Highly efficient CRISPR-mediated gene editing in a rotifer.

Rotifers have been studied in the laboratory and field for over 100 years in investigations of microevolution, ecological dynamics, and ecotoxicology. In recent years, rotifers have emerged as a model system for modern studies of the molecular mechanisms of genome evolution, development, DNA repair, aging, life history strategy, and desiccation tolerance. However, a lack of gene editing tools and transgenic strains has limited the ability to link genotype to phenotype and dissect molecular mechanisms. To facilitate genetic manipulation and the creation of reporter lines in rotifers, we developed a protocol for highly efficient, transgenerational, CRISPR-mediated gene editing in the monogonont rotifer Brachionus manjavacas by microinjection of Cas9 protein and synthetic single-guide RNA into the vitellaria of young amictic (asexual) females. To demonstrate the efficacy of the method, we created knockout mutants of the developmental gene vasa and the DNA mismatch repair gene mlh3. More than half of mothers survived injection and produced offspring. Genotyping these offspring and successive generations revealed that most carried at least 1 CRISPR-induced mutation, with many apparently mutated at both alleles. In addition, we achieved precise CRISPR-mediated knock-in of a stop codon cassette in the mlh3 locus, with half of injected mothers producing F2 offspring with an insertion of the cassette. Thus, this protocol produces knockout and knock-in CRISPR/Cas9 editing with high efficiency, to further advance rotifers as a model system for biological discovery.

The transcriptome of acute dehydration in myeloid leukemic cells.

Human myeloid leukemia cells (HL-60/S4) exposed to hyperosmotic stress with sucrose undergo dehydration and cell shrinkage. Interphase chromatin and mitotic chromosomes congeal, exhibiting altered phase separation (demixing) of chromatin proteins. To investigate changes in the transcriptome, we exposed HL-60/S4 cells to hyperosmotic sucrose stress (~600 milliOsmolar) for 30 and 60 minutes. We employed RNA-Seq of polyA mRNA to identify genes with increased or decreased transcript levels relative to untreated control cells (i.e., differential gene expression). These genes were examined for over-representation of Gene Ontology (GO) terms.  In stressed cells, multiple GO terms associated with transcription, translation, mitochondrial function and proteosome activity, as well as "replication-dependent histones", were over-represented among genes with increased transcript levels; whereas, genes with decreased transcript levels were over-represented with transcription repressors. The transcriptome profiles of hyperosmotically-stressed cells suggest acquisition of cellular rebuilding, a futile homeostatic response, as these cells are ultimately doomed to a dehydrated death.

Genomic evidence for ameiotic evolution in the bdelloid rotifer Adineta vaga.

Loss of sexual reproduction is considered an evolutionary dead end for metazoans, but bdelloid rotifers challenge this view as they appear to have persisted asexually for millions of years. Neither male sex organs nor meiosis have ever been observed in these microscopic animals: oocytes are formed through mitotic divisions, with no reduction of chromosome number and no indication of chromosome pairing. However, current evidence does not exclude that they may engage in sex on rare, cryptic occasions. Here we report the genome of a bdelloid rotifer, Adineta vaga (Davis, 1873), and show that its structure is incompatible with conventional meiosis. At gene scale, the genome of A. vaga is tetraploid and comprises both anciently duplicated segments and less divergent allelic regions. However, in contrast to sexual species, the allelic regions are rearranged and sometimes even found on the same chromosome. Such structure does not allow meiotic pairing; instead, we find abundant evidence of gene conversion, which may limit the accumulation of deleterious mutations in the absence of meiosis. Gene families involved in resistance to oxidation, carbohydrate metabolism and defence against transposons are significantly expanded, which may explain why transposable elements cover only 3% of the assembled sequence. Furthermore, 8% of the genes are likely to be of non-metazoan origin and were probably acquired horizontally. This apparent convergence between bdelloids and prokaryotes sheds new light on the evolutionary significance of sex.

Evolutionary diversity and novelty of DNA repair genes in asexual Bdelloid rotifers.

