Ewsa-dependent regulation of Runx2 during zebrafish skeletogenesis

Ewing’s sarcoma is the second most common malignant bone cancer found in adolescents, and the genetic hallmark of this disease is the presence of the aberrant chimeric fusion protein EWS/FLI1. This fusion is induced by the t(11; 22) chromosomal translocation of EWS and FLI1, and is the most prominent and common characteristic of Ewing sarcoma tumors found in approximately 85-90% of reported cases. EWS/FLI1 has been shown to directly bind to and inhibit the function of endogenous EWS in a dominant manner. In this study, we seek to increase our understanding of the role of endogenous EWS during development and skeletogenesis to gain insight into the pathogenesis of the disease. Previously, we demonstrated the role of a zebrafish EWS homolog Ewsa by analyzing the phenotype of a maternal zygotic homozygous ewsa/ewsa mutant line (MZ ewsa/ewsa) of zebrafish null for Ewsa protein. Prehypertrophic chondrocytes of Meckel’s cartilage in 4dpf MZ ewsa/ewsa mutants fail to completely differentiate into hypertrophic chondrocytes, followed by structural defects in craniofacial bones (dentary and basihyal bones) at the adult stage. Based on these results, we sought to understand EWS’s involvement in skeletogenesis by asking if Ewsa regulates activity of critical transcription factors involved in chondrocyte development. We have previously shown that Ewsa directs chondrocyte differentiation through modulation of chondrogenesis master transcription factor Sox9. Runx2 is also known to play a critical role in differentiation of both chondrocytes and osteoblasts, so we have addressed whether and how Ewsa regulates Runx2 expression and transcriptional activity. We discovered that Runx2 protein expression dramatically increases in craniofacial chondrocytes of 4-6dpf MZ ewsa/ewsa compared to wt/wt embryos. We have also observed premature mineralization in MZ ewsa/ewsa embryos at 6dpf and 10dpf. MZ ewsa/ewsa fish also display a decrease in expression of collagen10a1, a hypertrophic-specific Runx2 target gene. These data together suggests that Ewsa regulates Runx2 expression and transcriptional activity during chondrogenesis and regulates mineralization of these domains. Additionally, our lab has previously discovered that adult MZ ewsa/ewsa fish display aberrant curved spines. Based on these results we asked how Ewsa is involved in the formation of the axial skeleton. Since Collagen2a1 is a critical component of notochord development as both a precursor to intervertebral disc (IVD) formation and as a component of notochord sheath extracellular matrix (ECM) we have previously asked if Ewsa regulates the collagen2a1a gene. Ewsa interacts with the col2a1a gene and col2a1 is upregulated in the notochord MZ ewsa/ewsa fish which suggests that the notochord sheath ECM is misregulated in mutant fish. Additionally, our analysis also revealed that notochord cells have failed to intercalate 5-10dpf and remain in a single layer. Based on our previous data and the data in this report, we hypothesize that Ewsa regulates axis development through regulation of collagen2a1 expression, which in turn allows for normal notochord sheath ECM distribution, which in turn allows for notochord cell intercalation and later IVD and vertebral body formation. This is the first in vivo evidence for regulation of RUNX2 by EWS during chondrogenesis and axial skeleton development

Ewing Sarcoma Eswa Protein Regulates Chondrogenesis of Meckel's Cartilage through Modulation of Sox9 in Zebrafish

Ewing sarcoma is the second most common skeletal (bone and cartilage) cancer in adolescents, and it is characterized by the expression of the aberrant chimeric fusion gene EWS/FLI1. Wild-type EWS has been proposed to play a role in mitosis, splicing and transcription. We have previously shown that EWS/FLI1 interacts with EWS, and it inhibits EWS activity in a dominant manner. Ewing sarcoma is a cancer that specifically develops in skeletal tissues, and although the above data suggests the significance of EWS, its role in chondrogenesis/skeletogenesis is not understood. To elucidate the function of EWS in skeletal development, we generated and analyzed a maternal zygotic (MZ) ewsa/ewsa line because the ewsa/wt and ewsa/ewsa zebrafish appeared to be normal and fertile. Compared with wt/wt, the Meckel’s cartilage of MZ ewsa/ewsa mutants had a higher number of craniofacial prehypertrophic chondrocytes that failed to mature into hypertrophic chondrocytes at 4 days post-fertilization (dpf). Ewsa interacted with Sox9, which is the master transcription factor for chondrogenesis. Sox9 target genes were either upregulated (ctgfa, ctgfb, col2a1a, and col2a1b) or downregulated (sox5, nog1, nog2, and bmp4) in MZ ewsa/ewsa embryos compared with the wt/wt zebrafish embryos. Among these Sox9 target genes, the chromatin immunoprecipitation (ChIP) experiment demonstrated that Ewsa directly binds to ctgfa and ctgfb loci. Consistently, immunohistochemistry showed that the Ctgf protein is upregulated in the Meckel’s cartilage of MZ ewsa/ewsa mutants. Together, we propose that Ewsa promotes the differentiation from prehypertrophic chondrocytes to hypertrophic chondrocytes of Meckel’s cartilage through inhibiting Sox9 binding site of the ctgf gene promoter. Because Ewing sarcoma specifically develops in skeletal tissue that is originating from chondrocytes, this new role of EWS may provide a potential molecular basis of its pathogenesis.This manuscript was supported by the Massman Family Ewing Sarcoma Research Fund, the Sarcoma Foundation of America, P20RR016475 / P20GM103418 and P20 RR032682-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Haplotype-resolved diverse human genomes and integrated analysis of structural variation.

Long-read and strand-specific sequencing technologies together facilitate the de novo assembly of high-quality haplotype-resolved human genomes without parent-child trio data. We present 64 assembled haplotypes from 32 diverse human genomes. These highly contiguous haplotype assemblies (average minimum contig length needed to cover 50% of the genome: 26 million base pairs) integrate all forms of genetic variation, even across complex loci. We identified 107,590 structural variants (SVs), of which 68% were not discovered with short-read sequencing, and 278 SV hotspots (spanning megabases of gene-rich sequence). We characterized 130 of the most active mobile element source elements and found that 63% of all SVs arise through homology-mediated mechanisms. This resource enables reliable graph-based genotyping from short reads of up to 50,340 SVs, resulting in the identification of 1526 expression quantitative trait loci as well as SV candidates for adaptive selection within the human population