KAT2 paralogs prevent dsRNA accumulation and interferon signaling to maintain intestinal stem cells.

Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and dsRIP-seq were employed to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions as well as maintaining intestinal health.

Long-read genome assemblies for the study of chromosome expansion: Drosophila kikkawai, Drosophila takahashii, Drosophila bipectinata, and Drosophila ananassae.

Flow cytometry estimates of genome sizes among species of Drosophila show a 3-fold variation, ranging from ∼127 Mb in Drosophila mercatorum to ∼400 Mb in Drosophila cyrtoloma. However, the assembled portion of the Muller F element (orthologous to the fourth chromosome in Drosophila melanogaster) shows a nearly 14-fold variation in size, ranging from ∼1.3 Mb to >18 Mb. Here, we present chromosome-level long-read genome assemblies for 4 Drosophila species with expanded F elements ranging in size from 2.3 to 20.5 Mb. Each Muller element is present as a single scaffold in each assembly. These assemblies will enable new insights into the evolutionary causes and consequences of chromosome size expansion.

Evolutionary dynamics between transposable elements and their host genomes: mechanisms of suppression and escape.

Transposable elements (TEs) are ubiquitous among eukaryotic species. Their evolutionary persistence is likely due to a combination of tolerogenic, evasive/antagonistic, and cooperative interactions with their host genomes. Here, we focus on metazoan species and review recent advances related to the harmful effects of TE insertions, including how epigenetic effects and TE-derived RNAs can damage host cells. We discuss new findings related to host pathways that silence TEs, such as the piRNA pathway and the APOBEC3 and Kruppel-associated box zinc finger gene families. Finally, we summarize novel strategies used by TEs to evade host silencing, including the Y chromosome as a permissive niche for TE mobilization and TE counterdefense strategies to block host silencing factors.

Long-read genome assemblies for the study of chromosome expansion: Drosophila kikkawai , Drosophila takahashii , Drosophila bipectinata , and Drosophila ananassae.

Flow cytometry estimates of genome sizes among species of Drosophila show a 3-fold variation, ranging from ∼127 Mb in Drosophila mercatorum to ∼400 Mb in Drosophila cyrtoloma . However, the assembled portion of the Muller F Element (orthologous to the fourth chromosome in Drosophila melanogaster ) shows a nearly 14-fold variation in size, ranging from ∼1.3 Mb to > 18 Mb. Here, we present chromosome-level long read genome assemblies for four Drosophila species with expanded F Elements ranging in size from 2.3 Mb to 20.5 Mb. Each Muller Element is present as a single scaffold in each assembly. These assemblies will enable new insights into the evolutionary causes and consequences of chromosome size expansion.

Mode and Tempo of 3D Genome Evolution in Drosophila.

Topologically associating domains (TADs) are thought to play an important role in preventing gene misexpression by spatially constraining enhancer-promoter contacts. The deleterious nature of gene misexpression implies that TADs should, therefore, be conserved among related species. Several early studies comparing chromosome conformation between species reported high levels of TAD conservation; however, more recent studies have questioned these results. Furthermore, recent work suggests that TAD reorganization is not associated with extensive changes in gene expression. Here, we investigate the evolutionary conservation of TADs among 11 species of Drosophila. We use Hi-C data to identify TADs in each species and employ a comparative phylogenetic approach to derive empirical estimates of the rate of TAD evolution. Surprisingly, we find that TADs evolve rapidly. However, we also find that the rate of evolution depends on the chromatin state of the TAD, with TADs enriched for developmentally regulated chromatin evolving significantly slower than TADs enriched for broadly expressed, active chromatin. We also find that, after controlling for differences in chromatin state, highly conserved TADs do not exhibit higher levels of gene expression constraint. These results suggest that, in general, most TADs evolve rapidly and their divergence is not associated with widespread changes in gene expression. However, higher levels of evolutionary conservation and gene expression constraints in TADs enriched for developmentally regulated chromatin suggest that these TAD subtypes may be more important for regulating gene expression, likely due to the larger number of long-distance enhancer-promoter contacts associated with developmental genes.

