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bioRxiv2017

Modest static pressure suppresses columnar epithelial cell proliferation in association with cell shape and cytoskeletal modifications

Intraluminal pressure elevation can cause degenerative disorders, such as ileus and hydronephrosis, and the threshold is fairly low and constant, 20-30 cm H2O. We previously devised a novel two-chamber culture system subjecting cells cultured on a semipermeable membrane to increased culture medium height (water pressure up to 60 cm H2O). Here, we cultured several different cell lines using the low static pressure-loadable two-chamber system, and examined cell growth, cell cycle, and cell morphology. Madin-Darby canine kidney (MDCK) columnar epithelial cells were growth-suppressed in a manner dependent on static water pressure ranging from 2-50 cm H2O, without cell cycle arrest at any specific phase. Two other types of columnar epithelial cells exhibited similar phenotypes. By contrast, spherical epithelial and mesenchymal cells were not growth-suppressed, even at 50 cm H2O. Phalloidin staining revealed that 50 cm H2O pressure load vertically flattened and laterally widened columnar epithelial cells and made actin fiber distribution sparse, without affecting total phalloidin intensity per cell. When the mucosal protectant irsogladine maleate (100 nM) was added to 50-cm-high culture medium, MDCK cells were reduced in volume and their doubling time shortened. Cell proliferation and morphology are known to be regulated by the Hippo signaling pathway, but a pressure load of 50 cm H2O did not alter the expression levels of Hippo signaling molecules in columnar epithelial cells, suggesting that this pathway was not involved in the pressure-induced phenotypes. RNA sequencing of MDCK cells showed that a 50 cm H2O pressure load upregulated keratin 14, an intermediate filament, 12-fold. This upregulation was confirmed at the protein level by immunofluorescence, suggesting a role in cytoskeletal reinforcement. These results provide evidence that cell morphology and the cytoskeleton are closely linked to cell growth. Pathological intraluminal pressure elevation may cause mucosal degeneration by acting directly on this linkage.\n\nSummaryWe provide evidence that columnar epithelial cells are growth-suppressed by pressure loads as low as 30 cm H2O, in association with cell-shape flattening and cytoskeletal alterations.

molecular biology↗

bioRxiv2017

Evaluation of Topramezone on Zebrafish Retinoid Signaling

Topramezone is a highly selective herbicide developed for broadleaf and grass weeds control in corn. In this study, the effects of topramezone on zebrafish, especially in retinoid signaling were investigated. Zebrafish embryos were treated with topramezone from 4 hours post-fertilization (hpf) to 144 hpf. Exposed to topramezone significantly reduced the retinal and retinoic levels compared to controls. The transcriptional expression levels of retinol dehydrogenase (rdh1), retinoic acid receptor subunit (raraa), retinal dehydrogenase (raldh2), retinol binding protein (rbp1a), and cellular retinoic acid binding protein (crabp1a and crabp2a) were significantly decreased. Our results suggested that topramezone significantly impaired zebrafish retinoid signaling during a short time exposure. However, treatment with topramezone significantly increased the mRNA expression levels of zfblue, zfrho, zfgr1, zfuv, and zfred. Our data demonstrated that topramezone treatment could interrupt retinoid signaling and further affect zebrafish eye development.

molecular biology↗

bioRxiv2017

Repositioning the Sm-binding site in S. cerevisiae telomerase RNA reveals RNP organizational flexibility and Sm-directed 3′-end formation

Telomerase RNA contains a template for synthesizing telomeric DNA by reverse transcription and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae, the telomerase subunits Est1 and Ku bind to the telomerase RNA, TLC1, and it has been shown that these proteins still function when their binding sites are repositioned within the RNA. TLC1 is also bound by the Sm7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)-) TLC1 isoform. Here, we first show that Sm7 can perform this function even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we tested the hypothesis that the location of the Sm7-binding site relative to the 3' end is contrastingly important. When we moved the Sm site to locations 5' of its native position, we observed that this stabilized shorter forms of poly(A)- TLC1 in a manner precisely corresponding to how far upstream the Sm site was moved. This provides strong evidence that the location of Sm7 binding to TLC1 controls where the mature poly(A)- 3' end is formed. In summary, our results show that Sm7 and the 3' end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm7 in telomerase RNA biogenesis.

