Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells.

The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.

Elevated tRNA(iMet) synthesis can drive cell proliferation and oncogenic transformation.

Characteristics of transformed and tumor cells include increased levels of protein synthesis and elevated expression of RNA polymerase (pol) III products, such as tRNAs and 5S rRNA. However, whether deregulated pol III transcription contributes to transformation has been unclear. Generating cell lines expressing an inducible pol III-specific transcription factor, Brf1, allowed us to raise tRNA and 5S rRNA levels specifically. Brf1 induction caused an increase in cell proliferation and oncogenic transformation, whereas depletion of Brf1 impeded transformation. Among the gene products induced by Brf1 is the tRNA(iMet) that initiates polypeptide synthesis. Overexpression of tRNA(iMet) is sufficient to stimulate cell proliferation and allow immortalized fibroblasts to form foci in culture and tumors in mice. The data indicate that elevated tRNA synthesis can promote cellular transformation.

Effects on prostate cancer cells of targeting RNA polymerase III.

RNA polymerase (pol) III occurs in two forms, containing either the POLR3G subunit or the related paralogue POLR3GL. Whereas POLR3GL is ubiquitous, POLR3G is enriched in undifferentiated cells. Depletion of POLR3G selectively triggers proliferative arrest and differentiation of prostate cancer cells, responses not elicited when POLR3GL is depleted. A small molecule pol III inhibitor can cause POLR3G depletion, induce similar differentiation and suppress proliferation and viability of cancer cells. This response involves control of the fate-determining factor NANOG by small RNAs derived from Alu short interspersed nuclear elements. Tumour initiating activity in vivo can be reduced by transient exposure to the pol III inhibitor. Untransformed prostate cells appear less sensitive than cancer cells to pol III depletion or inhibition, raising the possibility of a therapeutic window.

Direct regulation of tRNA and 5S rRNA gene transcription by Polo-like kinase 1.

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity.

Codon-Driven Translational Efficiency Is Stable across Diverse Mammalian Cell States.

Whether codon usage fine-tunes mRNA translation in mammals remains controversial, with recent papers suggesting that production of proteins in specific Gene Ontological (GO) pathways can be regulated by actively modifying the codon and anticodon pools in different cellular conditions. In this work, we compared the sequence content of genes in specific GO categories with the exonic genome background. Although a substantial fraction of variability in codon usage could be explained by random sampling, almost half of GO sets showed more variability in codon usage than expected by chance. Nevertheless, by quantifying translational efficiency in healthy and cancerous tissues in human and mouse, we demonstrated that a given tRNA pool can equally well translate many different sets of mRNAs, irrespective of their cell-type specificity. This disconnect between variations in codon usage and the stability of translational efficiency is best explained by differences in GC content between gene sets. GC variation across the mammalian genome is most likely a result of the interplay between genome repair and gene duplication mechanisms, rather than selective pressures caused by codon-driven translational rates. Consequently, codon usage differences in mammalian transcriptomes are most easily explained by well-understood mutational biases acting on the underlying genome.

Visualizing nationwide variation in medicare Part D prescribing patterns.

BACKGROUND: To characterize the regional and national variation in prescribing patterns in the Medicare Part D program using dimensional reduction visualization methods. METHODS: Using publicly available Medicare Part D claims data, we identified and visualized regional and national provider prescribing profile variation with unsupervised clustering and t-distributed stochastic neighbor embedding (t-SNE) dimensional reduction techniques. Additionally, we examined differences between regionally representative prescribing patterns for major metropolitan areas. RESULTS: Distributions of prescribing volume and medication diversity were highly skewed among over 800,000 Medicare Part D providers. Medical specialties had characteristic prescribing patterns. Although the number of Medicare providers in each state was highly correlated with the number of Medicare Part D enrollees, some states were enriched for providers with > 10,000 prescription claims annually. Dimension-reduction, hierarchical clustering and t-SNE visualization of drug- or drug-class prescribing patterns revealed that providers cluster strongly based on specialty and sub-specialty, with large regional variations in prescribing patterns. Major metropolitan areas had distinct prescribing patterns that tended to group by major geographical divisions. CONCLUSIONS: This work demonstrates that unsupervised clustering, dimension-reduction and t-SNE visualization can be used to analyze and visualize variation in provider prescribing patterns on a national level across thousands of medications, revealing substantial prescribing variation both between and within specialties, regionally, and between major metropolitan areas. These methods offer an alternative system-wide and pattern-centric view of such data for hypothesis generation, visualization, and pattern identification.

High-resolution mapping of transcriptional dynamics across tissue development reveals a stable mRNA-tRNA interface.

