Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

University of Arizona College of Medicine

Treatment of [M(II)(en)(3)][OTs](2) or methanolic ethylenediamine solutions containing transition metal p-toluenesulfonates (M(II) = Mn, Co) with aqueous K(4)M(IV)(CN)(8).2H(2)O or Cs(3)M(V)(CN)(8) (M(IV) = Mo, W; M(V) = Mo) affords crystalline clusters of [M(II)(en)(3)][cis-M(II)(en)(2)(OH(2))(mu-NC)M(IV)(CN)(7)].2H(2)O (M(IV) = Mo; M(II) = Mn, 1; Ni, 5; M(IV) = W; M(II) = Mn, 2; Ni, 6) and [cis-M(II)(en)(2)(OH(2))](2)[(mu-NC)(2)M(IV)(CN)(6)].4H(2)O (M(IV) = Mo; M(II) = Co, 3; Ni, 7; M(IV) = W; M(II) = Co, 4) stoichiometry. Each cluster contains cis-M(II)(en)(2)(OH(2))(mu-NC)(2+) units that likely result from dissociative loss of en from [M(II)(en)(3)](2+), affording cis-M(II)(en)(2)(OH(2))(2)(2+) intermediates that are trapped by M(IV)(CN)(8)(4-).

Synthesis and structural characterization of bi- and trimetallic octacyanometalate(IV) complexes: [Delta,Lambda-MII(en)3][cis-MII(en)2(OH2)][MIV(CN)8].2H2O and [cis-MII(en)2(OH2)]2[(mu-NC)2MIV(CN)6].4H2O (MII = Mn, Co, Ni; MIV = Mo, W).

Reactive lipid aldehydes are implicated in the pathogenesis of various oxidative stress-mediated diseases, including non-alcoholic steatohepatitis, atherosclerosis, Alzheimer's and cataract. In the present study, we sought to define which hepatic Aldh isoform plays a major role in detoxification of lipid-derived aldehydes, such as acrolein and HNE by enzyme kinetic and gene expression studies. The catalytic efficiencies for metabolism of acrolein by Aldh1a1 was comparable to that of Aldh3a1 (V(max)/K(m)=23). However, Aldh1a1 exhibits far higher affinity for acrolein (K(m)=23.2 μM) compared to Aldh3a1 (K(m)=464 μM). Aldh1a1 displays a 3-fold higher catalytic efficiency for HNE than Aldh3a1 (218 ml/min/mg vs 69 ml/min/mg). The endogenous Aldh1a1 gene was highly expressed in mouse liver and a liver-derived cell line (Hepa-1c1c7) compared to Aldh2, Aldh1b1 and Aldh3a1. Aldh1a1 mRNA levels was 34-fold and 73-fold higher than Aldh2 in mouse liver and Hepa-1c1c7 cells respectively. Aldh3a1 gene was absent in mouse liver, but moderately expressed in Hepa-1c1c7 cells compared to Aldh1a1. We demonstrated that knockdown of Aldh1a1 expression by siRNA caused Hepa-1c1c7 cells to be more sensitive to acrolein-induced cell death and resulted in increased accumulation of acrolein-protein adducts and caspase 3 activation. These results indicate that Aldh1a1 plays a major role in cellular defense against oxidative damage induced by reactive lipid aldehydes in mouse liver. We also noted that hepatic Aldh1a1 mRNA levels were significantly increased (≈3-fold) in acrolein-fed mice compared to control. In addition, hepatic cytosolic ALDH activity was induced by acrolein when 1mM NAD(+) was used as cofactor, suggesting an Aldh1a1-protective mechanism against acrolein toxicity in mice liver. Thus, mechanisms to induce Aldh1a1 gene expression may provide a useful rationale for therapeutic protection against oxidative stress-induced pathologies.

Murine hepatic aldehyde dehydrogenase 1a1 is a major contributor to oxidation of aldehydes formed by lipid peroxidation.

Expression of channels to specific neuronal sites can critically impact their function and regulation. Currently, the molecular mechanisms underlying this targeting and intracellular trafficking of transient receptor potential (TRP) channels remain poorly understood, and identifying proteins involved in these processes will provide insight into underlying mechanisms. Vision is dependent on the normal function of retinal depolarizing bipolar cells (DBCs), which couple a metabotropic glutamate receptor 6 to the TRP melastatin 1 (TRPM1) channel to transmit signals from photoreceptors. We report that the extracellular membrane-attached protein nyctalopin is required for the normal expression of TRPM1 on the dendrites of DBCs in mus musculus. Biochemical and genetic data indicate that nyctalopin and TRPM1 interact directly, suggesting that nyctalopin is acting as an accessory TRP channel subunit critical for proper channel localization to the synapse.