Sex-specific variation in R-loop formation in Drosophila melanogaster.

R-loops are three-stranded nucleotide structures consisting of a DNA:RNA hybrid and a displaced ssDNA non-template strand. Previous work suggests that R-loop formation is primarily determined by the thermodynamics of DNA:RNA binding, which are governed by base composition (e.g., GC skew) and transcription-induced DNA superhelicity. However, R-loops have been described at genomic locations that lack these properties, suggesting that they may serve other context-specific roles. To better understand the genetic determinants of R-loop formation, we have characterized the Drosophila melanogaster R-loop landscape across strains and between sexes using DNA:RNA immunoprecipitation followed by high-throughput sequencing (DRIP-seq). We find that R-loops are associated with sequence motifs that are G-rich or exhibit G/C skew, as well as highly expressed genes, tRNAs, and small nuclear RNAs, consistent with a role for DNA sequence and torsion in R-loop specification. However, we also find motifs associated with R-loops that are A/T-rich and lack G/C skew as well as a subset of R-loops that are enriched in polycomb-repressed chromatin. Differential enrichment analysis reveals a small number of sex-biased R-loops: while non-differentially enriched and male-enriched R-loops form at similar genetic features and chromatin states and contain similar sequence motifs, female-enriched R-loops form at unique genetic features, chromatin states, and sequence motifs and are associated with genes that show ovary-biased expression. Male-enriched R-loops are most abundant on the dosage-compensated X chromosome, where R-loops appear stronger compared to autosomal R-loops. R-loop-containing genes on the X chromosome are dosage-compensated yet show lower MOF binding and reduced H4K16ac compared to R-loop-absent genes, suggesting that H4K16ac or MOF may attenuate R-loop formation. Collectively, these results suggest that R-loop formation in vivo is not fully explained by DNA sequence and topology and raise the possibility that a distinct subset of these hybrid structures plays an important role in the establishment and maintenance of epigenetic differences between sexes.

A transposon expression burst accompanies the activation of Y-chromosome fertility genes during Drosophila spermatogenesis.

Transposable elements (TEs) must replicate in germline cells to pass novel insertions to offspring. In Drosophila melanogaster ovaries, TEs can exploit specific developmental windows of opportunity to evade host silencing and increase their copy numbers. However, TE activity and host silencing in the distinct cell types of Drosophila testis are not well understood. Here, we reanalyze publicly available single-cell RNA-seq datasets to quantify TE expression in the distinct cell types of the Drosophila testis. We develop a method for identification of TE and host gene expression modules and find that a distinct population of early spermatocytes expresses a large number of TEs at much higher levels than other germline and somatic components of the testes. This burst of TE expression coincides with the activation of Y chromosome fertility factors and spermatocyte-specific transcriptional regulators, as well as downregulation of many components of the piRNA pathway. The TEs expressed by this cell population are specifically enriched on the Y chromosome and depleted on the X chromosome, relative to other active TEs. These data suggest that some TEs may achieve high insertional activity in males by exploiting a window of opportunity for mobilization created by the activation of spermatocyte-specific and Y chromosome-specific transcriptional programs.

The nuclear receptor HNF4 drives a brush border gene program conserved across murine intestine, kidney, and embryonic yolk sac.

