Building genomes to understand biology.

Genetic manipulation is one of the central strategies that biologists use to investigate the molecular underpinnings of life and its diversity. Thus, advances in genetic manipulation usually lead to a deeper understanding of biological systems. During the last decade, the construction of chromosomes, known as synthetic genomics, has emerged as a novel approach to genetic manipulation. By facilitating complex modifications to chromosome content and structure, synthetic genomics opens new opportunities for studying biology through genetic manipulation. Here, we discuss different classes of genetic manipulation that are enabled by synthetic genomics, as well as biological problems they each can help solve.

"Lessons from the extremes: Epigenetic and genetic regulation in point monocentromere and holocentromere establishment on artificial chromosomes".

The formation of de novo centromeres on artificial chromosomes in humans (HACs) and fission yeast (SpYACs) has provided much insights to the epigenetic and genetic control on regional centromere establishment and maintenance. Similarly, the use of artificial chromosomes in point centromeric budding yeast Saccharomyces cerevisiae (ScYACs) and holocentric Caenorhabditis elegans (WACs) has revealed epigenetic regulation in the originally thought purely genetically-determined point centromeres and some centromeric DNA sequence features in holocentromeres, respectively. These relatively extreme and less characterized centromere organizations, on the endogenous chromosomes and artificial chromosomes, will be discussed and compared to the more well-studied regional centromere systems. This review will highlight some of the common epigenetic and genetic features in different centromere architectures, including the presence of the centromeric histone H3 variant, CENP-A or CenH3, centromeric and pericentric transcription, AT-richness and repetitiveness of centromeric DNA sequences.

Histone H3K9 and H4 Acetylations and Transcription Facilitate the Initial CENP-AHCP-3 Deposition and De Novo Centromere Establishment in Caenorhabditis elegans Artificial Chromosomes.

BACKGROUND: The centromere is the specialized chromatin region that directs chromosome segregation. The kinetochore assembles on the centromere, attaching chromosomes to microtubules in mitosis. The centromere position is usually maintained through cell cycles and generations. However, new centromeres, known as neocentromeres, can occasionally form on ectopic regions when the original centromere is inactivated or lost due to chromosomal rearrangements. Centromere repositioning can occur during evolution. Moreover, de novo centromeres can form on exogenously transformed DNA in human cells at a low frequency, which then segregates faithfully as human artificial chromosomes (HACs). How centromeres are maintained, inactivated and activated is unclear. A conserved histone H3 variant, CENP-A, epigenetically marks functional centromeres, interspersing with H3. Several histone modifications enriched at centromeres are required for centromere function, but their role in new centromere formation is less clear. Studying the mechanism of new centromere formation has been challenging because these events are difficult to detect immediately, requiring weeks for HAC selection. RESULTS: DNA injected into the Caenorhabditis elegans gonad can concatemerize to form artificial chromosomes (ACs) in embryos, which first undergo passive inheritance, but soon autonomously segregate within a few cell cycles, more rapidly and frequently than HACs. Using this in vivo model, we injected LacO repeats DNA, visualized ACs by expressing GFP::LacI, and monitored equal AC segregation in real time, which represents functional centromere formation. Histone H3K9 and H4 acetylations are enriched on new ACs when compared to endogenous chromosomes. By fusing histone deacetylase HDA-1 to GFP::LacI, we tethered HDA-1 to ACs specifically, reducing AC histone acetylations, reducing AC equal segregation frequency, and reducing initial kinetochroe protein CENP-AHCP-3 and NDC-80 deposition, indicating that histone acetylations facilitate efficient centromere establishment. Similarly, inhibition of RNA polymerase II-mediated transcription also delays initial CENP-AHCP-3 loading. CONCLUSIONS: Acetylated histones on chromatin and transcription can create an open chromatin environment, enhancing nucleosome disassembly and assembly, and potentially contribute to centromere establishment. Alternatively, acetylation of soluble H4 may stimulate the initial deposition of CENP-AHCP-3-H4 nucleosomes. Our findings shed light on the mechanism of de novo centromere activation.

Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity.

The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.

Human and mouse artificial chromosome technologies for studies of pharmacokinetics and toxicokinetics.

