Simultaneous Plate-Reader Characterization of Promoter Activity and Cell Growth in Engineered Mammalian Cells.

Automated high-throughput methods that support tracking of mammalian cell growth are currently needed to advance cell line characterization and identification of desired genetic components required for cell engineering. Here, we describe a high-throughput noninvasive assay based on plate reader measurements. The assay relies on the change in absorbance of the pH indicator phenol red. We show that its basic and acidic absorbance profiles can be converted into a cell growth index consistent with cell count profiles, and that, by adopting a computational pipeline and calibration measurements, it is possible to identify a conversion that enables prediction of cell numbers from plate measurements alone. The assay is suitable for growth characterization of both suspension and adherent cell lines when these are grown under different environmental conditions and treated with chemotherapeutic drugs. The method also supports characterization of stably engineered cell lines and identification of desired promoters based on fluorescence output.

Mammalian cell growth characterisation by a non-invasive plate reader assay.

Automated and non-invasive mammalian cell analysis is currently lagging behind due to a lack of methods suitable for a variety of cell lines and applications. Here, we report the development of a high throughput non-invasive method for tracking mammalian cell growth and performance based on plate reader measurements. We show the method to be suitable for both suspension and adhesion cell lines, and we demonstrate it can be adopted when cells are grown under different environmental conditions. We establish that the method is suitable to inform on effective drug treatments to be used depending on the cell line considered, and that it can support characterisation of engineered mammalian cells over time. This work provides the scientific community with an innovative approach to mammalian cell screening, also contributing to the current efforts towards high throughput and automated mammalian cell engineering.

Emergent expression of fitness-conferring genes by phenotypic selection.

Genotypic and phenotypic adaptation is the consequence of ongoing natural selection in populations and is key to predicting and preventing drug resistance. Whereas classic antibiotic persistence is all-or-nothing, here we demonstrate that an antibiotic resistance gene displays linear dose-responsive selection for increased expression in proportion to rising antibiotic concentration in growing Escherichia coli populations. Furthermore, we report the potentially wide-spread nature of this form of emergent gene expression (EGE) by instantaneous phenotypic selection process under bactericidal and bacteriostatic antibiotic treatment, as well as an amino acid synthesis pathway enzyme under a range of auxotrophic conditions. We propose an analogy to Ohm's law in electricity (V = IR), where selection pressure acts similarly to voltage (V), gene expression to current (I), and resistance (R) to cellular machinery constraints and costs. Lastly, mathematical modeling using agent-based models of stochastic gene expression in growing populations and Bayesian model selection reveal that the EGE mechanism requires variability in gene expression within an isogenic population, and a cellular "memory" from positive feedbacks between growth and expression of any fitness-conferring gene. Finally, we discuss the connection of the observed phenomenon to a previously described general fluctuation-response relationship in biology.

Initial cell density encodes proliferative potential in cancer cell populations.

Individual cells exhibit specific proliferative responses to changes in microenvironmental conditions. Whether such potential is constrained by the cell density throughout the growth process is however unclear. Here, we identify a theoretical framework that captures how the information encoded in the initial density of cancer cell populations impacts their growth profile. By following the growth of hundreds of populations of cancer cells, we found that the time they need to adapt to the environment decreases as the initial cell density increases. Moreover, the population growth rate shows a maximum at intermediate initial densities. With the support of a mathematical model, we show that the observed interdependence of adaptation time and growth rate is significantly at odds both with standard logistic growth models and with the Monod-like function that governs the dependence of the growth rate on nutrient levels. Our results (i) uncover and quantify a previously unnoticed heterogeneity in the growth dynamics of cancer cell populations; (ii) unveil how population growth may be affected by single-cell adaptation times; (iii) contribute to our understanding of the clinically-observed dependence of the primary and metastatic tumor take rates on the initial density of implanted cancer cells.

Human NORs, comprising rDNA arrays and functionally conserved distal elements, are located within dynamic chromosomal regions.

