Target-directed microRNA degradation regulates developmental microRNA expression and embryonic growth in mammals.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8, which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in the heart and lungs, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in cotranscribed clusters and examples in which TDMD underlies "arm switching," a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8-null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.

Acetate dependence of tumors.

Acetyl-CoA represents a central node of carbon metabolism that plays a key role in bioenergetics, cell proliferation, and the regulation of gene expression. Highly glycolytic or hypoxic tumors must produce sufficient quantities of this metabolite to support cell growth and survival under nutrient-limiting conditions. Here, we show that the nucleocytosolic acetyl-CoA synthetase enzyme, ACSS2, supplies a key source of acetyl-CoA for tumors by capturing acetate as a carbon source. Despite exhibiting no gross deficits in growth or development, adult mice lacking ACSS2 exhibit a significant reduction in tumor burden in two different models of hepatocellular carcinoma. ACSS2 is expressed in a large proportion of human tumors, and its activity is responsible for the majority of cellular acetate uptake into both lipids and histones. These observations may qualify ACSS2 as a targetable metabolic vulnerability of a wide spectrum of tumors.

Unconventional mechanism of action and resistance to rapalogs in renal cancer.

mTORC1 is aberrantly activated in renal cell carcinoma (RCC) and is targeted by rapalogs. As for other targeted therapies, rapalogs clinical utility is limited by the development of resistance. Resistance often results from target mutation, but mTOR mutations are rarely found in RCC. As in humans, prolonged rapalog treatment of RCC tumorgrafts (TGs) led to resistance. Unexpectedly, explants from resistant tumors became sensitive both in culture and in subsequent transplants in mice. Notably, resistance developed despite persistent mTORC1 inhibition in tumor cells. In contrast, mTORC1 became reactivated in the tumor microenvironment (TME). To test the role of the TME, we engineered immunocompromised recipient mice with a resistance mTOR mutation (S2035T). Interestingly, TGs became resistant to rapalogs in mTORS2035T mice. Resistance occurred despite mTORC1 inhibition in tumor cells and could be induced by coculturing tumor cells with mutant fibroblasts. Thus, enforced mTORC1 activation in the TME is sufficient to confer resistance to rapalogs. These studies highlight the importance of mTORC1 inhibition in nontumor cells for rapalog antitumor activity and provide an explanation for the lack of mTOR resistance mutations in RCC patients.

miR-26 suppresses adipocyte progenitor differentiation and fat production by targeting Fbxl19.

Fat storage in adult mammals is a highly regulated process that involves the mobilization of adipocyte progenitor cells (APCs) that differentiate to produce new adipocytes. Here we report a role for the broadly conserved miR-26 family of microRNAs (miR-26a-1, miR-26a-2, and miR-26b) as major regulators of APC differentiation and adipose tissue mass. Deletion of all miR-26-encoding loci in mice resulted in a dramatic expansion of adipose tissue in adult animals fed normal chow. Conversely, transgenic overexpression of miR-26a protected mice from high-fat diet-induced obesity. These effects were attributable to a cell-autonomous function of miR-26 as a potent inhibitor of APC differentiation. miR-26 blocks adipogenesis, at least in part, by repressing expression of Fbxl19, a conserved miR-26 target without a previously known role in adipocyte biology that encodes a component of SCF-type E3 ubiquitin ligase complexes. These findings have therefore revealed a novel pathway that plays a critical role in regulating adipose tissue formation in vivo and suggest new potential therapeutic targets for obesity and related disorders.

Hepatic ribosomal protein S6 (Rps6) insufficiency results in failed bile duct development and loss of hepatocyte viability; a ribosomopathy-like phenotype that is partially p53-dependent.

