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.

Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells.

Mouse embryonic stem cells (mESCs) are biased toward producing embryonic rather than extraembryonic endoderm fates. Here, we identify the mechanism of this barrier and report that the histone deacetylase Hdac3 and the transcriptional corepressor Dax1 cooperatively limit the lineage repertoire of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor Gata6. This restriction is opposed by the pluripotency transcription factors Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC transcriptional network.

Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2.

Developmental cell fate specification is a unidirectional process that can be reverted in response to injury or experimental reprogramming. Whether differentiation and de-differentiation trajectories intersect mechanistically is unclear. Here, we performed comparative screening in lineage-related mouse naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), and identified the constitutively expressed zinc finger transcription factor (TF) Zfp281 as a bidirectional regulator of cell state interconversion. We showed that subtle chromatin binding changes in differentiated cells translate into activation of the histone H3 lysine 9 (H3K9) methyltransferase Ehmt1 and stabilization of the zinc finger TF Zic2 at enhancers and promoters. Genetic gain-of-function and loss-of-function experiments confirmed a critical role of Ehmt1 and Zic2 downstream of Zfp281 both in driving exit from the ESC state and in restricting reprogramming of EpiSCs. Our study reveals that cell type-invariant chromatin association of Zfp281 provides an interaction platform for remodeling the cis-regulatory network underlying cellular plasticity.

Activity-dependent neuroprotective protein recruits HP1 and CHD4 to control lineage-specifying genes.

De novo mutations in ADNP, which encodes activity-dependent neuroprotective protein (ADNP), have recently been found to underlie Helsmoortel-Van der Aa syndrome, a complex neurological developmental disorder that also affects several other organ functions 1 . ADNP is a putative transcription factor that is essential for embryonic development 2 . However, its precise roles in transcriptional regulation and development are not understood. Here we show that ADNP interacts with the chromatin remodeller CHD4 and the chromatin architectural protein HP1 to form a stable complex, which we refer to as ChAHP. Besides mediating complex assembly, ADNP recognizes DNA motifs that specify binding of ChAHP to euchromatin. Genetic ablation of ChAHP components in mouse embryonic stem cells results in spontaneous differentiation concomitant with premature activation of lineage-specific genes and in a failure to differentiate towards the neuronal lineage. Molecularly, ChAHP-mediated repression is fundamentally different from canonical HP1-mediated silencing: HP1 proteins, in conjunction with histone H3 lysine 9 trimethylation (H3K9me3), are thought to assemble broad heterochromatin domains that are refractory to transcription. ChAHP-mediated repression, however, acts in a locally restricted manner by establishing inaccessible chromatin around its DNA-binding sites and does not depend on H3K9me3-modified nucleosomes. Together, our results reveal that ADNP, via the recruitment of HP1 and CHD4, regulates the expression of genes that are crucial for maintaining distinct cellular states and assures accurate cell fate decisions upon external cues. Such a general role of ChAHP in governing cell fate plasticity may explain why ADNP mutations affect several organs and body functions and contribute to cancer progression1,3,4. Notably, we found that the integrity of the ChAHP complex is disrupted by nonsense mutations identified in patients with Helsmoortel-Van der Aa syndrome, and this could be rescued by aminoglycosides that suppress translation termination 5 . Therefore, patients might benefit from therapeutic agents that are being developed to promote ribosomal read-through of premature stop codons6,7.

The BTB-domain transcription factor ZBTB2 recruits chromatin remodelers and a histone chaperone during the exit from pluripotency.

Transcription factors (TFs) harboring broad-complex, tramtrack, and bric-a-brac (BTB) domains play important roles in development and disease. These BTB domains are thought to recruit transcriptional modulators to target DNA regions. However, a systematic molecular understanding of the mechanism of action of this TF family is lacking. Here, we identify the zinc finger BTB-TF Zbtb2 from a genetic screen for regulators of exit from pluripotency and demonstrate that its absence perturbs embryonic stem cell differentiation and the gene expression dynamics underlying peri-implantation development. We show that ZBTB2 binds the chromatin remodeler Ep400 to mediate downstream transcription. Independently, the BTB domain directly interacts with nucleosome remodeling and deacetylase and histone chaperone histone regulator A. Nucleosome remodeling and deacetylase recruitment is a common feature of BTB TFs, and based on phylogenetic analysis, we propose that this is a conserved evolutionary property. Binding to UBN2, in contrast, is specific to ZBTB2 and requires a C-terminal extension of the BTB domain. Taken together, this study identifies a BTB-domain TF that recruits chromatin modifiers and a histone chaperone during a developmental cell state transition and defines unique and shared molecular functions of the BTB-domain TF family.

