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Results for “Molecular Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Longitudinal plasma metabolomics of aging and sex

Understanding how metabolites are longitudinally influenced by age and sex could facilitate the identification of metabolomic profiles and trajectories that indicate disease risk. We investigated the metabolomics of age and sex using longitudinal plasma samples from the Wisconsin Registry for Alzheimers Prevention (WRAP), a cohort of participants who were dementia free at enrollment. Metabolomic profiles were quantified for 2,316 fasting plasma samples among 1,187 participants, each with up to three study visits. Of 1,097 metabolites tested, 608 (55.4%) were associated with age and 680 (62.0%) with sex after correcting for multiple testing. Approximately twice as many metabolites were associated with age in stratified analyses of women versus men, and 63 metabolite trajectories significantly differed by sex, most notably including sphingolipids, which tended to increase in women and decrease in men with age. Using genome-wide genotyping, we also report the heritabilities of metabolites investigated, which ranged dramatically (0.2-99.2%); however, the median heritability of 36.2% suggests that many metabolites are highly influenced by a complex combination of genomic and environmental influences. These findings offer a more profound description of the aging process and may inform many new hypotheses regarding the role metabolites play in healthy and accelerated aging.

molecular biology↗

bioRxiv2018

Altered chromatin localization of hybrid lethality proteins in Drosophila

Understanding hybrid incompatibilities is a fundamental pursuit in evolutionary genetics. In crosses between Drosophila melanogaster females and Drosophila simulans males, the interaction of at least three genes is necessary for hybrid male lethality: Hmr mel, Lhr sim, and gfzf sim. All three hybrid incompatibility genes are chromatin associated factors. While HMR and LHR physically bind each other and function together in a single complex, the connection between either of these proteins and gfzf remains mysterious. Here, we investigate the allele specific chromatin binding patterns of gfzf. First, our cytological analyses show that there is little difference in protein localization of GFZF between the two species except at telomeric sequences. In particular, GFZF binds the telomeric retrotransposon repeat arrays, and the differential binding of GFZF at telomeres reflects the rapid changes in sequence composition at telomeres between D. melanogaster and D. simulans. Second, we investigate the patterns of GFZF and HMR co-localization and find that the two proteins do not normally co-localize in D. melanogaster. However, in inter-species hybrids, HMR shows extensive mis-localization to GFZF sites, and this altered localization requires the presence of gfzf sim. Third, we find by ChIP-Seq that over-expression of HMR and LHR within species is sufficient to cause HMR to mis-localize to GFZF binding sites, indicating that HMR has a natural low affinity for GFZF sites. Together, these studies provide the first insights into the different properties of gfzf between D. melanogaster and D. simulans as well as a molecular interaction between gfzf and Hmr in the form of altered protein localization.

molecular biology↗

bioRxiv2018

HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

The tumor suppressor BRCA2 is essential for homologous recombination, replication fork stability and DNA interstrand crosslink (ICL) repair in vertebrates. We show that a functionally uncharacterized protein, HSF2BP, is involved in a novel, direct and highly evolutionarily conserved interaction with BRCA2. Although HSF2BP was previously described as testis-specific, we find it is expressed in mouse ES cells, in human cancer cell lines, and in tumor samples. Elevated levels of HSF2BP sensitize human cells to ICL-inducing agents (mitomycin C and cisplatin) and PARP inhibitors, resulting in a phenotype characteristic of cells from Fanconi anemia (FA) patients. We biochemically recapitulate the suppression of ICL repair and establish that excess HSF2BP specifically compromises homologous recombination by preventing BRCA2 and RAD51 loading at the ICL. As increased ectopic expression of HSF2BP occurs naturally, we suggest that it can be considered as a causative agent in FA and a source of cancer-promoting genomic instability.

molecular biology↗

bioRxiv2018

AMPK Signaling Regulates Expression of Urea Cycle Enzymes in Response to Changes in Dietary Protein Intake