The brush border is comprised of microvilli surface protrusions on the apical surface of epithelia. This specialized structure greatly increases absorptive surface area and plays crucial roles in human health. However, transcriptional regulatory networks controlling brush border genes are not fully understood. Here, we identify that hepatocyte nuclear factor 4 (HNF4) transcription factor is a conserved and important regulator of brush border gene program in multiple organs, such as intestine, kidney and yolk sac. Compromised brush border gene signatures and impaired transport were observed in these tissues upon HNF4 loss. By ChIP-seq, we find HNF4 binds and activates brush border genes in the intestine and kidney. H3K4me3 HiChIP-seq identifies that HNF4 loss results in impaired chromatin looping between enhancers and promoters at gene loci of brush border genes, and instead enhanced chromatin looping at gene loci of stress fiber genes in the intestine. This study provides comprehensive transcriptional regulatory mechanisms and a functional demonstration of a critical role for HNF4 in brush border gene regulation across multiple murine epithelial tissues.

Three-dimensional interactions between enhancers and promoters during intestinal differentiation depend upon HNF4.

Cells in renewing tissues exhibit dramatic transcriptional changes as they differentiate. The contribution of chromatin looping to tissue renewal is incompletely understood. Enhancer-promoter interactions could be relatively stable as cells transition from progenitor to differentiated states; alternatively, chromatin looping could be as dynamic as the gene expression from their loci. The intestinal epithelium is the most rapidly renewing mammalian tissue. Proliferative cells in crypts of Lieberkühn sustain a stream of differentiated cells that are continually shed into the lumen. We apply chromosome conformation capture combined with chromatin immunoprecipitation (HiChIP) and sequencing to measure enhancer-promoter interactions in progenitor and differentiated cells of the intestinal epithelium. Despite dynamic gene regulation across the differentiation axis, we find that enhancer-promoter interactions are relatively stable. Functionally, we find HNF4 transcription factors are required for chromatin looping at target genes. Depletion of HNF4 disrupts local chromatin looping, histone modifications, and target gene expression. This study provides insights into transcriptional regulatory mechanisms governing homeostasis in renewing tissues.

Telomeric TART elements target the piRNA machinery in Drosophila.

Coevolution between transposable elements (TEs) and their hosts can be antagonistic, where TEs evolve to avoid silencing and the host responds by reestablishing TE suppression, or mutualistic, where TEs are co-opted to benefit their host. The TART-A TE functions as an important component of Drosophila telomeres but has also reportedly inserted into the Drosophila melanogaster nuclear export factor gene nxf2. We find that, rather than inserting into nxf2, TART-A has actually captured a portion of nxf2 sequence. We show that TART-A produces abundant Piwi-interacting small RNAs (piRNAs), some of which are antisense to the nxf2 transcript, and that the TART-like region of nxf2 is evolving rapidly. Furthermore, in D. melanogaster, TART-A is present at higher copy numbers, and nxf2 shows reduced expression, compared to the closely related species Drosophila simulans. We propose that capturing nxf2 sequence allowed TART-A to target the nxf2 gene for piRNA-mediated repression and that these 2 elements are engaged in antagonistic coevolution despite the fact that TART-A is serving a critical role for its host genome.

3D genome evolution and reorganization in the Drosophila melanogaster species group.

Topologically associating domains, or TADs, are functional units that organize chromosomes into 3D structures of interacting chromatin. TADs play an important role in regulating gene expression by constraining enhancer-promoter contacts and there is evidence that deletion of TAD boundaries leads to aberrant expression of neighboring genes. While the mechanisms of TAD formation have been well-studied, current knowledge on the patterns of TAD evolution across species is limited. Due to the integral role TADs play in gene regulation, their structure and organization is expected to be conserved during evolution. However, more recent research suggests that TAD structures diverge relatively rapidly. We use Hi-C chromosome conformation capture to measure evolutionary conservation of whole TADs and TAD boundary elements between D. melanogaster and D. triauraria, two early-branching species from the melanogaster species group which diverged ∼15 million years ago. We find that the majority of TADs have been reorganized since the common ancestor of D. melanogaster and D. triauraria, via a combination of chromosomal rearrangements and gain/loss of TAD boundaries. TAD reorganization between these two species is associated with a localized effect on gene expression, near the site of disruption. By separating TADs into subtypes based on their chromatin state, we find that different subtypes are evolving under different evolutionary forces. TADs enriched for broadly expressed, transcriptionally active genes are evolving rapidly, potentially due to positive selection, whereas TADs enriched for developmentally-regulated genes remain conserved, presumably due to their importance in restricting gene-regulatory element interactions. These results provide novel insight into the evolutionary dynamics of TADs and help to reconcile contradictory reports related to the evolutionary conservation of TADs and whether changes in TAD structure affect gene expression.

