Biology subjects

McMahon, A.

Publications and source records attributed to McMahon, A..

7 recordsLinked to original sources

bioRxiv2023

Cadherin Adhesion Complexes Direct Cell Aggregation in the Epithelial Transition of Wnt-Induced Nephron Progenitor Cells

In the developing mammalian kidney, nephron formation is initiated by a subset of nephron progenitor cells (NPCs). Wnt input activates a {beta}-catenin (Ctnnb1)-driven, transcriptional nephrogenic program. In conjunction, induced mesenchymal NPCs transition through a pre-tubular aggregate to an epithelial renal vesicle, the precursor for each nephron. How this critical mesenchymal-to-epithelial transition (MET) is regulated is unclear. In an in vitro mouse NPC culture model, activation of the Wnt pathway results in the aggregation of induced NPCs into closely-packed, cell clusters. Genetic removal of {beta}-catenin resulted in a failure of both Wnt pathway-directed transcriptional activation and the formation of aggregated cell clusters. Modulating extracellular Ca2+ levels showed cell-cell contacts were Ca2+-dependent, suggesting a role for cadherin (Cdh)-directed cell adhesion. Molecular analysis identified Cdh2, Cdh4 and Cdh11 in uninduced NPCs and the up-regulation of Cdh3 and Cdh4 accompanying the Wnt pathway-induced MET. Genetic removal of all four cadherins, and independent removal of -catenin, which couples Cdh-{beta}-catenin membrane complexes to the actin cytoskeleton, abolished cell aggregation in response to Wnt pathway activation. However, the {beta}-catenin driven inductive transcriptional program was unaltered. Together with the accompanying paper (Bugacov et al., submitted), these data demonstrate that distinct cellular activities of {beta}-catenin - transcriptional regulation and cell adhesion - combine in the mammalian kidney programs generating differentiated epithelial nephron precursors from mesenchymal nephron progenitors. Summary statementOur study highlights the role of Wnt-{beta}-catenin pathway regulation of cadherin-mediated cell adhesion in the mesenchymal to epithelial transition of induced nephron progenitor cells.

developmental biology↗

bioRxiv2023

Canonical Wnt transcriptional complexes are essential for induction of nephrogenesis but not maintenance or proliferation of nephron progenitors

Wnt regulated transcriptional programs are associated with both the maintenance of mammalian nephron progenitor cells (NPC) and their induction, initiating the process of nephrogenesis. How opposing transcriptional roles are regulated remain unclear. Using an in vitro model replicating in vivo events, we examined the requirement for canonical Wnt transcriptional complexes in NPC regulation. In canonical transcription, Lef/Tcf DNA binding proteins associate the transcriptional co-activator {beta}-catenin. Wnt signaling is readily substituted by CHIR99021, a small molecule antagonist of glycogen synthase kinase-3{beta} (GSK3{beta}). GSK3{beta} inhibition blocks Gsk{beta}-dependent turnover of {beta}-catenin, enabling formation of Lef/Tcf/{beta}-catenin transcriptional complexes, and enhancer-mediated transcriptional activation. Removal of {beta}-catenin activity from NPCs under cell expansion conditions (low CHIR) demonstrated a non-transcriptional role for {beta}-catenin in the CHIR-dependent proliferation of NPCs. In contrast, CHIR-mediated induction of nephrogenesis, on switching from low to high CHIR, was dependent on Lef/Tcf and {beta}-catenin transcriptional activity. These studies point to a non-transcriptional mechanism for {beta}-catenin in regulation of NPCs, and potentially other stem progenitor cell types. Further, analysis of the {beta}-catenin-directed transcriptional response provides new insight into induction of nephrogenesis. Summary StatementThe study provides a mechanistic understanding of Wnt/ {beta}-catenin activity in self-renewal and differentiation of mammalian nephron progenitors.

developmental biology↗

bioRxiv2023

Direct androgen receptor regulation of sexually dimorphic gene expression in the mammalian kidney

Mammalian organs exhibit distinct physiology, disease susceptibility and injury responses between the sexes. In the mouse kidney, sexually dimorphic gene activity maps predominantly to proximal tubule (PT) segments. Bulk RNA-seq data demonstrated sex differences were established from 4 and 8 weeks after birth under gonadal control. Hormone injection studies and genetic removal of androgen and estrogen receptors demonstrated androgen receptor (AR) mediated regulation of gene activity in PT cells as the regulatory mechanism. Interestingly, caloric restriction feminizes the male kidney. Single-nuclear multiomic analysis identified putative cis-regulatory regions and cooperating factors mediating PT responses to AR activity in the mouse kidney. In the human kidney, a limited set of genes showed conserved sex-linked regulation while analysis of the mouse liver underscored organ-specific differences in the regulation of sexually dimorphic gene expression. These findings raise interesting questions on the evolution, physiological significance, and disease and metabolic linkage, of sexually dimorphic gene activity.

developmental biology↗

bioRxiv2022

APE1 assembles biomolecular condensates to promotethe ATR-Chk1 DNA damage response in nucleolus

