Methionine metabolism is essential for SIRT1‐regulated mouse embryonic stem cell maintenance and embryonic development

Methionine metabolism is critical for epigenetic maintenance, redox homeostasis, and animal development. However, the regulation of methionine metabolism remains unclear. Here, we provide evidence that SIRT1, the most conserved mammalian NAD⁺‐dependent protein deacetylase, is critically involved in modulating methionine metabolism, thereby impacting maintenance of mouse embryonic stem cells (mESCs) and subsequent embryogenesis. We demonstrate that SIRT1‐deficient mESCs are hypersensitive to methionine restriction/depletion‐induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S‐adenosylmethionine. This reduction markedly decreases methylation levels of histones, resulting in dramatic alterations in gene expression profiles. Mechanistically, we discover that the enzyme converting methionine to S‐adenosylmethionine in mESCs, methionine adenosyltransferase 2a (MAT2a), is under control of Myc and SIRT1. Consistently, SIRT1 KO embryos display reduced Mat2a expression and histone methylation and are sensitive to maternal methionine restriction‐induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice. Our findings uncover a novel regulatory mechanism for methionine metabolism and highlight the importance of methionine metabolism in SIRT1‐mediated mESC maintenance and embryonic development.     

MOV10 RNA Helicase Is a Potent Inhibitor of Retrotransposition in Cells

MOV10 protein, a putative RNA helicase and component of the RNA–induced silencing complex (RISC), inhibits retrovirus replication. We show that MOV10 also severely restricts human LINE1 (L1), Alu, and SVA retrotransposons. MOV10 associates with the L1 ribonucleoprotein particle, along with other RNA helicases including DDX5, DHX9, DDX17, DDX21, and DDX39A. However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains. MOV10 association with retrotransposons is further supported by its colocalization with L1 ORF1 protein in stress granules, by cytoplasmic structures associated with RNA silencing, and by the ability of MOV10 to reduce endogenous and ectopic L1 expression. The majority of the human genome is repetitive DNA, most of which is the detritus of millions of years of accumulated retrotransposition. Retrotransposons remain active mutagens, and their insertion can disrupt gene function. Therefore, the host has evolved defense mechanisms to protect against retrotransposition, an arsenal we are only beginning to understand. With homologs in other vertebrates, insects, and plants, MOV10 may represent an ancient and innate form of immunity against both infective viruses and endogenous retroelements.     

Host restriction of emerging high-pathogenic bunyaviruses via MOV10 by targeting viral nucleoprotein and blocking ribonucleoprotein assembly

Bunyavirus ribonucleoprotein (RNP) that is assembled by polymerized nucleoproteins (N) coating a viral RNA and associating with a viral polymerase can be both the RNA synthesis machinery and the structural core of virions. Bunyaviral N and RNP thus could be assailable targets for host antiviral defense; however, it remains unclear which and how host factors target N/RNP to restrict bunyaviral infection. By mass spectrometry and protein-interaction analyses, we here show that host protein MOV10 targets the N proteins encoded by a group of emerging high-pathogenic representatives of bunyaviruses including severe fever with thrombocytopenia syndrome virus (SFTSV), one of the most dangerous pathogens listed by World Health Organization, in RNA-independent manner. MOV10 that was further shown to be induced specifically by SFTSV and related bunyaviruses in turn inhibits the bunyaviral replication in infected cells in series of loss/gain-of-function assays. Moreover, animal infection experiments with MOV10 knockdown corroborated the role of MOV10 in restricting SFTSV infection and pathogenicity in vivo. Minigenome assays and additional functional and mechanistic investigations demonstrate that the anti-bunyavirus activity of MOV10 is likely achieved by direct impact on viral RNP machinery but independent of its helicase activity and the cellular interferon pathway. Indeed, by its N-terminus, MOV10 binds to a protruding N-arm domain of N consisting of only 34 amino acids but proving important for N function and blocks N polymerization, N-RNA binding, and N-polymerase interaction, disabling RNP assembly. This study not only advances the understanding of bunyaviral replication and host restriction mechanisms but also presents novel paradigms for both direct antiviral action of MOV10 and host targeting of viral RNP machinery.     