In the earliest stage of drug discovery/development, various cell-based models and animal models were used for the prediction of human pharmacokinetics and toxicokinetics. Unfortunately, drugs under development are often discontinued because their nonclinical results do not extrapolate to human clinical studies in relation to either safety or efficacy. Therefore, it is important to improve the time- and cost-effectiveness of drug development. This might be achieved by developing new technologies including pharmacokinetics and toxicokinetics models that use human and mouse artificial chromosome vectors (HACs/MACs). HACs/MACs are unique vectors with several advantages: 1) independent maintenance, 2) defined copy number and mitotically stable, 3) no silencing of the transgene, and 4) no limitation of DNA insertion size. This review provides information on the advantages and examples of the utility of various models based on the recent advances in HAC/MAC technologies, including multifunctional cell-based models for assaying drug-drug interactions, bidirectional permeability, and cytotoxicity, as well as fully genetically humanized mouse models. We also discuss the future prospects of these technologies to advance drug discovery. In summary, these technologies offer advantages over current conventional models and should improve the success rate of drug development related to efficacy and safety for humans.

Fungal artificial chromosomes for mining of the fungal secondary metabolome.

BACKGROUND: With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30-80 kb in size, breakthrough techniques are needed to characterize this SM wealth. RESULTS: Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery. CONCLUSION: The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery.

Generation of an artificial ring chromosome in Arabidopsis by Cre/LoxP-mediated recombination.

A eukaryotic chromosome consists of a centromere, two telomeres and a number of replication origins, and 'artificial chromosomes' may be created in yeast and mammals when these three elements are artificially joined and introduced into cells. Plant artificial chromosomes (PACs) have been suggested as new vectors for the development of new crops and as tools for basic research on chromosomes. However, indisputable PAC formation has not yet been confirmed. Here, we present a method for generating PACs in the model plant Arabidopsis thaliana using the Cre/LoxP and Activator/Dissociation element systems. The successfully generated PAC, designated AtARC1 (A. thaliana artificial ring chromosome 1), originated from a centromeric edge of the long arm of chromosome 2, but its size (2.85 Mb) is much smaller than that of the original chromosome (26.3 Mb). Although AtARC1 contains only a short centromere domain consisting of 180 bp repeats approximately 250 kb in length, compared with the 3 Mb domain on the original chromosome 2, centromere-specific histone H3 (HTR12) was detected on the centromeric region. This result supported the observed stability of the PAC during mitosis in the absence of selection, and transmission of the PAC to the next generation through meiosis. Because AtARC1 contains a unique LoxP site driven by the CaMV 35S promoter, it is possible to introduce a selectable marker and desired transgenes into AtARC1 at the LoxP site using Cre recombinase. Therefore, AtARC1 meets the criteria for a PAC and is a promising vector.

Isolation and characterization of SSR sequences from the genome and TAC clones of common wheat using the PCR technique.

We have developed the 2-step PCR method, a kind of suppression PCR procedure, to isolate simple sequence repeats (SSRs) from common wheat (Triticum aestivum L.) in a more convenient manner. This system requires neither genomic library screening nor the SSR-enrichment procedure. As a result, we designed 131 primer pairs based on isolated SSRs from not only genomic DNA, but also transformation-competent artificial chromosome (TAC) clones. It has been demonstrated that 34 of the 131 SSR markers developed were polymorphic among 8 wheat lines. Four of 34 polymorphic SSR markers were derived from TAC clones, indicating that this method could be applied to the targeted development of unique SSR markers in large genomic DNA libraries such as those composed of bacterial artificial chromosomes (BACs). A considerable number of isolated SSR clones had similarities with part of several long terminal repeats of retrotransposons (LTR-RTs) identified in various Triticeae genome sequences. Most of those SSRs showed smear amplification profiles, suggesting that a considerable number of dysfunctional SSRs originating from repetitive DNA components, especially LTR-RTs, might exist in the common wheat genome.

An unpaired mouse centromere passes consistently through male meiosis and does not significantly compromise spermatogenesis.

