Nucleic Acids Research

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A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor

чт, 2016-09-29 11:06

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 107 variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work.

Категории: Bioinformatics, Biology, Journals

Impact of donor-recipient phylogenetic distance on bacterial genome transplantation

чт, 2016-09-29 11:06

Genome transplantation (GT) allows the installation of purified chromosomes into recipient cells, causing the resulting organisms to adopt the genotype and the phenotype conferred by the donor cells. This key process remains a bottleneck in synthetic biology, especially for genome engineering strategies of intractable and economically important microbial species. So far, this process has only been reported using two closely related bacteria, Mycoplasma mycoides subsp. capri (Mmc) and Mycoplasma capricolum subsp. capricolum (Mcap), and the main factors driving the compatibility between a donor genome and a recipient cell are poorly understood. Here, we investigated the impact of the evolutionary distance between donor and recipient species on the efficiency of GT. Using Mcap as the recipient cell, we successfully transplanted the genome of six bacteria belonging to the Spiroplasma phylogenetic group but including species of two distinct genera. Our results demonstrate that GT efficiency is inversely correlated with the phylogenetic distance between donor and recipient bacteria but also suggest that other species-specific barriers to GT exist. This work constitutes an important step toward understanding the cellular factors governing the GT process in order to better define and eventually extend the existing genome compatibility limit.

Категории: Bioinformatics, Biology, Journals

Editorial: CRISPR in Nucleic Acids Research

чт, 2016-09-29 11:06
Категории: Bioinformatics, Biology, Journals

Nucleic Acids Research: Editorial Board

чт, 2016-09-29 11:06
Категории: Bioinformatics, Biology, Journals

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чт, 2016-09-29 11:06
Категории: Bioinformatics, Biology, Journals

3.9 A structure of the nucleosome core particle determined by phase-plate cryo-EM

чт, 2016-09-29 11:06

The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining phase-plate imaging and single particle analysis to determine the structure of a small protein–DNA complex. To test the method, we made use of a 200 kDa Nucleosome Core Particle (NCP) reconstituted with 601 DNA for which a high-resolution X-ray crystal structure is known. We find that the phase plate provides a significant contrast enhancement that permits individual NCPs and DNA to be clearly identified in amorphous ice. The refined structure from 26,060 particles has an overall resolution of 3.9 Å and the density map exhibits structural features consistent with the estimated resolution, including clear density for amino acid side chains and DNA features such as the phosphate backbone. Our results demonstrate that phase-plate cryo-EM promises to become an important method to determine novel near-atomic resolution structures of small and challenging samples, such as nucleosomes in complex with nucleosome-binding factors.

Категории: Bioinformatics, Biology, Journals

An integrated, structure- and energy-based view of the genetic code

чт, 2016-09-29 11:06

The principles of mRNA decoding are conserved among all extant life forms. We present an integrative view of all the interaction networks between mRNA, tRNA and rRNA: the intrinsic stability of codon–anticodon duplex, the conformation of the anticodon hairpin, the presence of modified nucleotides, the occurrence of non-Watson–Crick pairs in the codon–anticodon helix and the interactions with bases of rRNA at the A-site decoding site. We derive a more information-rich, alternative representation of the genetic code, that is circular with an unsymmetrical distribution of codons leading to a clear segregation between GC-rich 4-codon boxes and AU-rich 2:2-codon and 3:1-codon boxes. All tRNA sequence variations can be visualized, within an internal structural and energy framework, for each organism, and each anticodon of the sense codons. The multiplicity and complexity of nucleotide modifications at positions 34 and 37 of the anticodon loop segregate meaningfully, and correlate well with the necessity to stabilize AU-rich codon–anticodon pairs and to avoid miscoding in split codon boxes. The evolution and expansion of the genetic code is viewed as being originally based on GC content with progressive introduction of A/U together with tRNA modifications. The representation we present should help the engineering of the genetic code to include non-natural amino acids.

Категории: Bioinformatics, Biology, Journals

Origins of tmRNA: the missing link in the birth of protein synthesis?

чт, 2016-09-29 11:06

The RNA world hypothesis refers to the early period on earth in which RNA was central in assuring both genetic continuity and catalysis. The end of this era coincided with the development of the genetic code and protein synthesis, symbolized by the apparition of the first non-random messenger RNA (mRNA). Modern transfer-messenger RNA (tmRNA) is a unique hybrid molecule which has the properties of both mRNA and transfer RNA (tRNA). It acts as a key molecule during trans-translation, a major quality control pathway of modern bacterial protein synthesis. tmRNA shares many common characteristics with ancestral RNA. Here, we present a model in which proto-tmRNAs were the first molecules on earth to support non-random protein synthesis, explaining the emergence of early genetic code. In this way, proto-tmRNA could be the missing link between the first mRNA and tRNA molecules and modern ribosome-mediated protein synthesis.

