Biology subjects

Schmitz, R. J.

Publications and source records attributed to Schmitz, R. J..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Stability of DNA methylation and chromatin accessibility in structurally diverse maize genomes

Accessible chromatin and unmethylated DNA are associated with many genes and cis-regulatory elements. Attempts to understand natural variation for accessible chromatin regions (ACRs) and unmethylated regions (UMRs) often rely upon alignments to a single reference genome. This limits the ability to assess regions that are absent in the reference genome assembly and monitor how nearby structural variants influence variation in chromatin state. In this study, de novo genome assemblies for four maize inbreds (B73, Mo17, Oh43 and W22) are utilized to assess chromatin accessibility and DNA methylation patterns in a pan-genome context. The number of UMRs and ACRs that can be identified is more accurate when chromatin data is aligned to the matched genome rather than a single reference genome. While there are UMRs and ACRs present within genomic regions that are not shared between genotypes, these features are substantially enriched within shared regions, as determined by chromosomal alignments. Characterization of UMRs present within shared genomic regions reveals that most UMRs maintain the unmethylated state in other genotypes with only a small number being polymorphic between genotypes. However, the majority of UMRs between genotypes only exhibit partial overlaps suggesting that the boundaries between methylated and unmethylated DNA are dynamic. This instability is not solely due to sequence variation as these partially overlapping UMRs are frequently found within genomic regions that lack sequence variation. The ability to compare chromatin properties among individuals with structural variation enables pan-epigenome analyses to study the sources of variation for accessible chromatin and unmethylated DNA. Article summaryRegions of the genome that have accessible chromatin or unmethylated DNA are often associated with cis-regulatory elements. We assessed chromatin accessibility and DNA methylation in four structurally diverse maize genomes. There are accessible or unmethylated regions within the non-shared portions of the genomes but these features are depleted within these regions. Evaluating the dynamics of methylation and accessibility between genotypes reveals conservation of features, albeit with variable boundaries suggesting some instability of the precise edges of unmethylated regions.

plant biology↗

bioRxiv2021

De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes

We report de novo genome assemblies, transcriptomes, annotations, and methylomes for the 26 inbreds that serve as the founders for the maize nested association mapping population. The data indicate that the number of pan-genes exceeds 103,000 and that the ancient tetraploid character of maize continues to degrade by fractionation to the present day. Excellent contiguity over repeat arrays and complete annotation of centromeres further reveal the locations and internal structures of major cytological landmarks. We show that combining structural variation with SNPs can improve the power of quantitative mapping studies. Finally, we document variation at the level of DNA methylation, and demonstrate that unmethylated regions are enriched for cis-regulatory elements that overlap QTL and contribute to changes in gene expression. One sentence summaryA multi-genome analysis of maize reveals previously unknown variation in gene content, genome structure, and methylation.

genomics↗

bioRxiv2020

Unusual predominance of maintenance DNA methylation in Spirodela polyrhiza

5-methylcytosine (5mC) is a modified base often described as necessary for the proper regulation of genes and transposons and for the maintenance of genome integrity in plants. However, the extent of this dogma is limited by the current phylogenetic sampling of land plant species diversity. Here, we report that a monocot plant, Spirodela polyrhiza, has lost CG gene body methylation, genome-wide CHH methylation, and the presence or expression of several genes in the highly conserved RNA-directed DNA methylation (RdDM) pathway. It has also lost the CHH methyltransferase CHROMOMETHYLASE 2. Consequently, the transcriptome is depleted of 24-nucleotide, heterochromatic, small interfering RNAs that act as guides for the deposition of 5mC to RdDM-targeted loci in all other currently sampled angiosperm genomes. Although the genome displays low levels of genome-wide 5mC primarily at LTR retrotransposons, CG maintenance methylation is still functional. In contrast, CHG methylation is weakly maintained even though H3K9me2 is present at loci dispersed throughout the euchromatin and highly enriched at regions likely demarcating pericentromeric regions. Collectively, these results illustrate that S. polyrhiza is maintaining CG and CHG methylation mostly at repeats in the absence of small RNAs. S. polyrhiza reproduces rapidly through clonal propagation in aquatic environments, which we hypothesize may enable low levels of maintenance methylation to persist in large populations. Significance StatementDNA methylation is a widespread chromatin modification that is regularly found in all plant species. By examining one aquatic duckweed species, Spirodela polyrhiza, we find that it has lost highly conserved genes involved in methylation of DNA at sites often associated with repetitive DNA, and within genes, however DNA methylation and heterochromatin is maintained during cell division at other sites. Consequently, small RNAs that normally guide methylation to silence repetitive DNA like retrotransposons are diminished. Despite the loss of a highly conserved methylation pathway, and the reduction of small RNAs that normally target repetitive DNA, transposons have not proliferated in the genome, perhaps due in part to the rapid, clonal growth lifestyle of duckweeds.