Human nucleolar organizer regions (NORs), containing ribosomal gene (rDNA) arrays, are located on the p-arms of acrocentric chromosomes (HSA13-15, 21, and 22). Absence of these p-arms from genome references has hampered research on nucleolar formation. Previously, we assembled a distal junction (DJ) DNA sequence contig that abuts rDNA arrays on their telomeric side, revealing that it is shared among the acrocentrics and impacts nucleolar organization. To facilitate inclusion into genome references, we describe sequencing the DJ from all acrocentrics, including three versions of HSA21, ∼3 Mb of novel sequence. This was achieved by exploiting monochromosomal somatic cell hybrids containing single human acrocentric chromosomes with NORs that retain functional potential. Analyses revealed remarkable DJ sequence and functional conservation among human acrocentrics. Exploring chimpanzee acrocentrics, we show that "DJ-like" sequences and abutting rDNA arrays are inverted as a unit in comparison to humans. Thus, rDNA arrays and linked DJs represent a conserved functional locus. We provide direct evidence for exchanges between heterologous human acrocentric p-arms, and uncover extensive structural variation between chromosomes and among individuals. These findings lead us to revaluate the molecular definition of NORs, identify novel genomic structural variation, and provide a rationale for the distinctive chromosomal organization of NORs.

Sirtuin 7 promotes 45S pre-rRNA cleavage at site 2 and determines the processing pathway.

In humans, ribosome biogenesis mainly occurs in nucleoli following two alternative pre-rRNA processing pathways differing in the order in which cleavages take place but not by the sites of cleavage. To uncover the role of the nucleolar NAD+-dependent deacetylase sirtuin 7 in the synthesis of ribosomal subunits, pre-rRNA processing was analyzed after sirtinol-mediated inhibition of sirtuin 7 activity or depletion of sirtuin 7 protein. We thus reveal that sirtuin 7 activity is a critical regulator of processing of 45S, 32S and 30S pre-rRNAs. Sirtuin 7 protein is primarily essential to 45S pre-rRNA cleavage at site 2, which is the first step of processing pathway 2. Furthermore, we demonstrate that sirtuin 7 physically interacts with Nop56 and the GAR domain of fibrillarin, and propose that this could interfere with fibrillarin-dependent cleavage. Sirtuin 7 depletion results in the accumulation of 5' extended forms of 32S pre-rRNA, and also influences the localization of fibrillarin. Thus, we establish a close relationship between sirtuin 7 and fibrillarin, which might determine the processing pathway used for ribosome biogenesis.

Functional Insulator Scanning of CpG Islands to Identify Regulatory Regions of Promoters Using CRISPR.

The ability to mutate a promoter in situ is potentially a very useful approach for gaining insights into endogenous gene regulation mechanisms. The advent of CRISPR/Cas systems has provided simple, efficient, and targeted genetic manipulation in eukaryotes, which can be applied to studying genome structure and function.The basic CRISPR toolkit comprises an endonuclease, Cas9, and a short DNA-targeting sequence, made up of a single guide RNA (sgRNA). The catalytic domains of Cas9 are rendered active upon dimerization of Cas9 with sgRNA, resulting in targeted double stranded DNA breaks. Among other applications, this method of DNA cleavage can be coupled to endogenous homology-directed repair (HDR) mechanisms for the generation of site-specific editing or knockin mutations, at both promoter regulatory and gene coding sequences.A well-characterized regulatory feature of promoter regions is the high abundance of CpGs. These CpG islands tend to be unmethylated, ensuring a euchromatic environment that promotes gene transcription. Here, we demonstrate CRISPR-mediated editing of two CpG islands located within the promoter region of the MDR1 gene (Multi Drug Resistance 1). Cas9 is used to generate double stranded breaks across multiple target sites, which are then repaired while inserting the beta globin (ß-globin) insulator, 5'HS5. Thus, we are screening through promoter regulatory sequences with a chromatin barrier element to identify functional regions via "insulator scanning." Transcriptional and functional assessment of MDR1 expression provides evidence of genome engineering. Overall, this method allows the scanning of CpG islands to identify their promoter functions.

From noise to synthetic nucleoli: can synthetic biology achieve new insights?

Synthetic biology aims to re-organise and control biological components to make functional devices. Along the way, the iterative process of designing and testing gene circuits has the potential to yield many insights into the functioning of the underlying chassis of cells. Thus, synthetic biology is converging with disciplines such as systems biology and even classical cell biology, to give a new level of predictability to gene expression, cell metabolism and cellular signalling networks. This review gives an overview of the contributions that synthetic biology has made in understanding gene expression, in terms of cell heterogeneity (noise), the coupling of growth and energy usage to expression, and spatiotemporal considerations. We mainly compare progress in bacterial and mammalian systems, which have some of the most-developed engineering frameworks. Overall, one view of synthetic biology can be neatly summarised as "creating in order to understand."