Defective ribosome biogenesis (RiBi) underlies a group of clinically diverse human diseases collectively known as the ribosomopathies, core manifestations of which include cytopenias and developmental abnormalities that are believed to stem primarily from an inability to synthesize adequate numbers of ribosomes and concomitant activation of p53. The importance of a correctly functioning RiBi machinery for maintaining tissue homeostasis is illustrated by the observation that, despite having a paucity of certain cell types in early life, ribosomopathy patients have an increased risk for developing cancer later in life. This suggests that hypoproliferative states trigger adaptive responses that can, over time, become maladaptive and inadvertently drive unchecked hyperproliferation and predispose to cancer. Here we describe an experimentally induced ribosomopathy in the mouse and show that a normal level of hepatic ribosomal protein S6 (Rps6) is required for proper bile duct development and preservation of hepatocyte viability and that its insufficiency later promotes overgrowth and predisposes to liver cancer which is accelerated in the absence of the tumor-suppressor PTEN. We also show that the overexpression of c-Myc in the liver ameliorates, while expression of a mutant hyperstable form of p53 partially recapitulates specific aspects of the hepatopathies induced by Rps6 deletion. Surprisingly, co-deletion of p53 in the Rps6-deficient background fails to restore biliary development or significantly improve hepatic function. This study not only reveals a previously unappreciated dependence of the developing liver on adequate levels of Rps6 and exquisitely controlled p53 signaling, but suggests that the increased cancer risk in ribosomopathy patients may, in part, stem from an inability to preserve normal tissue homeostasis in the face of chronic injury and regeneration.

Loss of Dis3l2 partially phenocopies Perlman syndrome in mice and results in up-regulation of Igf2 in nephron progenitor cells.

Loss of function of the DIS3L2 exoribonuclease is associated with Wilms tumor and the Perlman congenital overgrowth syndrome. LIN28, a Wilms tumor oncoprotein, triggers the DIS3L2-mediated degradation of the precursor of let-7, a microRNA that inhibits Wilms tumor development. These observations have led to speculation that DIS3L2-mediated tumor suppression is attributable to let-7 regulation. Here we examine new DIS3L2-deficient cell lines and mouse models, demonstrating that DIS3L2 loss has no effect on mature let-7 levels. Rather, analysis of Dis3l2-null nephron progenitor cells, a potential cell of origin of Wilms tumors, reveals up-regulation of Igf2, a growth-promoting gene strongly associated with Wilms tumorigenesis. These findings nominate a new potential mechanism underlying the pathology associated with DIS3L2 deficiency.

p53 genes function to restrain mobile elements.

Throughout the animal kingdom, p53 genes govern stress response networks by specifying adaptive transcriptional responses. The human member of this gene family is mutated in most cancers, but precisely how p53 functions to mediate tumor suppression is not well understood. Using Drosophila and zebrafish models, we show that p53 restricts retrotransposon activity and genetically interacts with components of the piRNA (piwi-interacting RNA) pathway. Furthermore, transposon eruptions occurring in the p53(-) germline were incited by meiotic recombination, and transcripts produced from these mobile elements accumulated in the germ plasm. In gene complementation studies, normal human p53 alleles suppressed transposons, but mutant p53 alleles from cancer patients could not. Consistent with these observations, we also found patterns of unrestrained retrotransposons in p53-driven mouse and human cancers. Furthermore, p53 status correlated with repressive chromatin marks in the 5' sequence of a synthetic LINE-1 element. Together, these observations indicate that ancestral functions of p53 operate through conserved mechanisms to contain retrotransposons. Since human p53 mutants are disabled for this activity, our findings raise the possibility that p53 mitigates oncogenic disease in part by restricting transposon mobility.

Mammalian acetate-dependent acetyl CoA synthetase 2 contains multiple protein destabilization and masking elements.

Besides contributing to anabolism, cellular metabolites serve as substrates or cofactors for enzymes and may also have signaling functions. Given these roles, multiple control mechanisms likely ensure fidelity of metabolite-generating enzymes. Acetate-dependent acetyl CoA synthetases (ACS) are de novo sources of acetyl CoA, a building block for fatty acids and a substrate for acetyltransferases. Eukaryotic acetate-dependent acetyl CoA synthetase 2 (Acss2) is predominantly cytosolic, but is also found in the nucleus following oxygen or glucose deprivation, or upon acetate exposure. Acss2-generated acetyl CoA is used in acetylation of Hypoxia-Inducible Factor 2 (HIF-2), a stress-responsive transcription factor. Mutation of a putative nuclear localization signal in endogenous Acss2 abrogates HIF-2 acetylation and signaling, but surprisingly also results in reduced Acss2 protein levels due to unmasking of two protein destabilization elements (PDE) in the Acss2 hinge region. In the current study, we identify up to four additional PDE in the Acss2 hinge region and determine that a previously identified PDE, the ABC domain, consists of two functional PDE. We show that the ABC domain and other PDE are likely masked by intramolecular interactions with other domains in the Acss2 hinge region. We also characterize mice with a prematurely truncated Acss2 that exposes a putative ABC domain PDE, which exhibits reduced Acss2 protein stability and impaired HIF-2 signaling. Finally, using primary mouse embryonic fibroblasts, we demonstrate that the reduced stability of select Acss2 mutant proteins is due to a shortened half-life, which is a result of enhanced degradation via a nonproteasome, nonautophagy pathway.