plant biology↗

bioRxiv2020

A cis-regulatory atlas in maize at single-cell resolution

Cis-regulatory elements (CREs) encode the genomic blueprints of spatiotemporal gene expression programs enabling highly specialized cell functions. To identify CREs at cell-type resolution in Zea mays, we implemented single-cell sequencing of Assay for Transposase Accessible Chromatin (scATAC-seq) in seedlings, embryonic roots, crown roots, axillary buds, and pistillate and staminate inflorescence. We describe 92 states of chromatin accessibility across 165,913 putative CREs and 52 known cell types. Patterns of transcription factor (TF) motif accessibility predicted cell identity with high accuracy, uncovered putative non-cell autonomous TFs, and revealed TF motifs underlying higher-order chromatin interactions. Comparison of maize and Arabidopsis thaliana developmental trajectories identified TF motifs with conserved patterns of accessibility. Cell type-specific CREs were enriched with enhancer activity, phenotype-associated genetic variants, and signatures of breeding-era selection. These data, along with companion software, Socrates, afford a comprehensive framework for understanding cellular heterogeneity, evolution, and cis-regulatory grammar of cell-type specification in a major crop.

genetics↗

bioRxiv2020

The essential role of Dnmt1 in gametogenesis in the large milkweed bug Oncopeltus fasciatus

Given the importance of DNA methylation in protection of the genome against transposable elements and transcriptional regulation in other taxonomic groups, the diversity in both levels and patterns of DNA methylation in the insects raises questions about its function and evolution. We show that the maintenance DNA methyltransferase, DNMT1, affects meiosis and is essential to fertility in milkweed bugs, Oncopeltus fasciatus, while DNA methylation is not required in somatic cells. Our results support the hypothesis that Dnmt1 is required for the transition of germ cells to gametes in O. fasciatus and that this function is conserved in male and female gametogenesis. They further suggest that DNMT1 has a function independent of DNA methylation in germ cells. Our results raise the question of how a gene so critical in fitness across multiple insect species can have diverged widely across the insect tree of life.

evolutionary biology↗

bioRxiv2020

Stable unmethylated DNA demarcates expressed genes and their cis-regulatory space in plant genomes

The genomic sequences of crops continue to be produced at a frenetic pace. However, it remains challenging to develop complete annotations of functional genes and regulatory elements in these genomes. Here, we explore the potential to use DNA methylation profiles to develop more complete annotations. Using leaf tissue in maize, we define [~]100,000 unmethylated regions (UMRs) that account for 5.8% of the genome; 33,375 UMRs are found greater than 2 kilobase pairs from genes. UMRs are highly stable in multiple vegetative tissues and they capture the vast majority of accessible chromatin regions from leaf tissue. However, many UMRs are not accessible in leaf (leaf-iUMRs) and these represent a set of genomic regions with potential to become accessible in specific cell types or developmental stages. Leaf-iUMRs often occur near genes that are expressed in other tissues and are enriched for transcription factor (TF) binding sites of TFs that are also not expressed in leaf tissue. The leaf-iUMRs exhibit unique chromatin modification patterns and are enriched for chromatin interactions with nearby genes. The total UMRs space in four additional monocots ranges from 80-120 megabases, which is remarkably similar considering the range in genome size of 271 megabases to 4.8 gigabases. In summary, based on the profile from a single tissue, DNA methylation signatures pinpoint both accessible regions and regions poised to become accessible or expressed in other tissues. UMRs provide powerful filters to distill large genomes down to the small fraction of putative functional genes and regulatory elements. Significance StatementCrop genomes can be very large with many repetitive elements and pseudogenes. Distilling a genome down to the relatively small fraction of regions that are functionally valuable for trait variation can be like looking for needles in a haystack. The unmethylated regions in a genome are highly stable during vegetative development and can reveal the locations of potentially expressed genes or cis-regulatory elements. This approach provides a framework towards complete annotation of genes and discovery of cis-regulatory elements using methylation profiles from only a single tissue.

genomics↗

bioRxiv2020

Cis-regulatory elements within TEs can influence expression of nearby maize genes