Abundance of urea cycle enzymes in the liver is regulated by the dietary protein intake. Although urea cycle enzyme levels rise in response to a high protein diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either control or high (HP) protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptome profiles. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways, and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated AMPK as a possible regulator. Primary human hepatocytes, treated with AICAR an activator of AMPK, were used to test whether AMPK regulates expression of urea cycle genes. The abundance of CPS1 and OTC mRNA increased in hepatocytes treated with AICAR, which supports a role for AMPK signaling in regulation of the urea cycle. Because AMPK is either a target of drugs used to treat type-2 diabetes, these drugs might increase the expression of urea cycle enzymes in patients with urea cycle disorders, which could be the basis of a new therapeutic approach.\n\nAuthor summaryIntegrated analysis of transcriptional and proteomic profiles of the liver tissue from mice fed different protein content diets revealed that AMPK signaling pathway regulates expression of urea cycle enzymes.

molecular biology↗

bioRxiv2018

Single step, high efficiency CRISPR-Cas9 genome editing in primary human disease-derived fibroblasts

Genome editing is a tool that has many applications including the validation of potential drug targets. However, performing genome editing in low passage, primary human cells with the greatest physiological relevance, is notoriously difficult. High editing efficiency is desired because it enables gene knock outs (KO) to be generated in bulk cellular populations and circumvents the problem of having to generate clonal cell isolates. Here, we describe a single step workflow enabling >90% KO generation in primary human lung fibroblasts via CRISPR ribonucleoprotein delivery, in the absence of antibiotic selection or clonal expansion. As proof of concept, we performed a disease relevant phenotypic assay measuring collagen deposition in response to TGF{beta} and demonstrated SMAD3 but not SMAD2 dependent deposition of type I collagen following knockout of each using our single step methodology. The optimization of this workflow can readily be transferred to other primary cell types.

molecular biology↗

bioRxiv2018

Miniaturization and optimization of 384-well compatible metagenomic sequencing library preparation

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.

molecular biology↗

bioRxiv2018

Bromodomain Protein, BRD4, Contributes to the Regulation of Alternative Splicing

Bromodomain protein 4 (BRD4) is an atypical kinase and a histone acetyl transferase (HAT) which plays an important role in chromatin remodeling and early transcriptional elongation. During transcription elongation, BRD4 travels with the elongation complex. Since most of the alternative splicing events take place co-transcriptionally, we asked if BRD4 plays a role in regulation of alternative splicing. We find that distinct patterns of alternative splicing are associated with conditional deletion of BRD4 during thymocyte differentiation in vivo. Similarly, depletion of BRD4 in T-ALL cells alters patterns of splicing. Most of the alternatively spliced events affected by BRD4 are usage of exon skipping. In an established insulin receptor minigene model of splicing, BRD4 over expression modulates alternative splicing. Importantly, as assessed by both immunoprecipitation (IP) and proximity ligation (PLA) assays, BRD4 interacts with components of the splicing machinery. BRD4 also co-localizes on chromatin with one of the splicing regulators. We propose that BRD4 contributes to patterns of alternative splicing through its interaction with the splicing machinery during transcription elongation.\n\nSignificance StatementThe bromodomain protein, BRD4, is a transcriptional and epigenetic regulator that plays a critical role in both cancer and inflammation. It has pleiotropic activities, including chromatin organization, transcriptional pause release and initiation. We now report that it also contributes to the regulation of alternative splicing. Taken together, these findings indicate that BRD4 functions to coordinate the various steps in gene expression.

molecular biology↗

bioRxiv2018

The detection of novel and resident marine non-indigenous species using environmental DNA metabarcoding of seawater and sediment

Environmental DNA (eDNA) surveys are increasingly being used for biodiversity monitoring, principally because they are sensitive and can provide high resolution community composition data. Despite considerable progress in recent years, eDNA studies examining how different environmental sample types can affect species detectability remain rare. Comparisons of environmental samples are especially important for providing best practice guidance on early detection and subsequent mitigation of non-indigenous species. Here we used eDNA metabarcoding of COI (cytochrome c oxidase subunit I) and 18S (nuclear small subunit ribosomal DNA) genes to compare community composition between sediment and water samples in artificial coastal sites across the United Kingdom. We first detected markedly different communities and a consistently greater number of distinct operational taxonomic units in sediment compared to water. We then compared our eDNA datasets with previously published rapid assessment biodiversity surveys and found excellent concordance among the different survey techniques. Finally, our eDNA surveys detected many non-indigenous species, including several newly introduced species, highlighting the utility of eDNA metabarcoding for both early detection and temporal / spatial monitoring of non-indigenous species. We conclude that careful consideration on environmental sample type is needed when conducting eDNA surveys, especially for studies assessing community change.

