Biology subjects

Gent, J. I.

Publications and source records attributed to Gent, J. I..

16 recordsLinked to original sources

bioRxiv2026

Maternal repression of imprinted genes in endosperm by gene body demethylation

Removal of TE-like DNA methylation from regulatory elements drives canonical genomic imprinting of both maternally and paternally expressed genes in endosperm (MEGs and PEGs). Here we report that removal of CG-only gene body methylation also drives imprinting in maize, resulting in maternally repressed PEGs. These CG body methylated PEGs express broadly across cell types and lack H3K27me3 except in endosperm, where maternal copies acquire it. We found that partial gene body demethylation is a widespread feature of endosperm genes, usually does not result in imprinted expression, and is mediated by DNA glycosylases/lyases (DNGs). These and other data indicate that gene body methylation can shield select genes from PRC2 repression, and its maternal removal by DNGs explains the majority of PEGs in maize and rice but has weak impact in Arabidopsis thaliana. We hypothesize that increased gene expression noise arising from gene body demethylation--both with and without imprinting--is beneficial in endosperm.

genetics↗

bioRxiv2025

TE-like DNA methylation of highly expressed endosperm genes in maize

Unlike the stable transcription of plant genes with CG gene body methylation alone, genes with methylation in TE-like CHG and CG contexts are poorly transcribed and often missanotated. In contrast, here we describe a set of TE-like methylated genes that are maternally demethylated and expressed toward the extreme high end of the spectrum in endosperm. They are enriched for short, secreted proteins, and about a third encode zeins, the major seed storage proteins of maize. Consistent with their dynamic expression in endosperm, they acquire either activating or repressive gene regulatory modifications when demethylated but are heterochromatic when methylated in other tissues. The majority are expressed both maternally and paternally, but a subset whose methylation/demethylation extends upstream of gene bodies into promoters are strongly imprinted. These and other data indicate that TE-like methylation and associated heterochromatin can be a signature of broad silencing but exceptionally high and specific gene expression in either pollen or endosperm.

molecular biology↗

bioRxiv2025

Antagonistic kinesin-14s within a single chromosomal drive haplotype

In maize, there are two meiotic drive systems that operate on large tandem repeat arrays called knobs that are found on chromosome arms. One meiotic drive haplotype, Abnormal chromosome 10 (Ab10), encodes two kinesin proteins that interact with two distinct tandem repeat arrays in a sequence-specific manner to confer meiotic drive. The kinesin KINDR associates with knob180 repeats while the kinesin TRKIN associates with TR-1 repeats. Prior data show that meiotic drive is conferred primarily by the KINDR/knob180 system, with the TRKIN/TR-1 system having little or no role. The second meiotic drive haplotype, K10L2, shows low levels of meiotic drive and only encodes the TRKIN/TR-1 system. Here we used long-read sequencing to assemble the K10L2 haplotype and showed that it has strong homology to an internal portion of the Ab10 haplotype. We also carried out CRISPR mutagenesis of Trkin to test the role of Trkin on Ab10 and K10L2. The data indicate that the Trkin gene on Ab10 does not improve drive or fitness but instead has a weak deleterious effect when paired with a normal chromosome 10. The deleterious effect is more severe when Ab10 is paired with K10L2: in this context functional Trkin on either chromosome nearly abolishes Ab10 drive. We modeled the effect of Trkin on Ab10 and found it should not persist in the population. We conclude that Trkin either confers an advantage to Ab10 in untested circumstances or that it is in the process of being purged from the Ab10 population. ARTICLE SUMMARYMendels first law states that paired chromosomes are transmitted through meiosis at equal frequencies. Some chromosome variants, however, are transmitted at higher frequencies in a process called meiotic drive. We wanted to know the function of a motor protein called TRKIN that is encoded on a maize meiotic drive chromosome. Surprisingly, we found that TRKIN provides no advantage to the meiotic driver and instead seems to be deleterious, suggesting it had a function in a wild ancestor but is now being purged from the population. The results illustrate how genomes are shaped by often-conflicting forces of selection and selfish genetic elements.

