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Inoue, K.

Publications and source records attributed to Inoue, K..

At least 55 records · Page 3Linked to original sources

Energy transfer in ubiquitous rhodopsin pumps with xanthophyll antennas

Energy transfer from light-harvesting ketocarotenoids to light-driven proton pumps xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to rhodopsin proton pumps from the abundant marine and freshwater photoheterotrophs have thus far failed3-5. Here, using functional metagenomics combined with chromophore extraction from the environment, we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet/blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas have a significant impact on rhodopsin phototrophy in the worlds lakes, seas and oceans.

microbiology↗

Bone marrow AdipoQ-lineage progenitors are a major cellular source of M-CSF that dominates bone marrow macrophage development, osteoclastogenesis and bone mass

M-CSF is a critical growth factor for myeloid lineage cells, including monocytes, macrophages and osteoclasts. Tissue resident macrophages in most organs rely on local M-CSF. However, it is unclear what specific cells in bone marrow produce M-CSF to maintain myeloid homeostasis. Here, we identify bone marrow AdipoQ-lineage progenitors, but not bone marrow mature adipocytes or peripheral adipose tissue, as a major cellular source of M-CSF, with these AdipoQ-lineage progenitors producing M-CSF at levels much higher than those produced by osteoblast lineage cells. Deficiency of M-CSF in bone marrow AdipoQ-lineage progenitors drastically reduces the generation of bone marrow macrophages and osteoclasts, leading to severe osteopetrosis in mice. Furthermore, the postmenopausal osteoporosis in a mouse model can be significantly alleviated by the lack of M-CSF in bone marrow AdipoQ-lineage progenitors. Our findings identify bone marrow AdipoQ-lineage progenitors as a major cellular source of M-CSF in bone marrow and reveal their crucial contribution to bone marrow macrophage development, osteoclastogenesis, bone homeostasis and pathological bone loss.

cell biology↗

IFNA pathway drives the more aggressive phenotype of KRASG12D-mutant pancreatic ductal adenocarcinomas via IFNAR1/STAT3 activation

Activating mutations of KRAS play critical roles in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC). Accumulating evidence indicates that distinct KRAS alleles associate with different prognoses, but the underlying mechanisms are not known. We established isogenic KRAS mutants (KRASG12D, KRASG12V, and KRASWT) using a KRASG12R patient-derived PDAC cell line by CRISPR/Cas9 knock-in. We used these isogenic cell lines, a collection of characterized human PDAC patient-derived cell lines, and murine PDAC models to study the role of these KRAS alleles in vitro and in vivo. We verified that the growth of KRASG12D cells is more aggressive compared to KRASG12V isogenic cells in vitro and in vivo using orthotopic mouse models. Signal transducer and activator of transcription (STAT) activation was the most significant difference between KRASG12D and KRASG12V isogenic PDACs. Furthermore, activation of interferon-alpha (IFNA)/IFNA receptor (IFNAR)1/STAT3 signaling in the cancer cells mediated the more aggressive phenotype of KRASG12D PDACs. Conversely, inhibition of IFNAR1 in patient-derived PDAC cells suppressed tumor growth. Finally, IFNAR1 blockade was also effective in murine PDAC models and induced a significant increase in survival when combined with immune checkpoint blockade therapy. We conclude that the IFNA pathway and IFNAR1/STAT3 axis contribute to a more aggressive tumor progression in human KRASG12D PDACs and that IFNAR1 inhibition is a potential therapeutic target for overcoming resistance to immunotherapy in PDAC. One Sentence SummaryIFNA pathway drives the more aggressive phenotype of KRASG12D-mutant pancreatic ductal adenocarcinomas via IFNAR1/STAT3 activation.

cancer biology↗

Inflammasome targeting with an NLRP3 agonist therapy is feasible but ineffective in murine hepatocellular carcinoma models with liver damage