BACKGROUND: Bdelloid rotifers are the oldest, most diverse and successful animal taxon for which males, hermaphrodites, and traditional meiosis are unknown. Their degenerate tetraploid genome, with 2-4 copies of most loci, includes thousands of genes acquired from all domains of life by horizontal transfer. Many bdelloid species thrive in ephemerally aquatic habitats by surviving desiccation at any life stage with no loss of fecundity or lifespan. Their unique genomic diversity and the intense selective pressure of desiccation provide an exceptional opportunity to study the evolution of diversity and novelty in genes involved in DNA repair. RESULTS: We used genomic data and RNA-Seq of the desiccation process in the bdelloid Adineta vaga to characterize DNA damage reversal, translesion synthesis, and the major DNA repair pathways: base, nucleotide, and alternate excision repair, mismatch repair (MMR), and double strand break repair by homologous recombination (HR) and classical non-homologous end joining (NHEJ). We identify multiple horizontally transferred DNA damage response genes otherwise unknown in animals (AlkD, Fpg, LigK UVDE), and the presence of genes often considered vertebrate specific, particularly in the NHEJ complex and X family polymerases. While 75-100% of genes involved in MMR and HR are present in 0-2 copies, genes involved in NHEJ, which are present in only a single copy in nearly all other animals, are retained in 3-8 copies. We present structural predictions and expression evidence of neo- or sub-functionalization of multiple copy genes involved in NHEJ and other repair processes. CONCLUSION: The horizontally-acquired genes and duplicated genes in BER and NHEJ suggest resilience to oxidative damage is conferred in part by increased DNA damage recognition and efficient end repair capabilities. The pattern of gene loss and retention in MMR and HR may facilitate recombination and gene conversion between divergent sequences, thus providing at least some of the benefits of sex. The unique retention and divergence of duplicates genes in NHEJ may be facilitated by the lack of efficient selection in the absence of meiotic recombination and independent assortment, and may contribute to the evolutionary success of bdelloids.

Genome-wide transcriptomics of aging in the rotifer Brachionus manjavacas, an emerging model system.

BACKGROUND: Understanding gene expression changes over lifespan in diverse animal species will lead to insights to conserved processes in the biology of aging and allow development of interventions to improve health. Rotifers are small aquatic invertebrates that have been used in aging studies for nearly 100 years and are now re-emerging as a modern model system. To provide a baseline to evaluate genetic responses to interventions that change health throughout lifespan and a framework for new hypotheses about the molecular genetic mechanisms of aging, we examined the transcriptome of an asexual female lineage of the rotifer Brachionus manjavacas at five life stages: eggs, neonates, and early-, late-, and post-reproductive adults. RESULTS: There are widespread shifts in gene expression over the lifespan of B. manjavacas; the largest change occurs between neonates and early reproductive adults and is characterized by down-regulation of developmental genes and up-regulation of genes involved in reproduction. The expression profile of post-reproductive adults was distinct from that of other life stages. While few genes were significantly differentially expressed in the late- to post-reproductive transition, gene set enrichment analysis revealed multiple down-regulated pathways in metabolism, maintenance and repair, and proteostasis, united by genes involved in mitochondrial function and oxidative phosphorylation. CONCLUSIONS: This study provides the first examination of changes in gene expression over lifespan in rotifers. We detected differential expression of many genes with human orthologs that are absent in Drosophila and C. elegans, highlighting the potential of the rotifer model in aging studies. Our findings suggest that small but coordinated changes in expression of many genes in pathways that integrate diverse functions drive the aging process. The observation of simultaneous declines in expression of genes in multiple pathways may have consequences for health and longevity not detected by single- or multi-gene knockdown in otherwise healthy animals. Investigation of subtle but genome-wide change in these pathways during aging is an important area for future study.

Phylogeny of Syndermata (syn. Rotifera): Mitochondrial gene order verifies epizoic Seisonidea as sister to endoparasitic Acanthocephala within monophyletic Hemirotifera.

