Scaffolding Subunit Research Articles

Convergent and divergent roles of the glucose-responsive kinase SNF4 in Candida tropicalis

ABSTRACT The sucrose non-fermenting 1 (SNF1) complex is a heterotrimeric protein kinase complex that is an ortholog of the mammalian AMPK complex and is evolutionally conserved in most eukaryotes. This complex contains a catalytic subunit (Snf1), a regulatory subunit (Snf4) and a scaffolding subunit (Sip1/Sip2/Gal73) in budding yeast. Although the function of AMPK has been well studied in Saccharomyces cerevisiae and Candida albicans, the role of AMPK in Candida tropicalis has never been investigated. In this study, we focused on SNF4 in C. tropicalis as this fungus cannot produce a snf1Δ mutant. We demonstrated that C. tropicalis SNF4 shares similar roles in glucose derepression and is necessary for cell wall integrity and virulence. The expression of both SNF1 and SNF4 was significantly induced when glucose was limited. Furthermore, snf4Δ strains exhibited high sensitivity to many surface-perturbing agents because the strains contained lower levels of glucan, chitin and mannan. Interestingly, in contrast to C. albicans sak1Δ and snf4Δ, C. tropicalis snf4Δ exhibited phenotypes for cell aggregation and pseudohypha production. These data indicate that SNF4 performs convergent and divergent roles in C. tropicalis and possibly other unknown roles in the C. tropicalis SNF1-SNF4 AMPK pathway.

Epigenetic regulation of embryonic ectoderm development in stem cell differentiation and transformation during ontogenesis.

Dynamic chromatin accessibility regulates stem cell fate determination and tissue homeostasis via controlling gene expression. As a histone-modifying enzyme that predominantly mediates methylation of lysine 27 in histone H3 (H3K27me1/2/3), Polycomb repressive complex 2 (PRC2) plays the canonical role in targeting developmental regulators during stem cell differentiation and transformation. Embryonic ectoderm development (EED), the core scaffold subunit of PRC2 and as an H3K27me3-recognizing protein, has been broadly implicated with PRC2 stabilization and allosterically stimulated PRC2. Accumulating evidences from experimental data indicate that EED-associating epigenetic modifications are indispensable for stem cell maintenance and differentiation into specific cell lineages. In this review, we discuss the most updated advances to summarize the structural architecture of EED and its contributions and underlying mechanisms to mediating lineage differentiation of different stem cells during epigenetic modification to expand our understanding of PRC2.

Phosphorylation of Influenza A Virus Matrix Protein 1 at Threonine 108 Controls Its Multimerization State and Functional Association with the STRIPAK Complex.

The influenza A virus (IAV)-encoded matrix protein 1 (M1) acts as a master regulator of virus replication and fulfills multiple structural and regulatory functions in different cell compartments. Therefore, the spatiotemporal regulation of M1 is achieved by different mechanisms, including its structural and pH-dependent flexibility, differential association with cellular factors, and posttranslational modifications. Here, we investigated the function of M1 phosphorylation at the evolutionarily conserved threonine 108 (T108) and found that its mutation to a nonphosphorylatable alanine prohibited virus replication. Absent T108, phosphorylation led to strongly increased self-association of M1 at the cell membrane and consequently prohibited its ability to enter the nucleus and to contribute to viral ribonucleoprotein nuclear export. M1 T108 phosphorylation also controls the binding affinity to the cellular STRIPAK (striatin-interacting phosphatases and kinases) complex, which contains different kinases and the phosphatase PP2A to shape phosphorylation-dependent signaling networks. IAV infection led to the redistribution of the STRIPAK scaffolding subunits STRN and STRN3 from the cell membrane to cytosolic and perinuclear clusters, where it colocalized with M1. Inactivation of the STRIPAK complex resulted in compromised M1 polymerization and IAV replication. IMPORTANCE Influenza viruses pose a major threat to human health and cause annual epidemics and occasional pandemics. Many virus-encoded proteins exert various functions in different subcellular compartments, as exemplified by the M1 protein, but the molecular mechanisms endowing the multiplicity of functions remain incompletely understood. Here, we report that phosphorylation of M1 at T108 is essential for virus replication and controls its propensity for self-association and nuclear localization. This phosphorylation also controls binding affinity of the M1 protein to the STRIPAK complex, which contributes to M1 polymerization and virus replication.

