TRMT10A dysfunction perturbs codon translation of initiator methionine and glutamine and impairs brain functions in mice.

In higher eukaryotes, tRNA methyltransferase 10A (TRMT10A) is responsible for N1-methylguanosine modification at position nine of various cytoplasmic tRNAs. Pathogenic mutations in TRMT10A cause intellectual disability, microcephaly, diabetes, and short stature in humans, and generate cytotoxic tRNA fragments in cultured cells; however, it is not clear how TRMT10A supports codon translation or brain functions. Here, we generated Trmt10a null mice and showed that tRNAGln(CUG) and initiator methionine tRNA levels were universally decreased in various tissues; the same was true in a human cell line lacking TRMT10A. Ribosome profiling of mouse brain revealed that dysfunction of TRMT10A causes ribosome slowdown at the Gln(CAG) codon and increases translation of Atf4 due to higher frequency of leaky scanning of its upstream open reading frames. Broadly speaking, translation of a subset of mRNAs, especially those for neuronal structures, is perturbed in the mutant brain. Despite not showing discernable defects in the pancreas, liver, or kidney, Trmt10a null mice showed lower body weight and smaller hippocampal postsynaptic densities, which is associated with defective synaptic plasticity and memory. Taken together, our study provides mechanistic insight into the roles of TRMT10A in the brain, and exemplifies the importance of universal tRNA modification during translation of specific codons.

TIF1ß activates leukemic transcriptional program in HSCs and promotes BCR::ABL1-induced myeloid leukemia.

TIF1ß/KAP1/TRIM28, a chromatin modulator, both represses and activates the transcription of genes in normal and malignant cells. Analyses of datasets on leukemia patients revealed that the expression level of TIF1ß was increased in patients with chronic myeloid leukemia at the blast crisis and acute myeloid leukemia. We generated a BCR::ABL1 conditional knock-in (KI) mouse model, which developed aggressive myeloid leukemia, and demonstrated that the deletion of the Tif1ß gene inhibited the progression of myeloid leukemia and showed longer survival than that in BCR::ABL1 KI mice, suggesting that Tif1ß drove the progression of BCR::ABL1-induced leukemia. In addition, the deletion of Tif1ß sensitized BCR::ABL1 KI leukemic cells to dasatinib. The deletion of Tif1ß decreased the expression levels of TIF1ß-target genes and chromatin accessibility peaks enriched with the Fosl1-binding motif in BCR::ABL1 KI stem cells. TIF1ß directly bound to the promoters of proliferation genes, such as FOSL1, in human BCR::ABL1 cells, in which TIF1ß and FOSL1 bound to adjacent regions of chromatin. Since the expression of Fosl1 was critical for the enhanced growth of BCR::ABL1 KI cells, Tif1ß and Fosl1 interacted to activate the leukemic transcriptional program in and cellular function of BCR::ABL1 KI stem cells and drove the progression of myeloid leukemia.

Chromatin modifier Hmga2 promotes adult hematopoietic stem cell function and blood regeneration in stress conditions.

The molecular mechanisms governing the response of hematopoietic stem cells (HSCs) to stress insults remain poorly defined. Here, we investigated effects of conditional knock-out or overexpression of Hmga2 (High mobility group AT-hook 2), a transcriptional activator of stem cell genes in fetal HSCs. While Hmga2 overexpression did not affect adult hematopoiesis under homeostasis, it accelerated HSC expansion in response to injection with 5-fluorouracil (5-FU) or in vitro treatment with TNF-α. In contrast, HSC and megakaryocyte progenitor cell numbers were decreased in Hmga2 KO animals. Transcription of inflammatory genes was repressed in Hmga2-overexpressing mice injected with 5-FU, and Hmga2 bound to distinct regions and chromatin accessibility was decreased in HSCs upon stress. Mechanistically, we found that casein kinase 2 (CK2) phosphorylates the Hmga2 acidic domain, promoting its access and binding to chromatin, transcription of anti-inflammatory target genes, and the expansion of HSCs under stress conditions. Notably, the identified stress-regulated Hmga2 gene signature is activated in hematopoietic stem progenitor cells of human myelodysplastic syndrome patients. In sum, these results reveal a TNF-α/CK2/phospho-Hmga2 axis controlling adult stress hematopoiesis.