molecular biology↗

bioRxiv2017

The Correlation between MicroRNA-199a and White Adipose Tissue in C57/BL6J Mice with High-Fat Diet

Understanding is emerging about microRNAs as mediators in the regulation of white adipose tissue (WAT) and obesity. The expression level of miR-199a in mice was investigated to test our hypothesis: miR-199a might be related to fat accumulation and try to find its target mRNA, which perhaps could propose strategies with a therapeutic potential affecting the fat storage. C57/BL6J mice were randomly assigned to either a control group or an obesity model group (n=10 in both groups). Control mice were fed a normal diet (fat: 10 kcal %) ad libitum for 12 weeks, and model mice were fed a high-fat diet (fat: 30 kcal %) ad libitum for 12 weeks to induce obesity. At the end of the experiment, body fat mass and the free fatty acids (FFAs) and triglycerides (TGs) in WAT were tested. Fat cell size was measured by hematoxylin-eosin (H&E) staining method. The fat mass of the model group was higher than that of the control group (P<0.05). In addition, the concentrations of the FFAs and TGs were higher (P<0.05) and the adipocyte count was lower (P<0.05) in the model group. We tested the expression levels of miR-199a and key adipogenic transcription factors, including peroxisome proliferator activated receptor gamma2 (PPAR{gamma}2), CCAAT/enhancer binding proteins alpha (C/EBP), adipocyte fatty acid-binding protein (aP2), and sterol regulatory element binding protein-1c (SREBP-1c). Up-regulated expression of miR-199a was observed in model group. Increased levels of miR-199a was accompanied by high expression levels of SREBP-1c. We found that the 3-UTR of SREBP-1c mRNA has a predicted binding site for miR-199a. Based on the current discoveries, we suggest that miR-199a may exert its action by binding to its target mRNA and cooperate with SREBP-1c to induce obesity. Therefore, if the predicted binding site is confirmed by further research, miR-199a may have therapeutic potential for obesity.\n\nAbbreviationsWAT, white adipose tissue; PPAR{gamma}2, peroxisome proliferator, activated receptor {gamma}2; C/EBP CCAAT/enhancer binding proteins ; aP2, adipocyte fatty acid-binding protein; SREBP-1c, sterol regulatory element binding protein-1c; HFD, high-fat diet.

molecular biology↗

bioRxiv2017

P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells

CRISPR/Cas9 has revolutionized our ability to engineer genomes and to conduct genome-wide screens in human cells. While some cell types are easily modified with Cas9, human pluripotent stem cells (hPSCs) poorly tolerate Cas9 and are difficult to engineer. Using a stable Cas9 cell line or transient delivery of ribonucleoproteins (RNPs) we achieved an average insertion or deletion efficiency greater than 80%. This high efficiency made it apparent that double strand breaks (DSBs) induced by Cas9 are toxic and kill most treated hPSCs. Cas9 toxicity creates an obstacle to the high-throughput use CRISPR/Cas9 for genome-engineering and screening in hPSCs. We demonstrated the toxic response is tp53-dependent and the toxic effect of tp53 severely reduces the efficiency of precise genome-engineering in hPSCs. Our results highlight that CRISPR-based therapies derived from hPSCs should proceed with caution. Following engineering, it is critical to monitor for tp53 function, especially in hPSCs which spontaneously acquire tp53 mutations.

molecular biology↗

bioRxiv2017

High throughput amplicon sequencing to assess within- and between-host genetic diversity in plant viruses

Molecular epidemiology approaches at the landscape scale require to study the genetic diversity of viral populations from numerous hosts and to characterize mixed infections. In such a context, high-throughput amplicon sequencing (HTAS) techniques create interesting opportunities as they allow identifying distinct variants within a same host while simultaneously genotyping a high number of samples. Validating variants produced by HTAS may, however, remain difficult due to biases occurring at different steps of the data-generating process (e.g. environmental contaminations and sequencing error). Here, we focused on Endive necrotic mosaic virus (ENMV), a member of family Potyviridae, genus Potyvirus to develop an HTAS approach and to characterize the genetic diversity at the intra- and inter-host levels from 430 samples collected over an area of 1660 km2 located in south-eastern France. We demonstrated how it is possible, by incorporating various controls in the experimental design and by performing independent sample replicates, to estimate potential biases in HTAS results and to implement an automated and robust variant calling procedure.\n\nHighlightsO_LIHigh-throughput amplicon sequencing to assess plant virus genetic diversity\nC_LIO_LIEstimating bias in high throughput amplicon sequencing results\nC_LIO_LIAutomated variant calling procedure for robust high throughput amplicon sequencing\nC_LI

