Biology subjects

Fakhouri, W. D.

Publications and source records attributed to Fakhouri, W. D..

4 recordsLinked to original sources

bioRxiv2025

TWIST1 Modulates Cilia Length, Endocytic Vesicle Dynamics, and Cell-Cell Junctions during Neural Tube Morphogenesis

BackgroundEndocytosis constitutes a fundamental cellular process governing development through coordinated regulation of plasma membrane remodeling and ciliogenesis, processes essential for cell shape changes and embryonic development. Although Twist1 null embryos display complete cranial neural tube closure defects and conditional knockout in neuroectoderm disrupts cranial neural crest cell fate determination and delamination, the function of TWIST1 in neural tube morphogenesis remains unknown. We investigated the basis underlying neuroectodermal morphological abnormalities in TWIST1 mutant embryos, specifically the formation of ectopic lateral bending points and cellular disorganization, by examining TWIST1 function in cilia formation, adherens junction integrity, and endocytic vesicle dynamics. ResultsImmunofluorescence analysis revealed that cytosolic TWIST1 colocalizes with {beta}-catenin and endocytic regulators LRP2 and RAB11B along the apical surface of cranial neuroectoderm. Twist1 knockout resulted in reduced ciliary length and number. Quantitative PCR and Western blot analyses demonstrated upregulation of RAB11B and {beta}-catenin at mRNA and protein levels in Twist1 mutants. This molecular dysregulation coincided with increased accumulation of apical endocytic vesicles and altered expression profiles of endocytic component genes, ultimately modifying the apical neuroectodermal cell-cell junctions. ConclusionOur findings establish TWIST1 as a regulator of neuroectodermal morphology, demonstrating its ability to modulate ciliogenesis, endocytic vesicle dynamics, and cell-cell integrity.

developmental biology↗

bioRxiv2022

Sequence-to-expression approach to identify etiological non-coding DNA variations in P53 and cMYC-driven diseases

Disease risk prediction based on DNA sequence and transcriptional profile can improve disease screening, prevention, and potential therapeutic approaches by revealing contributing genetic factors and altered networks. Despite identifying many disease-associated DNA variants through genome-wide association studies, distinguishing deleterious non-coding DNA variations remains poor for most common diseases. We previously reported that non-coding variations disrupting cis-overlapping motifs (CisOMs) of opposing transcription factors significantly affect enhancer activity. Analyzing publicly available ChIP-seq data for P53 and cMYC in human embryonic stem cells and mouse embryonic cells showed that [~]344-366 genomic regions are co-occupied by P53 and cMYC. We identified, on average, two CisOMs per region, suggesting that co-occupancy is evolutionarily conserved in vertebrates. Therefore, we designed in vitro experiments to uncover the significance of the co-occupancy and competitive binding and inhibition between P53 and cMYC on target gene expression. We found that treating U2OS cells with doxorubicin increased P53 protein level while reducing cMYC level. In contrast, no change in protein levels was observed in Raji cells. ChIP-seq analysis showed that 16-922 genomic regions were co-occupied by P53 and cMYC before and after treatment, and substitutions of cMYC signals by P53 were detected after doxorubicin treatment in U2OS. Around 187 expressed genes near co-occupied regions were altered at mRNA level according to RNA-seq data. We utilized a computational motif-matching approach to determine that changes in predicted P53 binding affinity by DNA variations in CisOMs of co-occupied elements significantly correlate with alterations in reporter gene expression. We performed a similar analysis using SNPs mapped in CisOMs for P53 and cMYC from ChIP-seq data in U2OS and Raji, and expression of target genes from the GTEx portal. We found a significant correlation between change in motif-predicted cMYC binding affinity by SNPs in CisOMs and gene expression. In conclusion, our study suggests a generally applicable approach to filter etiological non-coding variations associated with P53 and cMYC-dependent diseases. Author SummaryMost DNA variants associated with common complex diseases fall outside the protein-coding regions of the genome, making them hard to detect and relate to a function. Although many computational tools are available for prioritizing functional disease risk variants outside the protein-coding regions of the genome, the precision of prediction of these tools is mostly unreliable and hence not close to cancer risk prediction. This study brings to light a novel way to improve prediction accuracy of publicly available tools by integrating the impact of cis-overlapping binding sites of opposing cancer proteins, such as P53 and cMYC, in their analysis to filter out deleterious DNA variants outside the protein-coding regions of the human genome. Using a biology-based statistical approach, DNA variants within cis-overlapping motifs impacting the binding affinity of opposing transcription factors can significantly alter the expression of target genes and regulatory networks. This study brings us closer to developing a generally applicable approach capable of filtering etiological non-coding variations in co-occupied genomic regions of P53 and cMYC family members to improve disease risk assessment.