Co-inhibition of programmed cell death receptor-1 (PD-1) and vascular endothelial growth factor receptor (VEGFR) pathway has shown efficacy in hepatocellular carcinoma (HCC). NLRP3 is a component of the inflammasome involved in the initiation, development, and progression of multiple cancers. We examined whether adding an NLRP3 agonist to dual PD-1/VEGFR inhibitors is feasible and can address treatment resistance in orthotopic HCC in mice with underlying liver damage. Mice with established tumors were treated with an NLRP3 agonist alone, combination of anti-VEGFR2 or the multikinase inhibitor regorafenib with anti-PD1 antibodies, or their combination. In all models tested, NLRP3 agonist therapy showed acceptable toxicity but no effect on tumor growth delay, disease morbidity, or survival. Pharmacodynamic analyses confirmed the effects of NLRP3 agonist therapy on inflammasome, evidenced by a significant elevation in plasma levels of pro-inflammatory cytokines such as IL-1{beta}. However, these changes were not detectable in tumor tissues, where we detected increased expression of immunosuppressive markers IL-6, KC/GRO, CCL9, and IL-18, and immune checkpoint molecules (PD1, PD-L1, and CTLA-4) after NLRP3 agonist therapy. Thus, modulation of the inflammasome with a novel NLRP3 agonist was feasible in mice with orthotopic HCC and liver damage but did not enhance efficacy when combined with anti-PD1/VEGFR therapies.

immunology↗

Proton-transporting heliorhodopsins from marine giant viruses

Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-gated proton channel. V2HeR3 absorbs blue-green lights, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a proton accepting group of the Schiff base, whose mutations converted the protein into an outward proton pump. Three environmental viral HeRs from the same group, as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses EhV-201 and EhV-202 contain two and three HeRs, respectively. Except for V2HeR3 from EhV-202, none of these proteins exhibit ion-transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tools, like type-1 microbial rhodopsins.

biophysics↗

Saccharibacteria harness light energy using Type-1 rhodopsins that may rely on retinal sourced from microbial hosts

Microbial rhodopsins are a family of photoreceptive membrane proteins with a wide distribution across the Tree of Life. Within the Candidate Phyla Radiation (CPR), a diverse group of putatively episymbiotic bacteria, the genetic potential to produce rhodopsins appears to be confined to a small clade of organisms from sunlit environments. Here, we characterize the metabolic context and biophysical features of Saccharibacteria Type-1 rhodopsin sequences derived from metagenomic surveys and show that these proteins function as outward proton pumps. This provides one of the only known mechanisms by which CPR can generate a proton gradient for ATP synthesis. Intriguingly, Saccharibacteria do not encode the genetic machinery to produce all-trans-retinal, the chromophore essential for rhodopsin function, but their rhodopsins are able to rapidly uptake this cofactor when provided in experimental assays. We found consistent evidence for the capacity to produce retinal from {beta}-carotene in organisms co-occurring with Saccharibacteria, and this genetic potential was dominated by Actinobacteria, which are known hosts of Saccharibacteria in other habitats. If Actinobacteria serve as hosts for Saccharibacteria in freshwater environments, exchange of retinal for use by rhodopsin may be a feature of their associations.

microbiology↗

A stepwise route to domesticate rice by controlling seed shattering and panicle shape