Expression of TGFβ family factors and FGF2 in mouse and human embryonic stem cells maintained in different culture systems

Mouse and human embryonic stem cells are in different states of pluripotency (naive/ground and primed states). Mechanisms of signaling regulation in cells with ground and primed states of pluripotency are considerably different. In order to understand the contribution of endogenous and exogenous factors in the maintenance of a metastable state of the cells in different phases of pluripotency, we examined the expression of TGFβ family factors (ActivinA, Nodal, Lefty1, TGFβ1, GDF3, BMP4) and FGF2 initiating the appropriate signaling pathways in mouse and human embryonic stem cells (mESCs, hESCs) and supporting feeder cells. Quantitative real-time PCR analysis of gene expression showed that the expression patterns of endogenous factors studied were considerably different in mESCs and hESCs. The most significant differences were found in the levels of endogenous expression of TGFβ1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFβ1, and FGF2 for hESCs are feeder cells (mouse and human embryonic fibroblasts) expressing high levels of these factors, as well as low levels of BMP4. Thus, our data demonstrated that the in vitro maintenance of metastable state of undifferentiated pluripotent cells is achieved in mESCs and hESCs using different schemes of the regulations of ActivinA/Nodal/Lefty/Smad2/3 and BMP/Smad1/5/8 endogenous branches of TGFβ signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFβ family factors and FGF2 in the mESCs and hESCs. For the hESCs, enhanced activity of ActivinA/Nodal/Lefty/Smad2/3 signaling by exogenous factor stimulation is necessary to mitigate the effects of BMP/Smad1/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primed states of pluripotency demonstrate diverse involvement of this factor in the regulation of the pluripotent cell self-renewal.     

TRPC3 is required for the survival,pluripotency and neural differentiation of mouse embryonic stem cells(mESCs)

Transient receptor potential canonical subfamily member 3(TRPC3) is known to be important for neural development and the formation of neuronal networks. Here, we investigated the role of TRPC3 in undifferentiated mouse embryonic stem cells(mESCs) and during the differentiation of mESCs into neurons. CRISPR/Cas9-mediated knockout(KO) of TRPC3 induced apoptosis and the disruption of mitochondrial membrane potential both in undifferentiated mESCs and in those undergoing neural differentiation. In addition, TRPC3 KO impaired the pluripotency of mESCs. TRPC3 KO also dramatically repressed the neural differentiation of mESCs by inhibiting the expression of markers for neural progenitors, neurons, astrocytes and oligodendrocytes.Taken together, our new data demonstrate an important function of TRPC3 with regards to the survival, pluripotency and neural differentiation of mESCs.     

Pluripotency of embryonic stem cells lacking clathrin-mediated endocytosis cannot be rescued by restoring cellular stiffness

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cellular stiffness in contrast to differentiated cells, which are stiffer. We have previously shown that mESCs lacking the clathrin heavy chain (Cltc), an essential component for clathrin-mediated endocytosis (CME), display a loss of pluripotency and an enhanced expression of differentiation markers. However, it is not known whether physical properties such as cellular stiffness also change upon loss of Cltc, similar to what is seen in differentiated cells, and if so, how these altered properties specifically impact pluripotency. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking Cltc display higher Young''s modulus, indicative of greater cellular stiffness, compared with WT mESCs. The increase in stiffness was accompanied by the presence of actin stress fibers and accumulation of the inactive, phosphorylated, actin-binding protein cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young''s modulus to values similar to those obtained with WT mESCs. However, a rescue in the expression profile of pluripotency factors was not obtained. Additionally, whereas WT mouse embryonic fibroblasts could be reprogrammed to a state of pluripotency, this was inhibited in the absence of Cltc. This indicates that the presence of active CME is essential for the pluripotency of embryonic stem cells. Additionally, whereas physical properties may serve as a simple readout of the cellular state, they may not always faithfully recapitulate the underlying molecular fate.     

