Biology subjects

Jacobsen, S. E.

Publications and source records attributed to Jacobsen, S. E..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

MORC-1 is a key component of the C. elegans CSR-1 germline gene licensing mechanism

The Argonaute CSR-1 is essential for germline development in C. elegans. Mutation of csr-1 downregulates thousands of germline-expressed genes, leading to the model that the CSR-1-mediated small RNA pathway promotes, or "licenses," gene expression by an unknown mechanism. CSR-1 also silences a limited number of genes through its canonical endonucleolytic "slicer" activity. We show that the GHKL-type ATPase MORC-1, a CSR-1 slicing target, over-accumulates at CSR-1 "licensed" target genes in csr-1(-), which correlates with ectopic gain of H3K9me3, H3K36me3 loss, and gene downregulation. Loss of morc-1 rescues csr-1(-) defects, while overexpressing MORC-1 in the germline of wild-type worms is sufficient to cause sterility and downregulate CSR-1 targets. These results show that MORC-1 overexpression in csr-1(-) is a primary driver of the CSR-1-mediated gene licensing mechanism. One-Sentence SummaryMORC-1 acts downstream of CSR-1 to regulate germline chromatin states and is a key component of the gene licensing mechanism.

molecular biology↗

bioRxiv2024

Viral delivery of an RNA-guided genome editor for transgene-free plant germline editing

AbstractGenome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. But most viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here, we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.

plant biology↗

bioRxiv2023

MBD2 couples DNA methylation to Transposable Elements silencing during male gametogenesis.

DNA methylation is an essential component of transposable element (TE) silencing, yet the mechanism by which methylation causes transcriptional repression remains poorly understood1-5. Here we study the Arabidopsis thaliana Methyl-CpG Binding Domain (MBD) proteins MBD1, MBD2, and MBD4, and show that MBD2 acts as a transposable element (TE) repressor during male gametogenesis. MBD2 bound chromatin regions containing high levels of CG methylation, and MBD2 was capable of silencing the FWA gene when tethered to its promoter. MBD2 loss caused TE activation in the vegetative cell (VC) of mature pollen without affecting DNA methylation levels, demonstrating that MBD2-mediated silencing acts strictly downstream of DNA methylation. Loss of silencing in mbd2 became more significant in the mbd5 mbd6 or adcp1 mutant backgrounds, as well as in plants with chemically induced genome-wide DNA demethylation, suggesting that MBD2 acts redundantly with other silencing pathways to safeguard TEs from activation. Overall, our study identifies MBD2 as a novel methyl reader that acts downstream of DNA methylation to silence TEs during male gametogenesis.

plant biology↗

bioRxiv2023

Enhancers associated with unstable RNAs are rare in plants

Unstable transcripts have emerged as markers of active enhancers in vertebrates and shown to be involved in many cellular processes and medical disorders. However, their prevalence and role in plants is largely unexplored. Here, we comprehensively captured all actively initiating ("nascent") transcripts across diverse crops and other plants using capped small (cs)RNA-seq. We discovered that unstable transcripts are rare, unlike in vertebrates, and often originate from promoters. Additionally, many "distal" elements in plants initiate tissue-specific stable transcripts and are likely bone fide promoters of yet-unannotated genes or non-coding RNAs, cautioning against using genome annotations to infer "enhancers" or transcript stability. To investigate enhancer function, we integrated STARR-seq data. We found that annotated promoters, and other regions that initiate stable transcripts rather than unstable transcripts, function as stronger enhancers in plants. Our findings underscore the blurred line between promoters and enhancers and suggest that cis-regulatory elements encompass diverse structures and mechanisms in eukaryotes.

genomics↗

bioRxiv2023

ACD15, ACD21, and SLN regulate accumulation and mobility of MBD6 to silence genes and transposable elements.

DNA methylation mediates silencing of transposable elements and genes in part via recruitment of the Arabidopsis MBD5/6 complex, which contains the methyl-CpG-binding domain (MBD) proteins MBD5 and MBD6, and the J-domain containing protein SILENZIO (SLN). Here we characterize two additional complex members: -crystalline domain containing proteins ACD15 and ACD21. We show that they are necessary for gene silencing, bridge SLN to the complex, and promote higher order multimerization of MBD5/6 complexes within heterochromatin. These complexes are also highly dynamic, with the mobility of complex components regulated by the activity of SLN. Using a dCas9 system, we demonstrate that tethering the ACDs to an ectopic site outside of heterochromatin can drive massive accumulation of MBD5/6 complexes into large nuclear bodies. These results demonstrate that ACD15 and ACD21 are critical components of gene silencing complexes that act to drive the formation of higher order, dynamic assemblies. One-Sentence SummaryArabidopsis ACD21 and ACD15 drive accumulation of MBD5/6 complex silencing assemblies at methyl-CG sites and recruit SLN to maintain protein mobility in these assemblages.

cell biology↗

bioRxiv2023

H1 restricts euchromatin-associated methylation pathways from heterochromatic encroachment

Silencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT DNA methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localized to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localization in an h1 mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase, Pol V. Loss of H1 resulted in NRPE1 enrichment predominantly in heterochromatic TEs. Increased NRPE1 binding was associated with increased chromatin accessibility in h1, suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an h1/nrpe1 double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in h1 corresponded to loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposition correlated with increased methylation specifically in the CHH context. Additionally, we found that H1 similarly restricts the occupancy of the methylation reader, SUVH1, and polycomb-mediated H3K27me3. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways.

plant biology↗

bioRxiv2023

The MOM1 complex recruits the RdDM machinery via MORC6 to establish de novo DNA methylation.