A monophyletic origin of endoparasitic thorny-headed worms (Acanthocephala) and wheel-animals (Rotifera) is widely accepted. However, the phylogeny inside the clade, be it called Syndermata or Rotifera, has lacked validation by mitochondrial (mt) data. Herein, we present the first mt genome of the key taxon Seison and report conflicting results of phylogenetic analyses: while mt sequence-based topologies showed monophyletic Lemniscea (Bdelloidea+Acanthocephala), gene order analyses supported monophyly of Pararotatoria (Seisonidea+Acanthocephala) and Hemirotifera (Bdelloidea+Pararotatoria). Sequence-based analyses obviously suffered from substitution saturation, compositional bias, and branch length heterogeneity; however, we observed no compromising effects in gene order analyses. Moreover, gene order-based topologies were robust to changes in coding (genes vs. gene pairs, two-state vs. multistate, aligned vs. non-aligned), tree reconstruction methods, and the treatment of the two monogonont mt genomes. Thus, mt gene order verifies seisonids as sister to acanthocephalans within monophyletic Hemirotifera, while deviating results of sequence-based analyses reflect artificial signal. This conclusion implies that the complex life cycle of extant acanthocephalans evolved from a free-living state, as retained by most monogononts and bdelloids, via an epizoic state with a simple life cycle, as shown by seisonids. Hence, Acanthocephala represent a rare example where ancestral transitional stages have counterparts amongst the closest relatives.

Rotifers as experimental tools for investigating aging.

Comparative biogerontology has much to contribute to the study of aging. A broad range of aging rates has evolved to meet environmental challenges, and understanding these adaptations can produce valuable insights into aging. The supra Phylum Lophotrochozoa is particularly understudied and has several groups that have intriguing patterns of aging. Members of the lophotrochozoan phylum Rotifera are particularly useful for aging studies because cohort life tables can be conducted with them easily, and biochemical and genomic tools are available for examining aging mechanisms. This paper reviews a variety of caloric restriction regimens, small molecule inhibitors, and dietary supplements that extend rotifer lifespan, as well as important interactions between caloric restriction and genotype, antioxidant supplements, and TOR and JNK pathways, and the use of RNAi to identify key genes involved in modulating the aging response. Examples of how rapamycin and JNK inhibitor exposure keeps mortality rates low during the reproductive phase of the life cycle are presented, and the ease of conducting life table experiments to screen natural products from red algae for life extending effects is illustrated. Finally, experimental evolution to produce longer-lived rotifer individuals is demonstrated, and future directions to determine the genetic basis of aging are discussed.

VAMPS: a website for visualization and analysis of microbial population structures.

BACKGROUND: The advent of next-generation DNA sequencing platforms has revolutionized molecular microbial ecology by making the detailed analysis of complex communities over time and space a tractable research pursuit for small research groups. However, the ability to generate 105-108 reads with relative ease brings with it many downstream complications. Beyond the computational resources and skills needed to process and analyze data, it is difficult to compare datasets in an intuitive and interactive manner that leads to hypothesis generation and testing. RESULTS: We developed the free web service VAMPS (Visualization and Analysis of Microbial Population Structures, http://vamps.mbl.edu) to address these challenges and to facilitate research by individuals or collaborating groups working on projects with large-scale sequencing data. Users can upload marker gene sequences and associated metadata; reads are quality filtered and assigned to both taxonomic structures and to taxonomy-independent clusters. A simple point-and-click interface allows users to select for analysis any combination of their own or their collaborators' private data and data from public projects, filter these by their choice of taxonomic and/or abundance criteria, and then explore these data using a wide range of analytic methods and visualizations. Each result is extensively hyperlinked to other analysis and visualization options, promoting data exploration and leading to a greater understanding of data relationships. CONCLUSIONS: VAMPS allows researchers using marker gene sequence data to analyze the diversity of microbial communities and the relationships between communities, to explore these analyses in an intuitive visual context, and to download data, results, and images for publication. VAMPS obviates the need for individual research groups to make the considerable investment in computational infrastructure and bioinformatic support otherwise necessary to process, analyze, and interpret massive amounts of next-generation sequence data. Any web-capable device can be used to upload, process, explore, and extract data and results from VAMPS. VAMPS encourages researchers to share sequence and metadata, and fosters collaboration between researchers of disparate biomes who recognize common patterns in shared data.

