Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3.

Factors that sustain self-renewal of mouse embryonic stem cells (ESCs) are well described. In contrast, the machinery regulating exit from pluripotency is ill defined. In a large-scale small interfering RNA (siRNA) screen, we found that knockdown of the tumor suppressors Folliculin (Flcn) and Tsc2 prevent ESC commitment. Tsc2 lies upstream of mammalian target of rapamycin (mTOR), whereas Flcn acts downstream and in parallel. Flcn with its interaction partners Fnip1 and Fnip2 drives differentiation by restricting nuclear localization and activity of the bHLH transcription factor Tfe3. Conversely, enforced nuclear Tfe3 enables ESCs to withstand differentiation conditions. Genome-wide location and functional analyses showed that Tfe3 directly integrates into the pluripotency circuitry through transcriptional regulation of Esrrb. These findings identify a cell-intrinsic rheostat for destabilizing ground-state pluripotency to allow lineage commitment. Congruently, stage-specific subcellular relocalization of Tfe3 suggests that Flcn-Fnip1/2 contributes to developmental progression of the pluripotent epiblast in vivo.

Neural G0: a quiescent-like state found in neuroepithelial-derived cells and glioma.

Single-cell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA-seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescent-like state in neuroepithelial-derived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in non-dividing neural progenitors. Putative glioblastoma stem-like cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down-regulation of quiescence-associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescent-like state found in neuroepithelial-derived cells and gliomas.

Genome-wide RNAi screens in human brain tumor isolates reveal a novel viability requirement for PHF5A.

To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the plant homeodomain (PHD)-finger domain protein PHF5A as differentially required for GSC expansion, as compared with untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancer-specific roles in RNA splicing. We found that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3' splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown in GSCs and also in NSCs or fibroblasts overexpressing MYC. Furthermore, PHF5A inhibition compromised GSC tumor formation in vivo and inhibited growth of established GBM patient-derived xenograft tumors. Our results demonstrate a novel viability requirement for PHF5A to maintain proper exon recognition in brain tumor-initiating cells and may provide new inroads for novel anti-GBM therapeutic strategies.

Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis.

We developed a versatile, high-throughput genetic screening strategy by coupling gene mutagenesis and expression profiling technologies. Using a retroviral gene-trap vector optimized for efficient mutagenesis and cloning, we randomly disrupted genes in mouse embryonic stem (ES) cells and amplified them to construct a cDNA microarray. With this gene-trap array, we show that transcriptional target genes of platelet-derived growth factor (PDGF) can be efficiently and reliably identified in physiologically relevant cells and are immediately accessible to genetic studies to determine their in vivo roles and relative contributions to PDGF-regulated developmental processes. The same platform can be used to search for genes of specific biological relevance in a broad array of experimental settings, providing a fast track from gene identification to functional validation.

N6-methyladenosine mRNA marking promotes selective translation of regulons required for human erythropoiesis.

Many of the regulatory features governing erythrocyte specification, maturation, and associated disorders remain enigmatic. To identify new regulators of erythropoiesis, we utilize a functional genomic screen for genes affecting expression of the erythroid marker CD235a/GYPA. Among validating hits are genes coding for the N6-methyladenosine (m6A) mRNA methyltransferase (MTase) complex, including, METTL14, METTL3, and WTAP. We demonstrate that m6A MTase activity promotes erythroid gene expression programs through selective translation of ~300 m6A marked mRNAs, including those coding for SETD histone methyltransferases, ribosomal components, and polyA RNA binding proteins. Remarkably, loss of m6A marks results in dramatic loss of H3K4me3 marks across key erythroid-specific KLF1 transcriptional targets (e.g., Heme biosynthesis genes). Further, each m6A MTase subunit and a subset of their mRNAs targets are required for human erythroid specification in primary bone-marrow derived progenitors. Thus, m6A mRNA marks promote the translation of a network of genes required for human erythropoiesis.

Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms.

Receptor tyrosine kinases (RTKs) direct diverse cellular and developmental responses by stimulating a relatively small number of overlapping signaling pathways. Specificity may be determined by RTK expression patterns or by differential activation of individual signaling pathways. To address this issue we generated knock-in mice in which the extracellular domain of the mouse platelet-derived growth factor alpha receptor (PDGFalphaR) is fused to the cytosolic domain of Drosophila Torso (alpha(Tor)) or the mouse fibroblast growth factor receptor 1 (alpha(FR)). alpha(Tor) homozygous embryos exhibit significant rescue of neural crest and angiogenesis defects normally found in PDGFalphaR-null embryos yet fail to rescue skeletal or extraembryonic defects. This phenotype was associated with the ability of alpha(Tor) to stimulate the mitogen-activated protein (MAP) kinase pathway to near wild-type levels but failure to completely activate other pathways, such as phosphatidylinositol (PI) 3-kinase. The alpha(FR) chimeric receptor fails to rescue any aspect of the PDGFalphaR-null phenotype. Instead, alpha(FR) expression leads to a gain-of-function phenotype highlighted by ectopic bone development. The alpha(FR) phenotype was associated with a failure to limit MAP kinase signaling and to engage significant PI3-kinase response. These results suggest that precise regulation of divergent downstream signaling pathways is critical for specification of RTK function.