Nanopore sequencing and Hi-C scaffolding provide insight into the evolutionary dynamics of transposable elements and piRNA production in wild strains of Drosophila melanogaster.

Illumina sequencing has allowed for population-level surveys of transposable element (TE) polymorphism via split alignment approaches, which has provided important insight into the population dynamics of TEs. However, such approaches are not able to identify insertions of uncharacterized TEs, nor can they assemble the full sequence of inserted elements. Here, we use nanopore sequencing and Hi-C scaffolding to produce de novo genome assemblies for two wild strains of Drosophila melanogaster from the Drosophila Genetic Reference Panel (DGRP). Ovarian piRNA populations and Illumina split-read TE insertion profiles have been previously produced for both strains. We find that nanopore sequencing with Hi-C scaffolding produces highly contiguous, chromosome-length scaffolds, and we identify hundreds of TE insertions that were missed by Illumina-based methods, including a novel micropia-like element that has recently invaded the DGRP population. We also find hundreds of piRNA-producing loci that are specific to each strain. Some of these loci are created by strain-specific TE insertions, while others appear to be epigenetically controlled. Our results suggest that Illumina approaches reveal only a portion of the repetitive sequence landscape of eukaryotic genomes and that population-level resequencing using long reads is likely to provide novel insight into the evolutionary dynamics of repetitive elements.

Adenine Methylation in Drosophila Is Associated with the Tissue-Specific Expression of Developmental and Regulatory Genes.

N6-methyladenine (6mA or m6dA) is a DNA modification that has long been known to play an important role in a variety of biological functions in prokaryotes. This modification has only recently been described in eukaryotes, where it seems to have evolved species-specific functions ranging from nucleosome positioning to transposon repression. In Drosophila, 6mA has been shown to be important for enforcing the tissue specificity of neuronal genes in the brain and suppressing transposable element expression in the ovaries. In this study, we have analyzed the raw signal data from nanopore sequencing to identify 6mA positions in the D. melanogaster genome at single-base resolution. We find that this modification is enriched upstream from transcription start sites, within the introns and 3' UTRs of genes, as well as in simple repeats. These 6mA positions are enriched for sequence motifs that are recognized by known transcriptional activators involved in development, such as Bicoid and Caudal, and the genes that carry this modification are enriched for functions involved in development, regulation of transcription, and neuronal activity. These genes show high expression specificity in a variety of tissues besides the brain, suggesting that this modification may play a more general role in enforcing the specificity of gene expression across many tissues, throughout development, and between the sexes.

Improving Operating Room Efficiency: First Case On-Time Start Project.

BACKGROUND: Operating rooms (ORs) are costly to run, and multiple factors influence efficiency. The first case on-time start (FCOS) of an OR is viewed as a harbinger of efficiency for the daily schedule. Across 26 ORs of a large, academic medical center, only 49% of cases started on time in October 2011. METHODS: The Perioperative Services Department engaged an interdisciplinary Operating Room Committee to apply Six Sigma tools to this problem. The steps of this project included (1) problem mapping, (2) process improvements to preoperative readiness, (3) informatics support improvements, and (4) continuous measurement and feedback. RESULTS: By June 2013, there was a peak of 92% first case on-time starts across service lines, decreasing to 78% through 2014, still significantly above the preintervention level of 49% (p = .000). Delay minutes also significantly decreased through the study period (p = .000). Across 2013, the most common delay owners were the patient, the surgeon, the facility, and the anesthesia department. CONCLUSIONS: Continuous and sustained improvement of first case on-time starts is attributed to tracking the FCOS metric, establishing embedded process improvement resources and creating transparency of data. This article highlights success factors and barriers to program success and sustainability.