RanBP9 controls the oligomeric state of CTLH complex assemblies

The CTLH (C-terminal to lissencephaly-1 homology motif) complex is a multisubunit RING E3 ligase with poorly defined substrate specificity and flexible subunit composition. Two key subunits, muskelin and Wdr26, specify two alternative CTLH complexes that differ in quaternary structure, thereby allowing the E3 ligase to presumably target different substrates. With the aid of different biophysical and biochemical techniques, we characterized CTLH complex assembly pathways, focusing not only on Wdr26 and muskelin but also on RanBP9, Twa1, and Armc8β subunits, which are critical to establish the scaffold of this E3 ligase. We demonstrate that the ability of muskelin to tetramerize and the assembly of Wdr26 into dimers define mutually exclusive oligomerization modules that compete with nanomolar affinity for RanBP9 binding. The remaining scaffolding subunits, Armc8β and Twa1, strongly interact with each other and with RanBP9, again with nanomolar affinity. Our data demonstrate that RanBP9 organizes subunit assembly and prevents higher order oligomerization of dimeric Wdr26 and the Armc8β-Twa1 heterodimer through its tight binding. Combined, our studies define alternative assembly pathways of the CTLH complex and elucidate the role of RanBP9 in governing differential oligomeric assemblies, thereby advancing our mechanistic understanding of CTLH complex architectures.

The protein phosphatase 2A holoenzyme is a key regulator of starch metabolism and bradyzoite differentiation in Toxoplasma gondii

Phenotypic switching between tachyzoite and bradyzoite is the fundamental mechanism underpinning the pathogenicity and adaptability of the protozoan parasite Toxoplasma gondii. Although accumulation of cytoplasmic starch granules is a hallmark of the quiescent bradyzoite stage, the regulatory factors and mechanisms contributing to amylopectin storage in bradyzoites are incompletely known. Here, we show that T. gondii protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic subunit PP2A-C, a scaffold subunit PP2A-A and a regulatory subunit PP2A-B. Disruption of any of these subunits increased starch accumulation and blocked the tachyzoite-to-bradyzoite differentiation. PP2A contributes to the regulation of amylopectin metabolism via dephosphorylation of calcium-dependent protein kinase 2 at S679. Phosphoproteomics identified several putative PP2A holoenzyme substrates that are involved in bradyzoite differentiation. Our findings provide novel insight into the role of PP2A as a key regulator of starch metabolism and bradyzoite differentiation in T. gondii.

CTR9 drives osteochondral lineage differentiation of human mesenchymal stem cells via epigenetic regulation of BMP-2 signaling.

Cell fate determination of human mesenchymal stem/stromal cells (hMSCs) is precisely regulated by lineage-specific transcription factors and epigenetic enzymes. We found that CTR9, a key scaffold subunit of polymerase-associated factor complex (PAFc), selectively regulates hMSC differentiation to osteoblasts and chondrocytes, but not to adipocytes. An in vivo ectopic osteogenesis assay confirmed the essentiality of CTR9 in hMSC-derived bone formation. CTR9 counteracts the activity of Enhancer Of Zeste 2 (EZH2), the epigenetic enzyme that deposits H3K27me3, in hMSCs. Accordingly, CTR9 knockdown (KD) hMSCs gain H3K27me3 mark, and the osteogenic differentiation defects of CTR9 KD hMSCs can be partially rescued by treatment with EZH2 inhibitors. Transcriptome analyses identified bone morphology protein-2 (BMP-2) as a downstream effector of CTR9. BMP-2 secretion, membrane anchorage, and the BMP-SMAD pathway were impaired in CTR9 KD MSCs, and the effects were rescued by BMP-2 supplementation. This study uncovers an epigenetic mechanism engaging the CTR9-H3K27me3-BMP-2 axis to regulate the osteochondral lineage differentiation of hMSCs.

