Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation.

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.

A Translation-Activating Function of MIWI/piRNA during Mouse Spermiogenesis.

Spermiogenesis is a highly orchestrated developmental process during which chromatin condensation decouples transcription from translation. Spermiogenic mRNAs are transcribed earlier and stored in a translationally inert state until needed for translation; however, it remains largely unclear how such repressed mRNAs become activated during spermiogenesis. We previously reported that the MIWI/piRNA machinery is responsible for mRNA elimination during late spermiogenesis in preparation for spermatozoa production. Here we unexpectedly discover that the same machinery is also responsible for activating translation of a subset of spermiogenic mRNAs to coordinate with morphological transformation into spermatozoa. Such action requires specific base-pairing interactions of piRNAs with target mRNAs in their 3' UTRs, which activates translation through coupling with cis-acting AU-rich elements to nucleate the formation of a MIWI/piRNA/eIF3f/HuR super-complex in a developmental stage-specific manner. These findings reveal a critical role of the piRNA system in translation activation, which we show is functionally required for spermatid development.

Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis.

Genetic studies have elucidated critical roles of Piwi proteins in germline development in animals, but whether Piwi is an actual disease gene in human infertility remains unknown. We report germline mutations in human Piwi (Hiwi) in patients with azoospermia that prevent its ubiquitination and degradation. By modeling such mutations in Piwi (Miwi) knockin mice, we demonstrate that the genetic defects are directly responsible for male infertility. Mechanistically, we show that MIWI binds the histone ubiquitin ligase RNF8 in a Piwi-interacting RNA (piRNA)-independent manner, and MIWI stabilization sequesters RNF8 in the cytoplasm of late spermatids. The resulting aberrant sperm show histone retention, abnormal morphology, and severely compromised activity, which can be functionally rescued via blocking RNF8-MIWI interaction in spermatids with an RNF8-N peptide. Collectively, our findings identify Piwi as a factor in human infertility and reveal its role in regulating the histone-to-protamine exchange during spermiogenesis.

LARP7-Mediated U6 snRNA Modification Ensures Splicing Fidelity and Spermatogenesis in Mice.

U6 snRNA, as an essential component of the catalytic core of the pre-mRNA processing spliceosome, is heavily modified post-transcriptionally, with 2'-O-methylation being most common. The role of these modifications in pre-mRNA splicing as well as their physiological function in mammals have remained largely unclear. Here we report that the La-related protein LARP7 functions as a critical cofactor for 2'-O-methylation of U6 in mouse male germ cells. Mechanistically, LARP7 promotes U6 loading onto box C/D snoRNP, facilitating U6 2'-O-methylation by box C/D snoRNP. Importantly, ablation of LARP7 in the male germline causes defective U6 2'-O-methylation, massive alterations in pre-mRNA splicing, and spermatogenic failure in mice, which can be rescued by ectopic expression of wild-type LARP7 but not an U6-loading-deficient mutant LARP7. Our data uncover a novel role of LARP7 in regulating U6 2'-O-methylation and demonstrate the functional requirement of such modification for splicing fidelity and spermatogenesis in mice.

Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells.

Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.

In focus: molecular and cell biology research in China.

An interactive, intellectual environment with good funding opportunities is essential for the development and success of basic research. The fast-growing economy and investment in science, together with a visionary plan, have attracted foreign scholars to work in China, motivated world-class Chinese scientists to return and strengthened the country's international collaborations. As a result, molecular and cell biology research in China has evolved rapidly over the past decade.

Activation of vascular endothelial growth factor receptor-3 in macrophages restrains TLR4-NF-κB signaling and protects against endotoxin shock.