Continental-level population differentiation and environmental adaptation in the mushroom Suillus brevipes.

Recent advancements in sequencing technology allowed researchers to better address the patterns and mechanisms involved in microbial environmental adaptation at large spatial scales. Here we investigated the genomic basis of adaptation to climate at the continental scale in Suillus brevipes, an ectomycorrhizal fungus symbiotically associated with the roots of pine trees. We used genomic data from 55 individuals in seven locations across North America to perform genome scans to detect signatures of positive selection and assess whether temperature and precipitation were associated with genetic differentiation. We found that S. brevipes exhibited overall strong population differentiation, with potential admixture in Canadian populations. This species also displayed genomic signatures of positive selection as well as genomic sites significantly associated with distinct climatic regimes and abiotic environmental parameters. These genomic regions included genes involved in transmembrane transport of substances and helicase activity potentially involved in cold stress response. Our study sheds light on large-scale environmental adaptation in fungi by identifying putative adaptive genes and providing a framework to further investigate the genetic basis of fungal adaptation.

Clonal reproduction in fungi.

Research over the past two decades shows that both recombination and clonality are likely to contribute to the reproduction of all fungi. This view of fungi is different from the historical and still commonly held view that a large fraction of fungi are exclusively clonal and that some fungi have been exclusively clonal for hundreds of millions of years. Here, we first will consider how these two historical views have changed. Then we will examine the impact on fungal research of the concept of restrained recombination [Tibayrenc M, Ayala FJ (2012) Proc Natl Acad Sci USA 109 (48):E3305-E3313]. Using animal and human pathogenic fungi, we examine extrinsic restraints on recombination associated with bottlenecks in genetic variation caused by geographic dispersal and extrinsic restraints caused by shifts in reproductive mode associated with either disease transmission or hybridization. Using species of the model yeast Saccharomyces and the model filamentous fungus Neurospora, we examine intrinsic restraints on recombination associated with mating systems that range from strictly clonal at one extreme to fully outbreeding at the other and those that lie between, including selfing and inbreeding. We also consider the effect of nomenclature on perception of reproductive mode and a means of comparing the relative impact of clonality and recombination on fungal populations. Last, we consider a recent hypothesis suggesting that fungi thought to have the most severe intrinsic constraints on recombination actually may have the fewest.

Genetic isolation between two recently diverged populations of a symbiotic fungus.

Fungi are an omnipresent and highly diverse group of organisms, making up a significant part of eukaryotic diversity. Little is currently known about the drivers of fungal population differentiation and subsequent divergence of species, particularly in symbiotic, mycorrhizal fungi. Here, we investigate the population structure and environmental adaptation in Suillus brevipes (Peck) Kuntze, a wind-dispersed soil fungus that is symbiotic with pine trees. We assembled and annotated the reference genome for Su. brevipes and resequenced the whole genomes of 28 individuals from coastal and montane sites in California. We detected two clearly delineated coast and mountain populations with very low divergence. Genomic divergence was restricted to few regions, including a region of extreme divergence containing a gene encoding for a membrane Na(+) /H(+) exchanger known for enhancing salt tolerance in plants and yeast. Our results are consistent with a very recent split between the montane and coastal Su. brevipes populations, with few small genomic regions under positive selection and a pattern of dispersal and/or establishment limitation. Furthermore, we identify a putatively adaptive gene that motivates further functional analyses to link genotypes and phenotypes and shed light on the genetic basis of adaptive traits.

Non-allelic gene conversion enables rapid evolutionary change at multiple regulatory sites encoded by transposable elements.