Abstract B064: The role of PP2A-B56α in KRAS-mediated pancreatic tumorigenesis

Abstract Protein phosphatase 2A (PP2A) is a major serine-threonine phosphatase that regulates many cellular pathways including KRAS, whose oncogenic mutation is prevalent in 95% of patients with Pancreatic Ductal Adenocarcinoma (PDAC). Previous research has identified a decrease in global PP2A activity and an increase in the expression of PP2A inhibitors in PDAC cell lines, suggesting that suppression of PP2A activity may be pertinent in PDAC maintenance. Importantly, PP2A has low mutation rates in PDAC, making it a viable target for therapeutic reactivation. While PP2A has been shown to have global tumor suppressive capabilities, the regulation of specific pathways by PP2A can be altered based on PP2A holoenzyme composition. Therefore, there is a critical need to understand the mechanisms by which oncogenic KRAS can affect PP2A function and differential substrate targeting in PDAC. The PP2A holoenzyme consists of 3 subunits: the scaffolding subunit (A), the catalytic subunit (C), and the regulatory subunit (B). There are 16 different B subunits that can be incorporated into the PP2A holoenzyme that are responsible for substrate specificity. The B56α subunit of PP2A has been shown to negatively regulate cellular transformation. Our research aims to investigate the mechanisms by which PP2A-B56α is regulated through oncogenic KRAS and how suppression of B56α impacts the initiation and progression of PDAC. To determine how oncogenic KRAS alters the dynamics of PP2A-B56α and overall PP2A activity we utilized tet-inducible KRASG12D cell lines to allow direct manipulation of KRAS mutational activation. Using this system, we have identified time dependent alterations in cancerous inhibitor of PP2A (CIP2A) following induction of KRASG12D expression, indicating that PP2A suppression may be an early event in PDAC initiation. Consistent with this hypothesis, we characterized changes in the acceleration of PDAC formation in vivo using the Ptf1a-Cre; LSL- KRASG12D (KC) genetic mouse model combined with a B56α hypomorph model (KCBhm/hm). Our data show that the loss of B56α accelerates PDAC initiation, with an increase in pancreatic precursor lesion (PanIN) number and a decrease in healthy acinar area. In response to B56α loss, similar acceleration of acinar to ductal metaplasia (ADM) kinetics were observed in a 3D-cultured ADM Assay. Furthermore, when 3D-cultured acinar cells were treated with a small molecule activator of PP2A (SMAP), SMAP treatment resulted in smaller and fewer ductal structures, preventing the ADM process. Collectively, these data suggest that PP2A-B56α plays a regulatory role in cellular plasticity and loss contributes to PDAC initiation. Future studies will investigate how mutant KRAS-mediated CIP2A expression effects overall PP2A phosphatase activity and how subsequent sequestration of B56α contributes to development of PDAC. Together, these studies identify PP2A as a critical regulator of KRAS-induced cellular plasticity and support reactivation of PP2A as a novel therapeutic strategy in PDAC patients. Citation Format: Samantha L Tinsley, Rebecca A. Shelley, Gagan K. Mall, Ella Rose D. Chianis, Mary C. Thoma, Marina Pasca di Magliano, Goutham Narla, Rosalie C. Sears, Brittany L. Allen-Petersen. The role of PP2A-B56α in KRAS-mediated pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B064.

PP2A phosphatase regulates cell-type specific cytoskeletal organization to drive dendrite diversity.

Uncovering molecular mechanisms regulating dendritic diversification is essential to understanding the formation and modulation of functional neural circuitry. Transcription factors play critical roles in promoting dendritic diversity and here, we identify PP2A phosphatase function as a downstream effector of Cut-mediated transcriptional regulation of dendrite development. Mutant analyses of the PP2A catalytic subunit (mts) or the scaffolding subunit (PP2A-29B) reveal cell-type specific regulatory effects with the PP2A complex required to promote dendritic growth and branching in Drosophila Class IV (CIV) multidendritic (md) neurons, whereas in Class I (CI) md neurons, PP2A functions in restricting dendritic arborization. Cytoskeletal analyses reveal requirements for Mts in regulating microtubule stability/polarity and F-actin organization/dynamics. In CIV neurons, mts knockdown leads to reductions in dendritic localization of organelles including mitochondria and satellite Golgi outposts, while CI neurons show increased Golgi outpost trafficking along the dendritic arbor. Further, mts mutant neurons exhibit defects in neuronal polarity/compartmentalization. Finally, genetic interaction analyses suggest β-tubulin subunit 85D is a common PP2A target in CI and CIV neurons, while FoxO is a putative target in CI neurons.

