Unrealized potential from smaller institutions: Four strategies for advancing STEM diversity.

Diversity within science, technology, engineering, and mathematics (STEM) remains disturbingly low. Relative to larger, highly funded universities, smaller schools harbor more diverse student demographics and more limited resources. Here, we propose four strategies leveraging the unique advantages of smaller institutions to advance underrepresented scholars along STEM pathways.

Identification of a DNA N6-Adenine Methyltransferase Complex and Its Impact on Chromatin Organization.

DNA N6-adenine methylation (6mA) has recently been described in diverse eukaryotes, spanning unicellular organisms to metazoa. Here, we report a DNA 6mA methyltransferase complex in ciliates, termed MTA1c. It consists of two MT-A70 proteins and two homeobox-like DNA-binding proteins and specifically methylates dsDNA. Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression. We investigate the impact of 6mA on nucleosome occupancy in vitro by reconstructing complete, full-length Oxytricha chromosomes harboring 6mA in native or ectopic positions. We show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a diverged DNA N6-adenine methyltransferase and defines the role of 6mA in chromatin organization.

The architecture of a scrambled genome reveals massive levels of genomic rearrangement during development.

Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements. The remarkably encrypted genome architecture contains >3,500 scrambled genes, as well as >800 predicted germline-limited genes expressed, and some posttranslationally modified, during genome rearrangements. Gene segments for different somatic loci often interweave with each other. Single gene segments can contribute to multiple, distinct somatic loci. Terminal precursor segments from neighboring somatic loci map extremely close to each other, often overlapping. This genome assembly provides a draft of a scrambled genome and a powerful model for studies of genome rearrangement.

Genomes on the edge: programmed genome instability in ciliates.

Ciliates are an ancient and diverse group of microbial eukaryotes that have emerged as powerful models for RNA-mediated epigenetic inheritance. They possess extensive sets of both tiny and long noncoding RNAs that, together with a suite of proteins that includes transposases, orchestrate a broad cascade of genome rearrangements during somatic nuclear development. This Review emphasizes three important themes: the remarkable role of RNA in shaping genome structure, recent discoveries that unify many deeply diverged ciliate genetic systems, and a surprising evolutionary "sign change" in the role of small RNAs between major species groups.

Piwi-interacting RNAs protect DNA against loss during Oxytricha genome rearrangement.

Genome duality in ciliated protozoa offers a unique system to showcase their epigenome as a model of inheritance. In Oxytricha, the somatic genome is responsible for vegetative growth, whereas the germline contributes DNA to the next sexual generation. Somatic nuclear development removes all transposons and other so-called "junk" DNA, which comprise ~95% of the germline. We demonstrate that Piwi-interacting small RNAs (piRNAs) from the maternal nucleus can specify genomic regions for retention in this process. Oxytricha piRNAs map primarily to the somatic genome, representing the ~5% of the germline that is retained. Furthermore, injection of synthetic piRNAs corresponding to normally deleted regions leads to their retention in later generations. Our findings highlight small RNAs as powerful transgenerational carriers of epigenetic information for genome programming.

Regional sequence expansion or collapse in heterozygous genome assemblies.

High levels of heterozygosity present a unique genome assembly challenge and can adversely impact downstream analyses, yet is common in sequencing datasets obtained from non-model organisms. Here we show that by re-assembling a heterozygous dataset with variant parameters and different assembly algorithms, we are able to generate assemblies whose protein annotations are statistically enriched for specific gene ontology categories. While total assembly length was not significantly affected by assembly methodologies tested, the assemblies generated varied widely in fragmentation level and we show local assembly collapse or expansion underlying the enrichment or depletion of specific protein functional groups. We show that these statistically significant deviations in gene ontology groups can occur in seemingly high-quality assemblies, and result from difficult-to-detect local sequence expansion or contractions. Given the unpredictable interplay between assembly algorithm, parameter, and biological sequence data heterozygosity, we highlight the need for better measures of assembly quality than N50 value, including methods for assessing local expansion and collapse.

Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA.

Extrachromosomal circular DNA (eccDNA) is both a driver of eukaryotic genome instability and a product of programmed genome rearrangements, but its extent had not been surveyed in Oxytricha, a ciliate with elaborate DNA elimination and translocation during development. Here, we captured rearrangement-specific circular DNA molecules across the genome to gain insight into its processes of programmed genome rearrangement. We recovered thousands of circularly excised Tc1/mariner-type transposable elements and high confidence non-repetitive germline-limited loci. We verified their bona fide circular topology using circular DNA deep-sequencing, 2D gel electrophoresis and inverse polymerase chain reaction. In contrast to the precise circular excision of transposable elements, we report widespread heterogeneity in the circular excision of non-repetitive germline-limited loci. We also demonstrate that circular DNAs are transcribed in Oxytricha, producing rearrangement-specific long non-coding RNAs. The programmed formation of thousands of eccDNA molecules makes Oxytricha a model system for studying nucleic acid topology. It also suggests involvement of eccDNA in programmed genome rearrangement.

Capture of complete ciliate chromosomes in single sequencing reads reveals widespread chromosome isoforms.

BACKGROUND: Whole-genome shotgun sequencing, which stitches together millions of short sequencing reads into a single genome, ushered in the era of modern genomics and led to a rapid expansion of the number of genome sequences available. Nevertheless, assembly of short reads remains difficult, resulting in fragmented genome sequences. Ultimately, only a sequencing technology capable of capturing complete chromosomes in a single run could resolve all ambiguities. Even "third generation" sequencing technologies produce reads far shorter than most eukaryotic chromosomes. However, the ciliate Oxytricha trifallax has a somatic genome with thousands of chromosomes averaging only 3.2 kbp, making it an ideal candidate for exploring the benefits of sequencing whole chromosomes without assembly. RESULTS: We used single-molecule real-time sequencing to capture thousands of complete chromosomes in single reads and to update the published Oxytricha trifallax JRB310 genome assembly. In this version, over 50% of the completed chromosomes with two telomeres derive from single reads. The improved assembly includes over 12,000 new chromosome isoforms, and demonstrates that somatic chromosomes derive from variable rearrangements between somatic segments encoded up to 191,000 base pairs away. However, while long reads reduce the need for assembly, a hybrid approach that supplements long-read sequencing with short reads for error correction produced the most complete and accurate assembly, overall. CONCLUSIONS: This assembly provides the first example of complete eukaryotic chromosomes captured by single sequencing reads and demonstrates that traditional approaches to genome assembly can mask considerable structural variation.

Stress Adapted Mollusca and Nematoda Exhibit Convergently Expanded Hsp70 and AIG1 Gene Families.

We recently sequenced the genome of the first subterrestrial metazoan, the nematode Halicephalobus mephisto. A central finding was a dramatic expansion of genes encoding avrRpt2 induced gene (AIG1), and 70 kDa heat shock (Hsp70) domains. While the role of Hsp70 in thermotolerance is well established, the contribution of AIG1 is much more poorly characterized, though in plants some members of this family are heat-induced. Hypothesizing that this dual domain expansion may constitute a general biosignature of thermal stress adaptation, here we examine a number of genomes, finding that expansion of both AIG1 and Hsp70 is common in bivalves. Phylogenetic analysis reveals that the bivalve-specific Hsp70 protein expansion groups with H. mephisto sequences. Our identification of the same gene expansions in bivalves and a nematode implies that this biosignature may be a general stress adaptation strategy for protostomes, particularly those organisms that cannot escape their stressful environments. We hypothesize that the two families play largely complementary mechanistic roles, with Hsp70 directly refolding heat-denatured proteins while AIG1 promotes cellular and organismal survival by inhibiting apoptosis.

