FoxA transcription factors pattern gut tissue across animal phylogeny. Beyond their early patterning function, little is known about whether they control the terminal differentiation and/or function of the fully mature enteric nervous system, the intrinsic nervous system of the gut. We show here that the expression and function of the sole FoxA homolog, PHA-4, reach beyond its previously described pioneer factor roles in patterning the foregut. Through the engineering of neuron-specific -regulatory alleles, Cre-mediated cell-specific knockouts, and degron-mediated, temporally controlled PHA-4/FoxA removal in postmitotic neurons, we found that PHA-4/FoxA is required not only to initiate the terminal differentiation program of foregut-associated enteric neurons but also to maintain their functional properties throughout the life of the animal. Moreover, we discovered novel sites of expression of PHA-4/FoxA in extrapharyngeal enteric neurons that innervate the hindgut (AVL and DVB), a GABAergic interneuron that controls foregut function during sleep (RIS), and a peptidergic neuron (PVT) that we implicate here in controlling defecation behavior. We show that while PHA-4/FoxA is not required for the developmental specification of AVL, DVB, RIS, and PVT, it is required to enable these neurons to control enteric functions. Taken together, PHA-4/FoxA is the only transcription factor known to date that is expressed in and required for the proper function of all distinct types of enteric neurons in a nervous system.

High-grade serous ovarian cancer (HGSOC) is a highly lethal gynecologic malignancy in women. Women diagnosed with HGSOC initially respond to chemotherapy, but there is a >80% rate of relapse. There is thus a significant unmet need for new therapeutic targets for HGSOC. Estrogen receptor α (ERα) is a particularly attractive candidate, as ∼70% of HGSOC tumors stain positively for ERα and there are approved inhibitors that show limited toxicity. However, unlike the case for breast cancer, endocrine therapy for HGSOC has not shown consistently promising results. In this work, we show that missense mutant forms of p53, which occur in >60% of HGSOC, bind and inhibit ERα function and confer resistance to fulvestrant and elacestrant. Mechanistically, we show that mutant p53 predominantly inhibits one arm of the ERα pathway-the transactivation of jointly regulated ERα-SP1 target genes such as the mTOR regulator We show that silencing mutant p53 restores the ability of ERα to transactivate ERα-SP1 target genes and renders HGSOC markedly more sensitive to endocrine therapy. Consistent with this premise, we show that the p53 mutant Y220C refolding compound rezatapopt enhances fulvestrant response in a Y220C mutant cell line.

Precise control of microRNA (miRNA) expression is critical during development. An important mechanism of miRNA regulation is target-directed microRNA degradation (TDMD), a pathway in which the binding of miRNAs to specialized trigger RNAs induces ubiquitylation and decay of associated argonaute (AGO) proteins by the ZSWIM8 ubiquitin ligase. Concomitant release of miRNAs results in their rapid turnover. ZSWIM8-deficient mice exhibit reduced body size, cardiopulmonary and neurodevelopmental defects, and perinatal lethality. Despite widespread dysregulation of miRNAs in these animals, the vast majority of presumptive trigger RNAs that induce decay of ZSWIM8-regulated miRNAs remain undefined. Here, using AGO cross-linking and sequencing of hybrids (AGO-CLASH), a high-throughput method for identifying miRNA binding sites, we report the identification of as a TDMD trigger for miR-322-5p and and as TDMD triggers for miR-503-5p in mouse embryonic fibroblasts (MEFs). In mice, deletion of the miR-322-5p and miR-503-5p trigger sites in the and 3' UTRs, respectively, abrogated TDMD of these miRNAs and resulted in miR-322/503-dependent embryonic growth restriction, recapitulating a key feature of the phenotype. Thus, and act as triggers for degradation of miR-322-5p and miR-503-5p, revealing a noncoding function for these mRNAs as regulators of mammalian body size.