molecular biology↗

bioRxiv2018

The ghr-miR164 and GhNAC100 module participates in cotton plant defence against Verticillium dahliae

Previous reports have shown that many miRNAs were identified at the early induction stage during which Verticillium dahliae localizes at the root surface. In this study, we constructed two sRNA libraries of cotton root responses to this fungus at the later induction stage when the pathogen enters the root vascular tissue. We identified 71 known miRNAs and 378 novel miRNAs from two pathogen-induced sRNAs and the control libraries. Combined with degradome and sRNA sequencing, 178 corresponding miRNA target genes were identified, in which 40 target genes from differentially expressed miRNAs were primarily associated with oxidation-reduction and stress responses. More importantly, we characterized the ghr-miR164-GhNAC100 module in the response of the plant to V dahliae infection. A GUS fusion reporter showed that ghr-miR164 directly cleaved the mRNA of GhNAC100 in the post-transcriptional process. ghr-miR164-silencing increased the resistance of the plant to this fungus, while the knockdown of GhNAC100 elevated the susceptibility of the plant, indicating that ghr-miR164-GhNAC100 modulates plant defence through the post-transcriptional regulation. Our data documented that there are numerous miRNAs at the later induction stage that participate in the plant response to V. dahliae, suggesting that miRNAs play important roles in plant resistance to vascular disease.\n\nHighlightAccording to degradome and sRNA sequencings of cotton root in responses to Verticillium dahliae at the later induction stage, many miRNAs and corresponding targets including ghr-miR164-GhNAC100 module participate plant defence.

molecular biology↗

bioRxiv2018

Dissociation rate compensation mechanism for budding yeast pioneer transcription factors

Nucleosomes restrict the occupancy of most transcription factors (TF) by reducing binding and accelerating dissociation, while a small group of TFs have high affinities to nucleosome-embedded sites and facilitate nucleosome displacement. To mechanistically understand this process, we investigated two S. cerevisiae TFs, Reb1 and Cbf1. We show these factors bind their sites within nucleosomes with similar affinities to naked DNA, trapping a partially unwrapped nucleosome without histone eviction. Both the binding and dissociation rates of Reb1 and Cbf1 are significantly slower at the nucleosomal sites relative to DNA, demonstrating that the high affinities are achieved by increasing the dwell time on nucleosomes to compensate for reduced binding. Reb1 also shows slow migration rate in the yeast nuclei. These properties are similar to human pioneer factors (PFs), suggesting the mechanism of nucleosome targeting is conserved from yeast to human.

molecular biology↗

bioRxiv2018

Dynamic enhancer partitioning instructs activation of a growth regulator during exit from naïve pluripotency

During early mammalian development, the genome undergoes profound transitions in chromatin states, topological organization and recruitment of cis regulatory factors involved in transcriptional control. How these three layers of gene regulation interact is the matter of intense research. The Zdbf2 gene--which is involved in growth control--provides a valuable model to study this question: upon exit from naive pluripotency and prior to tissue differentiation, it undergoes a switch in usage from a distal to a proximal promoter, along with a switch in chromatin states, from polycomb to DNA methylation occupancy. Using an embryonic stem cell (ESC) culture system to mimic this period, we show here that four enhancers contribute to the Zdbf2 promoter switch, concomitantly with dynamic changes in chromosome architecture. Indeed, CTCF plays a key role in partitioning the locus in ESCs, to facilitate enhancer contact with the distal Zdbf2 promoter only. Partition relieving enhances proximal Zdbf2 promoter activity, as observed during differentiation or with mutants that lack local CTCF-based partition. Importantly, we show that CTCF-based regulation occurs independently of the polycomb and DNA methylation pathways. Our study reveals the importance of multi-layered regulatory frameworks to ensure proper spatio-temporal activation of developmentally important genes.