The genetic code is an abstraction of how mRNA codons and tRNA anticodons molecularly interact during protein synthesis; the stability and regulation of this interaction remains largely unexplored. Here, we characterized the expression of mRNA and tRNA genes quantitatively at multiple time points in two developing mouse tissues. We discovered that mRNA codon pools are highly stable over development and simply reflect the genomic background; in contrast, precise regulation of tRNA gene families is required to create the corresponding tRNA transcriptomes. The dynamic regulation of tRNA genes during development is controlled in order to generate an anticodon pool that closely corresponds to messenger RNAs. Thus, across development, the pools of mRNA codons and tRNA anticodons are invariant and highly correlated, revealing a stable molecular interaction interlocking transcription and translation.

Transcription by RNA polymerase III: more complex than we thought.

RNA polymerase (Pol) III is highly specialized for the production of short non-coding RNAs. Once considered to be under relatively simple controls, recent studies using chromatin immunoprecipitation followed by sequencing (ChIP-seq) have revealed unexpected levels of complexity for Pol III regulation, including substantial cell-type selectivity and intriguing overlap with Pol II transcription. Here I describe these novel insights and consider their implications and the questions that remain.

Knobloch syndrome associated with Polymicrogyria and early onset of retinal detachment: two case reports.

BACKGROUND: Knobloch Syndrome (KS) is a rare congenital syndrome characterized by occipital skull defects and vitreoretinal degeneration. Retinal detachment (RD) often occurs at the end of the first decade of life or later. Aside from occipital skull defects, central nervous system abnormalities are uncommon. CASE PRESENTATIONS: We report on two siblings with KS. The first, a seven month old male, presented with nystagmus and was found to have a serous RD and a tessellated retinal appearance. His sister had a history of multiple visual abnormalities and had a similar retinal appearance although no signs of RD, but retina staphylomas. Genetic testing performed on both siblings showed a mutation in COL18A1, diagnostic of KS. MRI of both siblings demonstrated polymicrogyria but did not show occipital defects. CONCLUSIONS: Although several families with KS have been described previously, our case is noteworthy for several reasons. The RD observed in our first patient occurred at an early age, and we find evidence of only one patient with KS who had an RD identified at an earlier age. The findings of polymicrogyria are not characteristic of KS, and we found only a few previous reports of this association. Additionally, we review potential treatment options for this condition.

Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein.

BACKGROUND: BRF2 is a transcription factor required for synthesis of a small group of non-coding RNAs by RNA polymerase III. Overexpression of BRF2 can transform human mammary epithelial cells. In both breast and lung cancers, the BRF2 gene is amplified and overexpressed and may serve as an oncogenic driver. Furthermore, elevated BRF2 can be independently prognostic of unfavorable survival. Dietary soy isoflavones increase metastasis to lungs in a model of breast cancer and a recent study reported significantly increased cell proliferation in breast cancer patients who used soy supplementation. The soy isoflavone daidzein is a major food-derived phytoestrogen that is structurally similar to estrogen. The putative estrogenic effect of soy raises concern that high consumption of soy foods by breast cancer patients may increase tumor growth. METHODS: Expression of BRF2 RNA and protein was assayed in ER-positive or -negative human breast cancer cells after exposure to daidzein. We also measured mRNA stability, promoter methylation and response to the demethylating agent 5-azacytidine. In addition, expression was compared between mice fed diets enriched or deprived of isoflavones. RESULTS: We demonstrate that the soy isoflavone daidzein specifically stimulates expression of BRF2 in ER-positive breast cancer cells, as well as the related factor BRF1. Induction is accompanied by increased levels of non-coding RNAs that are regulated by BRF2 and BRF1. Daidzein treatment stabilizes BRF2 and BRF1 mRNAs and selectively decreases methylation of the BRF2 promoter. Functional significance of demethylation is supported by induction of BRF2 by the methyltransferase inhibitor 5-azacytidine. None of these effects are observed in an ER-negative breast cancer line, when tested in parallel with ER-positive breast cancer cells. In vivo relevance is suggested by the significantly elevated levels of BRF2 mRNA detected in female mice fed a high-isoflavone commercial diet. In striking contrast, BRF2 and BRF1 mRNA levels are suppressed in matched male mice fed the same isoflavone-enriched diet. CONCLUSIONS: The BRF2 gene that is implicated in cancer can be induced in human breast cancer cells by the isoflavone daidzein, through promoter demethylation and/or mRNA stabilization. Dietary isoflavones may also induce BRF2 in female mice, whereas the converse occurs in males.

Dioxin receptor and SLUG transcription factors regulate the insulator activity of B1 SINE retrotransposons via an RNA polymerase switch.