To increase our understanding of the interplay between transcription factor networks and the epigenetic landscape in early B-lymphoid development, we conducted combined SC-RNA/ATAC-seq analyses of bone marrow progenitor populations. Based on changes in DNA accessibility, we created a high-resolution model for B-cell development. Trend change analysis identified a rapid shift in DNA accessibility, resulting in the loss of T-lineage priming and the acquisition of the epigenetic landscape of B-lymphocytes in association with the activation of the B-lineage program. The epigenetic switch correlated strongly with the initiation of and transcription, as well as their functional activities. The importance of epigenetic silencing for the preservation of B-cell fate is supported by our finding that inhibition of the histone methylases EZH1 and EZH2 in pro-B cells allows for the activation of T-lineage genes and the generation of T-cell progenitors in response to Notch signaling. Our data reveal that B-lymphoid commitment is associated with a transcription factor-mediated, dose-dependent epigenetic switch, suppressing an inherent T-lineage potential in early lymphoid progenitors.

The Eyes Absent family of protein phosphatases (EYA1-4) is aberrantly expressed and tumor-promoting across many devastating cancers of neurological origin affecting both children and adults. It has recently been demonstrated that EYA1 and EYA4 promote tumor cell survival by increasing the active pool of Polo-like kinase 1 (PLK1) molecules. This discovery provides a rationale for the therapeutic combination of EYA inhibitors with direct, ATP-competitive PLK1 inhibitors. Here, we demonstrate potent loss of cell viability in response to combined EYA and PLK1 inhibition in cancer cell lines that overexpress EYA1 and/or EYA4, including in neuroblastoma and glioblastoma models. We identify decreases in PLK1 activity and RAD51 focus formation and increases in mitotic arrest and cell death as mechanistic contributors to combination sensitivity. Combined EYA and PLK1 inhibition is also effective in glioblastoma stem cell models that overexpress EYA1/EYA4 and specifically targets the cancer stem cell state. Finally, through multiomic correlational analysis, we identify high levels of the NuRD complex and SOX9 as contributors to combination treatment sensitivity. Overall, this work identifies a novel synthetic-lethal combination therapy with potential utility across a wide range of neurological cancers.

During neurodevelopment, a single progenitor cell can generate many different neuron types. As these neurons mature, they form unique morphologies, integrate into neural circuits, and contribute to behavior. However, the integration of these developmental events is understudied. Here, we show that the same transcription factor is important for both the generation of neuronal diversity and maintaining mature neuronal identity, providing novel insights into how the generation of neuronal identity and morphology are coordinated. We utilized a previously characterized larval locomotor circuit in , where activation of the moonwalker descending neuron (MDN) triggers backward locomotion via its presynaptic connection with the premotor neuron A18b. The MDN expresses the temporal transcription factor Hunchback (Hb), which has a well-characterized role in neural progenitors. Loss of Hb in the postmitotic MDN increases axon/dendrite branching, leading to additional functional synapses on A18b and increasing backward locomotion. We conclude that the endogenous function of Hb is to restrain axon/dendrite outgrowth, including limiting MDN-A18b synapses, thereby dampening backward locomotion. Our work provides insights into how a transcription factor can have different functions throughout life; that is, Hb generates neuronal diversity in the progenitor and regulates neuronal connectivity in the mature neuron to generate an appropriately tuned behavior.

One of the densest compartments in the cell is the dense fibrillar component (DFC) of the nucleolus, consisting mainly of nascent ribosomal RNA (rRNA), small nucleolar ribonucleoproteins (snoRNPs), and their chaperone, Nopp140 (gene name ). How this biomolecular condensate is formed and what underlies its structure and function are poorly understood, like those of most liquid-liquid phase-separated condensates. Although we established that Nopp140 is important for the cohesiveness of the DFC and for rRNA modification, it is not known how this is achieved. Here we demonstrate that Nopp140 concentrates intrinsically disordered and nuclear localization signal (NLS)-rich protein regions (IDRs), including a newly identified RNA polymerase I C-terminal domain (CTD) of the RNA polymerase I-associated factor PAF49. Altogether, this network of multivalent weak interactions forms the DFC, a liquid-liquid phase-separated biomolecular condensate that promotes rRNA modification. This local concentration of biomolecules ensures near-complete modification efficiency at some 200 nt in every one of the 10 million or so rRNAs per cell.