Asian rice (Oryza sativa L.) is consumed by more than half of the worlds population. Despite its global importance, the process of early rice domestication remains unclear. During domestication, wild rice (O. rufipogon Griff.) acquired non-seed-shattering behaviour, allowing humans to increase grain yield. Previous studies argued that the sh4 mutation triggered a reduction in seed shattering during rice domestication; but our experiments using wild introgression lines of O. rufipogon show that the domesticated sh4 allele alone is insufficient for shattering loss. Here, we identified the interaction between three key mutations associated with the interruption of abscission layer formation and panicle architecture that were causal in early rice domestication. An interruption of abscission layer formation requires both sh4 and qSH3 mutations, presenting an apparent barrier to the selection of shattering loss. We identified a causal single-nucleotide polymorphism at qSH3 within the seed-shattering gene OsSh1, which is conserved in indica and japonica subspecies but absent in the circum-aus group of rice. Through harvest experiments, we demonstrated that seed shattering alone did not significantly impact yield; rather yield increases were observed with closed panicle formation controlled by SPR3, which is further augmented by the integration of sh4 and qSH3 alleles. Complementary manipulation of seed shattering and panicle shape result in a mechanically stable panicle structure. We propose a stepwise route for the earliest phase of rice domestication, wherein selection of visible SPR3-controlled closed panicle morphology was instrumental in the sequential recruitment of sh4 and qSH3, which led to the loss of shattering. Significance StatementRice is one of the most important crops worldwide. Loss of seed shattering in domesticated rice, previously attributed to single mutations such as those in sh4, is considered the principal genetic change which resulted in yield increases. However, we show that sh4 alone is insufficient and other genes, such as qSH3, are required to cause abscission layer disruption. The evolution of non-seed-shattering therefore required multiple mutations. Furthermore, shattering loss in genetic backgrounds of wild rice does not correspondingly increase yields. We have identified an interaction in which a second trait, closed panicle formation controlled by SPR3, that both increases the yield and facilitates recruitment of sh4 and qSH3, which synergistically augment yield, leading to a stepwise model for rice domestication.

plant biology↗

Production of offspring from azoospermic mice with meiotic failure: Precise biparental meiosis within halved oocytes

While the large volume of mammalian oocytes is necessary for embryo development, it can lead to error-prone chromosomal segregation during meiosis. Conversely, we hypothesized that smaller oocytes would have a great unidentified potential to stabilize unstable meiosis and improve the development of the resultant embryos. Here, we show that reducing ooplasmic volume can rescue highly error-prone fertilization using primary spermatocytes by preventing segregation errors of chromosomes during biparental meiosis. High-resolution live-imaging analysis revealed that erroneous chromosome segregation occurred in most (90%) spermatocyte-injected oocytes of normal size, but could be ameliorated to 40% in halved oocytes. The birth rate improved remarkably from 1% to 19% (P < 0.0001). Importantly, this technique enabled the production of offspring from azoospermic mice with spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans. Thus, contrary to popular opinion, oocytes inherently have a strong potential for precise meiotic divisions, which can be evoked by reduction of the ooplasmic volume. Their potential might help rescue cases of untreatable human azoospermia with spermatocyte arrest.

developmental biology↗

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

ChRmine1, a recently-discovered bacteriorhodopsin-like cation-conducting channelrhodopsin1, 2, exhibits puzzling properties (unusually-large photocurrents, exceptional red-shift in action spectrum, and extreme light-sensitivity) that have opened up new opportunities in optogenetics1, 3-5. ChRmine and its homologs function as light-gated ion channels, but by primary sequence more closely resemble ion pump rhodopsins; the molecular mechanisms for passive channel conduction in this family of proteins, as well as the unusual properties of ChRmine itself, have remained mysterious. Here we present the cryo-electron microscopy structure of ChRmine at 2.0 [A] resolution. The structure reveals striking architectural features never seen before in channelrhodopsins including trimeric assembly, a short transmembrane-helix 3 unwound in the middle of the membrane, a prominently-twisting extracellular-loop 1, remarkably-large intracellular cavities and extracellular vestibule, and an unprecedented hydrophilic pore that extends through the center of the trimer, separate from the three individual monomer pores. Electrophysiological, spectroscopic, and computational analyses provide insight into conduction and gating of light-gated channels with these distinct design features, and point the way toward structure-guided creation of novel channelrhodopsins for optogenetic applications in biology.

biophysics↗

Characterization of heliorhodopsins detected via functional metagenomics in freshwater Actinobacteria, Chloroflexi and Archaea

