Description: Hereditary tyrosinemia type 1 (HT1) is a severe human autosomal recessive disorder caused by the deficiency of fumarylacetoacetate hydroxylase (FAH), an enzyme catalyzing the last step in the tyrosine degradation pathway. Lack of FAH causes accumulation of toxic metabolites (fumarylacetoacetate and succinylacetone) in blood and tissues, ultimately resulting in severe liver and kidney damage with onset that ranges from infancy to adolescence. This tissue damage is lethal but can be controlled by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which inhibits tyrosine catabolism upstream of the generation of fumarylacetoacetate and succinylacetone. Notably, in animals lacking FAH, transient withdrawal of NTBC can be used to induce liver damage and a concomitant regenerative response that stimulates the growth of healthy hepatocytes. Among other things, this model has raised tremendous interest for the in vivo expansion of human primary hepatocytes inside these animals and for exploring experimental gene therapy and cell-based therapies. Here, we report the generation of FAH knock-out rabbits via pronuclear stage embryo microinjection of transcription activator-like effector nucleases. FAH−/− rabbits exhibit phenotypic features of HT1 including liver and kidney abnormalities but additionally develop frequent ocular manifestations likely caused by local accumulation of tyrosine upon NTBC administration. We also show that allogeneic transplantation of wild-type rabbit primary hepatocytes into FAH−/− rabbits enables highly efficient liver repopulation and prevents liver insufficiency and death. Because of significant advantages over rodents and their ease of breeding, maintenance, and manipulation compared with larger animals including pigs, FAH−/− rabbits are an attractive alternative for modeling the consequences of HT1.

Description: Spermatogenesis is precisely controlled by complex gene expression programs and involves epigenetic reprogramming, including histone modification and DNA methylation. SET domain–containing 2 (SETD2) is the predominant histone methyltransferase catalyzing the trimethylation of histone H3 lysine 36 (H3K36me3) and plays key roles in embryonic stem cell differentiation and somatic cell development. However, its role in male germ cell development remains elusive. Here, we demonstrate an essential role of Setd2 for spermiogenesis, the final stage of spermatogenesis. Using RNA-seq, we found that, in postnatal mouse testes, Setd2 mRNA levels dramatically increase in 14-day-old mice. Using a germ cell–specific Setd2 knockout mouse model, we also found that targeted Setd2 knockout in germ cells causes aberrant spermiogenesis with acrosomal malformation before step 8 of the round-spermatid stage, resulting in complete infertility. Furthermore, we noted that the Setd2 deficiency results in complete loss of H3K36me3 and significantly decreases expression of thousands of genes, including those encoding acrosin-binding protein 1 (Acrbp1) and protamines, required for spermatogenesis. Our findings thus reveal a previously unappreciated role of the SETD2-dependent H3K36me3 modification in spermiogenesis and provide clues to the molecular mechanisms in epigenetic disorders underlying male infertility.

Description: The Hippo pathway plays important roles in controlling organ size and in suppressing tumorigenesis through large tumor suppressor kinase 1/2 (LATS1/2)–mediated phosphorylation of YAP/TAZ transcription co-activators. The kinase activity of LATS1/2 is regulated by phosphorylation in response to extracellular signals. Moreover, LATS2 protein levels are repressed by the ubiquitin–proteasome system in conditions such as hypoxia. However, the mechanism that removes the ubiquitin modification from LATS2 and thereby stabilizes the protein is not well understood. Here, using tandem affinity purification (TAP), we found that anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase complex, and USP9X, a deubiquitylase, specifically interact with LATS2. We also found that although APC1 co-localizes with LATS2 to intracellular vesicle structures, it does not regulate LATS2 protein levels and activity. In contrast, USP9X ablation drastically diminished LATS2 protein levels. We further demonstrated that USP9X deubiquitinates LATS2 and thus prevents LATS2 degradation by the proteasome. Furthermore, in pancreatic cancer cells, USP9X loss activated YAP and enhanced the oncogenic potential of the cells. In addition, the tumorigenesis induced by the USP9X ablation depended not only on LATS2 repression, but also on YAP/TAZ activity. We conclude that USP9X is a deubiquitylase of the Hippo pathway kinase LATS2 and that the Hippo pathway functions as a downstream signaling cascade that mediates USP9X's tumor-suppressive activity.