Thousands of RNA-cached copies of whole chromosomes are present in the ciliate Oxytricha during development.

The ciliate Oxytricha trifallax maintains two genomes: a germline genome that is active only during sexual conjugation and a transcriptionally active, somatic genome that derives from the germline via extensive sequence reduction and rearrangement. Previously, we found that long noncoding (lnc) RNA "templates"-telomere-containing, RNA-cached copies of mature chromosomes-provide the information to program the rearrangement process. Here we used a modified RNA-seq approach to conduct the first genome-wide search for endogenous, telomere-to-telomere RNA transcripts. We find that during development, Oxytricha produces long noncoding RNA copies for over 10,000 of its 16,000 somatic chromosomes, consistent with a model in which Oxytricha transmits an RNA-cached copy of its somatic genome to the sexual progeny. Both the primary sequence and expression profile of a somatic chromosome influence the temporal distribution and abundance of individual template RNAs. This suggests that Oxytricha may undergo multiple rounds of DNA rearrangement during development. These observations implicate a complex set of thousands of long RNA molecules in the wiring and maintenance of a highly elaborate somatic genome architecture.

Rapid molecular detection of macrolide resistance.

BACKGROUND: Emerging antimicrobial resistance is a significant threat to human health. However, methods for rapidly diagnosing antimicrobial resistance generally require multi-day culture-based assays. Macrolide efflux gene A, mef(A), provides resistance against erythromycin and azithromycin and is known to be laterally transferred among a wide range of bacterial species. METHODS: We use Recombinase Polymerase Assay (RPA) to detect the antimicrobial resistance gene mef(A) from raw lysates without nucleic acid purification. To validate these results we performed broth dilution assays to assess antimicrobial resistance to erythromycin and ampicillin (a negative control). RESULTS: We validate the detection of mef(A) in raw lysates of Streptococcus pyogenes, S. pneumoniae, S. salivarius, and Enterococcus faecium bacterial lysates within 7-10 min of assay time. We show that detection of mef(A) accurately predicts real antimicrobial resistance assessed by traditional culture methods, and that the assay is robust to high levels of spiked-in non-specific nucleic acid contaminant. The assay was unaffected by single-nucleotide polymorphisms within divergent mef(A) gene sequences, strengthening its utility as a robust diagnostic tool. CONCLUSIONS: This finding opens the door to implementation of rapid genomic diagnostics in a clinical setting, while providing researchers a rapid, cost-effective tool to track antibiotic resistance in both pathogens and commensal strains.

Chromosome fusions triggered by noncoding RNA.

Chromosomal fusions are common in normal and cancer cells and can produce aberrant gene products that promote transformation. The mechanisms driving these fusions are poorly understood, but recurrent fusions are widespread. This suggests an underlying mechanism, and some authors have proposed a possible role for RNA in this process. The unicellular eukaryote Oxytricha trifallax displays an exorbitant capacity for natural genome editing, when it rewrites its germline genome to form a somatic epigenome. This developmental process provides a powerful model system to directly test the influence of small noncoding RNAs on chromosome fusion events during somatic differentiation. Here we show that small RNAs are capable of inducing chromosome fusions in 4 distinct cases (out of 4 tested), including one fusion of 3 chromosomes. We further show that these RNA-mediated chromosome fusions are heritable over multiple sexual generations and that transmission of the acquired fusion is associated with endogenous production of novel piRNA molecules that target the fused junction. We also demonstrate the capacity of a long noncoding RNA (lncRNA) to induce chromosome fusion of 2 distal germline loci. These results underscore the ability of short-lived, aberrant RNAs to act as drivers of chromosome fusion events that can be stably transmitted to future generations.

The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes.

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor "silent" germline micronuclear genome by a process of "unscrambling" and fragmentation. The tiny macronuclear "nanochromosomes" typically encode single, protein-coding genes (a small portion, 10%, encode 2-8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.