The Human Leukocyte Antigen (HLA)-B27 Peptidome in Vivo, in Spondyloarthritis-susceptible HLA-B27 Transgenic Rats and the Effect of Erap1 Deletion.

HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used large scale quantitative mass spectrometry to identify over 15,000 unique HLA-B27 peptide ligands, isolated after immunoaffinity purification of the B27 molecules from the spleens of HLA-B27 transgenic rats. Heterozygous deletion of Erap1, which reduced the Erap1 level to less than half, had no qualitative or quantitative effects on the B27 peptidome. Homozygous deletion of Erap1 affected approximately one-third of the B27 peptidome but left most of the B27 peptidome unchanged, suggesting the possibility that some of the HLA-B27 immunopeptidome is not processed in the presence of Erap1. Deletion of Erap1 was permissive for the AS-like phenotype, increased mean peptide length and increased the frequency of C-terminal hydrophobic residues and of N-terminal Ala, Ser, or Lys. The presence of Erap1 increased the frequency of C-terminal Lys and Arg, of Glu and Asp at intermediate residues, and of N-terminal Gly. Several peptides of potential interest in AS pathogenesis, previously identified in human cell lines, were isolated. However, rats susceptible to arthritis had B27 peptidomes similar to those of non-susceptible rats, and no peptides were found to be uniquely associated with arthritis. Whether specific B27-bound peptides are required for AS pathogenesis remains to be determined. Data are available via ProteomeXchange with identifier PXD005502.

Mesolimbic neuropeptide W coordinates stress responses under novel environments.

Neuropeptide B (NPB) and neuropeptide W (NPW) are endogenous neuropeptide ligands for the G protein-coupled receptors NPBWR1 and NPBWR2. Here we report that the majority of NPW neurons in the mesolimbic region possess tyrosine hydroxylase immunoreactivity, indicating that a small subset of dopaminergic neurons coexpress NPW. These NPW-containing neurons densely and exclusively innervate two limbic system nuclei in adult mouse brain: the lateral bed nucleus of the stria terminalis and the lateral part of the central amygdala nucleus (CeAL). In the CeAL of wild-type mice, restraint stress resulted in an inhibition of cellular activity, but this stress-induced inhibition was attenuated in the CeAL neurons of NPW(-/-) mice. Moreover, the response of NPW(-/-) mice to either formalin-induced pain stimuli or a live rat (i.e., a potential predator) was abnormal only when they were placed in a novel environment: The mice failed to show the normal species-specific self-protective and aversive reactions. In contrast, the behavior of NPW(-/-) mice in a habituated environment was indistinguishable from that of wild-type mice. These results indicate that the NPW/NPBWR1 system could play a critical role in the gating of stressful stimuli during exposure to novel environments.

Interplay between ChREBP and SREBP-1c coordinates postprandial glycolysis and lipogenesis in livers of mice.

Lipogenesis in liver is highest in the postprandial state; insulin activates SREBP-1c, which transcriptionally activates genes involved in FA synthesis, whereas glucose activates carbohydrate-responsive element-binding protein (ChREBP), which activates both glycolysis and FA synthesis. Whether SREBP-1c and ChREBP act independently of one another is unknown. Here, we characterized mice with liver-specific deletion of ChREBP (L-Chrebp-/- mice). Hepatic ChREBP deficiency resulted in reduced mRNA levels of glycolytic and lipogenic enzymes, particularly in response to sucrose refeeding following fasting, a dietary regimen that elicits maximal lipogenesis. mRNA and protein levels of SREBP-1c, a master transcriptional regulator of lipogenesis, were also reduced in L-Chrebp-/- livers. Adeno-associated virus-mediated restoration of nuclear SREBP-1c in L-Chrebp-/- mice normalized expression of a subset of lipogenic genes, while not affecting glycolytic genes. Conversely, ChREBP overexpression alone failed to support expression of lipogenic genes in the livers of mice lacking active SREBPs as a result of Scap deficiency. Together, these data show that SREBP-1c and ChREBP are both required for coordinated induction of glycolytic and lipogenic mRNAs. Whereas SREBP-1c mediates insulin's induction of lipogenic genes, ChREBP mediates glucose's induction of both glycolytic and lipogenic genes. These overlapping, but distinct, actions ensure that the liver synthesizes FAs only when insulin and carbohydrates are both present.