Transposable elements (TEs) have the potential to create regulatory variation both through disruption of existing DNA regulatory elements and through creation of novel DNA regulatory elements. In a species with a large genome, such as maize, the many TEs interspersed with genes creates opportunities for significant allelic variation due to TE presence/absence polymorphisms among individuals. We used information on putative regulatory elements in combination with knowledge about TE polymorphisms in maize to identify TE insertions that interrupt existing accessible chromatin regions (ACRs) in B73 as well as examples of polymorphic TEs that contain ACRs among four inbred lines of maize including B73, Mo17, W22, and PH207. The TE insertions in three other assembled maize genomes (Mo17, W22 or PH207) that interrupt ACRs that are present in the B73 genome can trigger changes to the chromatin suggesting the potential for both genetic and epigenetic influences of these insertions. Nearly 20% of the ACRs located over 2kb from the nearest gene are located within an annotated TE. These are regions of unmethylated DNA that show evidence for functional importance similar to ACRs that are not present within TEs. Using a large panel of maize genotypes we tested if there is an association between the presence of TE insertions that interrupt, or carry, an ACR and the expression of nearby genes. TEs that carry ACRs exhibit an enrichment for being associated with higher expression of nearby genes, suggesting that these TEs may create novel regulatory elements. These analyses highlight the potential for TEs to rewire transcriptional responses in eukaryotic genomes. Data AvailabilityIn this study we utilize previously published datasets that are available through the following accessions: SRX4727413, SRR8738272, and SRR8740852.

plant biology↗

bioRxiv2020

The Gossypium longicalyx genome as a resource for cotton breeding and evolution

Cotton is an important crop that has made significant gains in production over the last century. Emerging pests such as the reniform nematode have threatened cotton production. The rare African diploid species Gossypium longicalyx is a wild species that has been used as an important source of reniform nematode immunity. While mapping and breeding efforts have made some strides in transferring this immunity to the cultivated polyploid species, the complexities of interploidal transfer combined with substantial linkage drag have inhibited progress in this area. Moreover, this species shares its most recent common ancestor with the cultivated A-genome diploid cottons, thereby providing insight into the evolution of long, spinnable fiber. Here we report a newly generated de novo genome assembly of G. longicalyx. This high-quality genome leveraged a combination of PacBio long-read technology, Hi-C chromatin conformation capture, and BioNano optical mapping to achieve a chromosome level assembly. The utility of the G. longicalyx genome for understanding reniform immunity and fiber evolution is discussed.

genomics↗

bioRxiv2019

The somatic genetic and epigenetic mutation rate in a wild long-lived perennial Populus trichocarpa

BackgroundPlants can transmit somatic mutations and epimutations to offspring, which in turn can affect fitness. Knowledge of the rate at which these variations arise is necessary to understand how plant development contributes to local adaption in an eco-evolutionary context, particularly in long-lived perennials. ResultsHere, we generated a new high-quality reference genome from the oldest branch of a wild Populus trichocarpa tree with two dominant stems which have been evolving independently for 330 years. By sampling multiple, age-estimated branches of this tree, we used a multi-omics approach to quantify age-related somatic changes at the genetic, epigenetic and transcriptional level. We show that the per-year somatic mutation and epimutation rates are lower than in annuals and that transcriptional variation is mainly independent of age divergence and cytosine methylation. Furthermore, a detailed analysis of the somatic epimutation spectrum indicates that transgenerationally heritable epimutations originate mainly from DNA methylation maintenance errors during mitotic rather than during meiotic cell divisions. ConclusionTaken together, our study provides unprecedented insights into the origin of nucleotide and functional variation in a long-lived perennial plant.

genomics↗

bioRxiv2019

AlphaBeta: Computational inference of epimutation rates and spectra from high-throughput DNA methylation data in plants