Atypical heat shock transcription factor HSF5 is critical for male meiotic prophase under non-stress conditions.

Meiotic prophase progression is differently regulated in males and females. In males, pachytene transition during meiotic prophase is accompanied by robust alteration in gene expression. However, how gene expression is regulated differently to ensure meiotic prophase completion in males remains elusive. Herein, we identify HSF5 as a male germ cell-specific heat shock transcription factor (HSF) for meiotic prophase progression. Genetic analyzes and single-cell RNA-sequencing demonstrate that HSF5 is essential for progression beyond the pachytene stage under non-stress conditions rather than heat stress. Chromatin binding analysis in vivo and DNA-binding assays in vitro suggest that HSF5 binds to promoters in a subset of genes associated with chromatin organization. HSF5 recognizes a DNA motif different from typical heat shock elements recognized by other canonical HSFs. This study suggests that HSF5 is an atypical HSF that is required for the gene expression program for pachytene transition during meiotic prophase in males.

Morc1 reestablishes H3K9me3 heterochromatin on piRNA-targeted transposons in gonocytes.

To maintain fertility, male mice re-repress transposable elements (TEs) that were de-silenced in the early gonocytes before their differentiation into spermatogonia. However, the mechanism of TE silencing re-establishment remains unknown. Here, we found that the DNA-binding protein Morc1, in cooperation with the methyltransferase SetDB1, deposits the repressive histone mark H3K9me3 on a large fraction of activated TEs, leading to heterochromatin. Morc1 also triggers DNA methylation, but TEs targeted by Morc1-driven DNA methylation only slightly overlapped with those repressed by Morc1/SetDB1-dependent heterochromatin formation, suggesting that Morc1 silences TEs in two different manners. In contrast, TEs regulated by Morc1 and Miwi2, the nuclear PIWI-family protein, almost overlapped. Miwi2 binds to PIWI-interacting RNAs (piRNAs) that base-pair with TE mRNAs via sequence complementarity, while Morc1 DNA binding is not sequence specific, suggesting that Miwi2 selects its targets, and then, Morc1 acts to repress them with cofactors. A high-ordered mechanism of TE repression in gonocytes has been identified.

Tdrd3-null mice show post-transcriptional and behavioral impairments associated with neurogenesis and synaptic plasticity.

The Topoisomerase 3B (Top3b) - Tudor domain containing 3 (Tdrd3) protein complex is the only dual-activity topoisomerase complex that can alter both DNA and RNA topology in animals. TOP3B mutations in humans are associated with schizophrenia, autism and cognitive disorders; and Top3b-null mice exhibit several phenotypes observed in animal models of psychiatric and cognitive disorders, including impaired cognitive and emotional behaviors, aberrant neurogenesis and synaptic plasticity, and transcriptional defects. Similarly, human TDRD3 genomic variants have been associated with schizophrenia, verbal short-term memory and educational attainment. However, the importance of Tdrd3 in normal brain function has not been examined in animal models. Here we generated a Tdrd3-null mouse strain and demonstrate that these mice display both shared and unique defects when compared to Top3b-null mice. Shared defects were observed in cognitive behaviors, synaptic plasticity, adult neurogenesis, newborn neuron morphology, and neuronal activity-dependent transcription; whereas defects unique to Tdrd3-deficient mice include hyperactivity, changes in anxiety-like behaviors, olfaction, increased new neuron complexity, and reduced myelination. Interestingly, multiple genes critical for neurodevelopment and cognitive function exhibit reduced levels in mature but not nascent transcripts. We infer that the entire Top3b-Tdrd3 complex is essential for normal brain function, and that defective post-transcriptional regulation could contribute to cognitive and psychiatric disorders.

Multiple genes in the Pate5-13 genomic region contribute to ADAM3 processing†.

