Quantifying the sequence-function relation in gene silencing by bacterial small RNAs

Sequence-function relations for small RNA (sRNA)-mediated gene silencing were quantified for the sRNA RyhB and some of its mRNA targets in Escherichia coli. Numerous mutants of RyhB and its targets were generated and their in vivo functions characterized at various levels of target and RyhB expression. Although a core complementary region is required for repression by RyhB, variations in the complementary sequences of the core region gave rise to a continuum of repression strengths, correlated exponentially with the computed free energy of RyhB-target duplex formation. Moreover, sequence variations in the linker region known to interact with the RNA chaperone Hfq also gave rise to a continuum of repression strengths, correlated exponentially with the computed energy cost of keeping the linker region open. These results support the applicability of the thermodynamic model in predicting sRNA-mRNA interaction and suggest that sequences at these locations may be used to fine-tune the degree of repression. Surprisingly, a truncated RyhB without the Hfq-binding region is found to repress multiple targets of the wild-type RyhB effectively, both in the presence and absence of Hfq, even though the former is required for the activity of wild-type RyhB itself. These findings challenge the commonly accepted model concerning the function of Hfq in gene silencing-both in providing stability to the sRNAs and in catalyzing the target mRNAs to take on active conformations-and raise the intriguing question of why many endogenous sRNAs subject their functions to Hfq-dependences.     

Connecting the epigenome,metabolome and proteome for a deeper understanding of disease

Epigenome-wide association studies (EWAS) identify genes that are dysregulated by the studied clinical endpoints, thereby indicating potential new diagnostic biomarkers, drug targets and therapy options. Combining EWAS with deep molecular phenotyping, such as approaches enabled by metabolomics and proteomics, allows further probing of the underlying disease-associated pathways. For instance, methylation of the TXNIP gene is associated robustly with prevalent type 2 diabetes and further with metabolites that are short-term markers of glycaemic control. These associations reflect TXNIP’s function as a glucose uptake regulator by interaction with the major glucose transporter GLUT1 and suggest that TXNIP methylation can be used as a read-out for the organism’s exposure to glucose stress. Another case is the association between DNA methylation of the AHRR and F2RL3 genes with smoking and a protein that is involved in the reprogramming of the bronchial epithelium. These examples show that associations between DNA methylation and intermediate molecular traits can open new windows into how the body copes with physiological challenges. This knowledge, if carefully interpreted, may indicate novel therapy options and, together with monitoring of the methylation state of specific methylation sites, may in the future allow the early diagnosis of impending disease. It is essential for medical practitioners to recognize the potential that this field holds in translating basic research findings to clinical practice. In this review, we present recent advances in the field of EWAS with metabolomics and proteomics and discuss both the potential and the challenges of translating epigenetic associations, with deep molecular phenotypes, to biomedical applications.     

Tyrosine phosphorylation sites on FRS2alpha responsible for Shp2 recruitment are critical for induction of lens and retina

Early development of the lens and retina depends upon reciprocal inductive interactions between the embryonic surface ectoderm and the underlying neuroepithelium of the optic vesicle. FGF signaling has been implicated in this signal exchange. The docking protein FRS2α is a major mediator of FGF signaling by providing a link between FGF receptors (FGFRs) and a variety of intracellular signaling pathways. After FGF stimulation, tyrosine-phosphorylated FRS2α recruits four molecules of the adaptor protein Grb2 and two molecules of the protein tyrosine phosphatase Shp2, resulting in activation of the Ras/extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase/Akt signaling pathways. In this report, we explore the role of signaling pathways downstream of FRS2α in eye development by analyzing the phenotypes of mice that carry point mutations in either the Grb2-(Frs2α^4F) or the Shp2-binding sites (Frs2α^2F) of FRS2α. Although Frs2α^4F/4F mice exhibited normal early eye development, all Frs2α^2F/2F embryos were defective in eye development and showed anophthalmia or microphthalmia. Consistent with the critical role of FRS2α in FGF signaling, the level of activated extracellular signal-regulated kinase in Frs2α^2F/2F embryos was significantly lower than that observed in wild-type embryos. Furthermore, expression of Pax6 and Six3, molecular markers for lens induction, were decreased in the Frs2α^2F/2F presumptive lens ectoderm. Similarly, the expression of Chx10 and Bmp4, genes required for retinal precursor proliferation and for lens development, respectively, was also decreased in the optic vesicles of Frs2α^2F/2F mice. These experiments demonstrate that intracellular signals that depend on specific tyrosine residues in FRS2α lie upstream of gene products critical for induction of lens and retina.     