genetics↗

bioRxiv2021

TWIST1 interacts with adherens junction proteins during neural tube formation and regulates fate transition in cranial neural crest cells

Cell fate determination is a necessary and tightly regulated process for producing different cell types and structures during development. Cranial neural crest cells (CNCCs) are unique to vertebrate embryos and emerge from the neural fold borders into multiple cell lineages that differentiate into bone, cartilage, neurons, and glial cells. We previously reported that Irf6 genetically interacts with Twist1 during CNCC-derived tissue formation. Here, we investigated the mechanistic role of Twist1 and Irf6 at early stages of craniofacial development. Our data indicates that TWIST1 interacts with /{beta}/{gamma}-CATENINS during neural tube closure, and Irf6 is involved in the structural integrity of the neural tube. Twist1 suppresses Irf6 and other epithelial genes in CNCCs during epithelial-to-mesenchymal transition (EMT) process and cell migration. Conversely, a loss of Twist1 leads to a sustained expression of epithelial and cell adhesion markers in migratory CNCCs. Disruption of TWIST1 phosphorylation in vivo leads to epidermal blebbing, edema, neural tube defects, and CNCC-derived structural abnormalities. Altogether, this study describes an uncharacterized function of Twist1 and Irf6 in the neural tube and CNCCs and provides new target genes of Twist1 involved in cytoskeletal remodeling. Furthermore, the association between DNA variations within TWIST1 putative enhancers and human facial morphology is also investigated. SUMMARY STATEMENTThis study uncovers a new function of Twist1 in neural tube development and epithelial-to-mesenchymal transition in cranial neural crest cells. Data further shows that Twist1-interacting Irf6 is involved in regulating neural tube integrity.

developmental biology↗

bioRxiv2019

Medication-related osteonecrosis of the jaws after tooth extraction in senescent female mice treated with zoledronic acid: microtomographic, histological and immunohistochemical characterization

Treatment with cumulative dosages of zoledronic acid (ZA) in elderly patients is a risk factor for the development of medication-related osteonecrosis of the jaws (MRONJ), mainly related to surgical triggers such as tooth extraction. However, animal models for the investigation and understanding of MRONJ pathophysiology in senescent and postmenopausal stages remains to be developed and characterized. The aim of this study was to analyze MRONJ development in senescent female mice treated with cumulative dosages of ZA. For this purpose, twenty 129/Sv female mice, 64 weeks old, were treated with 0.9% saline solution as Control group (n=10), and with ZA at 250{micro}g/Kg (n=10), once a week, starting 4 weeks before the upper right incisor extraction and until the end of the experimental time points (7 days and 21 days). At 7 and 21 days, specimens were harvested for microCT, histological, birefringence and immunohistochemical analysis. Clinically, an incomplete epithelialization was observed in ZA group at 7 days and a delayed bone matrix mineralization and collagen maturation at 7 and 21 days compared to the controls. Controls revealed sockets filled with mature bone at 21 days as observed by microCT and birefringence, while ZA group presented delayed bone deposition at 7 and 21 days, as well increased leukocyte infiltration and blood clot at 7 days, and increased bone sequestrum and empty osteocyte lacunae at 21 days (p<0.05). Also, ZA group presented decreased quantity TGFb+ and Runx-2+ cells at 7 days, and decreased quantity of TRAP+ osteoclasts compared to the control at 21 days (p<0.05). Togheter, these data demonstrate the usefulness of this model to understanding the pathophysiology of MRONJ.

immunology↗