molecular biology↗

bioRxiv2017

INO80 Chromatin Remodelling Coordinates Metabolic Homeostasis with Cell Division

Adaptive survival requires the coordination of nutrient availability with expenditure of cellular resources. For example, in nutrient-limited environments, 50% of all S. cerevisiae genes synchronize and exhibit periodic bursts of expression in coordination with respiration and cell division in the Yeast Metabolic Cycle (YMC). Despite the importance of metabolic and proliferative synchrony, the majority of YMC regulators are currently unknown. Here we demonstrate that the INO80 chromatin-remodelling complex is required to coordinate respiration and cell division with periodic gene expression. Specifically, INO80 mutants have severe defects in oxygen consumption and promiscuous cell division that is no longer coupled with metabolic status. In mutant cells, chromatin accessibility of periodic genes, including TORC-responsive genes, is relatively static, concomitant with severely attenuated gene expression. Collectively, these results reveal that the INO80 complex mediates metabolic signaling to chromatin in order to restrict proliferation to metabolically optimal states.

molecular biology↗

bioRxiv2017

Identification of disease resistance genes from a Chinese wild grapevine (Vitis davidii) by analysing grape transcriptomes and transgenic Arabidopsis

HighlightTranscription profiles showed that 20 candidate genes were obviously co-expressed at 12 hpi in 30 Vitis davidii.\n\nVdWRKY53 trancription factor enhanced the resisitance in grapevine and Arabidopsis.\n\nAbstractThe molecular mechanisms underlying disease tolerance in grapevines remain uncharacterized, even though there are substantial differences in the resistance of grapevine species to fungal and bacterial diseases. In this study, we identified genes and genetic networks involved in disease resistance in grapevines by comparing the transcriptomes of a strongly resistant clone of Chinese wild grapevine (Vitis davidii cv. Ciputao 941, DAC) and a susceptible clone of European grapevine (Vitis vinifera cv. Manicure Finger, VIM) before and after infection with white rot disease (Coniella diplodiella). Disease resistance-related genes were triggered in DAC approximately 12 hours post infection (hpi) with C. diplodiella. Twenty candidate resistant genes were co-expressed in DAC. One of these candidate genes, VdWRKY53 (GenBank accession KY124243), was over-expressed in transgenic Arabidopsis thaliana plants and was found to provide these plants with enhanced resistance to C. diplodiella, Pseudomonas syringae pv tomato PDC3000, and Golovinomyces cichoracearum. This result indicates that VdWRKY53 may be involved in nonspecific resistance via interaction with fungal and oomycete elicitor signals and the activation of defence gene expression. These results provide potential gene targets for molecular breeding to develop resistant grape cultivars.

molecular biology↗

bioRxiv2017

Accuracy through Subsampling of Protein EvolutioN: Analyzing and reconstructing protein divergence using an ensemble approach

Mapping the history of ancestor divergence can yield critical insight into protein function. However, reconstruction of this evolutionary history is subject to a great deal of uncertainty, as selection and alignment of extant descendants to represent each ancestor dramatically affects phylogenetic inference of all ancestral nodes. Although historically problematic and somewhat ignored, here we capitalize on that variability by subsampling descendants of each ancestor, generating an ensemble of reconstructions, and integrating over the resulting topological diversity to improve the accuracy of ancestor divergence reconstruction, above the level of subsampling. First, we demonstrate that reproducibility of an all-sequence, single-alignment reconstruction, measured by comparing topologies inferred from 90% subsamples, can be used to assess the likely accuracy of single-alignment reconstructions. We believe this relationship results from modulation of the likelihood landscape around the true topology induced by alignments of resampled sequences. Second, we hypothesize that topological features observed consistently across an ensemble of subsampled reconstructions best reflect true evolutionary history, despite the inaccuracy of each individual reconstruction due to the failure by substitution models to infer the correct sequence of mutations that produced the input sequences. We present a scoring function that evaluates topologies on the basis of their consistency with features extracted from an ensemble, and a reconstruction algorithm (ASPEN) that produces and ranks topologies according to that function. We test our methodology on 600 simulated protein families and the LacI transcription factor family, and find that ASPEN topologies are significantly more accurate than single-alignment reconstructions.