IntroductionHeritable changes in cytosine methylation can arise stochastically in plant genomes independently of DNA sequence alterations. These so-called spontaneous epimutations appear to be a byproduct of imperfect DNA methylation maintenance during mitotic or meitotic cell divisions. Accurate estimates of the rate and spectrum of these stochastic events are necessary to be able to quantify how epimutational processes shape methylome diversity in the context of plant evolution, development and aging. MethodHere we describe AlphaBeta, a computational method for estimating epimutation rates and spectra from pedigree-based high-throughput DNA methylation data. The approach requires that the topology of the pedigree is known, which is typically the case in the experimental construction of mutation accumulation lines (MA-lines) in sexually or clonally reproducing species. However, this method also works for inferring somatic epimutation rates in long-lived perennials, such as trees, using leaf methylomes and coring data as input. In this case, we treat the tree branching structure as an intra-organismal phylogeny of somatic lineages and leverage information about the epimutational history of each branch. ResultsTo illustrate the method, we applied AlphaBeta to multi-generational data from selfing- and asexually-derived MA-lines in Arabidopsis and dandelion, as well as to intra-generational leaf methylome data of a single poplar tree. Our results show that the epimutation landscape in plants is deeply conserved across angiosperm species, and that heritable epimutations originate mainly during somatic development, rather than from DNA methylation reinforcement errors during sexual reproduction. Finally, we also provide the first evidence that DNA methylation data, in conjunction with statistical epimutation models, can be used as a molecular clock for age-dating trees. ConclusionAlphaBeta faciliates unprecedented quantitative insights into epimutational processes in a wide range of plant systems. Software implementing our method is available as a Bioconductor R package at http://bioconductor.org/packages/3.10/bioc/html/AlphaBeta.html

genomics↗

bioRxiv2019

More than DNA methylation: does pleiotropy drive the complex pattern of evolution of Dnmt1?

DNA methylation is an important chromatin modification that can stably alter gene expression in cells and maintain genome integrity in plants and vertebrates. The function of DNA methylation outside of these well-studied systems, however, is unclear. Insects, in particular, represent an understudied group. Variation in the level of DNA methylation and gains and losses in the maintenance methyltransferase, DNMT1, across the insect tree of life suggests that there is much we dont understand about DMNT1 function and evolution. One constant across the studies examining patterns of Dnmt1 expression in insects is that expression is consistently high in reproductive tissues compared to somatic tissue. The explanation for this has been that DNMT1 is required in tissues that have high levels of cell division. Our previous study found that downregulation of Dnmt1 expression in the milkweed bug Oncopeltus fasciatus results in the expected reduction of DNA methylation, no global changes in gene expression reflecting changes in DNA methylation, and the loss of the ability to produce viable oocytes. Here, we show that females treated with ds-Dnmt1 RNA during larval development have a more extreme phenotype; they lack oocytes entirely but develop a normal somatic ovary. Our results indicate a specific role for DNMT1 in the formation of gametes and are consistent with data from other systems, including Tribolium castaneum, a species does not have DNA methylation. We propose that DNMT1 has multiple functional roles in addition to methylating DNA, which explains its complex patterns of evolution, and suggests that previous inferences of causation from associations are premature.

evolutionary biology↗

bioRxiv2019

Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

Base J, {beta}-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of promoters, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes important for host immune evasion. Our results provide the first direct mechanistic link between base J and regulation of Pol II termination and suggest a novel molecular model for the role of the CTD of Pol II in terminating polycistronic transcription in trypanosomatids.\n\nAuthor SummaryTrypanosoma brucei is an early-diverged parasitic protozoan that causes African sleeping sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J and H3.V leads to read-through transcription and the expression of downstream genes. We now identify a novel stable multimeric complex containing a J binding protein (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex regulates termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of specific proteins (including Pol II and termination factors) by PP1. These findings significantly expand our understanding of mechanisms underlying transcription termination in eukaryotes, including divergent organisms that utilize polycistronic transcription and novel epigenetic marks such as base J and H3.V. The studies also provide the first direct mechanistic link between J modification of DNA at termination sites and regulated Pol II termination and gene expression in kinetoplastids.

biochemistry↗

bioRxiv2019

Evolution of conserved noncoding sequences in Arabidopsis thaliana

Recent pangenome studies have revealed a large fraction of the gene content within a species exhibits presence-absence variation (PAV). However, coding regions alone provide an incomplete assessment of functional genomic sequence variation at the species level. Little to no attention has been paid to noncoding regulatory regions in pangenome studies, though these sequences directly modulate gene expression and phenotype. To uncover regulatory genetic variation, we generated chromosome-scale genome assemblies for thirty Arabidopsis thaliana accessions from multiple distinct habitats and characterized species level variation in Conserved Noncoding Sequences (CNS). Our analyses uncovered not only evidence for PAV and positional variation (PosV) but that diversity in CNS is non-random, with variants shared across different accessions. Using evolutionary analyses and chromatin accessibility data, we provide further evidence supporting roles for conserved and variable CNS in gene regulation. Characterizing species-level diversity in all functional genomic sequences may later uncover previously unknown mechanistic links between genotype and phenotype.