Multifunctional protein APE1/APEX1/HAP1/Ref-1 (designated as APE1) plays important roles in nuclease-mediated DNA repair and redox regulation in transcription. However, it is unclear how APE1 regulates the DNA damage response (DDR) pathways and influences genome integrity directly or indirectly. Here we show that siRNA-mediated APE1-knockdown or APE1 inhibitor treatment attenuates the ATR-Chk1 DDR under stress conditions in multiple immortalized cell lines. Congruently, APE1 overexpression (APE1-OE) activates the ATR DDR under unperturbed conditions, which is independent of APE1 nuclease and redox functions. Structural and functional analysis reveals a direct requirement of the extreme N-terminal 33 amino acids (NT33) within APE1 in the assembly of distinct biomolecular condensates in vitro and DNA/RNA-independent activation of the ATR DDR. Overexpressed APE1 co-localizes with nucleolar NPM1 and assembles biomolecular condensates in nucleoli in cancer but not non-malignant cells, which recruits ATR and its direct activator molecules TopBP1 and ETAA1. APE1 W119R mutant is deficient in nucleolar condensation and liquid-liquid phase separation and is incapable of activating nucleolar ATR DDR. Lastly, APE1-OE-induced nucleolar ATR DDR activation leads to compromised ribosomal RNA transcription and reduced cell viability. Taken together, we propose distinct mechanisms by which APE1 regulates ATR DDR pathways and functions in genome integrity maintenance.

molecular biology↗

bioRxiv2022

APE1 recruits ATRIP to ssDNA in an RPA-independent manner to promote the ATR DNA damage response

Cells have evolved the DNA damage response (DDR) pathways in response to DNA replication stress or DNA damage. In the ATR-Chk1 DDR pathway, it has been proposed that ATR is recruited to RPA-coated single-strand DNA (ssDNA) by direct ATRIP-RPA interaction. However, it remains elusive whether and how ATRIP is recruited to ssDNA in an RPA-independent manner. Here, we provide evidence that APE1 directly associates ssDNA to recruit ATRIP onto ssDNA in an RPA-independent fashion. The N-terminal motif within APE1 is required and sufficient for the APE1-ATRIP interaction in vitro and the distinct APE1-ATRIP interaction is required for ATRIP recruitment to ssDNA and the ATR-Chk1 DDR pathway activation in Xenopus egg extracts. In addition, APE1 directly associates with RPA70 and RPA32 via two distinct motifs. Taken together, our evidence identifies APE1 as a direct recruiter of ATRIP onto ssDNA independent of RPA in the activation of ATR DDR pathway. SummaryO_LIAPE1 associates with ssDNA and ATRIP directly via distinct motifs and thereby recruits ATRIP onto ssDNA independent of RPA to promote the ATR DDR. C_LIO_LIAPE1 interacts with RPA via distinct two motifs in vitro but such RPA-APE1 interaction is dispensable for ATRIP recruitment onto ssDNA. C_LI

molecular biology↗

bioRxiv2022

A community driven GWAS summary statistics standard

Summary statistics from genome-wide association studies (GWAS) represent a huge potential for research. A challenge for researchers in this field is the access and sharing of summary statistics data due to a lack of standards for the data content and file format. For this reason, the GWAS Catalog hosted a series of meetings in 2021 with summary statistics stakeholders to guide the development of a standard format. The key requirements from the stakeholders were for a standard that contained key data elements to be able to support a wide range of data analyses, required low bioinformatics skills for file access and generation, to have easily accessible metadata, and unambiguous and interoperable data. Here, we define the specifications for the first version of the GWAS-SSF format, which was developed to meet the requirements discussed with the community. GWAS-SSF consists of a tab-separated data file with well-defined fields and an accompanying metadata file.

bioinformatics↗

bioRxiv2021

High-throughput super-resolution analysis of influenza virus pleomorphism reveals insights into viral spatial organization

Many viruses form highly pleomorphic particles; in influenza, these particles range from spheres of ~ 100 nm in diameter to filaments of several microns in length. Virion structure is of interest, not only in the context of virus assembly, but also because pleomorphic variations may correlate with infectivity and pathogenicity. We have used fluorescence super-resolution microscopy combined with a rapid automated analysis pipeline to image many thousands of individual influenza virions, gaining information on their size, morphology and the distribution of membrane-embedded and internal proteins. We observed broad phenotypic variability in filament size, and Fourier transform analysis of super resolution images demonstrated no generalized common spatial frequency patterning of HA or NA on the virion surface, suggesting a model of virus particle assembly where the release of progeny filaments from cells occurs in a stochastic way. Finally, we showed that in long filaments, viral RNP complexes are located preferentially within Archetti bodies, suggesting that these structures may play a role in virus transmission. Our approach therefore offers exciting new insights into influenza virus morphology and represents a powerful technique that is easily extendable to the study of pleomorphism in other pathogenic viruses.

microbiology↗