Sperm proteins undergo post-translational modifications during sperm transit through the epididymis to acquire fertilizing ability. We previously reported that the genomic region coding Pate family genes is key to the proteolytic processing of the sperm membrane protein ADAM3 and male fertility. This region contains nine Pate family genes (Pate5-13), and two protein-coding genes (Gm27235 and Gm5916), with a domain structure similar to Pate family genes. Therefore, in this study, we aimed to identify key factors by narrowing the genomic region. We generated three knockout (KO) mouse lines using CRISPR/Cas9: single KO mice of Pate10 expressed in the caput epididymis; deletion KO mice of six caput epididymis-enriched genes (Pate5-7, 13, Gm27235, and Gm5916) (Pate7-Gm5916 KO); and deletion KO mice of four genes expressed in the placenta and epididymis (Pate8, 9, 11, and 12) (Pate8-12 KO). We observed that the fertility of only Pate7-Gm5916 KO males was reduced, whereas the rest remained unaffected. Furthermore, when the caput epididymis-enriched genes, Pate8 and Pate10 remained in Pate7-Gm5916 KO mice were independently deleted, both KO males displayed more severe subfertility due to a decrease in mature ADAM3 and a defect in sperm migration to the oviduct. Thus, our data showed that multiple caput epididymis-enriched genes within the region coding Pate5-13 cooperatively function to ensure male fertility in mice.

The IL-17-IL-17RA axis is required to promote osteosarcoma progression in mice.

Osteosarcoma is rare but is the most common bone tumor. Diagnostic tools such as magnetic resonance imaging development of chemotherapeutic agents have increased the survival rate in osteosarcoma patients, although 5-year survival has plateaued at 70%. Thus, development of new treatment approaches is needed. Here, we report that IL-17, a proinflammatory cytokine, increases osteosarcoma mortality in a mouse model with AX osteosarcoma cells. AX cell transplantation into wild-type mice resulted in 100% mortality due to ectopic ossification and multi-organ metastasis. However, AX cell transplantation into IL-17-deficient mice significantly prolonged survival relative to controls. CD4-positive cells adjacent to osteosarcoma cells express IL-17, while osteosarcoma cells express the IL-17 receptor IL-17RA. Although AX cells can undergo osteoblast differentiation, as can patient osteosarcoma cells, IL-17 significantly inhibited that differentiation, indicating that IL-17 maintains AX cells in the undifferentiated state seen in malignant tumors. By contrast, IL-17RA-deficient mice transplanted with AX cells showed survival comparable to wild-type mice transplanted with AX cells. Biopsy specimens collected from osteosarcoma patients showed higher expression of IL-17RA compared to IL-17. These findings suggest that IL-17 is essential to maintain osteosarcoma cells in an undifferentiated state and could be a therapeutic target for suppressing tumorigenesis.

4.5SH RNA counteracts deleterious exonization of SINE B1 in mice.

4.5SH RNA is a highly abundant, small rodent-specific noncoding RNA that localizes to nuclear speckles enriched in pre-mRNA-splicing regulators. To investigate the physiological functions of 4.5SH RNA, we have created mutant mice that lack the expression of 4.5SH RNA. The mutant mice exhibited embryonic lethality, suggesting that 4.5SH RNA is an essential species-specific noncoding RNA in mice. RNA-sequencing analyses revealed that 4.5SH RNA protects the transcriptome from abnormal exonizations of the antisense insertions of the retrotransposon SINE B1 (asB1), which would otherwise introduce deleterious premature stop codons or frameshift mutations. Mechanistically, 4.5SH RNA base pairs with complementary asB1-containing exons via the target recognition region and recruits effector proteins including Hnrnpm via its 5' stem loop region. The modular organization of 4.5SH RNA allows us to engineer a programmable splicing regulator to induce the skipping of target exons of interest. Our results also suggest the general existence of splicing regulatory noncoding RNAs.

Fibroblast growth factor 18 stimulates the proliferation of hepatic stellate cells, thereby inducing liver fibrosis.