Toll-like receptors (TLRs) are critical in mediating innate immune responses against infections. However, uncontrolled TLR-triggered inflammation is associated with endotoxin shock. To better understand the homeostatic mechanisms induced by TLR4 signaling, we screened a group of key cytokines, chemokines, growth factors, and their receptors for bacteria- or LPS-induced expression. The surface vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligand VEGF-C were upregulated in macrophages. VEGFR-3 ligation by VEGF-C significantly attenuated proinflammatory cytokine production. Notably, ablation of the ligand-binding domain or tyrosine kinase activity of VEGFR-3 rendered mice more sensitive to septic shock. VEGFR-3 restrained TLR4-NF-κB activation by regulating the PI3-kinase-Akt signaling pathway and SOCS1 expression. Aside from targeting lymphatic vessels, we suggest a key role of VEGFR-3 on macrophages to prevent infections that is complicated with lymphoedema. Thus, VEGFR-3-VEGF-C signaling represents a "self-control" mechanism during antibacterial innate immunity.

ECM1 is an essential factor for the determination of M1 macrophage polarization in IBD in response to LPS stimulation.

Inflammatory bowel disease (IBD) comprises chronic relapsing disorders of the gastrointestinal tract characterized pathologically by intestinal inflammation and epithelial injury. Here, we uncover a function of extracellular matrix protein 1 (ECM1) in promoting the pathogenesis of human and mouse IBD. ECM1 was highly expressed in macrophages, particularly tissue-infiltrated macrophages under inflammatory conditions, and ECM1 expression was significantly induced during IBD progression. The macrophage-specific knockout of ECM1 resulted in increased arginase 1 (ARG1) expression and impaired polarization into the M1 macrophage phenotype after lipopolysaccharide (LPS) treatment. A mechanistic study showed that ECM1 can regulate M1 macrophage polarization through the granulocyte-macrophage colony-stimulating factor/STAT5 signaling pathway. Pathological changes in mice with dextran sodium sulfate-induced IBD were alleviated by the specific knockout of the ECM1 gene in macrophages. Taken together, our findings show that ECM1 has an important function in promoting M1 macrophage polarization, which is critical for controlling inflammation and tissue repair in the intestine.

PHF7 is a novel histone H2A E3 ligase prior to histone-to-protamine exchange during spermiogenesis.

Epigenetic regulation, including histone-to-protamine exchanges, controls spermiogenesis. However, the underlying mechanisms of this regulation are largely unknown. Here, we report that PHF7, a testis-specific PHD and RING finger domain-containing protein, is essential for histone-to-protamine exchange in mice. PHF7 is specifically expressed during spermiogenesis. PHF7 deletion results in male infertility due to aberrant histone retention and impaired protamine replacement in elongated spermatids. Mechanistically, PHF7 can simultaneously bind histone H2A and H3; its PHD domain, a histone code reader, can specifically bind H3K4me3/me2, and its RING domain, a histone writer, can ubiquitylate H2A. Thus, our study reveals that PHF7 is a novel E3 ligase that can specifically ubiquitylate H2A through binding H3K4me3/me2 prior to histone-to-protamine exchange.

Structural basis for activity regulation of MLL family methyltransferases.

The mixed lineage leukaemia (MLL) family of proteins (including MLL1-MLL4, SET1A and SET1B) specifically methylate histone 3 Lys4, and have pivotal roles in the transcriptional regulation of genes involved in haematopoiesis and development. The methyltransferase activity of MLL1, by itself severely compromised, is stimulated by the three conserved factors WDR5, RBBP5 and ASH2L, which are shared by all MLL family complexes. However, the molecular mechanism of how these factors regulate the activity of MLL proteins still remains poorly understood. Here we show that a minimized human RBBP5-ASH2L heterodimer is the structural unit that interacts with and activates all MLL family histone methyltransferases. Our structural, biochemical and computational analyses reveal a two-step activation mechanism of MLL family proteins. These findings provide unprecedented insights into the common theme and functional plasticity in complex assembly and activity regulation of MLL family methyltransferases, and also suggest a universal regulation mechanism for most histone methyltransferases.