molecular biology↗

bioRxiv2017

Both DNA Polymerases δ and ε Contact Active and Stalled Replication Forks Differently

Three DNA polymerases (Pol , Pol {delta}, and Pol {varepsilon}) are responsible for eukaryotic genome duplication. When DNA replication stress is encountered, DNA synthesis stalls until the stress is ameliorated. However, it is not known whether there is a difference in the association of each polymerase with active and stalled replication forks. Here, we show that each DNA polymerase has distinct patterns of association with active and stalled replication forks. Pol is enriched at extending Okazaki fragments of active and stalled forks. In contrast, although Pol {delta} contacts the nascent lagging strands of active and stalled forks, it binds to only the matured (and not elongating) Okazaki fragments of stalled forks. Pol {varepsilon} has a greater contact with the nascent ssDNA of leading strand on active forks compared with stalled forks. We propose that the configuration of DNA polymerases at stalled forks facilitate resumption of DNA synthesis after stress removal.

molecular biology↗

bioRxiv2017

Roles of the Brca2 and Wapl complexes with Pds5 in sister chromatid cohesion, cohesin localization, and gene expression

The cohesin complex topologically encircles chromosomes and mediates sister chromatid cohesion to ensure accurate chromosome segregation upon cell division. Cohesin also participates in DNA repair and gene transcription. The Nipped-B - Mau2 protein complex loads cohesin onto chromosomes and the Pds5 - Wapl complex removes cohesin. Pds5 is also essential for sister chromatid cohesion, indicating that it has functions beyond cohesin removal. The Brca2 DNA repair protein interacts with Pds5, but the roles of this complex beyond DNA repair are unknown. Here we show that Brca2 opposes Pds5 function in sister chromatid cohesion by assaying precocious sister chromatid separation in metaphase spreads of cultured cells depleted for these proteins. By genome-wide chromatin immunoprecipitation we find that Pds5 facilitates SA cohesin subunit association with DNA replication origins and that Brca2 inhibits SA binding, mirroring their effects on sister chromatid cohesion. Cohesin binding is maximal at replication origins and extends outward to occupy active genes and regulatory sequences. Pds5 and Wapl, but not Brca2, limit the distance that cohesin extends from origins, thereby determining which active genes, enhancers and silencers bind cohesin. Using RNA-seq we find that Brca2, Pds5 and Wapl influence the expression of most genes sensitive to Nipped-B and cohesin, largely in the same direction. These findings demonstrate that Brca2 regulates sister chromatid cohesion and gene expression in addition to its canonical role in DNA repair and expand the known functions of accessory proteins in cohesins diverse functions.\n\nAuthor summaryThe cohesin protein complex has multiple functions in eukaryotic cells. It ensures that when a cell divides, the two daughter cells receive the correct number of chromosomes. It does this by holding together the sister chromatids that are formed when chromosomes are duplicated by DNA replication. Cohesin also helps repair damaged DNA, and to regulate genes important for growth and development. Even minor deficiencies in some proteins that regulate cohesin cause significant human birth defects. Here we investigated in Drosophila cells how three proteins, Pds5, Wapl and Brca2, determine where cohesin binds to chromosomes, control cohesins ability to hold sister chromatids together, and participate in gene expression. We find that Pds5 and Wapl work together, likely during DNA replication, to determine which genes bind cohesin by controlling how far cohesin spreads out along chromosomes. Pds5 is required for cohesin to hold sister chromatids together, and Brca2 counteracts this function. In contrast to the opposing roles in sister chromatid cohesion, Pds5 and Brca2 work together to facilitate control of gene expression by cohesin. Brca2 plays a critical role in DNA repair, and these studies expand the known roles for Brca2 by showing that it also regulates sister chromatid cohesion and gene expression. BRCA2 mutations in humans increase susceptibility to breast and ovarian cancer, and these findings raise the possibility that changes in chromosome segregation or gene expression might contribute to the increased cancer risk associated with these mutations.