Molecular architecture of the glycogen- committed PP1/PTG holoenzyme

The delicate alternation between glycogen synthesis and degradation is governed by the interplay between key regulatory enzymes altering the activity of glycogen synthase and phosphorylase. Among these, the PP1 phosphatase promotes glycogenesis while inhibiting glycogenolysis. PP1 is, however, a master regulator of a variety of cellular processes, being conveniently directed to each of them by scaffolding subunits. PTG, Protein Targeting to Glycogen, addresses PP1 action to glycogen granules. In Lafora disease, the most aggressive pediatric epilepsy, genetic alterations leading to PTG accumulation cause the deposition of insoluble polyglucosans in neurons. Here, we report the crystallographic structure of the ternary complex PP1/PTG/carbohydrate. We further refine the mechanism of the PTG-mediated PP1 recruitment to glycogen by identifying i) an unusual combination of recruitment sites, ii) their contributions to the overall binding affinity, and iii) the conformational heterogeneity of this complex by in solution SAXS analyses.

BAF (mSWI/SNF) complex regulates mediolateral cortical patterning in the developing forebrain.

Early forebrain patterning entails the correct regional designation of the neuroepithelium, and appropriate specification, generation, and distribution of neural cells during brain development. Specific signaling and transcription factors are known to tightly regulate patterning of the dorsal telencephalon to afford proper structural/functional cortical arealization and morphogenesis. Nevertheless, whether and how changes of the chromatin structure link to the transcriptional program(s) that control cortical patterning remains elusive. Here, we report that the BAF chromatin remodeling complex regulates the spatiotemporal patterning of the mouse dorsal telencephalon. To determine whether and how the BAF complex regulates cortical patterning, we conditionally deleted the BAF complex scaffolding subunits BAF155 and BAF170 in the mouse dorsal telencephalic neuroepithelium. Morphological and cellular changes in the BAF mutant forebrain were examined using immunohistochemistry and in situ hybridization. RNA sequencing, Co-immunoprecipitation, and mass spectrometry were used to investigate the molecular basis of BAF complex involvement in forebrain patterning. We found that conditional ablation of BAF complex in the dorsal telencephalon neuroepithelium caused expansion of the cortical hem and medial cortex beyond their developmental boundaries. Consequently, the hippocampal primordium is not specified, the mediolateral cortical patterning is compromised, and the cortical identity is disturbed in the absence of BAF complex. The BAF complex was found to interact with the cortical hem suppressor LHX2. The BAF complex suppresses cortical hem fate to permit proper forebrain patterning. We provide evidence that BAF complex modulates mediolateral cortical patterning possibly by interacting with the transcription factor LHX2 to drive the LHX2-dependent transcriptional program essential for dorsal telencephalon patterning. Our data suggest a putative mechanistic synergy between BAF chromatin remodeling complex and LHX2 in regulating forebrain patterning and ontogeny.