Bdelloid rotifers deploy horizontally acquired biosynthetic genes against a fungal pathogen.

Coevolutionary antagonism generates relentless selection that can favour genetic exchange, including transfer of antibiotic synthesis and resistance genes among bacteria, and sexual recombination of disease resistance alleles in eukaryotes. We report an unusual link between biological conflict and DNA transfer in bdelloid rotifers, microscopic animals whose genomes show elevated levels of horizontal gene transfer from non-metazoan taxa. When rotifers were challenged with a fungal pathogen, horizontally acquired genes were over twice as likely to be upregulated as other genes - a stronger enrichment than observed for abiotic stressors. Among hundreds of upregulated genes, the most markedly overrepresented were clusters resembling bacterial polyketide and nonribosomal peptide synthetases that produce antibiotics. Upregulation of these clusters in a pathogen-resistant rotifer species was nearly ten times stronger than in a susceptible species. By acquiring, domesticating, and expressing non-metazoan biosynthetic pathways, bdelloids may have evolved to resist natural enemies using antimicrobial mechanisms absent from other animals.

The mate recognition protein gene mediates reproductive isolation and speciation in the Brachionus plicatilis cryptic species complex.

BACKGROUND: Chemically mediated prezygotic barriers to reproduction likely play an important role in speciation. In facultatively sexual monogonont rotifers from the Brachionus plicatilis cryptic species complex, mate recognition of females by males is mediated by the Mate Recognition Protein (MRP), a globular glycoprotein on the surface of females, encoded by the mmr-b gene family. In this study, we sequenced mmr-b copies from 27 isolates representing 11 phylotypes of the B. plicatilis species complex, examined the mode of evolution and selection of mmr-b, and determined the relationship between mmr-b genetic distance and mate recognition among isolates. RESULTS: Isolates of the B. plicatilis species complex have 1-4 copies of mmr-b, each composed of 2-9 nearly identical tandem repeats. The repeats within a gene copy are generally more similar than are gene copies among phylotypes, suggesting concerted evolution. Compared to housekeeping genes from the same isolates, mmr-b has accumulated only half as many synonymous differences but twice as many non-synonymous differences. Most of the amino acid differences between repeats appear to occur on the outer face of the protein, and these often result in changes in predicted patterns of phosphorylation. However, we found no evidence of positive selection driving these differences. Isolates with the most divergent copies were unable to mate with other isolates and rarely self-crossed. Overall the degree of mate recognition was significantly correlated with the genetic distance of mmr-b. CONCLUSIONS: Discrimination of compatible mates in the B. plicatilis species complex is determined by proteins encoded by closely related copies of a single gene, mmr-b. While concerted evolution of the tandem repeats in mmr-b may function to maintain identity, it can also lead to the rapid spread of a mutation through all copies in the genome and thus to reproductive isolation. The mmr-b gene is evolving rapidly, and novel alleles may be maintained and increase in frequency via asexual reproduction. Our analyses indicate that mate recognition, controlled by MMR-B, may drive reproductive isolation and allow saltational sympatric speciation within the B. plicatilis cryptic species complex, and that this process may be largely neutral.

Evidence for degenerate tetraploidy in bdelloid rotifers.