The production of B cells is essential for a functional immune system. This process relies on the coordinated activity of a handful of transcription factors that act in part by modifying the chromatin landscape of lymphoid progenitors to allow the ordered expression of genes essential for B-cell development. In this issue of , Tingvall-Gustafsson and colleagues (doi:10.1101/gad.353002.125) have investigated the interplay of these transcriptional regulators with the chromatin state of developing lymphocytes at single-cell resolution. They pinpoint a rare population of progenitors where this epigenetic reprogramming occurs to simultaneously repress the expression of lineage-inappropriate genes and activate the B-cell program.

Compartmentalized regulation of RNAs is emerging as a key driver of developmental transitions, with RNA-binding proteins performing specialized functions in different subcellular compartments. The RNA-binding protein Mei2, which arrests mitotic proliferation and drives zygotic development in fission yeast, was shown to function in the nucleus to trigger meiotic divisions. Here, using compartment-restricted alleles, we report that Mei2 functions in the cytosol to arrest mitotic growth and initiate development. We found that Mei2 is a zygote-specific component of P-bodies that inhibits the translation of tethered mRNAs. Importantly, we show that P-bodies are necessary for Mei2-driven development. Phosphorylation of Mei2 by the inhibitory Pat1 kinase impedes P-body recruitment of both Mei2 and its target RNA. Finally, we establish that Mei2 recruitment to P-bodies and its cytosolic functions, including translational repression of tethered RNAs, depend on the RNA-binding domain of Mei2 that is dispensable for nuclear Mei2 roles. Collectively, our results dissect how distinct pools of an RNA-binding protein control developmental stages and implicate P-bodies as key regulators of gamete-to-zygote transition.

Pioneer transcription factors (TFs) such as SOX2 play critical roles in the control of stem cell identity and are dysregulated in many human cancers. For example, SOX2 regulates the self-renewal of neural stem cells (NSCs) and is typically highly expressed in glioblastoma stem cells (GSCs), where it is known to induce an immature NSC-like state. Here, we explored the regulation of SOX2 by phosphorylation during NSC division and identified an unexpected role for excessive SOX2 pioneer activity in driving mitotic damage. We found that SOX2 phosphorylation during mitosis is a key switch that prevents promiscuous chromatin binding across the genome. Without this regulatory control, excessive SOX2 in mitosis triggers chromatin opening, resulting in increased mitotic transit times and increased chromosomal damage. Therefore, elevated levels of SOX2 in cancers may have dual oncogenic roles: inducing stemness during interphase via its well-known transcriptional roles but simultaneously promoting chromosomal disruptions through unconstrained pioneer factor activity.

Stabilization of β-catenin on the dorsal side of the embryo is critical for the formation of the dorsal organizer. The novel transmembrane protein Huluwa (Hwa) has recently been identified as the maternal dorsal determinant responsible for β-catenin stabilization in dorsal organizer formation. The molecular mechanism by which Hwa induces WNT-independent β-catenin stabilization remains elusive. In this study, we demonstrate that the conserved PPNSP motif of Hwa is phosphorylated by GSK3 and that the phosphorylated PPNSP motif potently inhibits GSK3, leading to β-catenin stabilization. Notably, the phosphorylated PPNSP motif of Hwa has stronger GSK3 inhibitory activity than the phosphorylated PPPSP motif of LRP6. Molecular dynamics simulations suggest that the PPNpSP peptide has stronger affinity for GSK3 than the PPPpSP peptide, facilitated by the hydrogen bonding capacity of the asparagine residue. Consistent with Hwa's GSK3 inhibitory activity, Hwa enhances SIAH1-dependent degradation of AXIN. Hwa-induced β-catenin stabilization and AXIN degradation are significantly enhanced by oligomerization. Thus, Hwa stabilizes β-catenin through a molecular mechanism similar to that of LRP6 in mediating WNT signaling, representing a striking example of molecular convergence.