Silence is golden: gene silencing of V. cholerae during intestinal colonization delivers new aspects to the acid tolerance response

Bacterial pathogens of the gastrointestinal tract alter their expression profile upon ingestion by the host and activate a variety of factors enhancing colonization and virulence. However, gene silencing during infection might be as important as gene activation to achieve full colonization fitness. Thus, we developed and successfully applied a reporter technology to identify 101 in vivo repressed (ivr) genes of the bacterial pathogen Vibrio cholerae. In depth analysis of the in vivo repressed H⁺/Cl^? transporter ClcA revealed an inverse requirement along gastrointestinal colonization. ClcA could be linked to acid tolerance response required during stomach passage, but ClcA expression is detrimental during subsequent colonization of the lower intestinal tract as it exploits the proton-motive force in alkaline environments. The study summarized in this addendum demonstrates that constitutive expression of ivr genes can reduce intestinal colonization fitness of V. cholerae, highlighting the necessity to downregulate these genes in vivo.     

The p53 inhibitors MDM2/MDMX complex is required for control of p53 activity in vivo

There are currently two distinct models proposed to explain why both MDM2 and MDMX are required in p53 control, with a key difference centered on whether these two p53 inhibitors work together or independently. To test these two competing models, we generated knockin mice expressing a point mutation MDMX mutant (C462A) that is defective in MDM2 binding. This approach allowed a targeted disassociation of the MDM2/MDMX heterocomplex without affecting the ability of MDMX to bind to p53, and while leaving the MDM2 protein itself completely untouched. Significantly, Mdmx(C462A/C462A) homozygous mice died at approximately day 9.5 of embryonic development, as the result of a combination of apoptosis and decreased cell proliferation, as shown by TUNEL and BrdU incorporation assays, respectively. Interestingly, even though the MDMX mutant protein abundance was found slightly elevated in the Mdmx(C462A/C462A) homozygous embryos, both the abundance and activity of p53 were markedly increased. A p53-dependent death was demonstrated by the finding that concomitant deletion of p53 completely rescued the embryonic lethality in Mdmx(C462A/C462A) homozygous mice. Our data demonstrate that MDM2 and MDMX function as an integral complex in p53 control, providing insights into the nonredundant nature of the function of MDM2 and MDMX.     

Diabetes promotes cardiac stem cell aging and heart failure, which are prevented by deletion of the p66shc gene

Diabetes leads to a decompensated myopathy, but the etiology of the cardiac disease is poorly understood. Oxidative stress is enhanced with diabetes and oxygen toxicity may alter cardiac progenitor cell (CPC) function resulting in defects in CPC growth and myocyte formation, which may favor premature myocardial aging and heart failure. We report that in a model of insulin-dependent diabetes mellitus, the generation of reactive oxygen species (ROS) leads to telomeric shortening, expression of the senescent associated proteins p53 and p16INK4a, and apoptosis of CPCs, impairing the growth reserve of the heart. However, ablation of the p66shc gene prevents these negative adaptations of the CPC compartment, interfering with the acquisition of the heart senescent phenotype and the development of heart failure with diabetes. ROS elicit 3 cellular reactions: low levels activate cell growth, intermediate quantities trigger cell apoptosis, and high amounts initiate cell necrosis. CPC replication predominates in diabetic p66shc-/-, whereas CPC apoptosis and myocyte apoptosis and necrosis prevail in diabetic wild type. Expansion of CPCs and developing myocytes preserves cardiac function in diabetic p66shc-/-, suggesting that intact CPCs can effectively counteract the impact of uncontrolled diabetes on the heart. The recognition that p66shc conditions the destiny of CPCs raises the possibility that diabetic cardiomyopathy is a stem cell disease in which abnormalities in CPCs define the life and death of the heart. Together, these data point to a genetic link between diabetes and ROS, on the one hand, and CPC survival and growth, on the other.     