Beyond transcriptional silencing: is methylcytosine a widely conserved eukaryotic DNA elimination mechanism?

Methylation of cytosine DNA residues is a well-studied epigenetic modification with important roles in formation of heterochromatic regions of the genome, and also in tissue-specific repression of transcription. However, we recently found that the ciliate Oxytricha uses methylcytosine in a novel DNA elimination pathway important for programmed genome restructuring. Remarkably, mounting evidence suggests that methylcytosine can play a dual role in ciliates, repressing gene expression during some life-stages and directing DNA elimination in others. In this essay, I describe these recent advances in the DNA methylation field and discuss whether this unexpected novel role for methylcytosine in DNA elimination might be more widely conserved in eukaryotic biology, particularly in apoptotic pathways.

Evolution of a biological thermocouple by adaptation of cytochrome c oxidase in a subterrestrial metazoan.

In this study we report a naturally evolved temperature-sensing electrical regulator in the cytochrome c oxidase of the Devil Worm, Halicephalobus mephisto. This extremophile metazoan was isolated 1.3 km underground in a South African goldmine, where it adapted to heat and potentially to hypoxia, making its mitochondrial sequence a likely target of adaptational change. We obtained the full mitochondrial genome sequence of this organism, and show through dN/dS analysis statistically robust evidence of positive selection in H. mephisto cytochrome c oxidase subunits. Seventeen of these positively-selected amino acid substitutions were localized in proximity to the H- and K-pathway proton channels of the complex. Surprisingly, the H. mephisto cytochrome c oxidase proton pump completely shuts down at low temperatures (20°C) leading to approximately a 4.8-fold reduction in the transmembrane proton gradient voltage (ΔΨm) compared to optimal temperature (37°C). Direct measurement of oxygen consumption found a corresponding 4.7-fold drop at 20°C compared to 37°C. Correspondingly, the lifecycle of H. mephisto takes four-fold longer at the low temperature compared to higher. This elegant evolutionary adaptation creates a finely-tuned mitochondrial temperature sensor, allowing this ectothermic organism to maximize its reproductive success in varying environmental temperatures. Our study shows that evolutionary innovation may remodel core metabolism to make it more accurately map onto environmental variation.

Regulation of lin-4 miRNA expression, organismal growth and development by a conserved RNA binding protein in C. elegans.

Transcription and multiple processing steps are required to produce specific 22 nucleotide microRNAs (miRNAs) that can regulate the expression of target genes. In C. elegans, mature lin-4 miRNA accumulates at the end of the first larval stage to repress its direct targets lin-14 and lin-28, allowing the progression of several somatic cell types to later larval fates. In this study, we characterized the expression of endogenous lin-4 and found that temporally regulated independent transcripts, but not constitutive lin-4 containing RNAs derived from an overlapping gene, are processed to mature lin-4 miRNA. Through an RNAi screen, we identified a conserved RNA binding protein gene rbm-28 (R05H10.2), homologous to the human RBM28 and yeast Nop4p proteins, that is important for lin-4 expression in C. elegans. We also demonstrate that rbm-28 genetically interacts with the lin-4 developmental timing pathway and uncover a previously unrecognized role for lin-14 and lin-28 in coordinating organismal growth.

A Transcriptomic Pipeline Adapted for Genomic Sequence Discovery of Germline-Restricted Sequence in Zebra Finch, Taeniopygia guttata.

Songbirds have an unusual genomic element which is only found in their germline cells, known as the germline-restricted chromosome (GRC). Because germ cells contain both GRC and non-GRC (or A-chromosome) sequences, confidently identifying the GRC-derived elements from genome assemblies has proven difficult. Here, we introduce a new application of a transcriptomic method for GRC sequence identification. By adapting the Stringtie/Ballgown pipeline to use somatic and germline DNA reads, we find that the ratio of fragments per kilobase per million mapped reads can be used to confidently assign contigs to the GRC. Using this comparative coverage analysis, we successfully identify 733 contigs as high confidence GRC sequences (720 newly identified in this study) and 51 contigs which were validated using quantitative polymerase chain reaction. We also identified two new GRC genes, one hypothetical protein and one gene encoding an RNase H-like domain, and placed 16 previously identified but unplaced genes onto their host contigs. With the current focus on sequencing GRCs from different songbirds, our work adds to the genomic toolkit to identify GRC elements, and we provide a detailed protocol and GitHub repository at https://github.com/brachtlab/Comparative_Coverage_Analysis (last accessed May 12, 2021).