MBD5 and MBD6 stabilize the BAP1 complex and promote BAP1-dependent cancer

BackgroundBRCA1-associated protein 1 (BAP1) is an ubiquitin carboxy-terminal hydrolase, which forms a multi-protein complex with different epigenetic factors, such as ASXL1-3 and FOXK1/2. At the chromatin level, BAP1 catalyzes the removal of mono-ubiquitination on histone H2AK119 in collaboration with other subunits within the complex and functions as a transcriptional activator in mammalian cells. However, the crosstalk between different subunits and how these subunits impact BAP1’s function remains unclear.ResultsWe report the identification of the methyl-CpG-binding domain proteins 5 and 6 (MBD5 and MBD6) that bind to the C-terminal PHD fingers of the large scaffold subunits ASXL1-3 and stabilize the BAP1 complex at the chromatin. We further identify a novel Drosophila protein, the six-banded (SBA), as an ortholog of human MBD5 and MBD6, and demonstrate that the core modules of the BAP1 complex is structurally and functionally conserved from Drosophila (Calypso/ASX/SBA) to human cells (BAP1/ASXL/MBD). Dysfunction of the BAP1 complex induced by the misregulation/mutations in its subunit(s) are frequent in many human cancers. In BAP1-dependent human cancers, such as small cell lung cancer (SCLC), MBD6 tends to be a part of the predominant complex formed. Therefore, depletion of MBD6 leads to a global loss of BAP1 occupancy at the chromatin, resulting in a reduction of BAP1-dependent gene expression and tumor growth in vitro and in vivo.ConclusionsWe characterize MBD5 and MBD6 as important regulators of the BAP1 complex and maintain its transcriptional landscape, shedding light on the therapeutic potential of targeting MBD5 and MBD6 in BAP1-dependent human cancers.

A balancing act: interactions within NuA4/TIP60 regulate picNuA4 function in Saccharomyces cerevisiae and humans.

The NuA4 lysine acetyltransferase complex acetylates histone and nonhistone proteins and functions in transcription regulation, cell cycle progression, and DNA repair. NuA4 harbors an interesting duality in that its catalytic module can function independently and distinctly as picNuA4. At the molecular level, picNuA4 anchors to its bigger brother via physical interactions between the C-terminus of Epl1 and the HSA domain of Eaf1, the NuA4 central scaffolding subunit. This is reflected at the regulatory level, as picNuA4 can be liberated genetically from NuA4 by disrupting the Epl1-Eaf1 interaction. As such, removal of either Eaf1 or the Epl1 C-terminus offers a unique opportunity to elucidate the contributions of Eaf1 and Epl1 to NuA4 biology and in turn their roles in balancing picNuA4 and NuA4 activities. Using high-throughput genetic and gene expression profiling, and targeted functional assays to compare eaf1Δ and epl1-CΔ mutants, we found that EAF1 and EPL1 had both overlapping and distinct roles. Strikingly, loss of EAF1 or its HSA domain led to a significant decrease in the amount of picNuA4, while loss of the Epl1 C-terminus increased picNuA4 levels, suggesting starkly opposing effects on picNuA4 regulation. The eaf1Δ epl1-CΔ double mutants resembled the epl1-CΔ single mutants, indicating that Eaf1's role in picNuA4 regulation depended on the Epl1 C-terminus. Key aspects of this regulation were evolutionarily conserved, as truncating an Epl1 homolog in human cells increased the levels of other picNuA4 subunits. Our findings suggested a model in which distinct aspects of the Epl1-Eaf1 interaction regulated picNuA4 amount and activity.

The MLL3/4 complexes and MiDAC co-regulate H4K20ac to control a specific gene expression program.

The mitotic deacetylase complex MiDAC has recently been shown to play a vital physiological role in embryonic development and neurite outgrowth. However, how MiDAC functionally intersects with other chromatin-modifying regulators is poorly understood. Here, we describe a physical interaction between the histone H3K27 demethylase UTX, a complex-specific subunit of the enhancer-associated MLL3/4 complexes, and MiDAC. We demonstrate that UTX bridges the association of the MLL3/4 complexes and MiDAC by interacting with ELMSAN1, a scaffolding subunit of MiDAC. Our data suggest that MiDAC constitutes a negative genome-wide regulator of H4K20ac, an activity which is counteracted by the MLL3/4 complexes. MiDAC and the MLL3/4 complexes co-localize at many genomic regions, which are enriched for H4K20ac and the enhancer marks H3K4me1, H3K4me2, and H3K27ac. We find that MiDAC antagonizes the recruitment of UTX and MLL4 and negatively regulates H4K20ac, and to a lesser extent H3K4me2 and H3K27ac, resulting in transcriptional attenuation of associated genes. In summary, our findings provide a paradigm how the opposing roles of chromatin-modifying components, such as MiDAC and the MLL3/4 complexes, balance the transcriptional output of specific gene expression programs.