Complex genomes utilize insulators and boundary elements to help define spatial and temporal gene expression patterns. We report that a genome-wide B1 SINE (Short Interspersed Nuclear Element) retrotransposon (B1-X35S) has potent intrinsic insulator activity in cultured cells and live animals. This insulation is mediated by binding of the transcription factors dioxin receptor (AHR) and SLUG (SNAI2) to consensus elements present in the SINE. Transcription of B1-X35S is required for insulation. While basal insulator activity is maintained by RNA polymerase (Pol) III transcription, AHR-induced insulation involves release of Pol III and engagement of Pol II transcription on the same strand. B1-X35S insulation is also associated with enrichment of heterochromatin marks H3K9me3 and H3K27me3 downstream of B1-X35S, an effect that varies with cell type. B1-X35S binds parylated CTCF and, consistent with a chromatin barrier activity, its positioning between two adjacent genes correlates with their differential expression in mouse tissues. Hence, B1 SINE retrotransposons represent genome-wide insulators activated by transcription factors that respond to developmental, oncogenic, or toxicological stimuli.

Comparative statistical study of hourly precipitation determined by radar-based stage IV and ground-based methods in the North Central United States.

UNLABELLED: Detailed hourly precipitation data are required for long-range modeling of dispersion and wet deposition of particulate matter and water-soluble pollutants using the CALPUFF model. In sparsely populated areas such as the north central United States, ground-based precipitation measurement stations may be too widely spaced to offer a complete and accurate spatial representation of hourly precipitation within a modeling domain. The availability of remotely sensed precipitation data by satellite and the National Weather Service array of next-generation radars (NEXRAD) deployed nationally provide an opportunity to improve on the paucity of data for these areas. Before adopting a new method of precipitation estimation in a modeling protocol, it should be compared with the ground-based precipitation measurements, which are currently relied upon for modeling purposes. This paper presents a statistical comparison between hourly precipitation measurements for the years 2006 through 2008 at 25 ground-based stations in the north central United States and radar-based precipitation measurements available from the National Center for Environmental Predictions (NCEP) as Stage IV data at the nearest grid cell to each selected precipitation station. It was found that the statistical agreement between the two methods depends strongly on whether the ground-based hourly precipitation is measured to within 0.1 in/ hr or to within 0.01 in/hr. The results of the statistical comparison indicate that it would be more accurate to use gridded Stage IV precipitation data in a gridded dispersion model for a long-range simulation, than to rely on precipitation data interpolated between widely scattered rain gauges. IMPLICATIONS: The current reliance on ground-based rain gauges for precipitation events and hourly data for modeling of dispersion and wet deposition of particulate matter and water-soluble pollutants results in potentially large discontinuity in data coverage and the need to extrapolate data between monitoring stations. The use of radar-based precipitation data, which is available for the entire continental United States and nearby areas, would resolve these data gaps and provide a complete and accurate spatial representation of hourly precipitation within a large modeling domain.

Use of tRNA gene barriers improves stability of transgene expression in CHO cells.

Instability of transgene expression is a major challenge for the biopharmaceutical industry, which can impact yields and regulatory approval. Some tRNA genes (tDNAs) can resist epigenetic silencing, the principal mechanism of expression instability, and protect adjacent genes against the spread of repressive heterochromatin. We have taken two naturally occurring clusters of human tDNAs and tested their ability to reduce epigenetic silencing of transgenes integrated into the genome of Chinese hamster ovary (CHO) cells. We find sustained improvements in productivity both in adherent CHO-K1 cells and in an industrially relevant CHO-DG44 expression system (Apollo X, FUJIFILM Diosynth Biotechnologies). We conclude that specific tDNA clusters offer potential to mitigate the widespread problem of production instability.

Selective Occupation by E2F and RB of Loci Expressed by RNA Polymerase III.

In all cases tested, TFIIIB is responsible for recruiting pol III to its genetic templates. In mammalian cells, RB binds TFIIIB and prevents its interactions with both promoter DNA and pol III, thereby suppressing transcription. As TFIIIB is not recruited to its target genes when bound by RB, the mechanism predicts that pol III-dependent templates will not be occupied by RB; this contrasts with the situation at most genes controlled by RB, where it can be tethered by promoter-bound sequence-specific DNA-binding factors such as E2F. Contrary to this prediction, however, ChIP-seq data reveal the presence of RB in multiple cell types and the related protein p130 at many loci that rely on pol III for their expression, including RMRP, RN7SL, and a variety of tRNA genes. The sets of genes targeted varies according to cell type and growth state. In such cases, recruitment of RB and p130 can be explained by binding of E2F1, E2F4 and/or E2F5. Genes transcribed by pol III had not previously been identified as common targets of E2F family members. The data provide evidence that E2F may allow for the selective regulation of specific non-coding RNAs by RB, in addition to its influence on overall pol III output through its interaction with TFIIIB.