Rhodopsins are widespread in microbes residing in diverse aquatic environments across the globe. Recently, a new unusual rhodopsin family, the heliorhodopsins (HeRs), was discovered, distributed among diverse bacteria, archaea, eukarya and even viruses. Here, using functional metagenomics on samples from Lake HaHula and Ein Afek reserve, we found and characterized ten HeRs representing divergent members of the family. The expressed HeRs absorb light in the green and yellow wavelengths and originate from Actinobacteria, Chloroflexi and Archaea. The photocycle of the HeR from Chloroflexi revealed a low accumulation of the M-intermediate that we connect to the lack of two conserved histidine residues in transmembrane helices 1 and 2 in this protein. Another of HeR, from Actinobacteria, exhibited an unusually fast photocycle (166 ms, 5 times faster than HeR-48C12). To further explore the still unresolved question of the HeR function, we performed an analysis of protein families among genes neighboring HeRs, in our clones and thousands of other microbes. This analysis revealed a putative connection between HeRs and genes involved in oxidative stress. At the same time, very few protein families were found to distinguish genes surrounding prokaryotic HeRs from those surrounding rhodopsin pumps. The strongest association was found with the DegV family involved in activation of fatty acids and uncharacterized family DUF2177, which allowed us to hypothesize that HeRs are involved in membrane lipid remodeling. This work further establishes functional metagenomics as a simple and fruitful method of screening for new rhodopsins. SignificanceThe recently discovered divergent rhodopsin family of heliorhodopsins is abundant in freshwater environments. In this study, we sampled a habitat rich in dissolved organic matter to increase our chances of finding spectrally shifted rhodopsins. Using functional metagenomics, diverse heliorhodopsins absorbing green and yellow light were discovered. The metagenomic clones originated from diverse prokaryotic groups: Actinobacteria, Chloroflexi and even Archaea, emphasizing the versatility of the E. coli expression system used. Photocycles of representative heliorhodopsins were measured and exhibited diverse kinetic characteristics. Analysis of genes neighboring heliorhodopsins in diverse prokaryotes revealed their putative connection to membrane lipid re-modeling and oxidative stress. Our findings suggest that functional metagenomics is a productive method for the discovery of new and diverse rhodopsins.

microbiology↗

Coordination between growth and stress responses by DELLA in the liverwort Marchantia polymorpha

Plant survival depends on the optimal use of resources under variable environmental conditions. Among the mechanisms that mediate the balance between growth, differentiation and stress responses, the regulation of transcriptional activity by DELLA proteins stands out. In angiosperms, DELLA accumulation promotes defense against biotic and abiotic stress and represses cell division and expansion, while loss of DELLA function is associated with increased plant size and sensitivity towards stress1. Given that DELLA protein stability is dependent on gibberellin (GA) levels2, and GA metabolism is influenced by the environment3, this pathway is proposed to relay environmental information to the transcriptional programs that regulate growth and stress responses in angiosperms4,5. However, DELLA genes are also found in bryophytes, whereas canonical GA receptors appeared only in vascular plants6-10. Thus, it is not clear whether these regulatory functions of DELLA predated or emerged with typical GA signaling. Here we show that, as in vascular plants, the only DELLA in the liverwort Marchantia polymorpha also participates in the regulation of growth and key developmental processes, and promotes the tolerance towards oxidative stress. Moreover, part of these effects is likely caused by the conserved physical interaction with the MpPIF transcription factor. Therefore, we suggest that the role in the coordination of growth and stress responses was already encoded in DELLA from the common ancestor of land plants, and the importance of this function is justified by its conservation over the past 450 M years.