molecular biology↗

bioRxiv2017

Heterogeneity and Intrinsic Variation in Spatial Genome Organization

The genome is hierarchically organized in 3D space and its architecture is altered in differentiation, development and disease. Some of the general principles that determine global 3D genome organization have been established. However, the extent and nature of cell-to-cell and cell-intrinsic variability in genome architecture are poorly characterized. Here, we systematically probe the heterogeneity in genome organization in human fibroblasts by combining high-resolution Hi-C datasets and high-throughput genome imaging. Optical mapping of several hundred genome interaction pairs at the single cell level demonstrates low steady-state frequencies of colocalization in the population and independent behavior of individual alleles in single nuclei. Association frequencies are determined by genomic distance, higher-order chromatin architecture and chromatin environment. These observations reveal extensive variability and heterogeneity in genome organization at the level of single cells and alleles and they demonstrate the coexistence of a broad spectrum of chromatin and genome conformations in a cell population.

molecular biology↗

bioRxiv2017

Integrator Subunit 4 is a ‘Symplekin-like’ Scaffold that Associates with INTS9/11 to Form the Integrator Cleavage Module

Integrator (INT) is a transcriptional regulatory complex associated with RNA polymerase II that is required for the 3-end processing of both UsnRNAs and enhancer RNAs. Integrator subunits 9 (INTS9) and INTS11 constitute the catalytic core of INT and are paralogues of the cleavage and polyadenylation specificity factors CPSF100 and CPSF73. While CPSF73/100 are known to associate with a third protein called Symplekin, there is no paralog of Symplekin within INT raising the question of how INTS9/11 associate with the other INT subunits. Here, we have identified that INTS4 is a specific and conserved interaction partner of INTS9/11 that does not interact with either subunit individually. Although INTS4 has no significant homology with Symplekin, it possesses N-terminal HEAT repeats similar to Symplekin but also contains a {beta}-sheet rich C-terminal region, both of which are important to bind INTS9/11. We assess three functions of INT including UsnRNA 3-end processing, maintenance of Cajal body integrity, and formation of histone locus bodies to conclude that INTS4/9/11 are the most critical of the INT subunits for UsnRNA biogenesis. Altogether, these results indicate that INTS4/9/11 compose a heterotrimeric complex that likely represents the Integrator cleavage module responsible for its endonucleolytic activity.

molecular biology↗

bioRxiv2017

NmeCas9 is an intrinsically high-fidelity genome editing platform

BackgroundThe development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wild-type SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large (e.g. mammalian) genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large (~4.2 kb) open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs (e.g. from Staphylococcus aureus, Campylobacter jejuni, Geobacillus stearothermophilus and Neisseria meningitidis) are considerably smaller and therefore better suited for viral delivery.\n\nResultsHere we show that wild-type NmeCas9, when programmed with guide sequences of natural length (24 nucleotides), exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5-N4GATT-3), for NmeCas9 genome editing in human cells.\n\nConclusionsOur results show that NmeCas9 is a naturally high-fidelity genome editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

molecular biology↗

bioRxiv2017

COMPARISONS OF CODING AND NONCODING SEQUENCES TOINFER THE ORIGIN OF CODON USAGE BIAS

Codon usage bias is the preferential use of the subset of synonymous codons during translation. In this paper, the comparisons of normalized entropy and GC content between the sequence of coding regions of Escherichia coli k12 and noncoding regions (ncRNA, rRNA) of various organisms were done to shed light on the origin of the codon usage bias.The normalized entropy of the coding regions was found significantly higher than the noncoding regions, suggesting the role of the translation process in shaping codon usage bias. Further, when the position specific GC content of both coding and noncoding regions was analyzed, the GC2 content in coding regions was lower than GC1 and GC2 while in noncoding regions, the GC1, GC2, GC3 contents were approximately equal. This discrepancy is explained by the biased mutation coupled with the presence and absence of selection pressure. The accumulation of CG content occurs in the sequences due to mutation bias in DNA repair and recombination process. In noncoding regions, the mutation is harmful and thus, selected against while due to the degeneracy of codons in coding regions, a mutation in GC3 is neutral and hence, not selected. Thus, the accumulation of GC content occurs in coding regions, and thus codon usage bias occurs.

molecular biology↗

bioRxiv2017

DNA-PK facilitates HIV transcription by regulating the activity of RNA polymerase II and the recruitment of transcription machinery at HIV LTR