The exon junction complex (EJC) has roles in mRNA export and cytoplasmic quality control. However, the EJC is recruited to pre-mRNA by the spliceosome prior to the completion of splicing. When splicing is cotranscriptional, the EJC is deposited on nascent RNA early during synthesis, raising the question of whether the EJC regulates downstream RNA processing. Here we show, using long-read sequencing, that degron-mediated depletion of EJC component EIF4A3 leads to skipping of neighboring pairs of two or more exons on the same mRNA molecule. These data suggest that the entire "exon block" requires the EJC for inclusion. Introns flanking EJC-dependent exon blocks were longer and spliced after internal introns. In our working model, block exons are first spliced together to form a larger EJC-marked exon that promotes surrounding splicing events. Strikingly, analysis of 480 RNA binding protein knockdowns across two different human cell lines revealed block exons that are dependent on other splicing factors, indicating that coordinated splicing of adjacent exons is a general mechanism, of which the EJC is the dominant regulator. Cell type-specific coordinated splicing of adjacent exon pairs has been observed before. Here we identify the EJC as the main protein factor massively regulating this novel splicing mechanism in .

Polycomb chromatin domains are chromosomal regions decorated with histone H2A monoubiquitination at lysine 119 (H2Aub1) and histone H3 trimethylation at lysine 27 (H3K27me3). These domains are dynamically shaped through the actions of different Polycomb group protein complexes to control gene expression during development. To assess how different Polycomb group subcomplexes contribute to these histone modification profiles in embryos, we used mutants that abrogate their function. Canonical Polycomb repressive complex (PRC) 1 deposits low levels of H2Aub1 solely at Polycomb target genes, whereas variant PRC1 generates the bulk of H2Aub1 genome-wide. In late-stage embryos, PR-DUB-mediated deubiquitination effectuates a uniform low-level H2Aub1 profile across the genome. The combined activities of PRC2.1 and PRC2.2 drive the formation and maintenance of most H3K27me3 domains, but PRC2.1 is the limiting enzyme for creating such domains at HOX genes. Surprisingly, reduction in the H3K27me3 level and repression defects caused by removing PRC2.1 were largely rescued in animals also lacking PR-DUB, which showed extensive H2Aub1 accumulation at Polycomb targets that promoted compensatory H3K27me3 deposition by PRC2.2. Diversification of Polycomb protein complexes combined with feedback loop mechanisms involving histone modification cross-talk equips the system with the plasticity, adaptability, and buffering capacity needed to safeguard cell fate decisions during development.

The mechanisms governing the generation of neuronal subtypes at distinct times and proportions during human retinal development are poorly understood. While thyroid hormone (TH) signaling specifies cone photoreceptor subtypes, how this regulation changes over time remains unclear. To address this question, we studied the expression and function of type 3 iodothyronine deiodinase (DIO3), an enzyme that degrades TH, in human retinal organoids. We show that DIO3 is a master regulator of human photoreceptor developmental timing and cell fate stability. DIO3 is highly expressed in retinal progenitor cells (RPCs) and decreases as these cells asynchronously differentiate into neurons, progressively reducing TH degradation and increasing TH signaling. mutant organoids display precocious development of S cones, L/M cones, and rods; increased photoreceptor density; and subpopulations of photoreceptors that coexpress different opsin proteins. Our multiomics and chimeric organoid experiments show that cell-autonomous and non-cell-autonomous mechanisms locally coordinate and maintain DIO3 expression and TH signaling levels among cells. Computational modeling reveals a mechanism that couples TH levels and fate specification, providing robustness to photoreceptor development as compared with a probabilistic, cell-intrinsic mechanism. Based on our findings, we propose an hourglass-like mechanism in which the proportion of progenitors to neurons decreases over time to relieve TH degradation, triggering development of photoreceptor subtypes at specific times. Our study identifies how local regulation of thyroid hormone signaling influences neural cell fate specification, which may be a consideration for designing regenerative therapies.