ECM1 Prevents Activation of Transforming Growth Factor ß, Hepatic Stellate Cells, and Fibrogenesis in Mice.

BACKGROUND & AIMS: Activation of TGFB (transforming growth factor ß) promotes liver fibrosis by activating hepatic stellate cells (HSCs), but the mechanisms of TGFB activation are not clear. We investigated the role of ECM1 (extracellular matrix protein 1), which interacts with extracellular and structural proteins, in TGFB activation in mouse livers. METHODS: We performed studies with C57BL/6J mice (controls), ECM1-knockout (ECM1-KO) mice, and mice with hepatocyte-specific knockout of EMC1 (ECM1Δhep). ECM1 or soluble TGFBR2 (TGFB receptor 2) were expressed in livers of mice after injection of an adeno-associated virus vector. Liver fibrosis was induced by carbon tetrachloride (CCl4) administration. Livers were collected from mice and analyzed by histology, immunohistochemistry, in situ hybridization, and immunofluorescence analyses. Hepatocytes and HSCs were isolated from livers of mice and incubated with ECM1; production of cytokines and activation of reporter genes were quantified. Liver tissues from patients with viral or alcohol-induced hepatitis (with different stages of fibrosis) and individuals with healthy livers were analyzed by immunohistochemistry and in situ hybridization. RESULTS: ECM1-KO mice spontaneously developed liver fibrosis and died by 2 months of age without significant hepatocyte damage or inflammation. In liver tissues of mice, we found that ECM1 stabilized extracellular matrix-deposited TGFB in its inactive form by interacting with αv integrins to prevent activation of HSCs. In liver tissues from patients and in mice with CCl4-induced liver fibrosis, we found an inverse correlation between level of ECM1 and severity of fibrosis. CCl4-induced liver fibrosis was accelerated in ECM1Δhep mice compared with control mice. Hepatocytes produced the highest levels of ECM1 in livers of mice. Ectopic expression of ECM1 or soluble TGFBR2 in liver prevented fibrogenesis in ECM1-KO mice and prolonged their survival. Ectopic expression of ECM1 in liver also reduced the severity of CCl4-induced fibrosis in mice. CONCLUSIONS: ECM1, produced by hepatocytes, inhibits activation of TGFB and its activation of HSCs to prevent fibrogenesis in mouse liver. Strategies to increase levels of ECM1 in liver might be developed for treatment of fibrosis.

An essential function for beta-arrestin 2 in the inhibitory signaling of natural killer cells.

The inhibitory signaling of natural killer (NK) cells is crucial in the regulation of innate immune responses. Here we show that the association of KIR2DL1, an inhibitory receptor of NK cells, with beta-arrestin 2 mediated recruitment of the tyrosine phosphatases SHP-1 and SHP-2 to KIR2DL1 and facilitated 'downstream' inhibitory signaling. Consequently, the cytotoxicity of NK cells was higher in beta-arrestin 2-deficient mice but was inhibited in beta-arrestin 2-transgenic mice. Moreover, beta-arrestin 2-deficient mice were less susceptible than wild-type mice to mouse cytomegalovirus infection, an effect that was abolished by depletion of NK cells. Our findings identify a previously unknown mechanism by which the inhibitory signaling in NK cells is regulated.

Mediator MED23 plays opposing roles in directing smooth muscle cell and adipocyte differentiation.

The Mediator complex functions as a control center, orchestrating diverse signaling, gene activities, and biological processes. However, how Mediator subunits determine distinct cell fates remains to be fully elucidated. Here, we show that Mediator MED23 controls the cell fate preference that directs differentiation into smooth muscle cells (SMCs) or adipocytes. Med23 deficiency facilitates SMC differentiation but represses adipocyte differentiation from the multipotent mesenchymal stem cells. Gene profiling revealed that the presence or absence of Med23 oppositely regulates two sets of genes: the RhoA/MAL targeted cytoskeleton/SMC genes and the Ras/ELK1 targeted growth/adipogenic genes. Mechanistically, MED23 favors ELK1-SRF binding to SMC gene promoters for repression, whereas the lack of MED23 favors MAL-SRF binding to SMC gene promoters for activation. Remarkably, the effect of MED23 on SMC differentiation can be recapitulated in zebrafish embryogenesis. Collectively, our data demonstrate the dual, opposing roles for MED23 in regulating the cytoskeleton/SMC and growth/adipogenic gene programs, suggesting its "Ying-Yang" function in directing adipogenesis versus SMC differentiation.