Direct Detection of DNA and RNA on Carbon Fiber Microelectrodes Using Fast-Scan Cyclic Voltammetry.

DNA and RNA have been measured with many techniques but often with relatively long analysis times. In this study, we utilize fast-scan cyclic voltammetry (FSCV) for the subsecond codetection of adenine, guanine, and cytosine, first as free nucleosides, and then within custom synthesized oligos, plasmid DNA, and RNA from the nematode Caenorhabditis elegans. Previous studies have shown the detection of adenosine and guanosine with FSCV with high spatiotemporal resolution, while we have extended the assay to include cytidine and adenine, guanine, and cytosine in RNA and single- and double-stranded DNA (ssDNA and dSDNA). We find that FSCV testing has a higher sensitivity and yields higher peak oxidative currents when detecting shorter oligonucleotides and ssDNA samples at equivalent nucleobase concentrations. This is consistent with an electrostatic repulsion from negatively charged oxide groups on the surface of the carbon fiber microelectrode (CFME), the negative holding potential, and the negatively charged phosphate backbone. Moreover, as opposed to dsDNA, ssDNA nucleobases are not hydrogen-bonded to one another and thus are free to adsorb onto the surface of the carbon electrode. We also demonstrate that the simultaneous determination of nucleobases is not masked even in biologically complex serum samples. This is the first report demonstrating that FSCV, when used with CFMEs, is able to codetect nucleobases when polymerized into DNA or RNA and could potentially pave the way for future uses in clinical, diagnostic, or research applications.

Epigenetic Regulation of Neuregulin-1 Tunes White Adipose Stem Cell Differentiation.

Expansion of subcutaneous adipose tissue by differentiation of new adipocytes has been linked to improvements in metabolic health. However, an expandability limit has been observed wherein new adipocytes cannot be produced, the existing adipocytes become enlarged (hypertrophic) and lipids spill over into ectopic sites. Inappropriate ectopic storage of these surplus lipids in liver, muscle, and visceral depots has been linked with metabolic dysfunction. Here we show that Neuregulin-1 (NRG1) serves as a regulator of adipogenic differentiation in subcutaneous primary human stem cells. We further demonstrate that DNA methylation modulates NRG1 expression in these cells, and a 3-day exposure of stem cells to a recombinant NRG1 peptide fragment is sufficient to reprogram adipogenic cellular differentiation to higher levels. These results define a novel molecular adipogenic rheostat with potential implications for the expansion of adipose tissue in vivo.

The role of estrogens in the adipose tissue milieu.

One of the leading causes for the development of adverse metabolic effects, including type 2 diabetes, dyslipidemia, and cardiovascular diseases, is the accumulation of excess body weight, often measured by body mass index (BMI). Although BMI, calculated using weight and height, is the standard measure used to determine body adiposity in clinical and public health guidelines, an inherent limitation is that BMI does not distinguish where in the body adiposity is deposited. Central obesity, characterized by greater accumulation of adiposity in the abdominal region, has been associated with a higher risk of mortality, independent of BMI. Importantly, one of the determinants of body fat distribution is sex hormones. Both estrogens and androgens appear to directly and indirectly influence body fat distribution. Our review will focus specifically on the role of estrogens and their influence in determining body fat distribution and overall health of adipose tissues, and the role of epigenetic mechanisms in regulating the production and function of estrogens.