Hochman, M. | Search bioRxiv

bioRxiv2023

Hippo and Wnt pathways are impaired in Port Wine Birthmark-derived induced pluripotent stem cells and endothelial cells

AbstractO_ST_ABSBackgroundC_ST_ABSPort wine birthmark (PWB) is a congenital vascular malformation resulting from developmentally defective endothelial cells (ECs). Developing clinically relevant disease models for PWB studies is currently an unmet need. ObjectiveOur study aims to generate PWB-derived induced pluripotent stem cells (iPSCs) and iPSC-derived ECs that preserve disease-related phenotypes. MethodsPWB iPSCs were generated by reprogramming lesional dermal fibroblasts and differentiated into ECs. RNA-seq was performed to identify differentially expressed genes (DEGs) and enriched pathways. The functional phenotypes of iPSC-derived ECs were characterized by capillary-like structure (CLS) formation in vitro and Geltrex plug-in assay in vivo. ResultsHuman PWB and control iPSC lines were generated through reprogramming of dermal fibroblasts by introducing the "Yamanaka factors" (Oct3/4, Sox2, Klf4, c-Myc) into them; the iPSCs were successfully differentiated into ECs. These iPSCs and their derived ECs were validated by expression of a series of stem cell and EC biomarkers, respectively. PWB iPSC-derived ECs showed impaired CLS in vitro with larger perimeters and thicker branches as compared to control iPSC-derived ECs. In the plug-in assay, perfused human vasculature formed by PWB iPSC- derived ECs showed bigger perimeters and greater densities than those formed by control iPSC- derived ECs in severe combined immune deficient (SCID) mice. The transcriptome analysis showed that dysregulated pathways of stem cell differentiation, Hippo, Wnt, and focal adhesion persisted through differentiation of PWB iPSCs to ECs. Functional enrichment analysis showed that Hippo and Wnt pathway-related PWB DEGs are enriched for vasculature development, tube morphology, endothelium development, and EC differentiation. Further, members of the zinc finger (ZNF) gene family were overrepresented among the DEGs in PWB iPSCs. ZNF DEGs confer significant functions in transcriptional regulation, chromatin remodeling, protein ubiquitination, and retinoic acid receptor signaling. Furthermore, NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were dysregulated in PWB ECs as readouts of impaired differentiation. ConclusionsPWB iPSC-derived ECs render a novel and clinically-relevant disease model by retaining pathological phenotypes. Our data demonstrate multiple pathways, such as Hippo and Wnt, NF-kappa B, TNF, MAPK, and cholesterol metabolism, are dysregulated, which may contribute to the development of differentiation-defective ECs in PWB. Bulleted statementsO_ST_ABSWhat is already known about this topic?C_ST_ABSO_LIPort Wine Birthmark (PWB) is a congenital vascular malformation with an incidence rate of 0.1 - 0.3 % per live births. C_LIO_LIPWB results from developmental defects in the dermal vasculature; PWB endothelial cells (ECs) have differentiational impairments. C_LIO_LIPulse dye laser (PDL) is currently the preferred treatment for PWB; unfortunately, the efficacy of PDL treatment of PWB has not improved over the past three decades. C_LI What does this study add?O_LIInduced pluripotent stem cells (iPSCs) were generated from PWB skin fibroblasts and differentiated into ECs. C_LIO_LIPWB ECs recapitulated their pathological phenotypes such as forming enlarged blood vessels in vitro and in vivo. C_LIO_LIHippo and Wnt pathways were dysregulated in PWB iPSCs and ECs. C_LIO_LIZinc-finger family genes were overrepresented among the differentially expressed genes in PWB iPSCs. C_LIO_LIDysregulated NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were enriched in PWB ECs. C_LI What is the translational message?O_LITargeting Hippo and Wnt pathways and Zinc-finger family genes could restore the physiological differentiation of ECs. C_LIO_LITargeting NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways could mitigate the pathological progression of PWB. C_LIO_LIThese mechanisms may lead to the development of paradigm-shifting therapeutic interventions for PWB. C_LI

cell biology↗

bioRxiv2018

Nuclear TARBP2 drives oncogenic dysregulation of RNA splicing and decay

Post-transcriptional regulation of RNA stability is a key step in gene expression control. We describe a regulatory program, mediated by the double-stranded RNA binding protein TARBP2, that controls RNA stability in the nucleus. TARBP2 binding to pre-mRNAs results in increased intron retention, subsequently leading to targeted degradation of TARBP2-bound transcripts. This is mediated by TARBP2 recruitment of the m6A RNA methylation machinery to its target transcripts, where deposition of m6A marks influences the recruitment of splicing regulators, inhibiting efficient splicing. Interactions between TARBP2 and the nucleoprotein TPR then promote degradation of these TARBP2-bound transcripts by the nuclear exosome. Additionally, analysis of clinical gene expression datasets revealed a functional role for this TARBP2 pathway in lung cancer. Using xenograft mouse models, we find that TARBP2 impacts tumor growth in the lung, and that this function is dependent on TARBP2-mediated destabilization of ABCA3 and FOXN3. Finally, we establish the transcription factor ZNF143 as an upstream regulator of TARBP2 expression.\n\nRESEARCH HIGHLIGHTSO_LIThe RNA-binding protein TARBP2 controls the stability of its target transcripts in the nucleus\nC_LIO_LINuclear TARBP2 recruits the methyltransferase complex to deposit m6A marks on its target transcripts\nC_LIO_LITARBP2 and m6A-mediated interactions with splicing and nuclear RNA surveillance complexes result in target transcript intron retention and decay.\nC_LIO_LIIncreased TARBP2 expression is associated with lung cancer and promotes lung cancer growth in vivo.\nC_LIO_LIThe transcription factor ZNF143 drives oncogenic TARBP2 upregulation in lung cancer.\nC_LI

molecular biology↗

bioRxiv2017

Sequence, Structure and Context Preferences of Human RNA Binding Proteins

Production of functional cellular RNAs involves multiple processing and regulatory steps principally mediated by RNA binding proteins (RBPs). Here we present the affinity landscapes of 78 human RBPs using an unbiased assay that determines the sequence, structure, and context preferences of an RBP in vitro from deep sequencing of bound RNAs. Analyses of these data revealed several interesting patterns, including unexpectedly low diversity of RNA motifs, implying frequent convergent evolution of binding specificity toward a relatively small set of RNA motifs, many with low compositional complexity. Offsetting this trend, we observed extensive preferences for contextual features outside of core RNA motifs, including spaced \"bipartite\" motifs, biased flanking nucleotide context, and bias away from or towards RNA structure. These contextual features are likely to enable targeting of distinct subsets of transcripts by different RBPs that recognize the same core motif. Our results enable construction of \"RNA maps\" of RBP activity without requiring crosslinking-based assays, and provide unprecedented depth of information on the interaction of RBPs with RNA.

biochemistry↗