During vertebrate development, the segmentation clock drives oscillatory gene expression in the presomitic mesoderm (PSM), leading to the periodic formation of somites. Oscillatory gene expression is synchronized at the cell population level; inhibition of Delta-Notch signaling results in the loss of synchrony and the fusion of somites. However, it remains unclear how cell-cell signaling couples oscillatory gene expression and controls synchronization. Here, we report that synthetic cell-cell signaling using designed ligand-receptor pairs can induce synchronized oscillations in PSM organoids. Optogenetic assays uncovered that the intracellular domains of synthetic ligands play key roles in dynamic cell-cell communication. Oscillatory coupling using synthetic cell-cell signaling recovered the synchronized oscillation in PSM cells deficient for Delta-Notch signaling; nonoscillatory coupling did not induce recovery. This study reveals the mechanism by which ligand-receptor molecules coordinate the synchronization of the segmentation clock and provides a way to program temporal gene expression in organoids and artificial tissues.

Precise intercellular communication is critical for cellular decision-making. The segmentation clock is an oscillatory gene network regulating periodic segmentation of the presomitic mesoderm (PSM) in vertebrate embryos. Oscillations between neighboring cells are thought to be coupled by DELTA-NOTCH signaling. To directly test this experimentally, Isomura and colleagues (doi:10.1101/gad.352538.124) reconstituted this coupling using synthetic biology. They integrated a synthetic DELTA-NOTCH pathway into DELTA-deficient PSM organoids, which restored cell-cell communication. Additionally, optogenetic activation of the synthetic ligand further revealed that the dynamics of ligand presentation are crucial for effective communication. This work directly demonstrates the importance of oscillatory cell-cell signaling in development and provides a blueprint for using synthetic circuits in future studies.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer (CRPC). The molecular mechanisms underlying the progression of CRPC toward NEPC remain incompletely understood, and effective treatments remain to be discovered. Here, we report that loss of the nuclear receptor ERRγ promotes neuroendocrine differentiation in a Pten-deficient mouse model of prostate adenocarcinoma. These findings were recapitulated in advanced cellular and xenograft models of human prostate cancer. Critically, we show that ERRγ gain of function can reverse instilled NEPC features accompanied by suppression of growth and oncogenic metabolic reprogramming. Activation of a neuroendocrine transcriptional program enabled by ERRγ deficiency unveiled a targetable vulnerability exploited by the combined pharmacological inhibition of EZH2 and RET kinase that effectively inhibited the growth of ERRγ-deficient tumor organoids and cells. Collectively, our findings demonstrate that ERRγ downregulation facilitates prostate cancer adeno-to-neuroendocrine transformation and offer potential therapeutic strategies to prevent/treat the development of poor outcome NEPC.

Mitochondria play a crucial role in cellular energy metabolism and homeostasis and are strongly implicated in aging and age-related diseases. The outer mitochondrial membrane protein voltage-dependent anion channel (VDAC) plays multiple roles in mitochondrial homeostasis, including transport of metabolites, ATP, and Ca Dysregulation of VDAC levels has been associated with cancer, neurodegeneration, metabolic disorders, and aging. Previously, we demonstrated that elevated VDAC-1 levels in lead to increased mitochondrial permeability and reduced life span. Here we demonstrate that reduced VDAC-1 function extends life span through the activation of the mitochondrial unfolded protein response (UPR), a conserved stress response that maintains mitochondrial proteostasis and is linked to life span extension in multiple species. Leveraging unbiased genomic discovery, we identified genes encoding several proteins in the PeBoW complex as a critical mediator of UPR activation following VDAC-1 loss. More broadly, we demonstrated a universal requirement for several PeBoW component genes across diverse mitochondrial stressors in order to fully animate the UPR Our findings reveal a heretofore unappreciated role for PeBoW components in UPR induction and life span extension in response to mitochondrial stress, highlighting its essential function in mitochondrial quality control and longevity pathways.