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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 307 records · Page 17Linked to original sources

bioRxiv2016

Transcriptional activity of TRF2 is telomere length dependent

TRF2 is a telomere repeat binding factor crucial for telomere maintenance and genome stability. An emerging non-conventional role of TRF2 is as a transcriptional regulator through extra-telomeric bindings. Herein we report that increase in telomere length leads to sequestration of TRF2 at the telomeres leading to reduced extra-telomeric TRF2 occupancy genome wide. Decrease in TRF2 occupancy was found on multiple gene promoters in cells with elongated telomeres, including the cell cycle regulator kinase-p21. We found that TRF2 is a transcriptional repressor of p21, and, interestingly, TRF2-mediated regulatory control of p21 is telomere length dependent.

Molecular Biology↗

bioRxiv2016

Genome-wide specificity profiles of CRISPR-Cas Cpf1 nucleases in human cells

CRISPR-Cas Cpf1 nucleases have recently been described as an alternative genome-editing platform1, yet their activities and genome-wide specificities remain largely undefined. Here we show that two Cpf1 nucleases function robustly in human cells with on-target efficiencies comparable to those of the widely used Streptococcus pyogenes Cas9 (SpCas9)2-5. We also demonstrate that four to six bases at the 3 end of the short CRISPR RNA (crRNA) used to program Cpf1 are insensitive to single base mismatches but that many of the other bases within the crRNA targeting region are highly sensitive to single or double substitutions. Consistent with these results, GUIDE-seq and targeted deep sequencing analyses of two Cpf1 nucleases revealed no detectable off-target cleavage for over half of 20 different crRNAs we examined. Our results suggest that the two Cpf1 nucleases we characterized gen ...

Molecular Biology↗

bioRxiv2016

Diversity of Sericostomatidae (Trichoptera) caddisflies in the Montseny mountain range

Biodiversity is under threat by the ongoing global change and especially freshwater ecosystems are under threat by intensified land use, water abstraction and other anthropogenic stressors. In order to monitor the impacts that stressors have on freshwater biodiversity, it is important to know the current state of ecosystems and species living in them. This is often hampered by lacking knowledge on species and genetic diversity due to the fact that many taxa are complexes of several morphologically cryptic species. Lacking knowledge on species identity and ecology can lead to wrong biodiversity and stream quality assessments and molecular tools can greatly help resolving this problem. Here, we studied larvae of the caddisfly family Sericostomatidae in the Montseny mountains on the Iberian Peninsula. We expected to find cryptic species and that species would not occur in syntopy due to different ecological niches. We sampled 44 stream sites and sequenced 247 larval specimens for the barcoding region of the mitochondrial cytochrome c oxidase gene. A modeling approach was used to assess the bioclimatic preferences of the found species. Two molecular groups were identified. One could be assigned to Schizopelex furcifera and one to Sericostoma spp. We did find both taxa in syntopy in >50% of sampling sites and could show that the taxa prefer similar bioclimatic conditions. A reexamination of larval specimens showed that Sericostoma and Schizopelex larvae could not be unambiguously identified to the genus level. Overall, our results show the importance of including molecular tools into biodiversity assessments in order to correctly identify the species diversity of a region and to prevent wrong assessment results.

Molecular Biology↗

bioRxiv2016

Numerical and Spatial Patterning of Yeast Meiotic DNA Breaks by Tel1

The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and distribution. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to examine the contribution of the DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM) to this regulation in Saccharomyces cerevisiae. A tel1{Delta} mutant had globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tell mutation also increased Spo11-oligonucleotide levels, but mutating known Tel1 phosphotargets on Hop1 and Rec114 did not. Deep sequencing of Spo11 oligonucleotides from tel1{Delta} mutants demonstrated that Tel1 shapes the nonrandom DSB distribution in ways that are distinct but partially overlapping with previously described contributions of the recombination regulator Zip3. Finally, we uncover a context-dependent role for Tel1 in hotspot competition, in which an artificial DSB hotspot inhibits nearby hotspots. Evidence for Tel1-dependent competition involving strong natural hotspots is also provided.