Description: In aortic vascular smooth muscle (VSM), the canonical Wnt receptor LRP6 inhibits protein arginine (Arg) methylation, a new component of noncanonical Wnt signaling that stimulates nuclear factor of activated T cells (viz. NFATc4). To better understand how methylation mediates these actions, MS was performed on VSM cell extracts from control and LRP6-deficient mice. LRP6-dependent Arg methylation was regulated on 〉500 proteins; only 21 exhibited increased monomethylation (MMA) with concomitant reductions in dimethylation. G3BP1, a known regulator of arteriosclerosis, exhibited a 〉30-fold increase in MMA in its C-terminal domain. Co-transfection studies confirm that G3BP1 (G3BP is Ras-GAP SH3 domain–binding protein) methylation is inhibited by LRP6 and that G3BP1 stimulates NFATc4 transcription. NFATc4 association with VSM osteopontin (OPN) and alkaline phosphatase (TNAP) chromatin was increased with LRP6 deficiency and reduced with G3BP1 deficiency. G3BP1 activation of NFATc4 mapped to G3BP1 domains supporting interactions with RIG-I (retinoic acid inducible gene I), a stimulus for mitochondrial antiviral signaling (MAVS) that drives cardiovascular calcification in humans when mutated in Singleton-Merten syndrome (SGMRT2). Gain-of-function SGMRT2/RIG-I mutants increased G3BP1 methylation and synergized with osteogenic transcription factors (Runx2 and NFATc4). A chemical antagonist of G3BP, C108 (C108 is 2-hydroxybenzoic acid, 2-[1-(2-hydroxyphenyl)ethylidene]hydrazide CAS 15533-09-2), down-regulated RIG-I–stimulated G3BP1 methylation, Wnt/NFAT signaling, VSM TNAP activity, and calcification. G3BP1 deficiency reduced RIG-I protein levels and VSM osteogenic programs. Like G3BP1 and RIG-I deficiency, MAVS deficiency reduced VSM osteogenic signals, including TNAP activity and Wnt5-dependent nuclear NFATc4 levels. Aortic calcium accumulation is decreased in MAVS-deficient LDLR−/− mice fed arteriosclerotic diets. The G3BP1/RIG-I/MAVS relay is a component of Wnt signaling. Targeting this relay may help mitigate arteriosclerosis.

Description: ATP-binding cassette transporter A1 (ABCA1), which promotes cholesterol efflux from cells and inhibits inflammatory responses, is highly expressed in the kidney. Research has shown that exendin-4, a glucagon-like peptide-1 receptor (GLP-1R) agonist, promotes ABCA1 expression in multiple tissues and organs; however, the mechanisms underlying exendin-4 induction of ABCA1 expression in glomerular endothelial cells are not fully understood. In this study we investigated the effect of exendin-4 on ABCA1 in glomerular endothelial cells of diabetic kidney disease (DKD) and the possible mechanism. We observed a marked increase in glomerular lipid deposits in tissues of patients with DKD and diabetic apolipoprotein E knock-out (apoE−/−) mice by Oil Red O staining and biochemical analysis of cholesterol. We found significantly decreased ABCA1 expression in glomerular endothelial cells of diabetic apoE−/− mice and increased renal lipid, cholesterol, and inflammatory cytokine levels. Exendin-4 decreased renal cholesterol accumulation and inflammation and increased cholesterol efflux by up-regulating ABCA1. In human glomerular endothelial cells, GLP-1R-mediated signaling pathways (e.g. Ca2+/calmodulin-dependent protein kinase, cAMP/PKA, PI3K/AKT, and ERK1/2) were involved in cholesterol efflux and inflammatory responses by regulating ABCA1 expression. We propose that exendin-4 increases ABCA1 expression in glomerular endothelial cells, which plays an important role in alleviating renal lipid accumulation, inflammation, and proteinuria in mice with type 2 diabetes.

Description: Polyalanine (poly(A)) diseases are caused by the expansion of translated GCN triplet nucleotide sequences encoding poly(A) tracts in proteins. To date, nine human disorders have been found to be associated with poly(A) tract expansions, including congenital central hypoventilation syndrome and oculopharyngeal muscular dystrophy. Previous studies have demonstrated that unexpanded wild-type poly(A)-containing proteins localize to the cell nucleus, whereas expanded poly(A)-containing proteins primarily localize to the cytoplasm. Because most of these poly(A) disease proteins are transcription factors, this mislocalization causes cellular transcriptional dysregulation leading to cellular dysfunction. Correcting this faulty localization could potentially point to strategies to treat the aforementioned disorders, so there is a pressing need to identify the mechanisms underlying the mislocalization of expanded poly(A) protein. Here, we performed a glutathione S-transferase pulldown assay followed by mass spectrometry and identified eukaryotic translation elongation factor 1 α1 (eEF1A1) as an interacting partner with expanded poly(A)-containing proteins. Strikingly, knockdown of eEF1A1 expression partially corrected the mislocalization of the expanded poly(A) proteins in the cytoplasm and restored their functions in the nucleus. We further demonstrated that the expanded poly(A) domain itself can serve as a nuclear export signal. Taken together, this study demonstrates that eEF1A1 regulates the subcellular location of expanded poly(A) proteins and is therefore a potential therapeutic target for combating the pathogenesis of poly(A) diseases.