H3K4 methyltransferase Set1 is involved in maintenance of ergosterol homeostasis and resistance to Brefeldin A

Set1 is a conserved histone H3 lysine 4 (H3K4) methyltransferase that exists as a multisubunit complex. Although H3K4 methylation is located on many actively transcribed genes, few studies have established a direct connection showing that loss of Set1 and H3K4 methylation results in a phenotype caused by disruption of gene expression. In this study, we determined that cells lacking Set1 or Set1 complex members that disrupt H3K4 methylation have a growth defect when grown in the presence of the antifungal drug Brefeldin A (BFA), indicating that H3K4 methylation is needed for BFA resistance. To determine the role of Set1 in BFA resistance, we discovered that Set1 is important for the expression of genes in the ergosterol biosynthetic pathway, including the rate-limiting enzyme HMG-CoA reductase. Consequently, deletion of SET1 leads to a reduction in HMG-CoA reductase protein and total cellular ergosterol. In addition, the lack of Set1 results in an increase in the expression of DAN1 and PDR11, two genes involved in ergosterol uptake. The increase in expression of uptake genes in set1Δ cells allows sterols such as cholesterol and ergosterol to be actively taken up under aerobic conditions. Interestingly, when grown in the presence of ergosterol set1Δ cells become resistant to BFA, indicating that proper ergosterol levels are needed for antifungal drug resistance. These data show that H3K4 methylation impacts gene expression and output of a biologically and medically relevant pathway and determines why cells lacking H3K4 methylation have antifungal drug sensitivity.     

Human tumor p53 mutations are selected for in mouse embryonic fibroblasts harboring a humanized p53 gene

To date, there has been no way to examine induced human p53 gene mutations in cell cultures exposed to mutagenic factors, other than by restriction site analysis. Here, we used embryonic cells from our Hupki (human p53 knock-in) mouse strain to generate human p53 DNA-binding domain (DBD) mutations experimentally. Twenty cultures of untreated primary mouse Hupki fibroblasts and 20 short-wavelength UV light (UVC)-treated cultures (20J/m(2)) were passaged >20 times. Established Hupki embryonic fibroblast cell lines (HUFs) were genotyped by dideoxy DNA sequencing of p53 exons 4-9. Seven of the HUFs harbored point mutations in the humanized p53 DBD. Of the 9 mutations (6 single- and 1 triple-site mutation), 2 were at the most frequently mutated codons in human cancers (c.248 and c.273). The Affymetrix p53 GeneChip assay also readily identified the 6 single-base substitutions. All mutations in HUFs from UV-treated cultures were at dipyrimidine sites, including 3 nontranscribed strand C -->T transitions. The mutant HUFs were deficient in p53 transactivation function, and missense mutants had high levels of nuclear p53 protein. In a second experiment, primary Hupki cells were exposed to the carcinogen aristolochic acid I (AAI). Five of 10 cultures that became established within 2 months harbored p53 DBD mutations. All were transversions, including 4 A --> T substitutions on the nontranscribed strand, a hallmark of DNA mutation by AAI. We conclude that establishment of Hupki mouse fibroblasts in culture readily selects for p53 DBD mutations found in human tumors, providing a basis for generating experimental mutation patterns in human p53.     

Double-stranded RNA made in C. elegans neurons can enter the germline and cause transgenerational gene silencing