A role for nyctalopin, a small leucine-rich repeat protein, in localizing the TRP melastatin 1 channel to retinal depolarizing bipolar cell dendrites.

Complete congenital stationary night blindness (cCSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impairment of night vision, absence of the electroretinogram (ERG) b-wave, and variable degrees of involvement of other visual functions. We report here that mutations in GPR179, encoding an orphan G protein receptor, underlie a form of autosomal-recessive cCSNB. The Gpr179(nob5/nob5) mouse model was initially discovered by the absence of the ERG b-wave, a component that reflects depolarizing bipolar cell (DBC) function. We performed genetic mapping, followed by next-generation sequencing of the critical region and detected a large transposon-like DNA insertion in Gpr179. The involvement of GPR179 in DBC function was confirmed in zebrafish and humans. Functional knockdown of gpr179 in zebrafish led to a marked reduction in the amplitude of the ERG b-wave. Candidate gene analysis of GPR179 in DNA extracted from patients with cCSNB identified GPR179-inactivating mutations in two patients. We developed an antibody against mouse GPR179, which robustly labeled DBC dendritic terminals in wild-type mice. This labeling colocalized with the expression of GRM6 and was absent in Gpr179(nob5/nob5) mutant mice. Our results demonstrate that GPR179 plays a critical role in DBC signal transduction and expands our understanding of the mechanisms that mediate normal rod vision.

GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness.

Nyctalopin is a small leucine rich repeat proteoglycan (SLRP) whose function is critical for normal vision. The absence of nyctalopin results in the complete form of congenital stationary night blindness. Normally, glutamate released by photoreceptors binds to the metabotropic glutamate receptor type 6 (GRM6), which through a G-protein cascade closes the non-specific cation channel, TRPM1, on the dendritic tips of depolarizing bipolar cells (DBCs) in the retina. Nyctalopin has been shown to interact with TRPM1 and expression of TRPM1 on the dendritic tips of the DBCs is dependent on nyctalopin expression. In the current study, we used yeast two hybrid and biochemical approaches to investigate whether murine nyctalopin was membrane bound, and if so by what mechanism, and also whether the functional form was as a homodimer. Our results show that murine nyctalopin is anchored to the plasma membrane by a single transmembrane domain, such that the LRR domain is located in the extracellular space.

Topological analysis of small leucine-rich repeat proteoglycan nyctalopin.

Mutations in TRPM1 are found in humans with an autosomal recessive form of complete congenital stationary night blindness (cCSNB). The Trpm1(-/-) mouse has been an important animal model for this condition. Here we report a new mouse mutant, tvrm27, identified in a chemical mutagenesis screen. Genetic mapping of the no b-wave electroretinogram (ERG) phenotype of tvrm27 localized the mutation to a chromosomal region that included Trpm1. Complementation testing with Trpm1(-/-) mice confirmed a mutation in Trpm1. Sequencing identified a nucleotide change in exon 23, converting a highly conserved alanine within the pore domain to threonine (p.A1068T). Consistent with prior studies of Trpm1(-/-) mice, no anatomical changes were noted in the Trpm1(tvrm27/tvrm27) retina. The Trpm1(tvrm27/tvrm27) phenotype is distinguished from that of Trpm1(-/-) by the retention of TRPM1 expression on the dendritic tips of depolarizing bipolar cells (DBCs). While ERG b-wave amplitudes of Trpm1(+/-) heterozygotes are comparable to wild type, those of Trpm1(+/tvrm27) mice are reduced by 32%. A similar reduction in the response of Trpm1(+/tvrm27) DBCs to LY341495 or capsaicin is evident in whole cell recordings. These data indicate that the p.A1068T mutant TRPM1 acts as a dominant negative with respect to TRPM1 channel function. Furthermore, these data indicate that the number of functional TRPM1 channels at the DBC dendritic tips is a key factor in defining DBC response amplitude. The Trpm1(tvrm27/tvrm27) mutant will be useful for elucidating the role of TRPM1 in DBC signal transduction, for determining how Trpm1 mutations impact central visual processing, and for evaluating experimental therapies for cCSNB.