Rotifers of class Bdelloidea have evolved for millions of years apparently without sexual reproduction. We have sequenced 45- to 70-kb regions surrounding the four copies of the hsp82 gene of the bdelloid rotifer Philodina roseola, each of which is on a separate chromosome. The four regions comprise two colinear gene-rich pairs with gene content, order, and orientation conserved within each pair. Only a minority of genes are common to both pairs, also in the same orientation and order, but separated by gene-rich segments present in only one or the other pair. The pattern is consistent with degenerate tetraploidy with numerous segmental deletions, some in one pair of colinear chromosomes and some in the other. Divergence in 1,000-bp windows varies along an alignment of a colinear pair, from zero to as much as 20% in a pattern consistent with gene conversion associated with recombinational repair of DNA double-strand breaks. Although pairs of colinear chromosomes are a characteristic of sexually reproducing diploids and polyploids, a quite different explanation for their presence in bdelloids is suggested by the recent finding that bdelloid rotifers can recover and resume reproduction after suffering hundreds of radiation-induced DNA double-strand breaks per oocyte nucleus. Because bdelloid primary oocytes are in G(1) and therefore lack sister chromatids, we propose that bdelloid colinear chromosome pairs are maintained as templates for the repair of DNA double-strand breaks caused by the frequent desiccation and rehydration characteristic of bdelloid habitats.

Bacterial community dynamics during embryonic development of the little skate (Leucoraja erinacea).

BACKGROUND: Microbial transmission from parent to offspring is hypothesized to be widespread in vertebrates. However, evidence for this is limited as many evolutionarily important clades remain unexamined. There is currently no data on the microbiota associated with any Chondrichthyan species during embryonic development, despite the global distribution, ecological importance, and phylogenetic position of this clade. In this study, we take the first steps towards filling this gap by investigating the microbiota associated with embryonic development in the little skate, Leucoraja erinacea, a common North Atlantic species and popular system for chondrichthyan biology. METHODS: To assess the potential for bacterial transmission in an oviparous chondrichthyan, we used 16S rRNA amplicon sequencing to characterize the microbial communities associated with the skin, gill, and egg capsule of the little skate, at six points during ontogeny. Community composition was analyzed using the QIIME2 pipeline and microbial continuity between stages was tracked using FEAST. RESULTS: We identify site-specific and stage-specific microbiota dominated by the bacterial phyla Proteobacteria and Bacteroidetes. This composition is similar to, but distinct from, that of previously published data on the adult microbiota of other chondrichthyan species. Our data reveal that the skate egg capsule harbors a highly diverse bacterial community-particularly on the internal surface of the capsule-and facilitates intergenerational microbial transfer to the offspring. Embryonic skin and external gill tissues host similar bacterial communities; the skin and gill communities later diverge as the internal gills and skin denticles develop. CONCLUSIONS: Our study is the first exploration of the chondrichthyan microbiota throughout ontogeny and provides the first evidence of vertical transmission in this group.

The complex evolution of the metazoan HSP70 gene family.

The metazoan 70-kDa heat shock protein (HSP70) family contains several members localized in different subcellular compartments. The cytosolic members have been classified into inducible HSP70s and constitutive heat shock cognates (HSC70s), but their distinction and evolutionary relationship remain unclear because of occasional reports of "constitutive HSP70s" and the lack of cross-phylum comparisons. Here we provide novel insights into the evolution of these important molecular chaperones. Phylogenetic analyses of 125 full-length HSP70s from a broad range of phyla revealed an ancient duplication that gave rise to two lineages from which all metazoan cytosolic HSP70s descend. One lineage (A) contains a relatively small number of genes from many invertebrate phyla, none of which have been shown to be constitutively expressed (i.e., either inducible or unknown). The other lineage (B) included both inducible and constitutive genes from diverse phyla. Species-specific duplications are present in both lineages, and Lineage B contains well-supported phylum-specific clades for Platyhelminthes, Rotifera, Nematoda, Porifera/Cnidaria, and Chordata. Some genes in Lineage B have likely independently acquired inducibility, which may explain the sporadic distribution of "HSP70" or "HSC70" in previous phylogenetic analyses. Consistent with the diversification history within each group, inducible members show lower purifying selection pressure compared to constitutive members. These results illustrate the evolutionary history of the HSP70 family, encouraging us to propose a new nomenclature: "HSP70 + subcellular localization + linage + copy number in the organism + inducible or constitutive, if known." e.g., HSP70cA1i for cytosolic Lineage A, copy 1, inducible.

