Identification of Cellular Host Factors That Associate With LINE-1 ORF1p and the Effect of the Zinc Finger Antiviral Protein Zap on LINE-1 Retrotransposition.

Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. The human genome contains over 500,000 L1 sequences, which account for approximately 17% of human DNA. L1 sequences mobilize throughout the human genome by a copy-and-paste mechanism known as retrotransposition. Most genomic L1 sequences are incapable of mobility (i.e., retrotransposition) because they are either 5'-truncated, internally rearranged, and/or mutated; however, it is estimated that each human cell contains at least 80-100 intact L1 sequences that are retrotransposition capable. L1 retrotransposition is inherently mutagenic and on occasion can disrupt gene expression leading to diseases such as hemophilia A and cancer. Due to the mutagenic potential of L1 retrotransposition, it thus stands to reason that the host cell has evolved mechanisms to protect the cell from unabated retrotransposition. In this thesis I identified cellular host factors that associate with the first L1 open reading frame protein, ORF1p. I demonstrate that the zinc finger antiviral protein ZAP associates with L1 ORF1p and inhibits human L1 and Alu retrotransposition as well as the retrotransposition of LINE elements from mice and zebrafish. Molecular genetic, biochemical, and fluorescence microscopy data suggest that ZAP interacts with L1 RNA and reduces the expression of full-length L1 RNA and the L1-encoded proteins, thereby providing mechanistic insight into how ZAP may restrict retrotransposition. In addition to ZAP, I show that the ORF1p-associated cellular host factors MOV10, hnRNPL, and PAR-4 also inhibit L1 retrotransposition. Mechanistic data suggest that ZAP, MOV10, hnRNPL, and PAR-4 restrict L1 retrotransposition by distinct mechanisms, suggesting that each of these cellular host factors may target different post-transcriptional steps in the L1 retrotransposition cycle. Importantly, ZAP and MOV10 were first characterized as antiviral proteins due to their ability to suppress retroviral activity. Notably, several other host cell antiviral factors such as APOBEC3 proteins, TREX1, SAMHD1 and RNase L have recently been demonstrated to inhibit L1 retrotransposition. Thus, these data suggest that ZAP, MOV10 and perhaps other ORF1p-associated cellular host factors initially may have evolved to combat L1 and other endogenous retrotransposons and subsequently were co-opted as viral restriction factors.PHDCellular and Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113417/1/jmoldova_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113417/2/jmoldova_2.pd

Characterization of LINE-1 Ribonucleoprotein Particles

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1–encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination

Baseline characteristics of patients in the reduction of events with darbepoetin alfa in heart failure trial (RED-HF)

<p>Aims: This report describes the baseline characteristics of patients in the Reduction of Events with Darbepoetin alfa in Heart Failure trial (RED-HF) which is testing the hypothesis that anaemia correction with darbepoetin alfa will reduce the composite endpoint of death from any cause or hospital admission for worsening heart failure, and improve other outcomes.</p> <p>Methods and results: Key demographic, clinical, and laboratory findings, along with baseline treatment, are reported and compared with those of patients in other recent clinical trials in heart failure. Compared with other recent trials, RED-HF enrolled more elderly [mean age 70 (SD 11.4) years], female (41%), and black (9%) patients. RED-HF patients more often had diabetes (46%) and renal impairment (72% had an estimated glomerular filtration rate <60 mL/min/1.73 m2). Patients in RED-HF had heart failure of longer duration [5.3 (5.4) years], worse NYHA class (35% II, 63% III, and 2% IV), and more signs of congestion. Mean EF was 30% (6.8%). RED-HF patients were well treated at randomization, and pharmacological therapy at baseline was broadly similar to that of other recent trials, taking account of study-specific inclusion/exclusion criteria. Median (interquartile range) haemoglobin at baseline was 112 (106–117) g/L.</p> <p>Conclusion: The anaemic patients enrolled in RED-HF were older, moderately to markedly symptomatic, and had extensive co-morbidity.</p&gt

Phenome-wide association analysis of LDL-cholesterol lowering genetic variants in PCSK9

Abstract: Background: We characterised the phenotypic consequence of genetic variation at the PCSK9 locus and compared findings with recent trials of pharmacological inhibitors of PCSK9. Methods: Published and individual participant level data (300,000+ participants) were combined to construct a weighted PCSK9 gene-centric score (GS). Seventeen randomized placebo controlled PCSK9 inhibitor trials were included, providing data on 79,578 participants. Results were scaled to a one mmol/L lower LDL-C concentration. Results: The PCSK9 GS (comprising 4 SNPs) associations with plasma lipid and apolipoprotein levels were consistent in direction with treatment effects. The GS odds ratio (OR) for myocardial infarction (MI) was 0.53 (95% CI 0.42; 0.68), compared to a PCSK9 inhibitor effect of 0.90 (95% CI 0.86; 0.93). For ischemic stroke ORs were 0.84 (95% CI 0.57; 1.22) for the GS, compared to 0.85 (95% CI 0.78; 0.93) in the drug trials. ORs with type 2 diabetes mellitus (T2DM) were 1.29 (95% CI 1.11; 1.50) for the GS, as compared to 1.00 (95% CI 0.96; 1.04) for incident T2DM in PCSK9 inhibitor trials. No genetic associations were observed for cancer, heart failure, atrial fibrillation, chronic obstructive pulmonary disease, or Alzheimer’s disease – outcomes for which large-scale trial data were unavailable. Conclusions: Genetic variation at the PCSK9 locus recapitulates the effects of therapeutic inhibition of PCSK9 on major blood lipid fractions and MI. While indicating an increased risk of T2DM, no other possible safety concerns were shown; although precision was moderate