MOV10 as a novel telomerase-associated protein

A novel telomerase-associated protein was isolated from porcine testis. The 115-kDa protein, purified with telomerase activity, was molecular cloned using human cDNA library, and identified as MOV10. The expression levels of both MOV10 mRNA and MOV10 protein in cancer cells were 2-3 times higher than that of the normal cells, and MOV10 mRNA was highly expressed in human testis and ovary. The anti-MOV10 antibody precipitated the telomerase activity from cancer cell extracts, and inhibited the telomerase activity in vitro. Sf9-expressed MOV10 protein bound to G-rich strand of both single- and double-stranded telomere-sequenced DNA, but not to single C-rich strand. ChIP assay showed the binding of MOV10 to telomere region in vivo. These data suggest that MOV10 is involved in the progression of telomerase-catalyzing reaction via the interaction of telomerase protein and telomere DNA.     

Heritable L1 Retrotransposition Events During Development: Understanding Their Origins

The retrotransposon Long Interspersed Element 1 (LINE‐1 or L1) has played a major role in shaping the sequence composition of the mammalian genome. In our recent publication, “Heritable L1 retrotransposition in the mouse primordial germline and early embryo,” we systematically assessed the rate and developmental timing of de novo, heritable endogenous L1 insertions in mice. Such heritable retrotransposition events allow L1 to exert an ongoing influence upon genome evolution. Here, we place our findings in the context of earlier studies, and highlight how our results corroborate, and depart from, previous research based on human patient samples and transgenic mouse models harboring engineered L1 reporter genes. In parallel, we outline outstanding questions regarding the stage‐specificity, regulation, and functional impact of embryonic and germline L1 retrotransposition, and propose avenues for future research in this field.     

Id2诱导Myc表达促进小鼠胚胎干细胞自我更新

小鼠胚胎干细胞(mouse embryonic stem cells,m ESCs)分离自小鼠的囊胚内细胞团,在体外具有无限的自我更新能力和多向分化的潜能,因此拥有重大的社会和经济效益.前期人们发现,DNA结合抑制因子1(inhibitor of DNA binding 1,Id1)在含血清培养条件下,可以促进m ESCs自我更新,但其家族成员,如Id2和Id3,在m ESCs中的作用尚不清楚.本课题在m ESCs里分别上调Id2和Id3基因的表达,发现它们在含血清的培养条件下均具有促进m ESCs自我更新的能力,但Id2促进自我更新的能力大于Id3.通过转录组测序技术发现Id2上调c-Myc和n-Myc基因的表达水平.最后功能性验证实验证实,只有同时干扰c-Myc和n-Myc基因的表达才能够极大地削弱Id2维持m ESCs未分化状态的作用,表明Id2主要通过诱导Myc家族成员的表达来促进m ESCs的自我更新.本研究结果将扩大人们对干细胞多能性调控网络的认识,利于干细胞未来的基础研究和安全应用.     

Determination of L1 retrotransposition kinetics in cultured cells

L1 retrotransposons are autonomous retroelements that are active in the human and mouse genomes. Previously, we developed a cultured cell assay that uses a neomycin phosphotransferase (neo) retrotransposition cassette to determine relative retrotransposition frequencies among various L1 elements. Here, we describe a new retrotransposition assay that uses an enhanced green fluorescent protein (EGFP) retrotransposition cassette to determine retrotransposition kinetics in cultured cells. We show that retrotransposition is not detected in cultured cells during the first 48 h post-transfection, but then proceeds at a continuous high rate for at least 16 days. We also determine the relative retrotransposition rates of two similar human L1 retrotransposons, L1_RP and L1.3. L1_RP retrotransposed in the EGFP assay at a rate of ~0.5% of transfected cells/day, ~3-fold higher than the rate measured for L1.3. We conclude that the new assay detects near real time retrotransposition in a single cell and is sufficiently sensitive to differentiate retrotransposition rates among similar L1 elements. The EGFP assay exhibits improved speed and accuracy compared to the previous assay when used to determine relative retrotransposition frequencies. Furthermore, the EGFP cassette has an expanded range of experimental applications.