evolutionary biology↗

bioRxiv2025

Increased maize chromosome number by engineered chromosome fission

Activation of synthetic centromeres on chromosome 4 in maize leads to its breakage and formation of trisomic fragments called neochromosomes. A limitation of neochromosomes is their low and unpredictable transmission rates due to trisomy. Here we report that selecting for dicentric recombinants through male crosses uncovers stabilized chromosome 4 fission events which split it into 4a-4b complementary chromosome pairs, where 4a carries a native centromere and 4b a synthetic one. The cells rapidly stabilized chromosome ends by de novo telomere formation and the new centromeres spread among genes without altering their expression. When both 4a and 4b chromosomes were present in a homozygous state, they segregated through meiosis indistinguishably from wild-type, and gave rise to healthy plants with normal seed set. This work leverages synthetic centromeres to engineer chromosome fission, effectively raising the diploid chromosome number of maize from 20 to 22. TEASERUsing synthetic centromeres, we divide one maize chromosome into two chromosomes, and show that both chromosomes are fully functional.

synthetic biology↗

bioRxiv2024

MDR1 DNA glycosylase regulates the expression of genomically imprinted genes and helitrons

Targeted demethylation by DNA glycosylases (DNGs) results in differential methylation between parental alleles in the endosperm, which drives imprinted expression. Here, we performed RNA sequencing on endosperm derived from DNG mutant mdr1 and wild-type endosperm. Consistent with the role of DNA methylation in gene silencing, we find 96 gene and 86 TE differentially expressed (DE) transcripts that lost expression in the hypermethylated mdr1 mutant. Compared with other endosperm transcripts, the mdr1 targets are enriched for TEs (particularly Helitrons), and DE genes are depleted for both core genes and GO term assignments, suggesting that the majority of DE transcripts are TEs and pseudo-genes. By comparing DE genes to imprinting calls from prior studies, we find that the majority of DE genes have maternally biased expression, and approximately half of all maternally expressed genes (MEGs) are DE in this study. In contrast, no paternally expressed genes (PEGs) are DE. DNG-dependent imprinted genes are distinguished by maternal demethylation and expression primarily in the endosperm, so we also performed EM-seq on hybrids to identify maternal demethylation and utilized a W22 gene expression atlas to identify genes expressed primarily in the endosperm. Overall, approximately [2/3] of all MEGs show evidence of regulation by DNA glycosylases. Taken together, this study solidifies the role of MDR1 in the regulation of maternally expressed, imprinted genes and TEs and identifies subsets of genes with DNG-independent imprinting regulation. Significance StatementThis work investigates the transcriptome changes resulting from the loss of function of DNA glycosylase MDR1, revealing that, in wild-type endosperm, targets of MDR1 are expressed predominantly from the maternal allele and this expression is suppressed in mutants. Furthermore, by combining expression data, DNA methylation data, and developmental expression data, we are able to categorize all maternally expressed, imprinted genes based on DNA glycosylase dependent or independent regulatory methods.

plant biology↗

bioRxiv2024

Potent pollen gene regulation by DNA glycosylases in maize

Although DNA methylation primarily represses TEs, it also represses select genes that are methylated in plant body tissues but demethylated by DNA glycosylases (DNGs) in endosperm or pollen. Activity of either one of two DNGs, MDR1 or DNG102, is essential for pollen viability in maize. Using single-pollen mRNA sequencing on pollen segregating mutations in both genes, we identified 58 candidate DNG target genes that account for 11.1% of the wild-type transcriptome but are silent or barely detectable in the plant body (sporophyte). They are unusual in their tendency to lack introns but even more so in their having TE-like methylation in their CDS. The majority have predicted functions in cell wall modification, and they likely support the rapid tip growth characteristic of pollen tubes. These results suggest a critical role for DNA methylation and demethylation in regulating maize genes with potential for extremely high expression in pollen but constitutive silencing elsewhere.

genetics↗

bioRxiv2023

The maize striate leaves2 (sr2) gene encodes a conserved DUF3732 domain homologous to the rice yss1 gene

Maize striate leaves2 (sr2) is a mutant that causes white stripes on leaves that has been used in mapping studies for decades, though the underlying gene has not been identified. The sr2 locus has been previously mapped to small regions of the normal chromosome 10 (N10) and a rearranged variant called Abnormal Chromosome 10 (Ab10). A comparison of assembled genomes carrying N10 and Ab10 revealed only five candidate sr2 genes. Analysis of a stock carrying the sr2 reference allele (sr2-ref) showed that one of the five genes has a transposon insertion that disrupts its protein sequence and has a severe reduction in mRNA. An independent Mutator transposon insertion in the gene (sr2-Mu) failed to complement the sr2-ref mutation, and plants homozygous for sr2-Mu showed white striped leaf margins. The sr2 gene encodes a DUF3732 protein with strong homology to a rice gene with a similar mutant phenotype called young seedling stripe1 (yss1). These and other published data suggest that sr2 may have a function in plastid gene expression.