genomics↗

bioRxiv2018

Pericentromeric hypomethylation elicits an interferon response in an animal model of ICF syndrome

Pericentromeric satellite repeats are enriched in 5-methylcytosine (5mC). Loss of 5mC at these sequences is common in cancer and is a hallmark of Immunodeficiency, Centromere and Facial abnormalities (ICF) syndrome. While the general importance of 5mC is well-established, the specific functions of 5mC at pericentromeres are less clear. To address this deficiency, we generated a viable animal model of pericentromeric hypomethylation through mutation of the ICF-gene ZBTB24. Deletion of zebrafish zbtb24 caused a progressive loss of 5mC at pericentromeres and ICF-like phenotypes. Hypomethylation of these repeats triggered derepression of pericentromeric transcripts and activation of an interferon-based innate immune response. Injection of pericentromeric RNA is sufficient to elicit this response in wild-type embryos, and mutation of the MDA5-MAVS dsRNA-sensing machinery blocks the response in mutants. These findings identify activation of the innate immune system as an early consequence of pericentromeric hypomethylation, implicating derepression of pericentromeric transcripts as a trigger of autoimmunity.

molecular biology↗

bioRxiv2018

Gene-regulatory independent functions for insect DNA methylation

The function of cytosine (DNA) methylation in insects remains unknown. Here we provide evidence for the functional role of the maintenance DNA methyltransferase 1 (Dnmt1) in an insect using experimental manipulation. Through RNA interference (RNAi) we successfully post-transcriptionally knocked down Dnmt1 in ovarian tissue of the hemipteran Oncopeltus fasciatus (the large milkweed bug). Individuals depleted for Dnmt1, and subsequently DNA methylation, failed to reproduce. Eggs were inviable and declined in number, and nuclei structure of follicular epithelium was aberrant. Depletion of DNA methylation did not result in changes in gene or transposable element expression revealing an important function of DNA methylation seemingly not contingent on gene expression. Our work provides direct experimental evidence for a functional role of Dnmt1 and DNA methylation independent of gene expression in insects.

genetics↗

bioRxiv2018

R2C2: Improving nanopore read accuracy enables the sequencing of highly-multiplexed full-length single-cell cDNA

High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-read sequencing is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. However, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here we introduce and validate a new long-read ONT based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single cell samples.\n\nSignificance StatementSubtle changes in RNA transcript isoform expression can have dramatic effects on cellular behaviors in both health and disease. As such, comprehensive and quantitative analysis of isoform-level transcriptomes would open an entirely new window into cellular diversity in fields ranging from developmental to cancer biology. The R2C2 method we are presenting here is the first method with sufficient throughput and accuracy to make the comprehensive and quantitative analysis of RNA transcript isoforms in bulk and single cell samples economically feasible.

genomics↗

bioRxiv2017

Firefly genomes illuminate the origin and evolution of bioluminescence

Fireflies are among the best-studied of the bioluminescent organisms. Despite longterm interest in the biochemistry, neurobiology, and evolution of firefly flash signals and the widespread biotechnological applications of firefly luciferase, only a limited set of genes related to this complex trait have been described. To investigate the genetic basis of firefly bioluminescence, we generated a high-quality reference genome for the Big Dipper firefly Photinus pyralis, from which the first laboratory luciferase was cloned, using long-read (PacBio), short-read (Illumina), and Hi-C sequencing technologies. To facilitate comparative genomics, we also generated short-read genome assemblies for a Japanese firefly Aquatica lateralis and a bioluminescent click beetle, Ignelater luminosus. Analyses of these genomic datasets supports at least two independent gains of luminescence in beetles, and provides new insights into the evolution of beetle bioluminescence and chemical defenses that likely co-evolved over their 100 million years of evolution.

genomics↗

bioRxiv2017

Enhanced JBrowse plugins for epigenomics data visualization

New sequencing techniques require new visualization strategies, as is the case for epigenomics data such as DNA base modifications, small non-coding RNAs, and histone modifications. We present a set of plugins for the genome browser JBrowse that are targeted for epigenomics visualizations. Specifically, we have focused on visualizing DNA base modifications, small non-coding RNAs, stranded read coverage, and sequence motif density. Additionally, we present several plugins for improved user experience such as configurable, high-quality screenshots. In visualizing epigenomics with traditional genomics data, we see these plugins improving scientific communication and leading to discoveries within the field of epigenomics.

genomics↗