice as a regulator of thymocyte development. Further studies showed involvement of human orthologs in different processes related with viral replication, cellular proliferation, and differentiation. In recent years, a new role for human Slfn11 in DNA replication and chromatin remodeling was described. As commonly observed in many gene families, Slfn paralogs show a tissue-specific expression. This made it difficult to reach conclusions which can be valid in different biological models regarding the function of the different Schlafen proteins. In the present study, we investigate the involvement of SLFN5 in cell-cycle regulation and cell proliferation. A careful analysis of SLFN5 expression revealed that SLFN5 is highly expressed in proliferating tissues and that the protein is ubiquitously present in all the tissues and cell line models we analyzed. Very interestingly, SLFN5 expression oscillates during cell cycle, peaking during S phase. The fact that SLFN5 interacts with protein phosphatase 2A and that SLFN5 depletion causes cell cycle arrest and cellular apoptosis, suggests a direct involvement of this human paralog in cell cycle progression and cellular proliferation. We substantiated our in vitro and in cellulo results using Xenopus laevis oocytes to show that mRNA depletion of the unique Slfn gene present in Xenopus, whose protein sequence shares 80% of homology with SLFN5, recapitulates the phenotype observed in human cells preventing the resumption of meiosis during oocyte development.

R-spondin 3 deletion induces Erk phosphorylation to enhance Wnt signaling and promote bone formation in the appendicular skeleton.

Activation of Wnt signaling leads to high bone density. The R-spondin family of four secreted glycoproteins (Rspo1-4) amplifies Wnt signaling. In humans, RSPO3 variants are strongly associated with bone density. Here, we investigated the role of Rspo3 in skeletal homeostasis in mice. Using a comprehensive set of mouse genetic and mechanistic studies, we show that in the appendicular skeleton, Rspo3 haplo-insufficiency and Rspo3 targeted deletion in Runx2+ osteoprogenitors lead to an increase in trabecular bone mass, with increased number of osteoblasts and bone formation. In contrast and highlighting the complexity of Wnt signaling in the regulation of skeletal homeostasis, we show that Rspo3 deletion in osteoprogenitors results in the opposite phenotype in the axial skeleton, i.e., low vertebral trabecular bone mass. Mechanistically, Rspo3 deficiency impairs the inhibitory effect of Dkk1 on Wnt signaling activation and bone mass. We demonstrate that Rspo3 deficiency leads to activation of Erk signaling which in turn, stabilizes ß-catenin and Wnt signaling activation. Our data demonstrate that Rspo3 haplo-insufficiency/deficiency boosts canonical Wnt signaling by activating Erk signaling, to favor osteoblastogenesis, bone formation, and bone mass.

Mammalian N1-adenosine PARylation is a reversible DNA modification.

Poly-ADP-ribosylation (PARylation) is regarded as a protein-specific modification. However, some PARPs were recently shown to modify DNA termini in vitro. Here, we use ultrasensitive mass spectrometry (LC-MS/MS), anti-PAR antibodies, and anti-PAR reagents to show that mammalian DNA is physiologically PARylated and to different levels in primary tissues. Inhibition of PAR glycohydrolase (PARG) increases DNA PARylation, supporting that the modification is reversible. DNA PARylation requires PARP1 and in vitro PARP1 PARylates single-stranded DNA, while PARG reverts the modification. DNA PARylation occurs at the N1-position of adenosine residues to form N1-Poly(ADP-ribosyl)-deoxyadenosine. Through partial hydrolysis of mammalian gDNA we identify PAR-DNA via the diagnostic deamination product N1-ribosyl-deoxyinosine to occur in vivo. The discovery of N1-adenosine PARylation as a DNA modification establishes the conceptual and methodological framework to elucidate its biological relevance and extends the role of PARP enzymes.

In Vitro Binding of GADD45A to RNA:DNA Hybrids.

R-loops are three-stranded RNA:DNA hybrid structures that frequently form during transcription. While R-loop misregulation is associated with genome instability, cells also harness RNA-DNA hybrids in scheduled, "regulatory," R-loops to control gene expression. One regulatory role involves epigenetic gene regulation by the R-loop reader Growth Arrest and DNA Damage 45A (GADD45A). This small stress related protein promotes DNA demethylation by recruiting TET dioxygenase and Thymine DNA glycosylase to specific genomic loci. GADD45A requires adapters for its genomic localization. One such class of adapters are R-loops formed at certain CpG island promoters to which GADD45A binds directly, targets the demethylation machinery, and confers an open chromatin state. Here, we describe protocols for carrying out in vitro binding assays with GADD45A to RNA:DNA hybrids to biochemically study its direct binding to R-loops, specifically GADD45A pulldown and EMSA (electrophoretic mobility shift) assays.

CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia.