Molecular Biology↗

bioRxiv2016

Protocol: Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial immune system CRISPR (clustered regularly interspaced short palindromic repeats) has been adapted for genome-scale screening by combining Cas9 with guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom-or ready-made guide RNA libraries are constructed and packaged into lentivirus for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified from the screen, we further describe strategies for confirming the screening phenotype as well as genetic perturbation through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 6-10 weeks followed by 3-4 weeks of validation.

Molecular Biology↗

bioRxiv2016

A genetically encoded inhibitor of 53BP1 to stimulate homology-based gene editing

The expanding repertoire of programmable nucleases such as Cas9 brings new opportunities in genetic medicine1-3. In many cases, these nucleases are engineered to induce a DNA double-strand break (DSB) to stimulate precise genome editing by homologous recombination (HR). However, HR efficiency is nearly always hindered by competing DSB repair pathways such as non-homologous end-joining (NHEJ). HR is also profoundly suppressed in non-replicating cells, thus precluding the use of homology-based genome engineering in a wide variety4 of cell types. Here, we report the development of a genetically encoded inhibitor of 53BP1 (known as TP53BP1), a regulator of DSB repair pathway choice5. 53BP1 promotes NHEJ over HR by suppressing end resection, the formation of 3-prime single-stranded DNA tails, which is the rate-limiting step in HR initiation. 53BP1 also blocks the recruitment of the HR factor BRCA1 to DSB sites in G1 cells4,6. The inhibitor of 53BP1 (or i53) was identified through the screening of a massive combinatorial library of engineered ubiquitin variants by phage display7. i53 binds and occludes the ligand binding site of the 53BP1 Tudor domain with high affinity and selectivity, blocking its ability to accumulate at sites of DNA damage. i53 is a potent selective inhibitor of 53BP1 and enhances gene targeting and chromosomal gene conversion, two HR-dependent reactions. Finally, i53 can also activate HR in G1 cells when combined with the activation of end-resection and KEAP1 inhibition. We conclude that 53BP1 inhibition is a robust tool to enhance precise genome editing by canonical HR pathways.

Molecular Biology↗

bioRxiv2016

Quantum Transition in Zinc Atoms of Flavavirus Polymerase Triggers the Conformation of the Polymerase Motif F

Flavavirus RdRp contains two zinc atoms located in two specific zinc binding sites in the protein. Molecular dynamics experiments with flavavirus RdRp suggest that the conformation of the conservative motif F in the polymerase is highly sensitive to the bond lengths between the zinc atoms and their four coordinating atoms. In the experimental structures of flavavirus RdRp, motif F is presented in two different conformations. The polymerase acts as a catalyst only in one of the motif F conformations. We hypothesize that the second conformation is required for the formation of the viral replicative complex and that it is the quantum transition in the zinc atoms which slightly changes the bond lengths; thus assisting the switch between the two motif F conformations.

Molecular Biology↗

bioRxiv2016

Functional genetic characterization by CRISPR-Cas9 of two enhancers of FOXP2 in a child with speech and language impairment

Mutations in the coding region of FOXP2 are known to cause speech and language impairment. Microdeletions involving the region downstream the gene have been also associated to speech and cognitive deficits. We recently described a girl harbouring a complex chromosomal rearrangement with one breakpoint downstream the gene that might affect her speech and cognitive abilities via physical separation of distant regulatory DNA elements. In this study, we have used highly efficient targeted chromosomal deletions induced by the CRISPR/Cas9 genome editing tool to demonstrate the functionality of two enhancers, FOXP2-Eproximal and FOXP2-Edistal, located in the intergenic region between FOXP2 and its adjacent MDFIC gene. Deletion of any of these two functional enhancers in the neuroblastomic cell line SK-N-MC downregulates FOXP2 and decreases FOXP2 protein levels, conversely it upregulates MDFIC and increases MDFIC protein levels. This suggests that both regulatory elements may be shared between FOXP2 and MDFIC. We expect these findings contribute to a deeper understanding of how FOXP2 and MDFIC are regulated to pace neuronal development supporting speech and language.