Bap1 is essential for kidney function and cooperates with Vhl in renal tumorigenesis.

Why different species are predisposed to different tumor spectra is not well understood. In particular, whether the physical location of tumor suppressor genes relative to one another influences tumor predisposition is unknown. Renal cancer presents a unique opportunity to explore this question. Renal cell carcinoma (RCC) of clear-cell type (ccRCC), the most common type, begins with an intragenic mutation in the von Hippel-Lindau (VHL) gene and loss of 3p (where VHL is located). Chromosome 3p harbors several additional tumor suppressor genes, including BRCA1-associated protein-1 (BAP1). In the mouse, Vhl is on a different chromosome than Bap1. Thus, whereas loss of 3p in humans simultaneously deletes one copy of BAP1, loss of heterozygosity in the corresponding Vhl region in the mouse would not affect Bap1. To test the role of BAP1 in ccRCC development, we generated mice deficient for either Vhl or Vhl together with one allele of Bap1 in nephron progenitor cells. Six2-Cre;Vhl(F/F);Bap1(F/+) mice developed ccRCC, but Six2-Cre;Vhl(F/F) mice did not. Kidneys from Six2-Cre;Vhl(F/F);Bap1(F/+) mice resembled kidneys from humans with VHL syndrome, containing multiple lesions spanning from benign cysts to cystic and solid RCC. Although the tumors were small, they showed nuclear atypia and exhibited features of human ccRCC. These results provide an explanation for why VHL heterozygous humans, but not mice, develop ccRCC. They also explain why a mouse model of ccRCC has been lacking. More broadly, our data suggest that differences in tumor predisposition across species may be explained, at least in part, by differences in the location of two-hit tumor suppressor genes across the genome.

Insulin stimulation of SREBP-1c processing in transgenic rat hepatocytes requires p70 S6-kinase.

Insulin activates sterol regulatory element-binding protein-1c (SREBP-1c) in liver, thereby increasing fatty acid and triglyceride synthesis. We created a line of transgenic rats that produce epitope-tagged human SREBP-1c in liver under control of the constitutive apolipoprotein E promoter/enhancer. This system allows us to dissect the pathway by which insulin stimulates SREBP-1c processing without interference by the insulin-mediated increase in SREBP-1c mRNA. Rats are used because freshly isolated rat hepatocytes respond much more robustly to insulin than do mouse hepatocytes. The data reveal that insulin-mediated stimulation of SREBP-1c processing requires the mechanistic target of rapamycin complex 1 (mTORC1), which also is required for insulin-mediated SREBP-1c mRNA induction. However, in contrast to mRNA induction, insulin stimulation of SREBP-1c processing is blocked by an inhibitor of p70 S6-kinase. The data indicate that the pathways for insulin enhancement of SREBP-1c mRNA and proteolytic processing diverge after mTORC1. Stimulation of processing requires the mTORC1 target p70 S6-kinase, whereas induction of mRNA bypasses this enzyme. Insulin stimulation of both processes is blocked by glucagon. The transgenic rat system will be useful in further defining the molecular mechanism for insulin stimulation of lipid synthesis in liver in normal and diabetic states.

Deletion of ELOVL6 blocks the synthesis of oleic acid but does not prevent the development of fatty liver or insulin resistance.