molecular biology↗

bioRxiv2018

The mRNA export adaptor Yra1 contributes to DNA double-strand break repair through its C-box domain

Yra1 is an mRNA export adaptor involved in mRNA biogenesis and export in S. cerevisiae. Yra1 overexpression was recently shown to promote accumulation of DNA:RNA hybrids favoring DNA double strand breaks (DSB), cell senescence and telomere shortening, via an unknown mechanism. Yra1 was also identified at an HO-induced DSB and Yra1 depletion causes defects in DSB repair. Previous work from our laboratory showed that Yra1 ubiquitination by Tom1 is important for mRNA export. Interestingly, we found that Yra1 is also ubiquitinated by the SUMO-targeted ubiquitin ligases Slx5-Slx8 implicated in the interaction of irreparable DSB with nuclear pores. Here we show that Yra1 binds an HO-induced irreparable DSB. Importantly, a Yra1 mutant lacking the evolutionarily conserved C-box is not recruited to an HO-induced irreparable DSB and becomes lethal under DSB induction in a HO-cut reparable system. Together, the data provide evidence that Yra1 plays a crucial role in DSB repair via homologous recombination. Unexpectedly, while the Yra1 C-box is essential, Yra1 sumoylation and/or ubiquitination are dispensable in this process.

molecular biology↗

bioRxiv2018

Principles of Meiotic Chromosome Assembly

During meiotic prophase, chromosomes organise into a series of chromatin loops emanating from a proteinaceous axis, but the mechanisms of assembly remain unclear. Here we elucidate how this elaborate three-dimensional chromosome organisation is underpinned by genomic sequence in Saccharomyces cerevisiae. Entering meiosis, strong cohesin-dependent grid-like Hi-C interaction patterns emerge, reminiscent of mammalian interphase organisation, but with distinct regulation. Meiotic patterns agree with simulations of loop extrusion limited by barriers, yet are patterned by convergent transcription rather than binding of the mammalian interphase factor, CTCF, which is absent in S. cerevisiae--thereby both challenging and extending current paradigms of local chromosome organisation. While grid-like interactions emerge independently of meiotic chromosome synapsis, synapsis itself generates additional compaction that matures differentially according to telomere proximity and chromosome size. Collectively, our results elucidate fundamental principles of chromosome assembly and demonstrate the essential role of cohesin within this evolutionarily conserved process.

molecular biology↗

bioRxiv2018

Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins

Post-translational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate the transcription cycle. Crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac)--only found in higher eukaryotes--regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. We identified the regulator of pre-mRNA domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced CTD peptide binding to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins in isothermal calorimetry and molecular modeling experiments. Deacetylase inhibitors increased K7ac- and decreased S5-phosphorylated polymerases, consistent with acetylation-dependent S5 dephosphorylation by an RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p, but enhanced K7ac, indicating RPRD proteins recruit K7 deacetylases, including HDAC1. We also report auto-regulatory crosstalk between K7ac and S5p via RPRD proteins and their interactions with acetyl- and phospho-eraser proteins.

molecular biology↗

bioRxiv2018

Characterisation of the faecal virome of captive and wild Tasmanian devils using virus-like particles metagenomics and meta-transcriptomics