Dissecting cell death pathways in fed-batch bioreactors.

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells.

Coordinated control of the gene expression machinery.

Gene expression is a multi-step process starting from transcribing DNA through to the eventual production of proteins or RNA products. It is important that this process is controlled coordinately to ensure that all steps function in a concerted manner. Signal transduction pathways orchestrate such control and bring about wholesale changes in the gene expression profiles of cells that ultimately determine their phenotype. Recent studies on the MAP kinase and mTOR signaling pathways in mammalian cells have illustrated how integrated responses to signaling pathways are achieved. This occurs at both the transcriptional level, through the coordinate regulation of RNA polymerases I-III and downstream in the coordinate regulation of transcription with RNA processing and translation.

mTOR associates with TFIIIC, is found at tRNA and 5S rRNA genes, and targets their repressor Maf1.

Synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III is regulated by the mTOR pathway in mammalian cells. The mTOR kinase localizes to tRNA and 5S rRNA genes, providing an opportunity for direct control. Its presence at these sites can be explained by interaction with TFIIIC, a DNA-binding factor that recognizes the promoters of these genes. TFIIIC contains a TOR signaling motif that facilitates its association with mTOR. Maf1, a repressor that binds and inhibits pol III, is phosphorylated in a mTOR-dependent manner both in vitro and in vivo at serine 75, a site that contributes to its function as a transcriptional inhibitor. Proximity ligation assays confirm the interaction of mTOR with Maf1 and TFIIIC in nuclei. In contrast to Maf1 regulation in yeast, no evidence is found for nuclear export of Maf1 in response to mTOR signaling in HeLa cells. We conclude that mTOR associates with TFIIIC, is recruited to pol III-transcribed genes, and relieves their repression by Maf1.

Use of ubiquitous chromatin opening elements (UCOE) as tools to maintain transgene expression in biotechnology.

Amongst the most important outputs of the biopharmaceutical industry are recombinant proteins, many of which are produced by integrating transgenes into the genomes of mammalian cells. However, expression is highly variable and can be unstable during prolonged culture. This is often due to epigenetic mechanisms silencing the transgenes. To combat this problem, vectors have been engineered to include ubiquitous chromatin opening elements (UCOEs) that protect against silencing. Here, we recount the evidence that UCOEs can modify chromatin environments and benefit biomanufacturing.

Selection of tRNA Genes in Human Breast Tumours Varies Substantially between Individuals.

Abnormally elevated expression of tRNA is a common feature of breast tumours. Rather than a uniform increase in all tRNAs, some are deregulated more strongly than others. Elevation of particular tRNAs has been associated with poor prognosis for patients, and experimental models have demonstrated the ability of some tRNAs to promote proliferation or metastasis. Each tRNA isoacceptor is encoded redundantly by multiple genes, which are commonly dispersed across several chromosomes. An unanswered question is whether the consistently high expression of a tRNA in a cancer type reflects the consistent activation of the same members of a gene family, or whether different family members are activated from one patient to the next. To address this question, we interrogated ChIP-seq data to determine which tRNA genes were active in individual breast tumours. This revealed that distinct sets of tRNA genes become activated in individual cancers, whereas there is much less variation in the expression patterns of families. Several pathways have been described that are likely to contribute to increases in tRNA gene transcription in breast tumours, but none of these can adequately explain the observed variation in the choice of genes between tumours. Current models may therefore lack at least one level of regulation.

Utilizing the Un-Meeting model to advance innovative translational and team science.

Advances in translational science require innovative solutions, and engagement of productive transdisciplinary teams play a critical role. While various forms of scientific meetings have long provided venues for sharing scientific findings and generating new collaborations, many conferences lack opportunities for active discussions. We describe the use of an Un-Meeting to foster innovative translational science teams through engaged discussions across multidisciplinary groups addressing a shared theme. The Un-Meeting was delivered by the University of Rochester Center for Leading Innovation and Collaboration, the national coordinating center for the National Institutes of Health Clinical and Translational Science Awards (CTSA) program. This pilot CTSA program Un-Meeting focused on engaging translational scientists, policy-makers, community members, advocates, and public health professionals to address the opioid crisis. The participant-driven format leveraged lightning talks, attendee-led idea generation, and extensive breakout discussions to foster multidisciplinary networking. Results indicated participation by a broad set of attendees and a high level of networking during the meeting. These results, coupled with the growth of the Un-Meeting across the CTSA Consortium, provide practices and models to potentially advance team and translational science. While future work will further assess the impact of Un-Meetings, this format presents a promising approach to enhance translational science.