Elongation of very long chain fatty acid-like family member 6 (ELOVL6) is a fatty acyl elongase that performs the initial and rate-limiting condensing reaction required for microsomal elongation of long-chain fatty acids. Our previous in vitro studies suggested that ELOVL6 elongated long-chain saturated fatty acids and monounsaturated fatty acids with chain lengths of 12 to 16 carbons. Here, we describe the generation and phenotypic characterization of Elovl6(-/-) mice. As predicted from the in vitro studies, livers from Elovl6(-/-) mice accumulated palmitic (C16:0) and palmitoleic (C16:1, n-7) fatty acids and contained significantly less stearic (C18:0) and oleic (C18:1, n-9) acids, confirming that ELOVL6 is the only enzyme capable of elongating palmitate (C16:0). Unexpectedly, Elovl6(-/-) mice produced vaccenic acid (C18:1, n-7), the elongated product of palmitoleate (C16:1, n-7), suggesting that palmitoleate (C16:1, n-7) to vaccenate (C18:1, n-7) elongation was not specific to ELOVL6. The only detected consequence of deleting Elovl6(-/-) in mice was that their livers accumulated significantly more triglycerides than wild-type mice when fed a fat-free/high-carbohydrate diet. When mice were fed a high-fat diet or ELOVL6 was deleted in ob/ob mice, the absence of ELOVL6 did not alter the development of obesity, fatty liver, hyperglycemia, or hyperinsulinemia. Combined, these results suggest that palmitoleic (C16:1, n-7) and vaccenic (C18:1, n-7) acids can largely replace the roles of oleic acid (C18:1, n-9) in vivo and that the deletion of ELOVL6 does not protect mice from the development of hepatic steatosis or insulin resistance.

Autoimmune epididymoorchitis is essential to the pathogenesis of male-specific spondylarthritis in HLA-B27-transgenic rats.

OBJECTIVE: Male rats transgenic for HLA-B27 and human ß(2) -microglobulin (hß(2) m) spontaneously develop epididymoorchitis (EO) preceding the development of spondylarthritis (SpA). In the specific B27/hß(2) m-transgenic rat cross-strain (21-3 × 382-2)F(1) , only the males develop SpA, and neither sex develops gut inflammation. This study was undertaken to determine whether EO and SpA in male (21-3 × 382-2)F(1) rats are causally related. In addition, the primary characteristics of EO in this rat arthritis model were assessed. METHODS: Male B27/hß(2) m-transgenic (21-3 × 382-2)F(1) rats underwent bilateral, unilateral, or sham epididymoorchiectomy between ages 36 and 125 days. The castrated rats were given testosterone replacement. Alternatively, the 21-3 and 283-2 transgene loci were crossed with a transgene inducing aspermatogenesis. Rats were observed for the development of EO, arthritis, and spondylitis. RESULTS: In unmanipulated transgenic rats, inflammation was first evident in the ductuli efferentes (DE; ducts linking the rete testis to epididymis) as early as age 30 days. The inflammation was initially neutrophilic, and later became granulomatous. Antisperm and anti-testis cell antibodies appeared in the rat serum after age 70 days. Cells infiltrating the testes were predominantly CD4+ T cells and CD68+ or CD163+ macrophages. Quantitative polymerase chain reaction of the DE, epididymis, and testis showed elevations in the levels of interferon-γ, interleukin-10 (IL-10), and IL-17A. In addition, levels of IL-12A, IL-22, IL-23A, and IL-23 receptor were found to be elevated in the DE. Remarkably, castration of the rats before age 91 days completely prevented the subsequent onset of arthritis and spondylitis, as did transgene-induced azospermia. CONCLUSION: Autoimmune EO develops spontaneously in HLA-B27/hß(2) m-transgenic (21-3 × 283-2)F(1) rats at age 30 days, the age when antigen-positive meiotic germ cells first exit the testis. Persistent testicular inflammation and/or antigenic stimulation are essential prerequisites for the subsequent development of SpA. Thus, dysregulated innate immunity at immune-privileged sites may be an essential mechanism triggering the onset of SpA.

Soluble amyloid precursor protein (APP) regulates transthyretin and Klotho gene expression without rescuing the essential function of APP.

Amyloidogenic processing of the amyloid precursor protein (APP) generates a large secreted ectodomain fragment (APPsß), ß-amyloid (Aß) peptides, and an APP intracellular domain (AICD). Whereas Aß is viewed as critical for Alzheimer's disease pathogenesis, the role of other APP processing products remains enigmatic. Of interest, the AICD has been implicated in transcriptional regulation, and N-terminal cleavage of APPsß has been suggested to produce an active fragment that may mediate axonal pruning and neuronal cell death. We previously reported that mice deficient in APP and APP-like protein 2 (APLP2) exhibit early postnatal lethality and neuromuscular synapse defects, whereas mice with neuronal conditional deletion of APP and APLP2 are viable. Using transcriptional profiling, we now identify transthyretin (TTR) and Klotho as APP/APLP2-dependent genes whose expression is decreased in loss-of-function states but increased in gain-of-function states. Significantly, by creating an APP knockin allele that expresses only APPsß protein, we demonstrate that APPsß is not normally cleaved in vivo and is fully capable of mediating the APP-dependent regulation of TTR and Klotho gene expression. Despite being an active regulator of gene expression, APPsß did not rescue the lethality and neuromuscular synapse defects of APP and APLP2 double-KO animals. Our studies identify TTR and Klotho as physiological targets of APP that are regulated by soluble APPsß independent of developmental APP functions. This unexpected APP-mediated signaling pathway may play an important role in maintaining TTR and Klotho levels and their respective functions in Aß sequestration and aging.