The vacuolar morphology protein VAC14 plays an important role in sexual development in the filamentous ascomycete Sordaria macrospora

The multiprotein Fab1p/PIKfyve-complex regulating the abundance of the phospholipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is highly conserved among eukaryotes. In yeast/mammals, it is composed of the phosphatidylinositol 3-phosphate 5-kinase Fab1p/PIKfyve, the PtdIns(3,5)P2 phosphatase Fig4p/Sac3 and the scaffolding subunit Vac14p/ArPIKfyve. The complex is located to vacuolar membranes in yeast and to endosomal membranes in mammals, where it controls the synthesis and turnover of PtdIns(3,5)P2. In this study, we analyzed the role and function of the Fab1p/PIKfyve-complex scaffold protein SmVAC14 in the filamentous ascomycete Sordaria macrospora (Sm). We generated the Smvac14 deletion strain ∆vac14 and performed phenotypic analysis of the mutant. Furthermore, we conducted fluorescence microscopic localization studies of fluorescently labeled SmVAC14 with vacuolar and late endosomal marker proteins. Our results revealed that SmVAC14 is important for maintaining vacuolar size and appearance as well as proper sexual development in S. macrospora. In addition, SmVAC14 plays an important role in starvation stress response. Accordingly, our results propose that the turnover of PtdIns(3,5)P2 is of great significance for developmental processes in filamentous fungi.

O-094 Pathological/theoretical viewpoint

Abstract Endometriosis is a benign gynaecological disease. As the lifetime risk of developing an adenocarcinoma of the endometrium is about 2% in the general female population, there seems to be no reason to believe that the same type of mucosa should not undergo malignant derailments at a similar rate when displaced at ectopic sites. The endometrioid ovarian carcinomas and the clear cell ovarian carcinomas are the most frequent histotypes associated with endometriosis. However, determining whether the mere presence of endometrium at ectopic sites should be considered per se a premalignant condition seems crucial and constitutes the conceptual base of any strategy aimed at preventing and treating endometriosis-associated ovarian cancer. Lesions are defined “precancerous” based on definite epidemiologic, morphologic, molecular, and biologic criteria that imply the acquisition of genetic, karyotypic, structural, or functional changes in a cluster of cells that differentiate them from the surrounding normal tissue. In other words, premalignant lesions should reflect an intermediate stage along the pathway leading to cancer. The transformation of a normal cell into a cancer cell is due to the progressive acquisition of driver mutations. With the improvement of sequencing technology, several studies focusing on genomic features or molecular targets have been performed in endometriosis. In particular, studies have evaluated somatic cancer-associated mutations in endometriotic lesions without concurrent cancer to assess whether endometriosis might be considered as a precursor of cancer. It has been reported recently that several ovarian and deep endometriosis lesions harbour cancer-associated mutations. Cancer-driver mutations involve genes coding for AT-rich interactive domain-containing protein 1A (ARID1A), phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA), Kirsten rat sarcoma (KRAS), or protein phosphatase 2 scaffold subunit Aα (PPP2R1A). These mutations are quite frequent also in deep endometriosis and this result is surprising considering that deep infiltrating endometriotic lesions very rarely undergo malignant transformation. This poses some doubts on the real oncogenic potential of these mutations. On the other hand, a specific role seems to be played by the ovarian microenvironment in increasing the risk of malignant derailment. The chronic inflammatory environment of women with endometriosis may theoretically facilitate the transformation of abnormal endometrial cell into a malignant cell. The micro-environmental factors such as oxidative stress, immune cell dysfunction, steroid hormones, and stem cells required for malignant transformation have been found in endometriosis. Driver gene mutations have been identified at the genetic level also in the normal endometrium, which is the origin of endometriosis. The next issue is to clarify a trigger of carcinogenesis based on genomic lineage from normal endometrium to ovarian cancers via endometriosis. To address these issues, multiomics analysis combining genomics, transcriptomics, and proteomics will be necessary.

Nuclear pore complex acetylation regulates mRNA export and cell cycle commitment in budding yeast.