Liver fibrosis results from chronic liver injury triggered by factors such as viral infection, excess alcohol intake, and lipid accumulation. However, the mechanisms underlying liver fibrosis are not fully understood. Here, we demonstrate that the expression of fibroblast growth factor 18 (Fgf18) is elevated in mouse livers following the induction of chronic liver fibrosis models. Deletion of Fgf18 in hepatocytes attenuates liver fibrosis; conversely, overexpression of Fgf18 promotes liver fibrosis. Single-cell RNA sequencing reveals that overexpression of Fgf18 in hepatocytes results in an increase in the number of Lrat+ hepatic stellate cells (HSCs), thereby inducing fibrosis. Mechanistically, FGF18 stimulates the proliferation of HSCs by inducing the expression of Ccnd1. Moreover, the expression of FGF18 is correlated with the expression of profibrotic genes, such as COL1A1 and ACTA2, in human liver biopsy samples. Thus, FGF18 promotes liver fibrosis and could serve as a therapeutic target to treat liver fibrosis.

STRA8-RB interaction is required for timely entry of meiosis in mouse female germ cells.

Meiosis is differently regulated in males and females. In females, germ cells initiate meiosis within a limited time period in the fetal ovary and undergo a prolonged meiotic arrest until puberty. However, how meiosis initiation is coordinated with the cell cycle to coincide with S phase remains elusive. Here, we demonstrate that STRA8 binds to RB via the LXCXE motif. Mutation of the RB-binding site of STRA8 in female mice delays meiotic entry, which consequently delays progression of meiotic prophase and leads to precocious depletion of the oocyte pool. Single-cell RNA-sequencing analysis reveals that the STRA8-RB interaction is required for S phase entry and meiotic gene activation, ensuring precise timing of meiosis initiation in oocytes. Strikingly, the results suggest STRA8 could sequester RB from E2F during pre-meiotic G1/S transition. This study highlights the gene regulatory mechanisms underlying the female-specific mode of meiotic initiation in mice.

Tmem161a regulates bone formation and bone strength through the P38 MAPK pathway.

Bone remodeling is an extraordinarily complex process involving a variety of factors, such as genetic, metabolic, and environmental components. Although genetic factors play a particularly important role, many have not been identified. In this study, we investigated the role of transmembrane 161a (Tmem161a) in bone structure and function using wild-type (WT) and Tmem161a-depleted (Tmem161aGT/GT) mice. Mice femurs were examined by histological, morphological, and bone strength analyses. Osteoblast differentiation and mineral deposition were examined in Tmem161a-overexpressed, -knockdown and -knockout MC3T3-e1 cells. In WT mice, Tmem161a was expressed in osteoblasts of femurs; however, it was depleted in Tmem161aGT/GT mice. Cortical bone mineral density, thickness, and bone strength were significantly increased in Tmem161aGT/GT mice femurs. In MC3T3-e1 cells, decreased expression of alkaline phosphatase (ALP) and Osterix were found in Tmem161a overexpression, and these findings were reversed in Tmem161a-knockdown or -knockout cells. Microarray and western blot analyses revealed upregulation of the P38 MAPK pathway in Tmem161a-knockout cells, which referred as stress-activated protein kinases. ALP and flow cytometry analyses revealed that Tmem161a-knockout cells were resistant to oxidative stress. In summary, Tmem161a is an important regulator of P38 MAPK signaling, and depletion of Tmem161a induces thicker and stronger bones in mice.

A specific plasma amino acid profile in the Insulin2Q104del Kuma mice at the diabetic state and reversal from hyperglycemia.

The metabolites in the plasma serve as potential biomarkers of disease. We previously established an early-onset diabetes mouse model, Ins2+/Q104del Kuma mice, under a severe immune-deficient (Rag-2/Jak3 double-deficient in BALB/c) background. Here, we revealed the differences in plasma amino acid profiles between Kuma and the wild-type mice. We observed an early reduction in glucogenic and ketogenic amino acids, a late increase in branched-chain amino acids (BCAAs) and succinyl CoA-related amino acids, and a trend of increasing ketogenic amino acids in Kuma mice than in the wild-type mice. Kuma mice exhibited hyperglucagonemia at high blood glucose, leading to perturbations in plasma amino acid profiles. The reversal of blood glucose by islet transplantation normalized the increases of the BCAAs and several aspects of the altered metabolic profiles in Kuma mice. Our results indicate that the Kuma mice are a unique animal model to study the link between plasma amino acid profile and the progression of diabetes for monitoring the therapeutic effects.