genetics↗

bioRxiv2023

RdDM mutants reduce histone methylation rather than DNA methylation at the paramutated maize b1 enhancer

Paramutation is the transfer of mitotically and meiotically heritable silencing information between two alleles. With paramutation at the maize booster1 (b1) locus, the low expressed B epiallele heritably changes the high expressed B-I epiallele into B with 100% frequency. This requires specific tandem repeats and multiple components of the RNA-directed DNA methylation (RdDM) pathway, including the RNA-dependent RNA polymerase (RdRP, mediator of paramutation1, MOP1), the second-largest subunit of RNA polymerase IV and V (NRP(D/E)2a, mediator of paramutation2, MOP2), and the largest subunit of RNA Polymerase IV (NRPD1, mediator of paramutation3, MOP3). Mutations in mop genes prevent paramutation and release silencing at the B epiallele. In this study we investigated the effect of mutations in mop1, mop2 and mop3 on chromatin structure and DNA methylation at the B epiallele, and especially the regulatory hepta-repeat 100 kb upstream of the b1 gene. We show that mutations in mop1 and mop3 result in decreased repressive histone modifications H3K9me2 and H3K27me2 at the hepta-repeat. Associated with this decrease are partial activation of the hepta-repeat enhancer function, formation of a multi-loop structure, and elevated b1 expression. In mop2 mutants, which do not show elevated b1 expression, H3K9me2 and H3K27me2 and a single-loop structure like in wild type B are retained. Surprisingly, high DNA methylation levels at the B hepta-repeat remains in all three mutants. Our results raise the possibility of MOP factors mediating RNA-directed histone methylation rather than RNA-directed DNA methylation at the b1 locus.

plant biology↗

bioRxiv2022

Differences in activity and stability drive transposable element variation in tropical and temperate maize.

Much of the profound interspecific variation in genome content has been attributed to transposable elements (TEs). To explore the extent of TE variation within species, we developed an optimized open-source algorithm, panEDTA, to de novo annotate TEs in a pan-genome context. We then generated a unified TE annotation for a maize pan-genome derived from 26 reference-quality genomes, which revealed an excess of 35.1 Mb of TE sequences per genome in tropical maize relative to temperate maize. A small number (n = 216) of TE families, mainly LTR retrotransposons, drive these differences. Evidence from the methylome, transcriptome, LTR age distribution, and LTR insertional polymorphisms revealed that 64.7% of the variability was contributed by LTR families that were young, less methylated, and more expressed in tropical maize, while 18.5% was driven by LTR families with removal or loss in temperate maize. This study demonstrates the use of a comprehensive pan-TE annotation to reveal the driving role of TEs in within-species genomic variation via their ongoing amplification and purging.

genomics↗

bioRxiv2022

Synthetic maize centromeres transmit chromosomes across generations

Centromeres are long, often repetitive regions of genomes that bind kinetochore proteins and ensure normal chromosome segregation. Engineering centromeres that function in vivo has proven to be difficult. Here we describe a LexA-CENH3 tethering approach that activates functional centromeres at maize synthetic repeat arrays containing LexO binding sites. The synthetic centromeres are sufficient to cause chromosome breakage and release of chromosome fragments that are passed through meiosis and into progeny. Several independent chromosomes were identified, each with newly created centromeres localized over the repeat arrays where they were directed. The new centromeres were self-sustaining and stably transmitted chromosomes to progeny in the absence of the LexA-CENH3 activator. Our results demonstrate the feasibility of using synthetic centromeres for karyotype engineering applications.

genetics↗

bioRxiv2021

The maize gene maternal depression of r1 (mdr1) encodes a DNA glycosylase with maternal and paternal fertility functions

Demethylation of transposons can activate expression of nearby genes and cause imprinted gene expression in endosperm, and it is hypothesized to lead to expression of transposon siRNAs that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting are its main function in endosperm.