De novo mutations in the X-linked TFE3 gene cause intellectual disability with pigmentary mosaicism and storage disorder-like features.

INTRODUCTION: Pigmentary mosaicism (PM) manifests by pigmentation anomalies along Blaschko's lines and represents a clue toward the molecular diagnosis of syndromic intellectual disability (ID). Together with new insights on the role for lysosomal signalling in embryonic stem cell differentiation, mutations in the X-linked transcription factor 3 (TFE3) have recently been reported in five patients. Functional analysis suggested these mutations to result in ectopic nuclear gain of functions. MATERIALS AND METHODS: Subsequent data sharing allowed the clustering of de novo TFE3 variants identified by exome sequencing on DNA extracted from leucocytes in patients referred for syndromic ID with or without PM. RESULTS: We describe the detailed clinical and molecular data of 17 individuals harbouring a de novo TFE3 variant, including the patients that initially allowed reporting TFE3 as a new disease-causing gene. The 12 females and 5 males presented with pigmentation anomalies on Blaschko's lines, severe ID, epilepsy, storage disorder-like features, growth retardation and recognisable facial dysmorphism. The variant was at a mosaic state in at least two male patients. All variants were missense except one splice variant. Eleven of the 13 variants were localised in exon 4, 2 in exon 3, and 3 were recurrent variants. CONCLUSION: This series further delineates the specific storage disorder-like phenotype with PM ascribed to de novo TFE3 mutation in exons 3 and 4. It confirms the identification of a novel X-linked human condition associated with mosaicism and dysregulation within the mechanistic target of rapamycin (mTOR) pathway, as well as a link between lysosomal signalling and human development.

Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage.

Drosophila neuroblasts and ovarian stem cells are well characterized models for stem cell biology. In both cell types, one daughter cell self-renews continuously while the other undergoes a limited number of divisions, stops to proliferate mitotically and differentiates. Whereas neuroblasts segregate the Trim-NHL (tripartite motif and Ncl-1, HT2A and Lin-41 domain)-containing protein Brain tumour (Brat) into one of the two daughter cells, ovarian stem cells are regulated by an extracellular signal from the surrounding stem cell niche. After division, one daughter cell looses niche contact. It undergoes 4 transit-amplifying divisions to form a cyst of 16 interconnected cells that reduce their rate of growth and stop to proliferate mitotically. Here we show that the Trim-NHL protein Mei-P26 (refs 7, 8) restricts growth and proliferation in the ovarian stem cell lineage. Mei-P26 expression is low in stem cells but is strongly induced in 16-cell cysts. In mei-P26 mutants, transit-amplifying cells are larger and proliferate indefinitely leading to the formation of an ovarian tumour. Like brat, mei-P26 regulates nucleolar size and can induce differentiation in Drosophila neuroblasts, suggesting that these genes act through the same pathway. We identify Argonaute-1, a component of the RISC complex, as a common binding partner of Brat and Mei-P26, and show that Mei-P26 acts by inhibiting the microRNA pathway. Mei-P26 and Brat have a similar domain composition that is also found in other tumour suppressors and might be a defining property of a new family of microRNA regulators that act specifically in stem cell lineages.

The tumor suppressor WT1 drives progenitor cell progression and epithelialization to prevent Wilms tumorigenesis in human kidney organoids.