The heterogeneous response of acute myeloid leukemia (AML) to current anti-leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)-enriched compartments with different self-renewal capacities. How these compartments self-renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial-to-mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+ CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co-activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC-enriched subsets in vivo and synergize with the Bcl-2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl-2 inhibition as LSC-directed therapy in this disease.

Uncoupling the BMP receptor antagonist function from the WNT agonist function of R-spondin 2 using the inhibitory peptide dendrimer RWd.

Signaling by bone morphogenetic proteins (BMPs) plays pivotal roles in embryogenesis, adult tissue homeostasis, and disease. Recent studies revealed that the well-established WNT agonist R-spondin 2 (RSPO2) is also a BMP receptor (BMP receptor type 1A) antagonist, with roles in early Xenopus embryogenesis and human acute myeloid leukemia (AML). To uncouple the BMP antagonist function from the WNT agonist function and to promote development of AML therapeutics, here we identified a 10-mer peptide (RW) derived from the thrombospondin 1 domain of RSPO2, which specifically prevents binding between RSPO2 and BMP receptor type 1A without altering WNT signaling. We also show that a corresponding RW dendrimer (RWd) exhibiting improved half-life relieves inhibition of BMP receptor signaling by RSPO2 in human AML cells, reduces cell growth, and induces differentiation. Moreover, microinjection of RWd in Xenopus embryos ventralizes the dorsoventral embryonic patterning by upregulating BMP signaling without affecting WNT signaling. Our study corroborates the function of RSPO2 as a BMP receptor antagonist and provides a proof of concept for pharmacologically uncoupling BMP antagonist from WNT agonist functions of RSPO2 using the inhibitor peptide RWd with enhanced target selectivity and limited side effects.

The role of Xenopus developmental biology in unraveling Wnt signalling and antero-posterior axis formation.

Wnt signalling plays an eminent role in development, stem cell growth, and tissue homeostasis. Much of what we know about Wnt signalling, we owe to research in developmental biology. Here I review some salient discoveries in the older literature, beginning with the Lithium experiments in sea urchin by Curt Herbst in the 1890ies, when unknown to him he observed the gradual effects of Wnt overactivation upon embryonic axis formation. After revisiting key discoveries into Wingless signalling in Drosophila, I examine the role that the Xenopus embryo has played as model system in this regard. Not only were components of the Wnt cascade dissected and secreted Wnt antagonists discovered in Xenopus, but it also played a key role in unveiling the evolutionary conserved role of Wnt signalling in primary body axis formation. I conclude that Xenopus developmental biology has played a major role in elucidating the mechanisms of embryonic Wnt signalling.

A MET-PTPRK kinase-phosphatase rheostat controls ZNRF3 and Wnt signaling.

Zinc and ring finger 3 (ZNRF3) is a transmembrane E3 ubiquitin ligase that targets Wnt receptors for ubiquitination and lysosomal degradation. Previously, we showed that dephosphorylation of an endocytic tyrosine motif (4Y motif) in ZNRF3 by protein tyrosine phosphatase receptor-type kappa (PTPRK) promotes ZNRF3 internalization and Wnt receptor degradation (Chang et al 2020). However, a responsible protein tyrosine kinase(s) (PTK) phosphorylating the 4Y motif remained elusive. Here we identify the proto-oncogene MET (mesenchymal-epithelial transition factor) as a 4Y kinase. MET binds to ZNRF3 and induces 4Y phosphorylation, stimulated by the MET ligand HGF (hepatocyte growth factor, scatter factor). HGF-MET signaling reduces ZNRF3-dependent Wnt receptor degradation thereby enhancing Wnt/ß-catenin signaling. Conversely, depletion or pharmacological inhibition of MET promotes the internalization of ZNRF3 and Wnt receptor degradation. We conclude that HGF-MET signaling phosphorylates- and PTPRK dephosphorylates ZNRF3 to regulate ZNRF3 internalization, functioning as a rheostat for Wnt signaling that may offer novel opportunities for therapeutic intervention.

RSPO2 inhibits BMP signaling to promote self-renewal in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a rapidly progressing cancer, for which chemotherapy remains standard treatment and additional therapeutic targets are requisite. Here, we show that AML cells secrete the stem cell growth factor R-spondin 2 (RSPO2) to promote their self-renewal and prevent cell differentiation. Although RSPO2 is a well-known WNT agonist, we reveal that it maintains AML self-renewal WNT independently, by inhibiting BMP receptor signaling. Autocrine RSPO2 signaling is also required to prevent differentiation and to promote self-renewal in normal hematopoietic stem cells as well as primary AML cells. Comprehensive datamining reveals that RSPO2 expression is elevated in patients with AML of poor prognosis. Consistently, inhibiting RSPO2 prolongs survival in AML mouse xenograft models. Our study indicates that in AML, RSPO2 acts as an autocrine BMP antagonist to promote cancer cell renewal and may serve as a marker for poor prognosis.