An animal that can transfer gene-regulatory information from somatic cells to germ cells may be able to communicate changes in the soma from one generation to the next. In the worm Caenorhabditis elegans, expression of double-stranded RNA (dsRNA) in neurons can result in the export of dsRNA-derived mobile RNAs to other distant cells. Here, we show that neuronal mobile RNAs can cause transgenerational silencing of a gene of matching sequence in germ cells. Consistent with neuronal mobile RNAs being forms of dsRNA, silencing of target genes that are expressed either in somatic cells or in the germline requires the dsRNA-selective importer SID-1. In contrast to silencing in somatic cells, which requires dsRNA expression in each generation, silencing in the germline is heritable after a single generation of exposure to neuronal mobile RNAs. Although initiation of inherited silencing within the germline requires SID-1, a primary Argonaute RDE-1, a secondary Argonaute HRDE-1, and an RNase D homolog MUT-7, maintenance of inherited silencing is independent of SID-1 and RDE-1, but requires HRDE-1 and MUT-7. Inherited silencing can persist for >25 generations in the absence of the ancestral source of neuronal dsRNA. Therefore, our results suggest that sequence-specific regulatory information in the form of dsRNA can be transferred from neurons to the germline to cause transgenerational silencing.The germline is separated from the rest of the body, or soma, during early development in most animals, consistent with the suggestion that environmental effects on soma throughout the lifetime of an animal cannot influence inheritance through the germline (1). However, some environmental changes can cause effects that last for three or more generations, even in the apparent absence of changes in the genotype (reviewed in ref. 2). These transgenerational epigenetic effects are presumably initiated either by direct changes within the ancestral germline or by the transfer of information from ancestral somatic cells to the ancestral germline. It is difficult to distinguish between these possibilities because complex ancestral changes that affect subsequent generations, such as diet (3–5) or endocrine disruption (6), perturb many genes in many tissues in ways that are as yet unclear. Manipulating the activity of a single gene in specific tissues and across generations can help distinguish between these possibilities. Such specific inactivation of a single gene can be achieved by using double-stranded RNA (dsRNA) to trigger RNA interference (RNAi) in the worm Caenorhabditis elegans (7).As in most animals, the C. elegans germline is set aside early in development—after four cell divisions (8). Gene silencing initiated through RNAi-related mechanisms within the C. elegans germline can last for many generations (9–13). Such transgenerational silencing can be triggered by both injected dsRNA (14–16) and ingested dsRNA (16–19). However, both injection and ingestion can deliver dsRNA directly into the fluid-filled body cavity that surrounds the germline, without entry into the cytosol of any somatic cell (20, 21). Thus, it remains unknown whether somatic cells in C. elegans can export signals for delivery into the germline to cause transgenerational gene silencing.The transfer of gene-specific information from one somatic tissue to another somatic tissue during RNAi has been observed in C. elegans (22). Such intertissue transfer of gene-regulatory information appears to occur through the transport of forms of dsRNA called mobile RNAs (23). Entry of these mobile RNAs into the cytosol requires the dsRNA-selective importer SID-1 (22, 24, 25). Consequently, when dsRNA is expressed in a variety of somatic tissues such as the gut, muscles, or neurons, SID-1–dependent silencing of genes of matching sequence is observed in other somatic tissues (20). Because gene silencing by mobile RNAs from neurons appears to be stronger than that by mobile RNAs from other somatic tissues (20), we examined whether neurons export mobile RNAs that can enter the germline to cause transgenerational gene silencing.Here, we show that neuronal mobile RNAs can enter both somatic and germ cells to trigger gene silencing. Although silencing in somatic tissues is not detectably inherited despite multigenerational exposure to neuronal mobile RNAs, silencing in the germline is inherited for many generations after a single generation of exposure to neuronal mobile RNAs.     

肝细胞癌mdm2基因表达及其与p53基因突变的关系

目的研究ｍｄｍ２基因在原发性肝细胞癌（ＨＣＣ）中的表达并探讨其与ｐ５３基因突变的关系．方法用银染ＰＣＲＳＳＣＰ法检测ｐ５３基因第５～８外显子的突变,原位杂交检测ｍｄｍ２基因ｍＲＮＡ的表达,ＳＡＢＣ法检测ｍｄｍ２蛋白的表达．结果３９３％（１１／２８）的病例有异常的电泳迁移率．ｐ５３基因突变与肿瘤的大小、分化及转移无关．原位杂交显示９例ＨＣＣ出现ｍｄｍ２基因ｍＲＮＡ增加,７例ＨＣＣ可检测到ｍｄｍ２蛋白表达,ｍｄｍ２基因表达与ＨＣＣ的大小、分化及是否转移无关．Ⅰ～Ⅱ级ＨＣＣ中ｍｄｍ２阳性表达率（１３３％）明显低于Ⅲ～Ⅳ级ＨＣＣ中的阳性表达率（５３８％）．１１例有ｐ５３基因突变的ＨＣＣ中,只有３例出现ｍｄｍ２基因表达,另外６例有ｍｄｍ２过表达的ＨＣＣ未见ｐ５３基因突变．ｐ５３基因突变的ＨＣＣ与ｐ５３基因无突变的ＨＣＣ相比,ｍｄｍ２基因表达阳性率无显著差别．结论ｐ５３基因突变和ｍｄｍ２基因表达在原发性ＨＣＣ的发病中起重要作用．ｍｄｍ２基因表达与ＨＣＣ的恶性程度相关．ｍｄｍ２基因表达与ｐ５３基因是否突变无关．