Despite the use of highly effective antiretroviral therapy (HAART), the presence of latent or transcriptionally silent proviruses prevents cure and eradication of HIV infection. These transcriptionally silent proviruses are well protected from both the immune system and HAART regimens. Thus, in order to tackle the problem of latent HIV reservoirs, it is a prerequisite to define all the pathways that regulate HIV transcription. We have previously reported that DNA-PK facilitates HIV transcription by interacting with the RNA polymerase II (RNAP II) complex recruited at HIV LTR. To extend those studies further, here we demonstrate that DNA-PK promotes HIV transcription by supporting it at several stages, including initiation, pause-release and elongation. We discovered that DNA-PK increases phosphorylation of RNAP II C-terminal domain (CTD) at serine 5 (Ser5) and serine 2 (Ser2) by both directly catalyzing and by augmenting the recruitment of P-TEFb at HIV LTR. We found that DNA-PK facilitates the establishment of euchromatin structure at HIV LTR, which further supports HIV gene expression. DNA-PK inhibition or knockdown leads to the severe impairment of HIV gene expression and conversion of euchromatin to heterochromatin at HIV LTR. It also profoundly restricts HIV replication and reactivation of latent provirus. DNA-PK promotes the recruitment of TRIM28 at LTR and facilitates the release of paused RNAP II through TRIM28 phosphorylation. The results were reproduced in cell lines belonging to both lymphoid and myeloid lineages and were confirmed in primary CD4+ T cells and peripheral blood mononuclear cells (PBMCs) from HIV-infected patients.\n\nIMPORTANCEOur results reveal the important role of DNA-PK in supporting HIV transcription, replication and latent proviral reactivation. Intriguingly, this study sheds light on an important pathway that affects HIV gene expression. These findings provide strong rationale for developing and using transcriptional inhibitors, such as DNA-PK inhibitors, as supplement to HAART regimens in order to further enhance their effectiveness and to suppress toxicity due to HIV proteins.

molecular biology↗

bioRxiv2017

CBF-1 promotes the establishment and maintenance of HIV latency by recruiting Polycomb repressive complexes, PRC1 and PRC2, at HIV LTR

The C-promoter binding factor-1 (CBF-1) is a potent and specific inhibitor of the HIV-1 LTR promoter. Here we demonstrate that the knockdown of endogenous CBF-1 in latently infected primary CD4+ T cells, using specific small hairpin RNAs (shRNA), resulted in the reactivation of latent HIV proviruses. Chromatin immunoprecipitation (ChIP) assays using latently infected primary T cells and Jurkat T-cell lines demonstrated that CBF-1 induces the establishment and maintenance of HIV latency by recruiting Polycomb Group (PcG/PRC) corepressor complexes or Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Knockdown of CBF-1 resulted in the dissociation of PRCs corepressor complexes enhancing the recruitment of RNA polymerase II (RNAP II) at HIV LTR. Knockdown of certain components of PRC1 and PRC2 also led to the reactivation of latent proviruses. Similarly, treatment of latently infected primary CD4+ T cells with the EZH2 inhibitor, 3-deazaneplanocin A (DZNep), led to their reactivation.

molecular biology↗

bioRxiv2017

POLD replicates both strands of small kilobase-long replication bubbles initiated at a majority of human replication origins

Error-prone mutants of polymerase epsilon (POLE*) or polymerase delta (POLD1*) induce a mutator phenotype in human cancers. Here we show that the rate of mutations introduced by POLD1* is elevated by 50%, while the rate of POLE*-induced mutations is decreased twofold, within one kilobase from replication origins. These results support a model in which POLD1 replicates both the leading and the lagging strands within a kilobase from an origin. The magnitude of the mutational bias suggests that the probability of an individual origin to initiate replication exceeds 50%, which is much higher than previous estimates. Using additional data from nascent DNA sequencing and Okazaki fragments sequencing (OK-seq) experiments, we showed that a majority of origins are firing at each replication round, but the initiated replication fork does not propagate further than 1Kb in both directions. Analyses based on mutational data and on OK-seq data concordantly suggest that only approximately a quarter of fired origins result in a processive replication fork. Taken together, our results provide a new model of replication initiation.

molecular biology↗