Suppression of miR-199a maturation by HuR is crucial for hypoxia-induced glycolytic switch in hepatocellular carcinoma.

Glucose metabolic reprogramming is a hallmark of cancer. Cancer cells rapidly adjust their energy source from oxidative phosphorylation to glycolytic metabolism in order to efficiently proliferate in a hypoxic environment, but the mechanism underlying this switch is still incompletely understood. Here, we report that hypoxia potently induces the RNA-binding protein HuR to specifically bind primary miR-199a transcript to block miR-199a maturation in hepatocellular carcinoma (HCC) cells. We demonstrate that this hypoxia-suppressed miR-199a plays a decisive role in limiting glycolysis in HCC cells by targeting hexokinase-2 (Hk2) and pyruvate kinase-M2 (Pkm2). Furthermore, systemically delivered cholesterol-modified agomiR-199a inhibits [(18)F]-fluorodeoxyglucose uptake and attenuates tumor growth in HCC tumor-bearing mice. These data reveal a novel mechanism of reprogramming of cancer energy metabolism in which HuR suppresses miR-199a maturation to link hypoxia to the Warburg effect and suggest a promising therapeutic strategy that targets miR-199a to interrupt cancerous aerobic glycolysis.

Molecular basis for the recognition of CCN1 by monoclonal antibody 093G9.

CCN1, also named Cyr61 (cysteine-rich protein 61), is the first identified member of the CCN family that is composed of 6 secreted extracellular matrix-associated glycoproteins. CCN1 has been demonstrated to participate in pathogenesis of rheumatoid arthritis through various pathways. A monoclonal antibody, namely, 093G9, is effective to antagonize the effects of CCN1 and hence has potential therapeutic benefits against rheumatoid arthritis. Here, we show that the epitope recognized by 093G9 is mapped to residues 77 to 80 of CCN1, and a cyclic peptide encompassing residues 75 to 81 of CCN1 displays high binding affinity for 093G9. The crystal structure of the 093G9 Fab in complex with the cyclic peptide was determined at 2.7 Å resolution, which reveals the intensive interactions between CCN1 and 093G9. Particularly, residues Asn79 and Phe80 of CCN1 are inserted into cavities mainly formed by residues of complementarity-determining region loop L3 and framework region L2 and by residues of complementarity-determining region loops H2 and H3, respectively, which contribute most of the interactions and therefore are critical for the recognition by 093G9. Together, these findings not only identify the epitope of CCN1 for 093G9 but also reveal the molecular mechanism of recognition and binding of CCN1 by 093G9.

A novel miR-155/miR-143 cascade controls glycolysis by regulating hexokinase 2 in breast cancer cells.

Cancer cells preferentially metabolize glucose through aerobic glycolysis. This phenomenon, known as the Warburg effect, is an anomalous characteristic of glucose metabolism in cancer cells. Chronic inflammation is a key promoting factor of tumourigenesis. It remains, however, largely unexplored whether and how pro-tumourigenic inflammation regulates glucose metabolism in cancer cells. Here, we show that pro-inflammatory cytokines promote glycolysis in breast cancer cells, and that the inflammation-induced miR-155 functions as an important mediator in this process. We further show that miR-155 acts to upregulate hexokinase 2 (hk2), through two distinct mechanisms. First, miR-155 promotes hk2 transcription by activation of signal transducer and activator of transcription 3 (STAT3), a transcriptional activator for hk2. Second, via targeting C/EBPß (a transcriptional activator for mir-143), miR-155 represses mir-143, a negative regulator of hk2, thus resulting in upregulation of hk2 expression at the post-transcriptional level. The miR-155-mediated hk2 upregulation also appears to operate in other types of cancer cells examined. We suggest that the miR-155/miR-143/HK2 axis may represent a common mechanism linking inflammation to the altered metabolism in cancer cells.