Two circular chromosomes of unequal copy number make up the mitochondrial genome of the rotifer Brachionus plicatilis.

The monogonont rotifer Brachionus plicatilis is an emerging model system for a diverse array of questions in limnological ecosystem dynamics, the evolution of sexual recombination, cryptic speciation, and the phylogeny of basal metazoans. We sequenced the complete mitochondrial genome of B. plicatilis sensu strictu NH1L and found that it is composed of 2 circular chromosomes, designated mtDNA-I (11,153 bp) and mtDNA-II (12,672 bp). Hybridization to DNA isolated from mitochondria demonstrated that mtDNA-I is present at 4 times the copy number of mtDNA-II. The only nucleotide similarity between the 2 chromosomes is a 4.9-kbp region of 99.5% identity including a transfer RNA (tRNA) gene and an extensive noncoding region that contains putative D-loop and control sequence. The mtDNA-I chromosome encodes 4 proteins (ATP6, COB, NAD1, and NAD2), 13 tRNAs, and the large and small subunit ribosomal RNAs; mtDNA-II encodes 8 proteins (COX1-3, NAD3-6, and NAD4L) and 9 tRNAs. Gene order is not conserved between B. plicatilis and its closest relative with a sequenced mitochondrial genome, the acanthocephalan Leptorhynchoides thecatus, or other sequenced mitochondrial genomes. Polymerase chain reaction assays and Southern hybridization to DNA from 18 strains of Brachionus suggest that the 2-chromosome structure has been stable for millions of years. The novel organization of the B. plicatilis mitochondrial genome into 2 nearly equal chromosomes of 4-fold different copy number may provide insight into the evolution of metazoan mitochondria and the phylogenetics of rotifers and other basal animal phyla.

Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure.

PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.

Support for the monophyletic origin of Gnathifera from phylogenomics.

The monophyletic origin of Spiralia within the metazoan tree of life is supported by many large-scale phylogenomic data. While there is now substantial molecular evidence for Lophotrochozoa being a monophyletic taxon within Spiralia, the phylogenetic affiliations of many other spiralian phyla remain unclear. Here we focus on the question of a monophyletic taxon Gnathifera, which was originally characterized by jaw morphology as comprising the taxa Rotifera, Acanthocephala and Gnathostomulida. Based on a large-scale molecular sequence dataset of 11,146 amino acid residues, we reconstructed phylogenetic trees of spiralian phyla using maximum-likelihood and Bayesian approaches. We obtain the first phylogenomic evidence for the clade Gnathifera, linking Syndermata (Rotifera+Acanthocephala) with Gnathostomulida. Furthermore, our data support recent findings concerning the paraphyly of Eurotatoria.

Scientists' warning to humanity: microorganisms and climate change.

In the Anthropocene, in which we now live, climate change is impacting most life on Earth. Microorganisms support the existence of all higher trophic life forms. To understand how humans and other life forms on Earth (including those we are yet to discover) can withstand anthropogenic climate change, it is vital to incorporate knowledge of the microbial 'unseen majority'. We must learn not just how microorganisms affect climate change (including production and consumption of greenhouse gases) but also how they will be affected by climate change and other human activities. This Consensus Statement documents the central role and global importance of microorganisms in climate change biology. It also puts humanity on notice that the impact of climate change will depend heavily on responses of microorganisms, which are essential for achieving an environmentally sustainable future.

Congeneric variability in lifespan extension and onset of senescence suggest active regulation of aging in response to low temperature.