ST1 is an artificial mini-chromosome approximately 4.5 Mb in size containing mouse minor and major satellite DNA, human alphoid DNA and sequences derived from interval 5 of the human Y chromosome. Here we have measured the mitotic and meiotic transmission of ST1 and have used the mini-chromosome to define the ability of mice to monitor the presence of unpaired centromeres during meiosis. ST1 is mitotically stable, remaining intact and autonomous in mice for many generations. Female mice efficiently transmit ST1 to their offspring at a frequency approaching 50%. Male mice also reliably transmit the mini-chromosome, though to only 20% of their offspring. Presence of ST1 in males is not associated with any compromise in the output of the seminiferous epithelium nor with histological or immunocytochemical evidence of increased apoptosis, outcomes predicted for a synapsis checkpoint. These data indicate that the presence of an unpaired centromere is not sufficient to arrest male meiosis, implying that univalents are normally eliminated by a mechanism other than a tension-sensitive spindle checkpoint.

Disruption of a novel MFS transporter gene, DIRC2, by a familial renal cell carcinoma-associated t(2;3)(q35;q21).

Previously, we described a family with a significantly increased predisposition for renal cell cancer co-segregating with a t(2;3)(q35;q21) chromosomal translocation. Several primary tumors of the clear cell type from different family members were analyzed at a molecular level. Loss of the derivative chromosome 3 was consistently found. In addition, different somatic Von Hippel Lindau (VHL) gene mutations were observed in most of the tumors analyzed, even within the same patient. Based on these results a multistep tumorigenesis model was proposed in which (non-disjunctional) loss of the derivative chromosome 3 represents an early event and somatic mutation of the VHL gene represents a late event related to tumor progression. More recently, however, we noted that these two anomalies were absent in at least one early-stage tumor sample that we tested. Similar results were obtained in another family with renal cell cancer and t(3;6)(q12;q15), thus suggesting that another genetic event may precede these two oncogenetic steps. We speculate that deregulation of a gene(s) located at or near the translocation breakpoint may act as such. In order to identify such genes, a detailed physical map encompassing the 3q21 breakpoint region was constructed. Through a subsequent positional cloning effort we found that this breakpoint targets a hitherto unidentified gene, designated DIRC2 (disrupted in renal cancer 2). Computer predictions of the putative DIRC2 protein showed significant homology to different members of the major facilitator superfamily (MFS) of transporters. Based on additional DIRC2 expression and mutation analyses, we propose that the observed gene disruption may result in haplo-insufficiency and, through this mechanism, in the onset of tumor growth.

Alteration of chromosome numbers by generation of minichromosomes -- is there a lower limit of chromosome size for stable segregation?

Practical applications of minichromosomes, generated by de novo composition or by truncation of natural chromosomes, rely on stable transmission of these chromosomes. Functional centromeres, telomeres and replication origins are recognized as prerequisites for minichromosome stability. However, it is not yet clear whether, and if yes, to what degree the chromatin content has a qualitative or quantitative impact on stable chromosome transmission. A small translocation chromosome, which arose after X-irradiation of a reconstructed field bean karyotype, comprised approximately 5% of the haploid metaphase complement and was found to consist of three pieces of duplicated chromatin and a wild-type centromere. This chromosome was stably transmitted through all meristematic and pollen grain mitoses but was frequently lost during meiosis (66% loss in hemizygous and 33% in homozygous condition). This minichromosome was only a little smaller than stably segregating translocation chromosomes (comprising approximately 6% of the genome) of a euploid field bean karyotype. The duplications specific for this minichromosome did not influence meiotic segregation when associated with non-duplicated chromatin of other chromosomes. In comparison with minichromosomes of other species, the possibility of a lower size limit for a stable chromosome transmission must therefore be considered which might be based, for instance, on insufficient lateral support of centromeres or on insufficient bivalent stability due to the incapability of chiasma formation.

Exploiting chromosomal and viral strategies: the design of safe and efficient non-viral gene transfer systems.

The development of gene transfer systems for therapeutic applications depends, to a large part, on an understanding of chromosomal elements mediating controlled gene expression, and DNA synthesis and maintenance. The integration of transgenes into chromosomes assures their faithful replication and segregation due to the natural functions of the host chromosome, but their expression is susceptible to inactivation by the host-cell apparatus, and they may also cause unwanted mutagenic effects. While episomal vectors are free from these shortcomings, progress in this field suffers from the lack of an in-depth understanding of the accessory functions, although a number of first-generation prototypes have been constructed in the past years. As an immediate solution, small non-viral circular episomal vectors are emerging which not only permit the study of the relevant components in a minimal gene-transfer system, but for which a considerable potential for therapeutic applications can be anticipated.