Acss2/HIF-2 signaling facilitates colon cancer growth and metastasis.

The microenvironment of solid tumors is characterized by oxygen and glucose deprivation. Acss2/HIF-2 signaling coordinates essential genetic regulators including acetate-dependent acetyl CoA synthetase 2 (Acss2), Creb binding protein (Cbp), Sirtuin 1 (Sirt1), and Hypoxia Inducible Factor 2α (HIF-2α). We previously shown in mice that exogenous acetate augments growth and metastasis of flank tumors derived from fibrosarcoma-derived HT1080 cells in an Acss2/HIF-2 dependent manner. Colonic epithelial cells are exposed to the highest acetate levels in the body. We reasoned that colon cancer cells, like fibrosarcoma cells, may respond to acetate in a pro-growth manner. In this study, we examine the role of Acss2/HIF-2 signaling in colon cancer. We find that Acss2/HIF-2 signaling is activated by oxygen or glucose deprivation in two human colon cancer-derived cell lines, HCT116 and HT29, and is crucial for colony formation, migration, and invasion in cell culture studies. Flank tumors derived from HCT116 and HT29 cells exhibit augmented growth in mice when supplemented with exogenous acetate in an Acss2/HIF-2 dependent manner. Finally, Acss2 in human colon cancer samples is most frequently localized in the nucleus, consistent with it having a signaling role. Targeted inhibition of Acss2/HIF-2 signaling may have synergistic effects for some colon cancer patients.

Target-directed microRNA degradation regulates developmental microRNA expression and embryonic growth in mammals.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8 , which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in heart and lung, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in co-transcribed clusters and examples in which TDMD underlies 'arm switching', a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8 null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.

FGF21 counteracts alcohol intoxication by activating the noradrenergic nervous system.

Animals that consume fermenting fruit and nectar are at risk of exposure to ethanol and the detrimental effects of inebriation. In this report, we show that the hormone FGF21, which is strongly induced by ethanol in murine and human liver, stimulates arousal from intoxication without changing ethanol catabolism. Mice lacking FGF21 take longer than wild-type littermates to recover their righting reflex and balance following ethanol exposure. Conversely, pharmacologic FGF21 administration reduces the time needed for mice to recover from ethanol-induced unconsciousness and ataxia. FGF21 did not counteract sedation caused by ketamine, diazepam, or pentobarbital, indicating specificity for ethanol. FGF21 mediates its anti-intoxicant effects by directly activating noradrenergic neurons in the locus coeruleus region, which regulates arousal and alertness. These results suggest that this FGF21 liver-brain pathway evolved to protect against ethanol-induced intoxication and that it might be targeted pharmaceutically for treating acute alcohol poisoning.

The master negative regulator REST/NRSF controls adult neurogenesis by restraining the neurogenic program in quiescent stem cells.

Transcriptional regulation is a critical mechanism in the birth, specification, and differentiation of granule neurons in the adult hippocampus. One of the first negative-acting transcriptional regulators implicated in vertebrate development is repressor element 1-silencing transcription/neuron-restrictive silencer factor (REST/NRSF)--thought to regulate hundreds of neuron-specific genes--yet its function in the adult brain remains elusive. Here we report that REST/NRSF is required to maintain the adult neural stem cell (NSC) pool and orchestrate stage-specific differentiation. REST/NRSF recruits CoREST and mSin3A corepressors to stem cell chromatin for the regulation of pro-neuronal target genes to prevent precocious neuronal differentiation in cultured adult NSCs. Moreover, mice lacking REST/NRSF specifically in NSCs display a transient increase in adult neurogenesis that leads to a loss in the neurogenic capacity of NSCs and eventually diminished granule neurons. Our work identifies REST/NRSF as a master negative regulator of adult NSC differentiation and offers a potential molecular target for neuroregenerative approaches.