Molecular Biology↗

bioRxiv2016

The fail-safe mechanism of post-transcriptional silencing of unspliced HAC1 mRNA

HAC1 encodes a transcription factor that is the central effector of the unfolded protein response (UPR) in budding yeast. When the UPR is inactive, HAC1 mRNA is stored as an unspliced isoform in the cytoplasm and no Hac1 protein is detectable. Intron removal is both necessary and sufficient to relieve the post-transcriptional silencing of HAC1 mRNA, yet the precise mechanism by which the intron prevents Hac1 protein accumulation has remained elusive. Here, we show that a combination of inhibited translation initiation and accelerated protein degradation--both dependent on the intron--prevents the accumulation of Hac1 protein when the UPR is inactive. Functionally, both components of this fail-safe silencing mechanism are required to prevent ectopic production of Hac1 protein and concomitant activation of the UPR. Our results provide a mechanistic understanding of HAC1 regulation and reveal a novel strategy for complete post-transcriptional silencing of a cytoplasmic mRNA.

Molecular Biology↗

bioRxiv2016

A Dynamic Mode of Mitotic Bookmarking by Transcription Factors

During mitosis, transcription is shut off, chromatin condenses, and most transcription factors (TFs) are reported to be excluded from chromosomes. How do daughter cells re-establish the original transcription program? Recent discoveries that a select set of TFs remain bound on mitotic chromosomes suggest a potential mechanism for maintaining transcriptional programs through the cell cycle termed mitotic bookmarking. Here we report instead that many TFs remain associated with chromosomes, and that the exclusion previously described is largely a fixation artifact. In particular, most TFs we tested are significantly enriched on mitotic chromosomes. Studies with Sox2 reveal that this mitotic interaction is more dynamic than in interphase and requires both DNA binding and nuclear import. Furthermore, this dynamic mode results from lack of transcriptional activation rather than decreased accessibility of underlying DNA sequences in mitosis. The nature of the cross-linking artifact prompts careful re-examination of the role of TFs in mitotic bookmarking.\n\nHighlightsO_LIMany transcription factors bind to mitotic chromosomes\nC_LIO_LISox2 mitotic interaction is dynamic and requires DNA binding and nuclear import\nC_LIO_LIDNA remains highly accessible in mitotic chromosomes\nC_LIO_LIFormaldehyde-based cross-linking leads to mis-localization of TFs\nC_LI

Molecular Biology↗

bioRxiv2016

A global view of meiotic double-strand break end resection

The DNA double-strand breaks that initiate homologous recombination during meiosis are subject to extensive 5'[->]3' exonucleolytic processing. This resection is a central and conserved feature of recombination, yet its mechanism is poorly understood. Using a purpose-made deep-sequencing method, we mapped meiotic resection endpoints genome-wide at high spatial resolution in Saccharomyces cerevisiae. Generating full-length resection tracts requires Exo1 exonuclease activity and the DNA-damage responsive kinase Tel1, but not the helicase Sgs1. Tel1 is also required for efficient and timely initiation of resection. We find that distributions of resection endpoints at individual genomic loci display pronounced heterogeneity that reflects a tendency for nucleosomes to block Exo1 in vivo, yet modeling experiments indicate that Exo1 digests chromatin with high apparent processivity and at rates approaching those for naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a determining feature of the meiotic resection landscape.

Molecular Biology↗

bioRxiv2016

Removing bias against short sequences enables northern blotting to better complement RNA-seq for the study of small RNAs

Changes in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that northern blotting of small RNAs is biased against short sequences, and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm C. elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq. When using northern blotting during these studies, we discovered that miRNA-length probes can have a ~360-fold bias against detecting even synthetic sequences that are 8 nt shorter. By using shorter probes and by performing hybridization and washes at low temperatures, we greatly reduced this bias to enable equivalent detection of 24 nt to 14 nt RNAs. Our protocol can better discriminate RNAs that differ by a single nucleotide and can detect specific miRNAs present in total RNA from C. elegans. This improved northern blotting is particularly useful to obtain a measure of small RNA integrity, analyze products of RNA processing or turnover, and analyze functional RNAs that are shorter than typical miRNAs.

Molecular Biology↗

bioRxiv2016

High-resolution phenotypic landscape of the RNA Polymerase II trigger loop

The active site of multicellular RNA polymerases have a \"trigger loop\" (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three major mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.