plant biology↗

Heliorhodopsin evolution is driven by photosensory promiscuity in monoderms

The ability to harness Suns electromagnetic radiation by channeling it into high-energy phosphate bonds empowered microorganisms to tap into a cheap and inexhaustible source of energy. Lifes billion-years history of metabolic innovations led to the emergence of only two biological complexes capable of harvesting light: one based on rhodopsins and the other on (bacterio)chlorophyll. Rhodopsins encompass the most diverse and abundant photoactive proteins on Earth and were until recently canonically split between type-1 (microbial rhodopsins) and type-2 (animal rhodopsins) families. Unexpectedly, the long-lived type-1/type-2 dichotomy was recently amended through the discovery of heliorhodopsins (HeRs) (Pushkarev et al. 2018), a novel and exotic family of rhodopsins (i.e. type-3) that evaded recognition in our current homology-driven scrutiny of lifes genomic milieu. Here, we bring to resolution the debated monoderm/diderm occurrence patterns by conclusively showing that HeR distribution is restricted to monoderms. Furthermore, through investigating protein domain fusions, contextual genomic information, and gene co-expression data we show that HeRs likely function as generalised light-dependent switches involved in the mitigation of light-induced oxidative stress and metabolic circuitry regulation. We reason that HeRs ability to function as sensory rhodopsins is corroborated by their photocycle dynamics (Pushkarev et al. 2018) and that their presence and function in monoderms is likely connected to the increased sensitivity to light-induced damage of these organisms (Maclean et al. 2009).

microbiology↗

Transport engineering for improving production and secretion of valuable alkaloids in Escherichia coli

Metabolic engineering of microorganisms to produce specialized plant metabolites has been established. However, these methods are limited by low productivity and the intracellular accumulation of metabolites. Here, we aimed to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. We established a reticuline-producing Escherichia coli strain and introduced a multidrug and toxic compound extrusion transporter, Arabidopsis AtDTX1, into it. AtDTX1 was selected due to its suitable expression in E. coli and its reticuline-transport activity. Expression of AtDTX1 significantly enhanced reticuline production by 11-fold; produced reticuline was secreted into the medium. AtDTX1 expression conferred high plasmid stability, and up- or downregulated genes associated with biological processes including metabolic pathways for reticuline biosynthesis, leading to a high production and secretion of reticuline. The successful application of a transporter for alkaloid production suggests that the transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.

synthetic biology↗

Machine learning based cellular contractile force detection: a new approach to predict cell mechanics by images

Combining experiments with artificial intelligence algorithms, we propose a new machine learning based approach to extract the cellular force distributions from the microscope images. The full process can be divided into three steps. First, we culture the cells on a special substrate allowing to measure both the cellular traction force on the substrate and the corresponding substrate wrinkles simultaneously. The cellular forces are obtained using the traction force microscopy (TFM), at the same time that cell-generated contractile forces wrinkle their underlying substrate. Second, the wrinkle positions are extracted from the microscope images. Third, we train the machine learning system with GAN (generative adversarial network) by using sets of corresponding two images, the traction field and the input images (raw microscope images or extracted wrinkle images), as the training data. The network understands the way to convert the input images of the substrate wrinkles to the traction distribution from the training. After sufficient training, the network is utilized to predict the cellular forces just from the input images. Our system provides a powerful tool to evaluate the cellular forces efficiently because the forces can be predicted just by observing the cells under the microscope, which is a way simpler method compared to the TFM experiment. Additionally, the machine learning based approach presented here has the profound potential for being applied to diverse cellular assays for studying mechanobiology of cells. Significance StatementCell-generated forces are indispensable determinants of fundamental cell functions such as motility and cell division. As such, quantifying how the forces change upon perturbations to the cells such as gene mutations and drug administration is of profound importance. Here we present a novel machine learning based system that allows for efficient estimations of the forces that are determined only by "observing" microscope images. Given that the cellular traction forces are regulated downstream of diverse signaling pathways, our system - that helps significantly improve the throughput of the measurements - presents a new, high throughput platform for real time analysis of the effects of a massive number of genetic and molecular perturbations on the forces and resulting cell mechanics.

biophysics↗

Comparative neurotranscriptomics reveal widespread species differences associated with bonding

