Biology subjects

Bready, D.

Publications and source records attributed to Bready, D..

6 recordsLinked to original sources

bioRxiv2025

A developmentally regulated long-range enhancer-promoter contact mediates human neural development

SOX2 is a core pluripotency factor in human embryonic stem cells (hESCs), but upon differentiation to the three germ layers, its expression is preserved selectively in neuroectoderm. The mechanisms regulating SOX2 transcription in distinct developmental stages remain incompletely understood. Here, we demonstrate that a distant enhancer 550 kb from the human SOX2 locus is selectively activated in neural stem cells (NSCs) and establishes long-range contact with the SOX2 gene. CRISPR-Cas9 excision of the enhancer has no effect in hESCs but reduces SOX2 transcription in NSCs and impairs neuroectodermal differentiation and forebrain specification in teratomas and cerebral organoids. CRISPR excision of a CTCF recognition motif adjacent to the enhancer does not affect enhancer activation in neuroectoderm but reduces chromatin looping and SOX2 transcription to partially reproduce phenotypes seen with enhancer deletion. Our findings indicate that the development of the human nervous system depends on a developmentally regulated long-range contact between a distant enhancer and the SOX2 locus.

developmental biology↗

bioRxiv2023

The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their functional role in gliomagenesis and their potential as treatment targets. To identify therapeutically targetable opportunities among aGPCR family members in unbiased fashion, we analyzed expression levels of all aGPCRs in GBM and non-neoplastic brain tissue. Using bulk and single cell transcriptomic and proteomic data, we show that CD97 (ADGRE5), an aGPCR previously implicated in GBM pathogenesis, is the most promising aGPCR target in GBM, by virtue of its abundance in all GBM tumors and its de novo expression profile in GBM compared to normal brain tissue and neural progenitors. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGC) in vitro and in vivo. Transcriptomic and metabolomic data from PDGCs suggest that CD97 promotes glycolytic metabolism. The oncogenic and metabolic effects of CD97 are mediated by the MAPK pathway. Activation of MAPK signaling depends on phosphorylation of the cytosolic C-terminus of CD97 and recruitment of {beta}-arrestin. Using single-cell RNA-sequencing and biochemical assays, we demonstrate that THY1/CD90 is the most likely CD97 ligand in GBM. Lastly, we show that targeting of PDGCs with an anti-CD97 antibody-drug conjugate in vitro selectively kills tumor cells but not human astrocytes or neural stem cells. Our studies identify CD97 as an important regulator of tumor metabolism in GBM, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it for therapeutic purposes.

cancer biology↗

bioRxiv2023

Modulation of GPR133 (ADGRD1) Signaling by its Intracellular Interaction Partner Extended Synaptotagmin 1 (ESYT1)

GPR133 (ADGRD1) is an adhesion G protein-coupled receptor that signals through Gs and is required for growth of glioblastoma (GBM), an aggressive brain malignancy. The regulation of GPR133 signaling is incompletely understood. Here, we use proximity biotinylation proteomics to identify ESYT1, a Ca2+-dependent mediator of endoplasmic reticulum-plasma membrane bridge formation, as an intracellular interactor of GPR133. ESYT1 knockdown or knockout increases GPR133 signaling, while its overexpression has the opposite effect, without altering GPR133 levels in the plasma membrane. The GPR133-ESYT1 interaction requires the Ca2+-sensing C2C domain of ESYT1. Thapsigargin-mediated increases in cytosolic Ca2+ relieve signaling-suppressive effects of ESYT1 by promoting ESYT1-GPR133 dissociation. ESYT1 knockdown or knockout in GBM impairs tumor growth in vitro, suggesting functions of ESYT1 beyond the interaction with GPR133. Our findings suggest a novel mechanism for modulation of GPR133 signaling by increased cytosolic Ca2+, which reduces the signaling-suppressive interaction between GPR133 and ESYT1 to raise cAMP levels.