BackgroundThe Tasmanian devil is an endangered carnivorous marsupial threatened by devil facial tumour disease (DFTD). While research on DFTD has been extensive, little is known about the viruses present in devils, and whether any of these are of potential conservation relevance for this endangered species.\n\nMethodsUsing both metagenomics based on virus-like particle (VLP) enrichment and sequence-independent amplification (VLP metagenomics), and meta-transcriptomics based on bulk RNA sequencing, we characterised and compared the faecal viromes of captive and wild Tasmanian devils.\n\nResultsA total of 54 devil faecal samples collected from captive (n = 2) and wild (n = 4) populations were processed for virome characterisation using both approaches. We detected many novel, highly divergent viruses, including vertebrate viruses, bacteriophage and other dietary associated plant and insect viruses. In total, 18 new vertebrate viruses, including novel sapelovirus, astroviruses, bocaviruses, papillomaviruses and gammaherpesvirus were identified, as well as known mammalian pathogens including rabbit haemorrhagic disease virus 2 (RHDV2). Captive devils showed significantly lower levels of viral diversity than wild devils. Comparison of the two methodological approaches revealed substantial differences in the number and types of viruses detected, with meta-transcriptomics mainly identifying RNA viruses, and VLP metagenomics largely identifying DNA viruses.\n\nConclusionThis study has greatly expanded our knowledge of eukaryotic viruses in the Tasmanian devil and provides important baseline information that will contribute to the conservation and captive management of this endangered species. In addition, our results showed that a combination of VLP metagenomics and meta-transcriptomics may be a more comprehensive approach to virome characterisation than either method alone.

molecular biology↗

bioRxiv2018

Bud13 Promotes a Type I Interferon Response By Countering Intron Retention in Irf7

Intron retention (IR) has emerged as an important mechanism of gene expression control. Despite this, the factors that control IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified Bud13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in Bud13 led to increased IR, decreased mature Irf7 transcript and protein levels, and consequently to a dampened type I interferon response. This impairment of Irf7 production in Bud13-deficient cells compromised their ability to withstand VSV infection. Global analysis of Bud13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a Bud13-dependent manner. Deficiency of Bud13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, Bud13 facilitates the expression of genes at which IR occurs.

molecular biology↗

bioRxiv2018

Proteotoxic stress-induced Nrf1 transcriptional program requires a functional TIP60 complex

In response to inhibition of the cellular proteasome, the transcription factor Nrf1 (also called NFE2L1) induces transcription of proteasome subunit genes resulting in the restoration of proteasome activity and thus enabling the cells to mitigate the proteotoxic stress. To identify novel regulators of Nrf1, we performed an RNA interference screen and discovered that the AAA+ ATPase RUVBL1 is necessary for its transcriptional activity. Given that RUVBL1 is part of different multi-subunit complexes that play key roles in transcription, we dissected this phenomenon further and found that the TIP60 chromatin regulatory complex is essential for Nrf1-dependent transcription of proteasome genes. Consistent with these observations, Nrf1, RUVBL1, and TIP60 proteins were co-recruited to the promoter regions of proteasome genes after proteasome inhibitor treatments. More importantly, depletion of RUVBL1 or TIP60 in various cancer cells sensitized them to cell death induced by proteasome inhibition. Our study provides a framework for manipulating the Nrf1-TIP60 axis to alter proteasome function in various human diseases including cancer.

molecular biology↗

bioRxiv2018

TADs pair homologous chromosomes to promote interchromosomal gene regulation

Homologous chromosomes colocalize to regulate gene expression in processes including genomic imprinting and X-inactivation, but the mechanisms driving these interactions are poorly understood. In Drosophila, homologous chromosomes pair throughout development, promoting an interchromosomal gene regulatory mechanism called transvection. Despite over a century of study, the molecular features that facilitate chromosome-wide pairing are unknown. The \"button\" model of pairing proposes that specific regions along chromosomes pair with a higher affinity than their surrounding regions, but only a handful of DNA elements that drive homologous pairing between chromosomes have been described. Here, we identify button loci interspersed across the fly genome that have the ability to pair with their homologous sequences. Buttons are characterized by topologically associated domains (TADs), which drive pairing with their endogenous loci from multiple locations in the genome. Fragments of TADs do not pair, suggesting a model in which combinations of elements interspersed along the full length of a TAD are required for pairing. Though DNA-binding insulator proteins are not associated with pairing, buttons are enriched for insulator cofactors, suggesting that these proteins may mediate higher order interactions between homologous TADs. Using a TAD spanning the spinelessd gene as a paradigm, we find that pairing is necessary but not sufficient for transvection. spineless pairing and transvection are cell-type-specific, suggesting that local buttoning and unbuttoning regulates transvection efficiency between cell types. Together, our data support a model in which specialized TADs button homologous chromosomes together to facilitate cell-type-specific interchromosomal gene regulation.

molecular biology↗