Lifespan extension under low temperature is well conserved across both endothermic and exothermic taxa, but the mechanism underlying this change in aging is poorly understood. Low temperature is thought to decrease metabolic rate, thus slowing the accumulation of cellular damage from reactive oxygen species, although recent evidence suggests involvement of specific cold-sensing biochemical pathways. We tested the effect of low temperature on aging in 11 strains of Brachionus rotifers, with the hypothesis that if the mechanism of lifespan extension is purely thermodynamic, all strains should have a similar increase in lifespan. We found differences in change in median lifespan ranging from a 6% decrease to a 100% increase, as well as differences in maximum and relative lifespan extension and in mortality rate. Low temperature delays reproductive senescence in most strains, suggesting an extension of healthspan, even in strains with little to no change in lifespan. The combination of low temperature and caloric restriction in one strain resulted in an additive lifespan increase, indicating these interventions may work via non- or partially-overlapping pathways. The known low temperature sensor TRPA1 is present in the rotifer genome, but chemical TRPA1 agonists did not affect lifespan, suggesting that this gene may be involved in low temperature sensation but not in chemoreception in rotifers. The congeneric variability in response to low temperature suggests that the mechanism of low temperature lifespan extension is an active genetic process rather than a passive thermodynamic one and is dependent upon genotype.

Transcriptomes reflect the phenotypes of undifferentiated, granulocyte and macrophage forms of HL-60/S4 cells.

To understand the chromatin changes underlying differential gene expression during induced differentiation of human leukemic HL-60/S4 cells, we conducted RNA-Seq analysis on quadruplicate cultures of undifferentiated, granulocytic- and macrophage-differentiated cell forms. More than half of mapped genes exhibited altered transcript levels in the differentiated cell forms. In general, more genes showed increased mRNA levels in the granulocytic form and in the macrophage form, than showed decreased levels. The majority of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in genes that exhibited differential transcript levels after either RA or TPA treatment. Changes in transcript levels for groups of genes with characteristic protein phenotypes, such as genes encoding cytoplasmic granular proteins, nuclear envelope and cytoskeletal proteins, cell adhesion proteins, and proteins involved in the cell cycle and apoptosis illustrate the profound differences among the various cell states. In addition to the transcriptome analyses, companion karyotyping by M-FISH of undifferentiated HL-60/S4 cells revealed a plethora of chromosome alterations, compared with normal human cells. The present mRNA profiling provides important information related to nuclear shape changes (e.g., granulocyte lobulation), deformability of the nuclear envelope and linkage between the nuclear envelope and cytoskeleton during induced myeloid chromatin differentiation.

Nucleosome repositioning during differentiation of a human myeloid leukemia cell line.

Cell differentiation is associated with changes in chromatin organization and gene expression. In this study, we examine chromatin structure following differentiation of the human myeloid leukemia cell line (HL-60/S4) into granulocytes with retinoic acid (RA) or into macrophage with phorbol ester (TPA). We performed ChIP-seq of histone H3 and its modifications, analyzing changes in nucleosome occupancy, nucleosome repeat length, eu-/heterochromatin redistribution and properties of epichromatin (surface chromatin adjacent to the nuclear envelope). Nucleosome positions changed genome-wide, exhibiting a specific class of alterations involving nucleosome loss in extended (∼1kb) regions, pronounced in enhancers and promoters. Genes that lost nucleosomes at their promoters showed a tendency to be upregulated. On the other hand, nucleosome gain did not show simple effects on transcript levels. The average genome-wide nucleosome repeat length (NRL) did not change significantly with differentiation. However, we detected an approximate 10 bp NRL decrease around the haematopoietic transcription factor (TF) PU.1 and the architectural protein CTCF, suggesting an effect on NRL proximal to TF binding sites. Nucleosome occupancy changed in regions associated with active promoters in differentiated cells, compared with untreated HL-60/S4 cells. Epichromatin regions revealed an increased GC content and high nucleosome density compared with surrounding chromatin. Epichromatin showed depletion of major histone modifications and revealed enrichment with PML body-associated genes. In general, chromatin changes during HL-60/S4 differentiation appeared to be more localized to regulatory regions, compared with genome-wide changes among diverse cell types studied elsewhere.