Категории: Bioinformatics, Biology, Journals

Evoking picomolar binding in RNA by a single phosphorodithioate linkage

чт, 2016-09-29 11:06

RNA aptamers are synthetic oligonucleotide-based affinity molecules that utilize unique three-dimensional structures for their affinity and specificity to a target such as a protein. They hold the promise of numerous advantages over biologically produced antibodies; however, the binding affinity and specificity of RNA aptamers are often insufficient for successful implementation in diagnostic assays or as therapeutic agents. Strong binding affinity is important to improve the downstream applications. We report here the use of the phosphorodithioate (PS2) substitution on a single nucleotide of RNA aptamers to dramatically improve target binding affinity by ~1000-fold (from nanomolar to picomolar). An X-ray co-crystal structure of the α-thrombin:PS2-aptamer complex reveals a localized induced-fit rearrangement of the PS2-containing nucleotide which leads to enhanced target interaction. High-level quantum mechanical calculations for model systems that mimic the PS2 moiety and phenylalanine demonstrate that an edge-on interaction between sulfur and the aromatic ring is quite favorable, and also confirm that the sulfur analogs are much more polarizable than the corresponding phosphates. This favorable interaction involving the sulfur atom is likely even more significant in the full aptamer-protein complexes than in the model systems.

Категории: Bioinformatics, Biology, Journals

Backmasking in the yeast genome: encoding overlapping information for protein-coding and RNA degradation

чт, 2016-09-29 11:06

Backmasking is a recording technique used to hide a sound or message in a music track in reverse, meaning that it is only audible when the record is played backwards. Analogously, the compact yeast genome encodes for diverse sources of information such as overlapping coding and non-coding transcripts, and protein-binding sites on the two complementary DNA strands. Examples are the consensus binding site sequences of the RNA-binding proteins Nrd1 and Nab3 that target non-coding transcripts for degradation. Here, by examining the overlap of stable (SUTs, stable unannotated transcripts) and unstable (CUTs, cryptic unstable transcripts) transcripts with protein-coding genes, we show that the predicted Nrd1 and Nab3-binding site sequences occur at differing frequencies. They are always depleted in the sense direction of protein-coding genes, thus avoiding degradation of the transcript. However in the antisense direction, predicted binding sites occur at high frequencies in genes with overlapping unstable ncRNAs (CUTs), so limiting the availability of non-functional transcripts. In contrast they are depleted in genes with overlapping stable ncRNAs (SUTs), presumably to avoid degrading the non-coding transcript. The protein-coding genes maintain similar amino-acid contents, but they display distinct codon usages so that Nrd1 and Nab3-binding sites can arise at differing frequencies in antisense depending on the overlapping transcript type. Our study demonstrates how yeast has evolved to encode multiple layers of information—protein-coding genes in one strand and the relative chance of degrading antisense RNA in the other strand—in the same regions of a compact genome.

Категории: Bioinformatics, Biology, Journals

The interplay between chromosome stability and cell cycle control explored through gene-gene interaction and computational simulation

чт, 2016-09-29 11:06

Chromosome stability models are usually qualitative models derived from molecular-genetic mechanisms for DNA repair, DNA synthesis, and cell division. While qualitative models are informative, they are also challenging to reformulate as precise quantitative models. In this report we explore how (A) laboratory experiments, (B) quantitative simulation, and (C) seriation algorithms can inform models of chromosome stability. Laboratory experiments were used to identify 19 genes that when over-expressed cause chromosome instability in the yeast Saccharomyces cerevisiae. To better understand the molecular mechanisms by which these genes act, we explored their genetic interactions with 18 deletion mutations known to cause chromosome instability. Quantitative simulations based on a mathematical model of the cell cycle were used to predict the consequences of several genetic interactions. These simulations lead us to suspect that the chromosome instability genes cause cell-cycle perturbations. Cell-cycle involvement was confirmed using a seriation algorithm, which was used to analyze the genetic interaction matrix to reveal an underlying cyclical pattern. The seriation algorithm searched over 1014 possible arrangements of rows and columns to find one optimal arrangement, which correctly reflects events during cell cycle phases. To conclude, we illustrate how the molecular mechanisms behind these cell cycle events are consistent with established molecular interaction maps.