A cyclic pyrrole-imidazole polyamide reduces pathogenic RNA in CAG/CTG triplet repeat neurological disease models.

Expansion of CAG and CTG (CWG) triplet repeats causes several inherited neurological diseases. The CWG repeat diseases are thought to involve complex pathogenic mechanisms through expanded CWG repeat-derived RNAs in a noncoding region and polypeptides in a coding region, respectively. However, an effective therapeutic approach has not been established for the CWG repeat diseases. Here, we show that a CWG repeat DNA-targeting compound, cyclic pyrrole-imidazole polyamide (CWG-cPIP), suppressed the pathogenesis of coding and noncoding CWG repeat diseases. CWG-cPIP bound to the hairpin form of mismatched CWG DNA, interfering with transcription elongation by RNA polymerase through a preferential activity toward repeat-expanded DNA. We found that CWG-cPIP selectively inhibited pathogenic mRNA transcripts from expanded CWG repeats, reducing CUG RNA foci and polyglutamine accumulation in cells from patients with myotonic dystrophy type 1 (DM1) and Huntington's disease (HD). Treatment with CWG-cPIP ameliorated behavioral deficits in adeno-associated virus-mediated CWG repeat-expressing mice and in a genetic mouse model of HD, without cytotoxicity or off-target effects. Together, we present a candidate compound that targets expanded CWG repeat DNA independently of its genomic location and reduces both pathogenic RNA and protein levels. CWG-cPIP may be used for the treatment of CWG repeat diseases and improvement of clinical outcomes.

ANGPTL2-mediated epigenetic repression of MHC-I in tumor cells accelerates tumor immune evasion.

Loss or downregulation of major histocompatibility complex class I (MHC-I) contributes to tumor immune evasion. We previously demonstrated that angiopoietin-like protein 2 (ANGPTL2) promotes tumor progression using a Xp11.2 translocation renal cell carcinoma (tRCC) mouse model. However, molecular mechanisms underlying ANGPTL2 tumor-promoting activity in the tRCC model remained unclear. Here, we report that ANGPTL2 deficiency in renal tubular epithelial cells slows tumor progression in the tRCC mouse model and promotes activated CD8+ T-cell infiltration of kidney tissues. We also found that Angptl2-deficient tumor cells show enhanced interferon γ-induced expression of MHC-I and increased susceptibility to CD8+ T-cell-mediated anti-tumor immune responses. Moreover, we provide evidence that the ANGPTL2-α5ß1 integrin pathway accelerates polycomb repressive complex 2-mediated repression of MHC-I expression in tumor cells. These findings suggest that ANGPTL2 signaling in tumor cells contributes to tumor immune evasion and that suppressing that signaling in tumor cells could serve as a potential strategy to facilitate tumor elimination by T-cell-mediated anti-tumor immunity.

Tdrd3-null mice show post-transcriptional and behavioral impairments associated with neurogenesis and synaptic plasticity.

The Topoisomerase 3B (Top3b) - Tudor domain containing 3 (Tdrd3) protein complex is the only dual-activity topoisomerase complex in animals that can alter the topology of both DNA and RNA. TOP3B mutations in humans are associated with schizophrenia, autism and cognitive disorders; and Top3b-null mice exhibit several phenotypes observed in animal models of psychiatric and cognitive disorders, including impairments in cognitive and emotional behaviors, aberrant neurogenesis and synaptic plasticity, and transcriptional defects. Similarly, human TDRD3 genomic variants have been associated with schizophrenia, verbal shorten-memory and learning, and educational attainment. However, the importance of Tdrd3 in normal brain function has not been examined in animal models. Here we built a Tdrd3-null mouse strain and demonstrate that these mice display both shared and unique defects when compared to Top3b-null mice. Shared defects were observed in cognitive behaviors, synaptic plasticity, adult neurogenesis, newborn neuron morphology, and neuronal activity-dependent transcription; whereas defects unique to Tdrd3-deficient mice include hyperactivity, changes in anxiety-like behaviors, increased new neuron complexity, and reduced myelination. Interestingly, multiple genes critical for neurodevelopment and cognitive function exhibit reduced levels in mature but not nascent transcripts. We infer that the entire Top3b-Tdrd3 complex is essential for normal brain function, and that defective post-transcriptional regulation could contribute to cognitive impairment and psychiatric disorders.