cell biology↗

bioRxiv2022

PTK7 is a positive allosteric modulator of GPR133 (ADGRD1) signaling in GBM

GPR133 (ADGRD1), an adhesion G protein-coupled receptor, supports growth of glioblastoma, a brain malignancy. We demonstrated that GPR133 is intramolecularly cleaved, and that dissociation of its N-terminal and C-terminal fragments (NTF and CTF) at the plasma membrane correlates with increased receptor signaling. However, how the extracellular interactome of GPR133 in glioblastoma modulates signaling remains unknown. Here, we use affinity purification and mass spectrometry to identify extracellular binding partners of GPR133 in patient-derived glioblastoma cells. We show that the transmembrane protein PTK7 binds the GPR133 NTF and its expression in trans increases GPR133 signaling. This effect requires the intramolecular cleavage of GPR133 and PTK7s anchoring in the plasma membrane. The GPR133-PTK7 interaction facilitates orthosteric activation of GPR133 by soluble peptide mimicking the endogenous tethered Stachel agonist, suggesting PTK7 binding allosterically enhances accessibility of GPR133s orthosteric Stachel binding pocket. GPR133 and PTK7 are expressed in adjacent cells in glioblastoma, where their knockdown phenocopies each other. We propose that this novel ligand-receptor interaction is relevant to the pathogenesis of glioblastoma, as well as physiological processes in several tissues.

cell biology↗

bioRxiv2021

The H3K36me2 writer-reader dependency in H3K27M-DIPG

The lysine-to-methionine mutation at residue 27 of histone H3 (H3K27M) is a driving mutation in Diffuse Intrinsic Pontine Glioma (DIPG), a highly aggressive form of pediatric brain tumor with no effective treatment and little chance of survival. H3K27M reshapes the epigenome through a global inhibition of PRC2 catalytic activity, the placement of methylation at lysine 27 of histone H3 (H3K27me2/3), promoting oncogenesis of DIPG. As a consequence, a histone modification H3K36me2, antagonistic to H3K27me2/3, is aberrantly elevated. Here, we investigate the role of H3K36me2 in H3K27M-DIPG by tackling its upstream catalyzing enzymes (writers) and downstream binding factors (readers). We determine that NSD1 and NSD2 are the key writers for H3K36me2. Loss of NSD1/2 in H3K27M-DIPG impedes cellular proliferation in vitro and tumorigenesis in vivo, and disrupts tumor-promoting gene expression programs. Further, we demonstrate that LEDGF and HDGF2 are the main readers that mediate the pro-tumorigenic effects downstream of NSD1/2-H3K36me2. Treatment with a chemically modified peptide mimicking endogenous H3K36me2 dislodges LEDGF/HDGF2 from chromatin and specifically inhibits the proliferation of H3K27M-DIPG. Together, our results indicate a functional pathway of NSD1/2-H3K36me2-LEDGF/HDGF2 as an acquired dependency in H3K27M-DIPG and suggest a possibility to target this pathway for therapeutic interventions.

cancer biology↗

bioRxiv2020

Dissociation of the intramolecularly cleaved N- and C-terminal fragments of the adhesion G protein-coupled receptor GPR133 (ADGRD1) increases canonical signaling

GPR133 (ADGRD1), an adhesion G protein-coupled receptor (GPCR), is necessary for growth of glioblastoma (GBM), a brain malignancy. The extracellular N-terminus of GPR133 is thought to be autoproteolytically cleaved into an N-terminal and a C-terminal fragment (NTF and CTF). Nevertheless, the role of this cleavage in receptor activation remains unclear. Here, we show that the wild-type (WT) receptor is cleaved after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant (H543R) in patient-derived GBM cultures and HEK293T cells. However, the resulting NTF and CTF remain non-covalently bound until the receptor is trafficked to the plasma membrane, where we find NTF-CTF dissociation. Using a fusion of the hPAR1 receptor N-terminus and the CTF of GPR133, we demonstrate that thrombin-induced cleavage and shedding of the hPAR1 NTF increases receptor signaling. This study supports a model where dissociation of the NTF at the plasma membrane promotes GPR133 activation. Highlights- GPR133 is intramolecularly cleaved in patient-derived GBM cultures - Cleaved GPR133 signals at higher efficacy than the uncleavable GPR133 H543R mutant - The N- and C-terminal fragments (NTF and CTF) of GPR133 dissociate at the plasma membrane - Acute thrombin-induced cleavage of the human PAR1 NTF from the GPR133 CTF increases signaling eTOC BlurbFrenster et al. demonstrate intramolecular cleavage of the adhesion GPCR GPR133 in glioblastoma and HEK293T cells. The resulting N- and C-terminal fragments dissociate at the plasma membrane to increase canonical signaling. The findings suggest dissociation of GPR133s N-terminus at the plasma membrane represents a major mechanism of receptor activation.

cell biology↗