Nuclear pore complexes (NPCs) mediate communication between the nucleus and the cytoplasm, and regulate gene expression by interacting with transcription and mRNA export factors. Lysine acetyltransferases (KATs) promote transcription through acetylation of chromatin‐associated proteins. We find that Esa1, the KAT subunit of the yeast NuA4 complex, also acetylates the nuclear pore basket component Nup60 to promote mRNA export. Acetylation of Nup60 recruits the mRNA export factor Sac3, the scaffolding subunit of the Transcription and Export 2 (TREX‐2) complex, to the nuclear basket. The Esa1‐mediated nuclear export of mRNAs in turn promotes entry into S phase, which is inhibited by the Hos3 deacetylase in G1 daughter cells to restrain their premature commitment to a new cell division cycle. This mechanism is not only limited to G1/S‐expressed genes but also inhibits the expression of the nutrient‐regulated GAL1 gene specifically in daughter cells. Overall, these results reveal how acetylation can contribute to the functional plasticity of NPCs in mother and daughter yeast cells. In addition, our work demonstrates dual gene expression regulation by the evolutionarily conserved NuA4 complex, at the level of transcription and at the stage of mRNA export by modifying the nucleoplasmic entrance to nuclear pores.

MEDB-42. GermlineElp1 deficiency promotes genomic instability and survival of granule neuron progenitors primed for SHH medulloblastoma pathogenesis

Germline loss-of-function (LOF) mutations in Elongator complex protein 1 (ELP1) are found in 15-20% of childhood SHH medulloblastoma (MB) and are exceedingly rare in non-SHH-MB or other cancers. ELP1 germline carriers that develop SHH-MB harbor frequent somatic PTCH1 mutations and universally sustain loss-of-heterozygosity of the remaining ELP1 allele through chromosome 9q deletion. ELP1 functions as a scaffolding subunit of the Elongator complex that is required for posttranscriptional modification of tRNAs and maintenance of efficient translational elongation and protein homeostasis. However, the molecular, biochemical, and cellular mechanisms by which ELP1/Elongator LOF contribute to SHH-MB tumorigenesis remain largely unknown. Herein, we report that mice harboring germline Elp1 monoallelic loss (i.e., Elp1+/-) exhibit hallmark features of malignant predisposition in developing cerebellar granule neuron progenitors (GNPs), the lineage-of-origin for SHH-MB. Elp1+/- GNPs are characterized by increased replication stress-induced DNA damage, upregulation of the homologous recombination repair pathway, aberrant cell cycle, and attenuation of p53-dependent apoptosis. CRISPR/Cas9-mediated Elp1 and Ptch1 gene targeting in mouse GNPs reproduces highly penetrant SHH-MB tumors recapitulating the molecular and phenotypic features of patient tumors. Reactivation of the p53 pathway through MDM2 and PAK4 inhibitors promotes selective cell death in patient-derived xenograft tumors (PDX) harboring deleterious ELP1 mutations. Together, our findings reveal that germline Elp1 deficiency heightens genomic instability and survival in GNPs, providing a mechanistic model for the subgroup-restricted pattern of predisposition and malignancy associated with pathogenic ELP1 germline carriers. These results provide rationale for further preclinical studies evaluating drugs that overcome p53 pathway inhibition in ELP1-associated SHH-MB and a renewed outlook for improving treatment options for affected children and their families.*, # Contributed equally