genetics↗

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

Resetting of 24-nt siRNA landscape is initiated in the unicellular zygote in rice

BackgroundThe zygote, a totipotent stem cell, constitutes a critical stage of the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells -- the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Due to technical challenges, the epigenetic changes that accompany the zygotic transition are poorly understood. ResultsHere, we characterized the small RNA transcriptome of rice zygotes. We found widespread redistribution of 24-nt siRNAs relative to gametes, including absence of sperm signature siRNAs, reduction at egg signature siRNA loci, and upregulation at seedling signature siRNA loci. Loci with reduced siRNAs in zygote relative to egg were gene-distal and heterochromatic, while loci with increased siRNAs relative to egg had a similar genomic distribution to canonical siRNA loci. Although both egg and zygote siRNA loci had higher mCHH level in wildtype than in drm2 embryo, zygote but not egg siRNA loci were associated with hypermethylation in mature embryo. A small fraction of siRNA loci (~1%) called siren loci accounted for 60% of all siRNAs within zygote siRNA loci, that likely arose from maternal carryover as they had similarly abundant siRNAs in egg; these siren loci were not associated with embryo hypermethylation. ConclusionsTaken together, our results indicate re-distribution of siRNAs in rice zygotes towards the canonical vegetative profile, that are consistent with the initiation of resetting of the gametic epigenome before the first embryonic division.

plant biology↗

bioRxiv2020

Gapless assembly of maize chromosomes using long read technologies

Creating gapless telomere-to-telomere assemblies of complex genomes is one of the ultimate challenges in genomics. We used long read technologies and an optical map based approach to produce a maize genome assembly composed of only 63 contigs. The B73-Ab10 genome includes gapless assemblies of chromosome 3 (236 Mb) and chromosome 9 (162 Mb), multiple highly repetitive centromeres and heterochromatic knobs, and 53 Mb of the Ab10 meiotic drive haplotype.

bioinformatics↗

bioRxiv2019

Reprogramming of 24nt siRNAs in rice gametes

Gametes constitute a critical stage of the plant life cycle, during which the genome undergoes reprogramming in preparation for embryogenesis. Here we characterized the small RNA transcriptomes of egg cells and sperm cells from rice to elucidate genome-wide distributions of 24nt siRNAs, which are a hallmark of RNA-directed DNA methylation (RdDM) in plants and are typically concentrated at boundaries of heterochromatin. We found that 24nt siRNAs were depleted from heterochromatin boundaries in both gametes, reminiscent of siRNA patterns in DDM1-type nucleosome remodeler mutants. In sperm, 24nt siRNAs were spread across broad heterochromatic regions, while in eggs, 24nt siRNAs were concentrated at a smaller number of heterochromatic loci throughout the genome, which were shared with vegetative tissues and sperm. In both gametes, patterns of CHH methylation, typically a strong indicator of RdDM, were similar to vegetative tissues, although lower in magnitude. These findings indicate that the small RNA transcriptome undergoes large-scale re-programming in both male and female gametes, which is not correlated with recruitment of DNA methyltransferases in gametes and suggestive of unexplored regulatory activities of gamete small RNAs in seeds after fertilization.

molecular biology↗

bioRxiv2018

Loss of mCHH islands in maize chromomethylase and DDM1-type nucleosome remodeler mutants

Plants make use of three types of DNA methylation, each characterized by distinct DNA methyltransferases. One type, RNA-directed DNA methylation (RdDM), is guided by siRNAs to the edges of transposons that are close to genes, areas called mCHH islands in maize. Another type, chromomethylation, is guided by histone H3 lysine 9 methylation to heterochromatin across the genome. We examined DNA methylation and small RNA expression in plant tissues that were mutant for both copies of the genes encoding chromomethylases as well as mutants for both copies of the genes encoding DDM1-type nucleosome remodelers, which facilitate chromomethylation. Both sets of double mutants were nonviable but produced embryos and endosperm. RdDM was severely compromised in the double mutant embryos, both in terms of DNA methylation and siRNAs. Loss of 24nt siRNA from mCHH islands was coupled with a gain of 21, 22, and 24nt siRNAs in heterochromatin. These results reveal a requirement for both chromomethylation and DDM1-type nucleosome remodeling for RdDM in mCHH islands, which we hypothesize is due to dilution of RdDM components across the genome when heterochromatin is compromised.

genetics↗