Transposable elements (TEs) allow rewiring of regulatory networks, and the recent amplification of the ISX element dispersed 77 functional but suboptimal binding sites for the dosage compensation complex to a newly formed X chromosome in Drosophila. Here we identify two linked refining mutations within ISX that interact epistatically to increase binding affinity to the dosage compensation complex. Selection has increased the frequency of this derived haplotype in the population, which is fixed at 30% of ISX insertions and polymorphic among another 41%. Sharing of this haplotype indicates that high levels of gene conversion among ISX elements allow them to 'crowd-source' refining mutations, and a refining mutation that occurs at any single ISX element can spread in two dimensions: horizontally across insertion sites by non-allelic gene conversion, and vertically through the population by natural selection. These results describe a novel route by which fully functional regulatory elements can arise rapidly from TEs and implicate non-allelic gene conversion as having an important role in accelerating the evolutionary fine-tuning of regulatory networks.

Structural variation among wild and industrial strains of Penicillium chrysogenum.

Strain selection and strain improvement are the first, and arguably most important, steps in the industrial production of biological compounds by microorganisms. While traditional methods of mutagenesis and selection have been effective in improving production of compounds at a commercial scale, the genetic changes underpinning the altered phenotypes have remained largely unclear. We utilized high-throughput Illumina short read sequencing of a wild Penicillium chrysogenum strain in order to make whole genome comparisons to a sequenced improved strain (WIS 54-1255). We developed an assembly-free method of identifying chromosomal rearrangements and validated the in silico predictions with a PCR-based assay and Sanger sequencing. Despite many rounds of mutagen treatment and artificial selection, WIS 54-1255 differs from its wild progenitor at only one of the identified rearrangements. We suggest that natural variants predisposed for high penicillin production were instrumental in the success of WIS 54-1255 as an industrial strain. In addition to finding a previously published inversion in the penicillin biosynthesis cluster, we located several genes related to penicillin production associated with these rearrangements. By comparing the configuration of rearrangement events among several historically important strains known to be high penicillin producers to a collection of recently isolated wild strains, we suggest that wild strains with rearrangements similar to those in known high penicillin producers may be viable candidates for further improvement efforts.

Discovering functions of unannotated genes from a transcriptome survey of wild fungal isolates.

Most fungal genomes are poorly annotated, and many fungal traits of industrial and biomedical relevance are not well suited to classical genetic screens. Assigning genes to phenotypes on a genomic scale thus remains an urgent need in the field. We developed an approach to infer gene function from expression profiles of wild fungal isolates, and we applied our strategy to the filamentous fungus Neurospora crassa. Using transcriptome measurements in 70 strains from two well-defined clades of this microbe, we first identified 2,247 cases in which the expression of an unannotated gene rose and fell across N. crassa strains in parallel with the expression of well-characterized genes. We then used image analysis of hyphal morphologies, quantitative growth assays, and expression profiling to test the functions of four genes predicted from our population analyses. The results revealed two factors that influenced regulation of metabolism of nonpreferred carbon and nitrogen sources, a gene that governed hyphal architecture, and a gene that mediated amino acid starvation resistance. These findings validate the power of our population-transcriptomic approach for inference of novel gene function, and we suggest that this strategy will be of broad utility for genome-scale annotation in many fungal systems. IMPORTANCE Some fungal species cause deadly infections in humans or crop plants, and other fungi are workhorses of industrial chemistry, including the production of biofuels. Advances in medical and industrial mycology require an understanding of the genes that control fungal traits. We developed a method to infer functions of uncharacterized genes by observing correlated expression of their mRNAs with those of known genes across wild fungal isolates. We applied this strategy to a filamentous fungus and predicted functions for thousands of unknown genes. In four cases, we experimentally validated the predictions from our method, discovering novel genes involved in the metabolism of nutrient sources relevant for biofuel production, as well as colony morphology and starvation resistance. Our strategy is straightforward, inexpensive, and applicable for predicting gene function in many fungal species.