BackgroundPair bonding with a reproductive partner is rare among mammals but is an important feature of human social behavior. Decades of research on monogamous prairie voles (Microtus ochrogaster), along with comparative studies using the related non-bonding meadow vole (M. pennsylvanicus), have revealed many of the neural and molecular mechanisms necessary for pair-bond formation in that species. However, these studies have largely focused on just a few neuromodulatory systems. To test the hypothesis that neural gene expression differences underlie differential capacities to bond, we performed RNA-sequencing on tissue from three brain regions important for bonding and other social behaviors across bond-forming prairie voles and non-bonding meadow voles. We examined gene expression in the amygdala, hypothalamus, and combined ventral pallidum/nucleus accumbens in virgins and at three time points after mating to understand species differences in gene expression at baseline, in response to mating, and during bond formation. ResultsWe first identified species and brain region as the factors most strongly associated with gene expression in our samples. Next, we found gene categories related to cell structure, translation and metabolism that differed in expression across species in virgins, as well as categories associated with cell structure, synaptic and neuroendocrine signaling, and transcription and translation that varied among the focal regions in our study. Additionally, we identified genes that were differentially expressed across species after mating in each of our regions of interest. These include genes involved in regulating transcription, neuron structure, and synaptic plasticity. Finally, we identified modules of co-regulated genes that were strongly correlated with brain region in both species, and modules that were correlated with post-mating time points in prairie voles but not meadow voles. ConclusionsThese results reinforce the importance of pre-mating differences that confer the ability to form pair bonds in prairie voles but not promiscuous species such as meadow voles. Gene ontology analysis supports the hypothesis that pair-bond formation involves transcriptional regulation, and changes in neuronal structure. Together, our results expand knowledge of the genes involved in the pair bonding process and open new avenues of research in the molecular mechanisms of bond formation.

neuroscience↗

Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs reportedly work as light-driven inward H+ pumps, as xenorhodopsin. Here we report the crystal structure of SzR AM_5_00977 at 2.1 [A] resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 in SzR are shorter than those in the other microbial rhodopsins. Thus, E81 is located near the cytosol, playing a critical role in the inward H+ release. We suggested the H+ is not metastably trapped in E81 and released through the water-mediated transport network from the retinal Schiff base to the cytosol. Moreover, most residues on the H+ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H+ release mechanisms.

biochemistry↗

An SNP variant MT1-MMP with a defect in its collagenolytic activity confers the fibrotic phenotype of Dupuytren's Disease

Dupuytrens Disease (DD) is a common fibroproliferative disease of the palmar fascia. We previously identified a strong association with a non-synonymous variant (rs1042704, pD273N) in MMP14 (encoding MT1-MMP). We investigated the functional consequences of this variant, and demonstrated that the variant MT1-MMP (MT1-N273) exhibits only 17% of cell surface collagenolytic activity compared to the ancestral enzyme (MT1-D273). Cells expressing both MT1-D273 and MT1-N273 in a 1:1 ratio, mimicking the heterozygous state, possess 38% of the collagenolytic activity compared to the cells expressing MT1-D273, suggesting that MT1-N273 acts in a dominant negative manner. Consistent with this hypothesis, patient-derived DD myofibroblasts expressing MT1-N273 demonstrated around 30% of full collagenolytic activity regardless of the heterozygous or homozygous state. 3D-molecular envelope modelling using small angle X-ray scattering demonstrated altered positioning of the catalytic domain within dimeric molecules. Taken together, our data suggest that rs1042704 directly contributes to the fibrotic phenotype of DD.

biochemistry↗

Elements at the 5' end of Xist harbor SPEN-independent transcriptional antiterminator activity

The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5' end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for the production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of multiple downstream polyadenylation sequences. Readthrough required Repeat A and the ~750 nucleotides downstream but did not require SPEN. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a transgene comprising Xists first 5.5 kilobases robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5' end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

molecular biology↗