Neuraminidase of Influenza A Virus Binds Lysosome-Associated Membrane Proteins Directly and Induces Lysosome Rupture.

UNLABELLED: As a recycling center, lysosomes are filled with numerous acid hydrolase enzymes that break down waste materials and invading pathogens. Recently, lysosomal cell death has been defined as "lysosomal membrane permeabilization and the consequent leakage of lysosome contents into cytosol." Here, we show that the neuraminidase (NA) of H5N1 influenza A virus markedly deglycosylates and degrades lysosome-associated membrane proteins (LAMPs; the most abundant membrane proteins of lysosome), which induces lysosomal rupture, and finally leads to cell death of alveolar epithelial carcinoma A549 cells and human tracheal epithelial cells. The NA inhibitors peramivir and zanamivir could effectively block the deglycosylation of LAMPs, inhibit the virus cell entry, and prevent cell death induced by the H5N1 influenza virus. The NA of seasonal H1N1 virus, however, does not share these characteristics. Our findings not only reveal a novel role of NA in the early stage of the H5N1 influenza virus life cycle but also elucidate the molecular mechanism of lysosomal rupture crucial for influenza virus induced cell death. IMPORTANCE: The integrity of lysosomes is vital for maintaining cell homeostasis, cellular defense and clearance of invading pathogens. This study shows that the H5N1 influenza virus could induce lysosomal rupture through deglycosylating lysosome-associated membrane proteins (LAMPs) mediated by the neuraminidase activity of NA protein. NA inhibitors such as peramivir and zanamivir could inhibit the deglycosylation of LAMPs and protect lysosomes, which also further interferes with the H5N1 influenza virus infection at early stage of life cycle. This work is significant because it presents new concepts for NA's function, as well as for influenza inhibitors' mechanism of action, and could partially explain the high mortality and high viral load after H5N1 virus infection in human beings and why NA inhibitors have more potent therapeutic effects for lethal avian influenza virus infections at early stage.

Deficiency of a beta-arrestin-2 signal complex contributes to insulin resistance.

Insulin resistance, a hallmark of type 2 diabetes, is a defect of insulin in stimulating insulin receptor signalling, which has become one of the most serious public health threats. Upon stimulation by insulin, insulin receptor recruits and phosphorylates insulin receptor substrate proteins, leading to activation of the phosphatidylinositol-3-OH kinase (PI(3)K)-Akt pathway. Activated Akt phosphorylates downstream kinases and transcription factors, thus mediating most of the metabolic actions of insulin. Beta-arrestins mediate biological functions of G-protein-coupled receptors by linking activated receptors with distinct sets of accessory and effecter proteins, thereby determining the specificity, efficiency and capacity of signals. Here we show that in diabetic mouse models, beta-arrestin-2 is severely downregulated. Knockdown of beta-arrestin-2 exacerbates insulin resistance, whereas administration of beta-arrestin-2 restores insulin sensitivity in mice. Further investigation reveals that insulin stimulates the formation of a new beta-arrestin-2 signal complex, in which beta-arrestin-2 scaffolds Akt and Src to insulin receptor. Loss or dysfunction of beta-arrestin-2 results in deficiency of this signal complex and disturbance of insulin signalling in vivo, thereby contributing to the development of insulin resistance and progression of type 2 diabetes. Our findings provide new insight into the molecular pathogenesis of insulin resistance, and implicate new preventive and therapeutic strategies against insulin resistance and type 2 diabetes.