Категории: Bioinformatics, Biology, Journals

Preferential extension of short telomeres induced by low extracellular pH

чт, 2016-09-29 11:06

The majority of tumor cells overcome proliferative limit by expressing telomerase. Whether or not telomerase preferentially extends the shortest telomeres is still under debate. When human cancer cells are cultured at neutral pH, telomerase extends telomeres in telomere length-independent manner. However, the microenvironment of tumor is slightly acidic, and it is not yet known how this influences telomerase action. Here, we examine telomere length homeostasis in tumor cells cultured at pHe 6.8. The results indicate that telomerase preferentially extends short telomeres, such that telomere length distribution narrows and telomeres become nearly uniform in size. After growth at pHe 6.8, the expression of telomerase, TRF1, TRF2 and TIN2 decreases, and the abundance of Cajal bodies decreases. Therefore, telomerase are insufficient for extending every telomere and shorter telomeres bearing less shelterin proteins are more accessible for telomerase recruitment. The findings support the ‘protein-counting mechanism’ in which extended and unextended state of telomere is determined by the number of associated shelterin proteins and the abundance of telomerase. Decreased expression of telomerase and preferential extension of short telomeres have important implications for tumor cell viability, and generate a strong rationale for research on telomerase-targeted anti-cancer therapeutics.

Категории: Bioinformatics, Biology, Journals

Human mutations affect the epigenetic/bookmarking function of HNF1B

чт, 2016-09-29 11:06

Bookmarking factors are transcriptional regulators involved in the mitotic transmission of epigenetic information via their ability to remain associated with mitotic chromatin. The mechanisms through which bookmarking factors bind to mitotic chromatin remain poorly understood. HNF1β is a bookmarking transcription factor that is frequently mutated in patients suffering from renal multicystic dysplasia and diabetes. Here, we show that HNF1β bookmarking activity is impaired by naturally occurring mutations found in patients. Interestingly, this defect in HNF1β mitotic chromatin association is rescued by an abrupt decrease in temperature. The rapid relocalization to mitotic chromatin is reversible and driven by a specific switch in DNA-binding ability of HNF1β mutants. Furthermore, we demonstrate that importin-β is involved in the maintenance of the mitotic retention of HNF1β, suggesting a functional link between the nuclear import system and the mitotic localization/translocation of bookmarking factors. Altogether, our studies have disclosed novel aspects on the mechanisms and the genetic programs that account for the mitotic association of HNF1β, a bookmarking factor that plays crucial roles in the epigenetic transmission of information through the cell cycle.

Категории: Bioinformatics, Biology, Journals

BAP18 coactivates androgen receptor action and promotes prostate cancer progression

чт, 2016-09-29 11:06

BPTF associated protein of 18 kDa (BAP18) has been reported as a component of MLL1-WDR5 complex. However, BAP18 is an uncharacterized protein. The detailed biological functions of BAP18 and underlying mechanisms have not been defined. Androgen receptor (AR), a member of transcription factor, plays an essential role in prostate cancer (PCa) and castration-resistant prostate cancer (CRPC) progression. Here, we demonstrate that BAP18 is identified as a coactivator of AR in Drosophilar experimental system and mammalian cells. BAP18 facilitates the recruitment of MLL1 subcomplex and AR to androgen-response element (ARE) of AR target genes, subsequently increasing histone H3K4 trimethylation and H4K16 acetylation. Knockdown of BAP18 attenuates cell growth and proliferation of PCa cells. Moreover, BAP18 depletion results in inhibition of xenograft tumor growth in mice even under androgen-depletion conditions. In addition, our data show that BAP18 expression in clinical PCa samples is higher than that in benign prostatic hyperplasia (BPH). Our data suggest that BAP18 as an epigenetic modifier regulates AR-induced transactivation and the function of BAP18 might be targeted in human PCa to promote tumor growth and progression to castration-resistance.