Construction of synthetic nucleoli and what it tells us about propagation of sub-nuclear domains through cell division.

The cell nucleus is functionally compartmentalized into numerous membraneless and dynamic, yet defined, bodies. The cell cycle inheritance of these nuclear bodies (NBs) is poorly understood at the molecular level. In higher eukaryotes, their propagation is challenged by cell division through an "open" mitosis, where the nuclear envelope disassembles along with most NBs. A deeper understanding of the mechanisms involved can be achieved using the engineering principles of synthetic biology to construct artificial NBs. Successful biogenesis of such synthetic NBs demonstrates knowledge of the basic mechanisms involved. Application of this approach to the nucleolus, a paradigm of nuclear organization, has highlighted a key role for mitotic bookmarking in the cell cycle propagation of NBs.

Construction of synthetic nucleoli in human cells reveals how a major functional nuclear domain is formed and propagated through cell division.

Human cell nuclei are functionally organized into structurally stable yet dynamic bodies whose cell cycle inheritance is poorly understood. Here, we investigate the biogenesis and propagation of nucleoli, sites of ribosome biogenesis and key regulators of cellular growth. Nucleolar and cell cycles are intimately connected. Nucleoli disappear during mitosis, reforming around prominent uncharacterized chromosomal features, nucleolar organizer regions (NORs). By examining the effects of UBF depletion on both endogenous NORs and synthetic pseudo-NORs, we reveal its essential role in maintaining competency and establishing a bookmark on mitotic NORs. Furthermore, we demonstrate that neo-NORs, UBF-binding site arrays coupled with rDNA transcription units, direct the de novo biogenesis of functional compartmentalized neonucleoli irrespective of their site of chromosomal integration. For the first time, we establish the sequence requirements for nucleolar biogenesis and provide proof that this is a staged process where UBF-dependent mitotic bookmarking precedes function-dependent nucleolar assembly.

Fibrillarin and Nop56 interact before being co-assembled in box C/D snoRNPs.

Small nucleolar RNAs play crucial roles in ribosome biogenesis. They guide folding, site-specific nucleotide modifications and participate in cleavage of precursor ribosomal RNAs. To better understand how the biogenesis of the box C/D small nucleolar RNPs (snoRNPs) occur in a cellular context, we used a new approach based on the possibility of relocalizing a given nuclear complex by adding an affinity tag for B23 to one component of this complex. We selectively delocalized each core box C/D protein, namely 15.5kD, Nop56, Nop58 and fibrillarin, and analyzed the effect of such changes on other components of the box C/D snoRNPs. We show that modifying the localization and the mobility of core box C/D proteins impairs their association with box C/D snoRNPs. In addition, we demonstrate that fibrillarin and Nop56 directly interact in vivo. This interaction, indispensable for the association of both proteins with the box C/D snoRNPs, does not involve the glycine- and arginine-rich domain or the RNA-binding domain but the alpha-helix domain of fibrillarin. In addition, no RNA seems required to maintain fibrillarin-Nop56 interaction.

Involvement of SIRT7 in resumption of rDNA transcription at the exit from mitosis.

Sirtuins, also designated class III histone deacetylases, are implicated in the regulation of cell division, apoptosis, DNA damage repair, genomic silencing and longevity. The nucleolar Sirtuin7 (SIRT7) was reported to be involved in the regulation of ribosomal gene (rDNA) transcription, but there are no data concerning the regulation of SIRT7 during the cell cycle. Here we have analyzed the behavior of endogenous SIRT7 during mitosis, while rDNA transcription is repressed. SIRT7 remains associated with nucleolar organizer regions, as does the RNA polymerase I machinery. SIRT7 directly interacts with the rDNA transcription factor UBF. Moreover, SIRT7 is phosphorylated via the CDK1-cyclin B pathway during mitosis and dephosphorylated by a phosphatase sensitive to okadaic acid at the exit from mitosis before onset of rDNA transcription. Interestingly, dephosphorylation events induce a conformational modification of the carboxy-terminal region of SIRT7 before the release of mitotic repression of rDNA transcription. As SIRT7 activity is required to resume rDNA transcription in telophase, we propose that this conformational modification regulates onset of rDNA transcription.