Molecular Biology↗

bioRxiv2016

RNase H enables efficient repair of R-loop induced DNA damage

R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in budding yeast, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair. Our results indicate that persistent R-loops in the rDNA induce damage that is slowly repaired by break-induced replication (BIR). Furthermore, R-loop induced BIR at the rDNA leads to lethal repair intermediates when RNA polymerase I elongation is compromised. We present a model to explain how removal of R-loops by RNase H is critical in ensuring the efficient repair of R-loop induced DNA damage by pathways other than BIR.

Molecular Biology↗

bioRxiv2016

ChIP-exo interrogation of Crp, DNA, and RNAP holoenzyme interactions

Numerous in vitro studies have yielded a refined picture of the structural and molecular associations between Cyclic-AMP receptor protein (Crp), the DNA motif, and RNA polymerase (RNAP) holoenzyme. In this study, high-resolution ChIP-exonuclease (ChIP-exo) was applied to study Crp binding in vivo and at genome-scale. Surprisingly, Crp was found to provide little to no protection of the DNA motif under activating conditions. Instead, Crp demonstrated binding patterns that closely resembled those generated by {sigma}70. The binding patterns of both Crp and {sigma}70 are indicative of RNAP holoenzyme DNA footprinting profiles associated with stages during transcription initiation that occur post-recruitment. This is marked by a pronounced advancement of the template strand footprint profile to the +20 position relative to the transcription start site and a multimodal distribution on the nontemplate strand. This trend was also observed in the familial transcription factor, Fnr, but full protection of the motif was seen in the repressor ArcA. Given the time-scale of ChIP studies and that the rate-limiting step in transcription initiation is typically post recruitment, we propose a hypothesis where Crp is absent from the DNA motif but remains associated with RNAP holoenzyme post-recruitment during transcription initiation. The release of Crp from the DNA motif may be a result of energetic changes that occur as RNAP holoenzyme traverses the various stable intermediates towards elongation complex formation.

Molecular Biology↗

bioRxiv2016

ARGLU1 is a Glucocorticoid Receptor Coactivator and Splicing Modulator Important in Stress Hormone Signaling and Brain Development

Prolonged exposure to glucocorticoid stress hormones precipitates mood and cognitive disorders. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies found that the glutamate rich C-terminus coactivates the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and RNA. RNA-seq of neuronal cells {+/-}siARGLU1found significant changes in the expression and alternative splicing of distinct genes involved in neurogenesis. Loss of ARGLU1 was embryonic lethal in mice, and knockdown in zebrafish caused neurodevelopmental and heart defects. Treatment with dexamethasone, a GR activator, also induced changes in the pattern of alternatively spliced genes, highlighting an underappreciated global mechanism of glucocorticoid action in neuronal cells. Thus, in addition to its basal role, ARGLU1 links glucocorticoid-mediated transcription and alternative splicing in neural cells, providing new avenues from which to investigate the molecular underpinnings of cognitive stress disorders.

Molecular Biology↗

bioRxiv2016

Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament

In this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3 polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3 polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3 polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3 polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

Molecular Biology↗

bioRxiv2016

Fast Antibiotic Susceptibility Testing (FASTest) based on single cell growth rate measurements.

The emergence and spread of antibiotic resistant bacteria is a global threat to human health. The problem is aggravated by unnecessary and incorrect use of broad-spectrum antibiotics. One way to provide correct treatment and to slow down the development of antibiotic resistance is to assay the susceptibility profile of the infecting bacteria before treatment is initiated and let this information guide the choice of antibiotic. However, current methods for Antibiotics Susceptibility Testing (AST) are too slow for point of care application. Here we present a fast AST, fASTest, that rapidly captures individual bacterial cells in nanofluidics channels and monitors their response to different antibiotics based on direct imaging. By averaging the growth rate over many cells, we determined the susceptibility to several antibiotics in less than 25 min even at cell densities as low as 104 CFU/mL. The short time scale, high sensitivity and high specificity make the method practically useful for guiding antibiotic treatment in, for example, urinary tract infections.\n\nOne Sentence SummaryIndividual bacterial cells can be captured and imaged in a microfluidic device to determine how their growth rate responds to antibiotic treatment in a few minutes.

Molecular Biology↗