Категории: Bioinformatics, Biology, Journals

G9a promotes proliferation and inhibits cell cycle exit during myogenic differentiation

чт, 2016-09-29 11:06

Differentiation of skeletal muscle cells, like most other cell types, requires a permanent exit from the cell cycle. The epigenetic programming underlying these distinct cellular states is not fully understood. In this study, we provide evidence that the lysine methyltransferase G9a functions as a central axis to regulate proliferation and differentiation of skeletal muscle cells. Transcriptome analysis of G9a knockdown cells revealed deregulation of many cell cycle regulatory genes. We demonstrate that G9a enhances cellular proliferation by two distinct mechanisms. G9a blocks cell cycle exit via methylation-dependent transcriptional repression of the MyoD target genes p21Cip/Waf1 and Rb1. In addition, it activates E2F1-target genes in a methyltransferase activity-independent manner. We show that G9a is present in the E2F1/PCAF complex, and enhances PCAF occupancy and histone acetylation marks at E2F1-target promoters. Interestingly, G9a preferentially associates with E2F1 at the G1/S phase and with MyoD at the G2/M phase. Our results provide evidence that G9a functions both as a co-activator and a co-repressor to enhance cellular proliferation and inhibit myogenic differentiation.

Категории: Bioinformatics, Biology, Journals

Heat shock represses rRNA synthesis by inactivation of TIF-IA and lncRNA-dependent changes in nucleosome positioning

чт, 2016-09-29 11:06

Attenuation of ribosome biogenesis in suboptimal growth environments is crucial for cellular homeostasis and genetic integrity. Here, we show that shutdown of rRNA synthesis in response to elevated temperature is brought about by mechanisms that target both the RNA polymerase I (Pol I) transcription machinery and the epigenetic signature of the rDNA promoter. Upon heat shock, the basal transcription factor TIF-IA is inactivated by inhibition of CK2-dependent phosphorylations at Ser170/172. Attenuation of pre-rRNA synthesis in response to heat stress is accompanied by upregulation of PAPAS, a long non-coding RNA (lncRNA) that is transcribed in antisense orientation to pre-rRNA. PAPAS interacts with CHD4, the adenosine triphosphatase subunit of NuRD, leading to deacetylation of histones and movement of the promoter-bound nucleosome into a position that is refractory to transcription initiation. The results exemplify how stress-induced inactivation of TIF-IA and lncRNA-dependent changes of chromatin structure ensure repression of rRNA synthesis in response to thermo-stress.

Категории: Bioinformatics, Biology, Journals

FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function

чт, 2016-09-29 11:06

Forkhead box A1 (FOXA1) is an FKHD family protein that plays pioneering roles in lineage-specific enhancer activation and gene transcription. Through genome-wide location analyses, here we show that FOXA1 expression and occupancy are, in turn, required for the maintenance of these epigenetic signatures, namely DNA hypomethylation and histone 3 lysine 4 methylation. Mechanistically, this involves TET1, a 5-methylcytosine dioxygenase. We found that FOXA1 induces TET1 expression via direct binding to its cis-regulatory elements. Further, FOXA1 physically interacts with the TET1 protein through its CXXC domain. TET1 thus co-occupies FOXA1-dependent enhancers and mediates local DNA demethylation and concomitant histone 3 lysine 4 methylation, further potentiating FOXA1 recruitment. Consequently, FOXA1 binding events are markedly reduced following TET1 depletion. Together, our results suggest that FOXA1 is not only able to recognize but also remodel the epigenetic signatures at lineage-specific enhancers, which is mediated, at least in part, by a feed-forward regulatory loop between FOXA1 and TET1.

Категории: Bioinformatics, Biology, Journals

ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells

чт, 2016-09-29 11:06

ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.

Категории: Bioinformatics, Biology, Journals

Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex

чт, 2016-09-29 11:06

We have purified a minimal core human Ino80 complex from recombinant protein expressed in insect cells. The complex comprises one subunit each of an N-terminally truncated Ino80, actin, Arp4, Arp5, Arp8, Ies2 and Ies6, together with a single heterohexamer of the Tip49a and Tip49b proteins. This core complex has nucleosome sliding activity that is similar to that of endogenous human and yeast Ino80 complexes and is also inhibited by inositol hexaphosphate (IP6). We show that IP6 is a non-competitive inhibitor that acts by blocking the stimulatory effect of nucleosomes on the ATPase activity. The IP6 binding site is located within the C-terminal region of the Ino80 subunit. We have also prepared complexes lacking combinations of Ies2 and Arp5/Ies6 subunits that reveal regulation imposed by each of them individually and synergistically that couples ATP hydrolysis to nucleosome sliding. This coupling between Ies2 and Arp5/Ies6 can be overcome in a bypass mutation of the Arp5 subunit that is active in the absence of Ies2. These studies reveal several underlying mechanisms for regulation of ATPase activity involving a complex interplay between these protein subunits and IP6 that in turn controls nucleosome sliding.

Категории: Bioinformatics, Biology, Journals