Brazilian cave heritage under siege

info:eu-repo/semantics/publishe

Schizophrenia-associated somatic copy-number variants from 12,834 cases reveal recurrent NRXN1 and ABCB11 disruptions

While germline copy-number variants (CNVs) contribute to schizophrenia (SCZ) risk, the contribution of somatic CNVs (sCNVs)—present in some but not all cells—remains unknown. We identified sCNVs using blood-derived genotype arrays from 12,834 SCZ cases and 11,648 controls, filtering sCNVs at loci recurrently mutated in clonal blood disorders. Likely early-developmental sCNVs were more common in cases (0.91%) than controls (0.51%, p = 2.68e−4), with recurrent somatic deletions of exons 1–5 of the NRXN1 gene in five SCZ cases. Hi-C maps revealed ectopic, allele-specific loops forming between a potential cryptic promoter and non-coding cis-regulatory elements upon 5′ deletions in NRXN1. We also observed recurrent intragenic deletions of ABCB11, encoding a transporter implicated in anti-psychotic response, in five treatment-resistant SCZ cases and showed that ABCB11 is specifically enriched in neurons forming mesocortical and mesolimbic dopaminergic projections. Our results indicate potential roles of sCNVs in SCZ risk

The Zinc-Finger Antiviral Protein ZAP Inhibits LINE and Alu Retrotransposition

<div><p>Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements.</p></div

The effect of ZAP-S on L1 RNA and L1 protein expression.

<p><i>(A) Schematic of pJM101/L1</i>.<i>3Δneo</i>: Bold black lines indicate the approximate location of probes (5UTR99 and ORF2_5804) used in the northern blot experiments. pJM101/L1.3Δneo is expressed from a pCEP4 vector. A CMV promoter augments L1 expression and an SV40 polyadenylation signal (pA) is located downstream of the native L1 polyadenylation signal. <i>(B) Results of northern blots</i>: Top panel: HeLa cells were co-transfected with pJM101/L1.3Δneo and either the indicated ZAP-S expression plasmids or an empty pCEP4 vector. Northern blot images depict the effect of ZAP-S overexpression on polyadenylated L1 RNA levels. The constructs transfected into HeLa cells are indicated above each lane. UTF indicates untransfected HeLa cells and serves as a negative control. Probes (5UTR99 and ORF2_5804) are indicated in the top left corner of the respective blots. The black arrow indicates the position of the full-length L1 RNA. The blue and yellow arrows indicate shorter L1 RNA species. The experiment was repeated three times with similar results. Actin served as a loading control. RNA size standards (~kb) are shown at the right of the blot image. Bottom panel: Quantification of northern blot bands. The X-axis indicates the cDNA expression construct that was co-transfected with pJM101/L1.3Δneo. The Y-axis indicates relative band intensity normalized to pCEP4 controls (100%). Black bars represent the full-length L1 band. Blue and yellow bars represent the smaller L1 RNA bands, corresponding to the colored arrows, respectively, in the top panel. The results are the average of three independent experiments. Error bars represent standard deviations. <i>(C) Schematic of pJBM2TE1</i>: The construct contains a T7 epitope tag on the carboxyl-terminus of ORF1p and a TAP tag on the carboxyl-terminus of ORF2p. An <i>mneoI</i> retrotransposition indicator cassette is present in the 3’ UTR. pJMB2TE1 is expressed from a pCEP4 backbone, which has been modified to contain a puromycin selectable marker. A CMV promoter augments L1 expression and an SV40 polyadenylation signal (pA) is located downstream of the native L1 polyadenylation signal. <i>(D) ZAP-S decreases the accumulation of the L1-encoded proteins</i>: HeLa cells were co-transfected with pJBM2TE1 and the plasmids indicated above each lane. UTF indicates untransfected HeLa cells and serves as a negative control. Depicted are western blots using whole cell lysates (WCL, top panel) or RNP fractions (RNP, bottom panel). Blue arrows indicate the positions of ORF2p, ORF1p, ZAP-S, and ZAP-S/∆72–372. The eIF3 protein is used as a loading control. Representative images are shown. The experiments were repeated three times with similar results.</p