Growth factor independence 1B (GFI1B) coordinates assembly of transcriptional repressor complexes comprised of corepressors and histone-modifying enzymes to control gene expression programs governing lineage allocation in hematopoiesis. Enforced expression of GFI1B in K562 erythroleukemia cells favors erythroid over megakaryocytic differentiation, providing a platform to define molecular determinants of binary fate decisions triggered by GFI1B. We deployed proteome-wide proximity labeling to identify factors whose inclusion in GFI1B complexes depends upon GFI1B’s obligate effector, lysine-specific demethylase 1 (LSD1). We show that GFI1B preferentially recruits core and putative elements of the BRAF-histone deacetylase (HDAC) (BHC) chromatin-remodeling complex (LSD1, RCOR1, HMG20A, HMG20B, HDAC1, HDAC2, PHF21A, GSE1, ZMYM2, and ZNF217) in an LSD1-dependent manner to control acquisition of erythroid traits by K562 cells. Among these elements, depletion of both HMG20A and HMG20B or of GSE1 blocks GFI1B-mediated erythroid differentiation, phenocopying impaired differentiation brought on by LSD1 depletion or disruption of GFI1B-LSD1 binding. These findings demonstrate the central role of the GFI1B-LSD1 interaction as a determinant of BHC complex recruitment to enable cell fate decisions driven by GFI1B.

Pediatric cancer is a leading cause of death idraw n children and adolescents. Improvements in pediatric cancer treatment that include the alleviation of longterm adverse effects require a deeper understanding of the genetic, epigenetic, and developmental factors driving these cancers. Here, we review how the unique attributes of the zebrafish model system in embryology, imaging, and scalability have been used to identify new mechanisms of tumor initiation, progression, and relapse and for drug discovery. We focus on zebrafish models of leukemias, neural tumors and sarcomasthe most common and difficult childhood cancers to treat. Childhood and Adolescent CancerPediatric cancer is one of the leading causes of death in children and adolescents (age 0-19 years) and is mainly comprised of leukemias, cancers of the nervous system, and sarcomas [1]. Recent genomic profiling efforts to better understand the etiology of this group of diseases have enabled the stratification of tumor types based on molecular signatures and have led to the identification of potential genetic drivers and cooperating molecular events that underlie the development of different pediatric cancers. Most pediatric malignancies are mutationally quiet and are thought to be driven by a single driver gene, a fusion oncoprotein, or structural/copy number alterations [2,3]. In contrast, adult tumors frequently exhibit high mutational burdens, likely due to a longer period of mutational acquisition under selective pressure [2,3]. Despite these differences, however, pediatric cancer treatments are still largely modeled after treatments designed for the adult version of the disease and can cause debilitating, long-term side effects when administered to children. The development of robust preclinical pediatric cancer models that accurately recapitulate these diseases will ultimately be necessary for the design of more precise, targeted therapies to improve outcomes for pediatric cancer patients. Here, we discuss how the use of zebrafish has advanced the pediatric cancer field as a preclinical model for gene and drug discovery. HighlightsZebrafish develop tumors that are histologically and genetically similar to human tumors. Zebrafish enable the rapid identification of molecular drivers of tumor development and can be used to model cancers with unknown cells of origin using heatshock and β-actin promoters.Zebrafish models are amenable to highthroughput drug screening through larval drug submersion approaches, as well as transplantation of primary patient tumors into immunocompromised lines.Tumor-cell dynamics can be visualized in vivo throughout the lifetime of the animal by coupling oncogenes to fluorescent markers.

RNA splicing factors are essential for the viability of all eukaryotic cells; however, in metazoans some cell types are exquisitely sensitive to disruption of splicing factors. Neuronal cells represent one such cell type, and defects in RNA splicing factors can lead to neurodegenerative diseases. The basis for this tissue selectivity is not well understood owing to difficulties in analyzing the consequences of splicing factor defects in whole-animal systems. Here, we use zebrafish mutants to show that loss of spliceosomal components, including splicing factor 3b, subunit 1 (sf3b1), causes increased DNA double-strand breaks and apoptosis in embryonic neurons. Moreover, these mutants show a concomitant accumulation of R-loops, which are non-canonical nucleic acid structures that promote genomic instability. Dampening R-loop formation by conditional induction of ribonuclease H1 in sf3b1 mutants reduced neuronal DNA damage and apoptosis. These findings show that splicing factor dysfunction leads to R-loop accumulation and DNA damage that sensitizes embryonic neurons to apoptosis. Our results suggest that diseases associated with splicing factor mutations could be susceptible to treatments that modulate R-loop levels.