Interplay between TPR nucleoporin and TREX‐2 complex in mRNA export

Nuclear pore complexes (NPCs) are conduits between the cytoplasm and the nucleus. NPCs consist of roughly 35 distinct nucleoporins with filaments extending from both the nuclear and cytoplasmic faces of the pore. Nucleoporins regulate many processes, including nucleocytoplasmic trafficking, protein modification, chromatin remodeling and transcription, as well as mRNA processing and export. Our current research is focused on basket nucleoporin TPR, which works as a docking platform for other proteins, including transport receptors and subunits of the TRanscription and EXport 2 (TREX‐2) complex. We found that Auxin Inducible Degron (AID)‐mediated TPR elimination specifically disrupts TREX‐2 complex recruitment to NPCs, and results in rapid and profound changes in transcriptomic profiles of TREX‐2‐dependent RNAs and retention of those RNAs within the nucleus. The molecular mechanism through which TPR controls TREX‐2‐dependent gene expression is not clear.We are currently working in two ways to understand TREX‐2‐dependent RNA export and how TPR facilitates this pathway. First, we are examining the components of TREX‐2. GANP is a major scaffold subunit of the TREX‐2 complex, and loss of GANP disrupts the association of TREX‐2 complex subunits to the NPC. We have AID‐tagged other TREX‐2 complex members (PCID2, ENY2, CETN2) and are analyzing how their loss impacts TREX‐2 targeting, transcriptomic profiles and RNA retention. Our findings suggest that GANP or PCID2 depletion rapidly leads to polyA RNA retention inside the nucleus, but this was not the case after depletion of ENY2 or CETN2. Further comparison of TREX‐2 complex subunit profiles to each other should elucidate the sequence of their actions during mRNP maturation.Second, we are investigating the point(s) at which TPR controls TREX‐2‐dependent gene expression: TPR may help to remodel mRNP particles within the nucleus, to recruit ready‐to‐export mRNP particles to the nuclear envelope, or it may perform both of these functions. To distinguish between these possibilities, we have analyzed a TPR mutant that lacks an NPC recruitment domain, to see whether it can rescue any part of TPR’s function in this pathway without recruiting TREX‐2 to the NPC. We found that this TPR mutant resides inside the nucleus and can not restore normal export of polyA RNA. We speculate that TPR preferentially acts as a landing platform for TREX‐2‐bound mRNAs at the nuclear pore and may help with quality control of the transcripts. To explore if TPR‐dependent transcripts have unique signatures, we are analyzing mRNA length, GC content and intron context in other AID‐tagged nucleoporins and comparing transcriptomic profiles after their loss in comparison to TPR.Alltogether, these data will help us to unfold the role of TREX2 complex subunits in targeting GANP‐TPR dependent transcripts to the pore and to gain a better understanding of how TPR controls gene expression.

Elp1 is required for development of visceral sensory peripheral and central circuitry.

ABSTRACTCardiovascular instability and a blunted respiratory drive in hypoxic conditions are hallmark features of the genetic sensory and autonomic neuropathy, familial dysautonomia (FD). FD results from a mutation in the gene ELP1, the encoded protein of which is a scaffolding subunit of the six-subunit Elongator complex. In mice, we and others have shown that Elp1 is essential for the normal development of neural crest-derived dorsal root ganglia sensory neurons. Whether Elp1 is also required for development of ectodermal placode-derived visceral sensory receptors, which are required for normal baroreception and chemosensory responses, has not been investigated. Using mouse models for FD, we here show that the entire circuitry underlying baroreception and chemoreception is impaired due to a requirement for Elp1 in the visceral sensory neuron ganglia, as well as for normal peripheral target innervation, and in their central nervous system synaptic partners in the medulla. Thus, Elp1 is required in both placode- and neural crest-derived sensory neurons, and its reduction aborts the normal development of neuronal circuitry essential for autonomic homeostasis and interoception. This article has an associated First Person interview with the first author of the paper.

Regulation of XPO5 phosphorylation by PP2A in hepatocellular carcinoma.

Exportin 5 (XPO5) is a shuttle protein that mediates precursor miRNA (pre‐miRNA) export from the nucleus to the cytoplasm, an important step in miRNA maturation. We previously demonstrated that XPO5 was phosphorylated by ERK kinase and subsequently underwent conformation change by the peptidyl‐prolyl isomerase Pin1, leading to the reduced miRNA expression in hepatocellular carcinoma (HCC). Protein phosphorylation modification serves as a reversible regulatory mechanism precisely governed by protein kinases and phosphatases. Here we identified that the phosphatase PP2A catalyzed XPO5 dephosphorylation. PP2A holoenzyme is a ternary complex composed of a catalytic subunit, a scaffold subunit, and a regulatory subunit that determines substrate specificity. In this study, we characterized the involvement of B55β subunit in XPO5 dephosphorylation that favored the distribution of XPO5 into the cytoplasm and promoted miRNA expression, leading to HCC inhibition in vitro and in vivo. Our study demonstrates the regulatory role of B55β‐containing PP2A in miRNA expression and may shed light on HCC pathogenesis.