ZNF131 suppresses centrosome fragmentation in glioblastoma stem-like cells through regulation of HAUS5.

Zinc finger domain genes comprise ~3% of the human genome, yet many of their functions remain unknown. Here we investigated roles for the vertebrate-specific BTB domain zinc finger gene ZNF131 in the context of human brain tumors. We report that ZNF131 is broadly required for Glioblastoma stem-like cell (GSC) viability, but dispensable for neural progenitor cell (NPC) viability. Examination of gene expression changes after ZNF131 knockdown (kd) revealed that ZNF131 activity notably promotes expression of Joubert Syndrome ciliopathy genes, including KIF7, NPHP1, and TMEM237, as well as HAUS5, a component of Augmin/HAUS complex that facilitates microtubule nucleation along the mitotic spindle. Of these genes only kd of HAUS5 displayed GSC-specific viability loss. Critically, HAUS5 ectopic expression was sufficient to suppress viability defects of ZNF131 kd cells. Moreover, ZNF131 and HAUS5 kd phenocopied each other in GSCs, each causing: mitotic arrest, centrosome fragmentation, loss of Augmin/HAUS complex on the mitotic spindle, and loss of GSC self-renewal and tumor formation capacity. In control NPCs, we observed centrosome fragmentation and lethality only when HAUS5 kd was combined with kd of HAUS2 or HAUS4, demonstrating that the complex is essential in NPCs, but that GSCs have heightened requirement. Our results suggest that GSCs differentially rely on ZNF131-dependent expression of HAUS5 as well as the Augmin/HAUS complex activity to maintain the integrity of centrosome function and viability.

Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells.

To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

BuGZ is required for Bub3 stability, Bub1 kinetochore function, and chromosome alignment.

During mitosis, the spindle assembly checkpoint (SAC) monitors the attachment of kinetochores (KTs) to the plus ends of spindle microtubules (MTs) and prevents anaphase onset until chromosomes are aligned and KTs are under proper tension. Here, we identify a SAC component, BuGZ/ZNF207, from an RNAi viability screen in human glioblastoma multiforme (GBM) brain tumor stem cells. BuGZ binds to and stabilizes Bub3 during interphase and mitosis through a highly conserved GLE2p-binding sequence (GLEBS) domain. Inhibition of BuGZ results in loss of both Bub3 and its binding partner Bub1 from KTs, reduction of Bub1-dependent phosphorylation of centromeric histone H2A, attenuation of KT-based Aurora B kinase activity, and lethal chromosome congression defects in cancer cells. Phylogenetic analysis indicates that BuGZ orthologs are highly conserved among eukaryotes, but are conspicuously absent from budding and fission yeasts. These findings suggest that BuGZ has evolved to facilitate Bub3 activity and chromosome congression in higher eukaryotes.

Cancer-Specific requirement for BUB1B/BUBR1 in human brain tumor isolates and genetically transformed cells.

UNLABELLED: To identify new candidate therapeutic targets for glioblastoma multiforme, we combined functional genetics and glioblastoma network modeling to identify kinases required for the growth of patient-derived brain tumor-initiating cells (BTIC) but that are dispensable to proliferating human neural stem cells (NSC). This approach yielded BUB1B/BUBR1, a critical mitotic spindle checkpoint player, as the top-scoring glioblastoma lethal kinase. Knockdown of BUB1B inhibited expansion of BTIC isolates, both in vitro and in vivo, without affecting proliferation of NSCs or astrocytes. Mechanistic studies revealed that BUB1B's GLE2p-binding sequence (GLEBS) domain activity is required to suppress lethal kinetochore-microtubule (KT-MT) attachment defects in glioblastoma isolates and genetically transformed cells with altered sister KT dynamics, which likely favor KT-MT instability. These results indicate that glioblastoma tumors have an added requirement for BUB1B to suppress lethal consequences of altered KT function and further suggest that sister KT measurements may predict cancer-specific sensitivity to BUB1B inhibition and perhaps other mitotic targets that affect KT-MT stability. SIGNIFICANCE: Currently, no effective therapies are available for glioblastoma, the most frequent and aggressive brain tumor. Our results suggest that targeting the GLEBS domain activity of BUB1B may provide a therapeutic window for glioblastoma, as the GLEBS domain is nonessential in untransformed cells. Moreover, the results further suggest that sister KT distances at metaphase may predict sensitivity to anticancer therapeutics targeting KT function.