Depolarizing bipolar cell dysfunction due to a Trpm1 point mutation.

Long Interspersed Nuclear Element-1 (LINE-1 or L1) is an autonomous, mobile element within the human genome that transposes via a "copy and paste" mechanism and relies upon L1-encoded endonuclease and reverse transcriptase (RT) activities to compromise genome integrity. L1 has been implicated in various forms of cancer, but its role in the regulation of the oncogenic phenotype is not understood. The present studies were conducted to evaluate mechanisms of genetic regulatory control in HepG2 cells by human L1, or a D702Y mutant deficient in RT activity, and their influence on cellular phenotype. Forced expression of synthetic L1 ORF1p and ORF2p was associated with formation of cytoplasmic foci and minor association with the nuclear compartment. While de novo L1 mobilizations were only identified in cells expressing wild type L1, and were absent in the D702Y mutant, changes in gene expression profiles involved RT dependent as well as RT independent mechanisms. Synthetic L1 altered the expression of 24 in silico predicted genetic targets; ten of which showed RT-dependence, ten RT-independence, and four reciprocal regulatory control by both wild type and RT mutant. Of five targets examined, only VCAM1 and PTPRB colocalized with newly retrotransposed wild type L1. Biological discretization to partition patterns of gene expression into unique frequencies identified adhesion, inflammation, and cellular metabolism as key processes targeted for molecular interference with disruption of epithelial-to-mesenchymal programming seen irrespective of the RT phenotype. These findings establish L1 as a key regulator of genome plasticity and EMT via mechanisms independent of RT activity.

Reprogramming of the HepG2 genome by long interspersed nuclear element-1.

Genetic mutations and gross structural defects in the DNA sequence permanently alter genetic loci in ways that significantly disrupt gene function. In sharp contrast, genes modified by aberrant epigenetic modifications remain structurally intact and are subject to partial or complete reversal of modifications that restore the original (i.e. non-diseased) state. Such reversibility makes epigenetic modifications ideal targets for therapeutic intervention. The epigenome of cancer cells is extensively modified by specific hypermethylation of the promoters of tumor suppressor genes relative to the extensive hypomethylation of repetitive sequences, overall loss of acetylation, and loss of repressive marks at microsatellite/repeat regions. In this review, we discuss emerging therapies targeting specific epigenetic modifications or epigenetic modifying enzymes either alone or in combination with other treatment regimens. The limitations posed by cancer treatments elicit unintended epigenetic modifications that result in exacerbation of tumor progression are also discussed. Lastly, a brief discussion of the specificity restrictions posed by epigenetic therapies and ways to address such limitations is presented.

The promise and failures of epigenetic therapies for cancer treatment.

Long interspersed nuclear elements (Line-1 or L1s) account for ~17% of the human genome. While the majority of human L1s are inactive, ~80-100 elements remain retrotransposition competent and mobilize through RNA intermediates to different locations within the genome. De novo insertions of L1s account for polymorphic variation of the human genome and disruption of target loci at their new location. In the present study, fluorescence in situ hybridization and DNA sequencing were used to characterize retrotransposition profiles of L1(RP) in cultured human HepG2 cells. While expression of synthetic L1(RP) was associated with full-length and truncated insertions throughout the entire genome, a strong preference for gene-poor regions, such as those found in chromosome 13 was observed for full-length insertions. These findings shed light into L1 targeting mechanisms within the human genome and question the putative randomness of L1 retrotransposition.

De novo LINE-1 retrotransposition in HepG2 cells preferentially targets gene poor regions of chromosome 13.

Long Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms. We have previously shown that the regulatory region of human and murine L1s interact with retinoblastoma (RB) proteins to effect retroelement silencing. The present studies were conducted to identify the corepressor complex responsible for RB-mediated silencing of L1.

Pasano Bojang

Division of Pulmonary,
Allergy,
Critical Care and Sleep Medicine,
Division of Pulmonary, Allergy, Critical Care and Sleep Medicine

University of Arizona College of Medicine

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Pasano Bojang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Pulmonary,Allergy,Critical Care and Sleep Medicine,Division of Pulmonary, Allergy, Critical Care and Sleep Medicine

University of Arizona College of Medicine

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Division of Pulmonary,
Allergy,
Critical Care and Sleep Medicine,
Division of Pulmonary, Allergy, Critical Care and Sleep Medicine