A gain-of-function mutation in microRNA 142 is sufficient to cause the development of T-cell leukemia in mice.

MicroRNAs (miRNAs) play a crucial role in regulating gene expression. MicroRNA expression levels fluctuate, and point mutations and methylation occur in cancer cells; however, to date, there have been no reports of carcinogenic point mutations in miRNAs. MicroRNA 142 (miR-142) is frequently mutated in patients with follicular lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia (CLL), and acute myeloid leukemia/myelodysplastic syndrome (AML/MDS). To understand the role of miR-142 mutation in blood cancers, the CRISPR-Cas9 system was utilized to successfully generate miR-142-55A>G mutant knock-in (Ki) mice, simulating the most frequent mutation in patients with miR-142 mutated AML/MDS. Bone marrow cells from miR-142 mutant heterozygous Ki mice were transplanted, and we found that the miR-142 mutant/wild-type cells were sufficient for the development of CD8+ T-cell leukemia in mice post-transplantation. RNA-sequencing analysis in hematopoietic stem/progenitor cells and CD8+ T-cells revealed that miR-142-Ki/+ cells had increased expression of the mTORC1 activator, a potential target of wild-type miR-142-3p. Notably, the expression of genes involved in apoptosis, differentiation, and the inhibition of the Akt-mTOR pathway was suppressed in miR-142-55A>G heterozygous cells, indicating that these genes are repressed by the mutant miR-142-3p. Thus, in addition to the loss of function due to the halving of wild-type miR-142-3p alleles, mutated miR-142-3p gained the function to suppress the expression of distinct target genes, sufficient to cause leukemogenesis in mice.

The testis-, epididymis-, or seminal vesicle-enriched genes Aldoart2, Serpina16, Aoc1l3, and Pate14 are not essential for male fertility in mice.

Spermatozoa released from the testis acquire fertilizing ability by translocating thorough the epididymis. Further, accessory gland secretions ejaculated into the female reproductive tract along with spermatozoa are also required to ensure male fecundity, such as the maintenance of proper sperm count and inhibition of premature sperm capacitation in the uterus. Here, we focus on a testis-enriched gene "Aldoart2", an epididymis-enriched gene "Serpina16", and seminal vesicle-enriched genes "Aoc1l3" and "Pate14" which were thought to be important for male fertility based on the previous studies. We independently deleted almost the entire protein-coding sequence of these genes in mice using CRISPR/Cas9. There were no overt defects in the histology and the sperm morphology and motility of any knockout (KO) mice. Further, Aoc1l3 and Pate14 KO males were able to form copulatory plugs. Finally, female mice that mated with these KO males delivered pups at a comparable level with the control males. Given our data, we demonstrated that the four genes predominantly expressed in the testis, epididymis, or seminal vesicle are independently dispensable for male fertility.

Generation of transgenic mice expressing a FRET biosensor, SMART, that responds to necroptosis.

Necroptosis is a regulated form of cell death involved in various pathological conditions, including ischemic reperfusion injuries, virus infections, and drug-induced tissue injuries. However, it is not fully understood when and where necroptosis occurs in vivo. We previously generated a Forster resonance energy transfer (FRET) biosensor, termed SMART (the sensor for MLKL activation by RIPK3 based on FRET), which monitors conformational changes of MLKL along with progression of necroptosis in human and murine cell lines in vitro. Here, we generate transgenic (Tg) mice that express the SMART biosensor in various tissues. The FRET ratio is increased in necroptosis, but not apoptosis or pyroptosis, in primary cells. Moreover, the FRET signals are elevated in renal tubular cells of cisplatin-treated SMART Tg mice compared to untreated SMART Tg mice. Together, SMART Tg mice may provide a valuable tool for monitoring necroptosis in different types of cells in vitro and in vivo.