Wilms tumor is the most widespread kidney cancer in children and frequently associated with homozygous loss of the tumor suppressor WT1. Pediatric tumorigenesis is largely inaccessible in humans. Here, we develop a human kidney organoid model for Wilms tumor formation and show that deletion of WT1 during organoid development induces overgrowth of kidney progenitor cells at the expense of differentiating glomeruli and tubules. Functional and gene expression analyses demonstrate that absence of WT1 halts progenitor cell progression at a pre-epithelialized cell state and recapitulates the transcriptional changes detected in a subgroup of Wilms tumor patients with ectopic myogenesis. By "transplanting" WT1 mutant cells into wild-type kidney organoids, we find that their propagation requires an untransformed microenvironment. This work defines the role of WT1 in kidney progenitor cell progression and tumor suppression, and establishes human kidney organoids as a phenotypic model for pediatric tumorigenesis.

Fragile X protein functions with lgl and the par complex in flies and mice.

Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss of function for the Fragile X Mental Retardation 1 gene (FMR1). FMR1 protein (FMRP) has specific mRNA targets and is thought to be involved in their transport to subsynaptic sites as well as translation regulation. We report a saturating genetic screen of the Drosophila autosomal genome to identify functional partners of dFmr1. We recovered 19 mutations in the tumor suppressor lethal (2) giant larvae (dlgl) gene and 90 mutations at other loci. dlgl encodes a cytoskeletal protein involved in cellular polarity and cytoplasmic transport and is regulated by the PAR complex through phosphorylation. We provide direct evidence for a Fmrp/Lgl/mRNA complex, which functions in neural development in flies and is developmentally regulated in mice. Our data suggest that Lgl may regulate Fmrp/mRNA sorting, transport, and anchoring via the PAR complex.

Sequential roles of Cdc42, Par-6, aPKC, and Lgl in the establishment of epithelial polarity during Drosophila embryogenesis.

How epithelial cells subdivide their plasma membrane into an apical and a basolateral domain is largely unclear. In Drosophila embryos, epithelial cells are generated from a syncytium during cellularization. We show here that polarity is established shortly after cellularization when Par-6 and the atypical protein kinase C concentrate on the apical side of the newly formed cells. Apical localization of Par-6 requires its interaction with activated Cdc42 and dominant-active or dominant-negative Cdc42 disrupt epithelial polarity, suggesting that activation of this GTPase is crucial for the establishment of epithelial polarity. Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Genetic and biochemical experiments suggest that phosphorylation by aPKC inactivates Lgl on the apical side. On the basolateral side, Lgl is active and excludes Par-6 from the cell cortex, suggesting that complementary cortical domains are maintained by mutual inhibition of aPKC and Lgl on opposite sides of an epithelial cell.

Lysosomal Signaling Licenses Embryonic Stem Cell Differentiation via Inactivation of Tfe3.

Self-renewal and differentiation of pluripotent murine embryonic stem cells (ESCs) is regulated by extrinsic signaling pathways. It is less clear whether cellular metabolism instructs developmental progression. In an unbiased genome-wide CRISPR/Cas9 screen, we identified components of a conserved amino-acid-sensing pathway as critical drivers of ESC differentiation. Functional analysis revealed that lysosome activity, the Ragulator protein complex, and the tumor-suppressor protein Folliculin enable the Rag GTPases C and D to bind and seclude the bHLH transcription factor Tfe3 in the cytoplasm. In contrast, ectopic nuclear Tfe3 represses specific developmental and metabolic transcriptional programs that are associated with peri-implantation development. We show differentiation-specific and non-canonical regulation of Rag GTPase in ESCs and, importantly, identify point mutations in a Tfe3 domain required for cytoplasmic inactivation as potentially causal for a human developmental disorder. Our work reveals an instructive and biomedically relevant role of metabolic signaling in licensing embryonic cell fate transitions.

Phosphorylation-induced autoinhibition regulates the cytoskeletal protein Lethal (2) giant larvae.

During asymmetric cell division, cell fate determinants localize asymmetrically and segregate into one of the two daughter cells. In Drosophila neuroblasts, the asymmetric localization of cell fate determinants to the basal cell cortex requires aPKC. aPKC localizes to the apical cell cortex and phosphorylates the cytoskeletal protein Lethal (2) giant larvae (Lgl). Upon phosphorylation, Lgl dissociates from the cytoskeleton and becomes inactive. Here, we show that phosphorylation regulates Lgl by allowing an autoinhibitory interaction of the N terminus with the C terminus of the protein. We demonstrate that interaction with the cytoskeleton is mediated by a C-terminal domain while the N terminus is not required. Instead, the N terminus can bind to the C terminus and can compete for binding to the cytoskeleton. Interaction between the N- and C-terminal domains requires phosphorylation of Lgl by aPKC. Our results suggest that unphosphorylated, active Lgl exists in an open conformation that interacts with the cytoskeleton while phosphorylation changes the protein to an autoinhibited state.

Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction.

BACKGROUND: Shot (previously named Kakapo), is a Drosophila Plakin family member containing both Actin binding and microtubule binding domains. In Drosophila, it is required for a wide range of processes, including axon extension, dendrite formation, axonal terminal arborization at the neuromuscular junction, tendon cell development, and adhesion of wing epithelium. RESULTS: To address how Shot exerts its activity at the molecular level, we investigated the molecular interactions of Shot with candidate proteins in mature larval tendon cells. We show that Shot colocalizes with EB1/APC1 and with a compact microtubule array extending between the muscle-tendon junction and the cuticle. Shot forms a protein complex with EB1 via its C-terminal EF-hands and GAS2-containing domains. In tendon cells with reduced Shot activity, EB1/APC1 dissociate from the muscle-tendon junction, and the microtubule array elongates. The resulting tendon cell, although associated with the muscle and the cuticle ends, loses its stress resistance and elongates. CONCLUSIONS: Our results suggest that Shot mediates tendon stress resistance by the organization of a compact microtubule network at the muscle-tendon junction. This is achieved by Shot association with the cytoplasmic faces of the basal hemiadherens junction and with the EB1/APC1 complex.

Charting Developmental Dissolution of Pluripotency.

The formation of tissues and organs during metazoan development begs fundamental questions of cellular plasticity: How can the very same genome program have diverse cell types? How do cell identity programs unfold during development in space and time? How can defects in these mechanisms cause disease and also provide opportunities for therapeutic intervention? And ultimately, can developmental programs be exploited for bioengineering tissues and organs? Understanding principle designs of cellular identity and developmental progression is crucial for providing answers. Here, I will discuss how the capture of embryonic pluripotency in murine embryonic stem cells (ESCs) in vitro has allowed fundamental insights into the molecular underpinnings of a developmental cell state and how its ordered disassembly during differentiation prepares for lineage specification.

The tumor suppressors Brat and Numb regulate transit-amplifying neuroblast lineages in Drosophila.

In both vertebrates and insects, neurons typically arise from neural stem cells or terminally dividing intermediate progenitors. Here, we describe another mode of neurogenesis where neural stem cells generate secondary precursors that undergo multiple rounds of self-renewing transit-amplifying divisions. We identify the Posterior Asense-Negative (PAN) neuroblasts, which do not express the transcription factors Asense or Prospero. PAN neuroblasts rely on the segregating determinants Numb and Brat to generate smaller, secondary neuroblasts that in turn give rise to ganglion mother cells (GMCs) and neurons throughout larval development. In brat or numb mutants, misspecified secondary neuroblasts are unable to produce differentiated progeny and initiate tumor-like overgrowth. In prospero mutants, however, tumors arise from GMCs while secondary neuroblasts are correctly specified. Our data describe a transit-amplifying lineage in the Drosophila nervous system and suggest that different vulnerabilities in intermediate cell types can affect the outcome of tumor suppressor loss in stem cell lineages.

Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells.

How stem cells generate both differentiating and self-renewing daughter cells is unclear. Here, we show that Drosophila larval neuroblasts-stem cell-like precursors of the adult brain-regulate proliferation by segregating the growth inhibitor Brat and the transcription factor Prospero into only one daughter cell. Like Prospero, Brat binds and cosegregates with the adaptor protein Miranda. In larval neuroblasts, both Brat and Prospero are required to inhibit self-renewal in one of the two daughter cells. While Prospero regulates cell-cycle gene transcription, Brat acts as a posttranscriptional inhibitor of dMyc. In brat or prospero mutants, both daughter cells grow and behave like neuroblasts leading to the formation of larval brain tumors. Similar defects are seen in lethal giant larvae (lgl) mutants where Brat and Prospero are not asymmetric. We have identified a molecular mechanism that may control self-renewal and prevent tumor formation in other stem cells as well.