MOM1 is an Arabidopsis factor previously shown to mediate transcriptional silencing independent of major DNA methylation changes. Here we found that MOM1 localizes with sites of RNA-directed DNA methylation (RdDM). Tethering MOM1 with artificial zinc finger to unmethylated FWA promoter led to establishment of DNA methylation and FWA silencing. This process was blocked by mutations in components of the Pol V arm of the RdDM machinery, as well as by mutation of MORC6. We found that at some endogenous RdDM sites, MOM1 is required to maintain DNA methylation and a closed chromatin state. In addition, efficient silencing of newly introduced FWA transgenes was impaired by mutation of MOM1 or mutation of genes encoding the MOM1 interacting PIAL1/2 proteins. In addition to RdDM sites, we identified a group of MOM1 peaks at active chromatin near genes that colocalized with MORC6. These findings demonstrate a multifaceted role of MOM1 in genome regulation.

plant biology↗

bioRxiv2023

Arabidopsis TRB proteins function in H3K4me3 demethylation by recruiting JMJ14

Arabidopsis telomeric repeat binding factors (TRBs) can bind telomeric DNA sequences to protect telomeres from degradation. TRBs can also recruit Polycomb Repressive Complex 2 (PRC2) to deposit tri-methylation of H3 lysine 27 (H3K27me3) over certain target loci. Here, we demonstrate that TRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger H3K4me3 demethylation at some loci. The trb1/2/3 triple mutant and the jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their target genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger (TRB-ZF) successfully triggers target gene silencing, as well as H3K27me3 deposition, and H3K4me3 removal. Interestingly, JMJ14 is predominantly recruited to ZF off-target sites with low levels of H3K4me3, which is accompanied with TRB-ZFs triggered H3K4me3 removal at these loci. These results suggest that TRB proteins coordinate PRC2 and JMJ14 activities to repress target genes via H3K27me3 deposition and H3K4me3 removal.

plant biology↗

bioRxiv2022

A gene silencing screen uncovers diverse tools for targeted gene repression in Arabidopsis

DNA methylation has been utilized for target gene silencing in plants, however its not well-understood whether other silencing pathways can be also used to manipulate gene expression. Here we performed a gain of function screen for proteins that could silence a target gene when fused to an artificial zinc finger. We uncovered many proteins that suppressed gene expression either through the establishment of DNA methylation, or via DNA methylation-independent processes including histone H3K27me3 deposition, H3K4me3 demethylation, H3K9, H3K14, H3K27, and H4K16 deacetylation, inhibition of RNA Polymerase II transcription elongation or Ser-5 dephosphorylation. The silencing fusion proteins also silenced many other genes with different efficacy, and a machine learning model could accurately predict the efficacy of each silencer based on various chromatin features of the target loci. These results provide a more comprehensive understanding of epigenetic regulatory pathways and provide an armament of tools for targeted manipulation of gene expression.

plant biology↗

bioRxiv2022

MORC proteins regulate transcription factor binding by mediating chromatin compaction in active chromatin regions

BackgroundThe Microrchidia (MORC) proteins are a family of evolutionarily conserved GHKL-type ATPases involved in chromatin compaction and gene silencing. Arabidopsis MORC proteins act in the RNA-directed DNA methylation (RdDM) pathway, where they act as molecular tethers to ensure the efficient establishment of RdDM and de novo gene silencing. However, MORC proteins also have RdDM-independent functions; although, their underlying mechanisms are unknown. ResultsIn this study, we examined regions of MORC binding where RdDM does not occur in order to shed light on the RdDM-independent functions of MORC proteins. We found that MORC proteins compact chromatin and reduce DNA accessibility to transcription factors (TFs), thereby repressing gene expression. We also found that MORC-mediated repression of gene expression was particularly important under conditions of stress. We showed that MORC proteins regulate TFs through either direct or indirect interactions, and these TFs can in some cases regulate their own transcription, resulting in feedforward loops. ConclusionsOur findings provide insights into the molecular mechanisms of MORC-mediated chromatin compaction and transcription regulation.

plant biology↗

bioRxiv2022

Genome editing in plants using the compact editor Cas{Phi}

CRISPR-Cas systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact Cas{Phi} nuclease is able to generate stably inherited gene edits in Arabidopsis, and that Cas{Phi} guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele we show that Cas{Phi} displays higher editing efficiency when the target locus is not DNA methylated, suggesting that Cas{Phi} is sensitive to chromatin environment. Importantly, two Cas{Phi} protein variants, vCas{Phi} and nCas{Phi}, both showed much higher editing efficiency relative to the wildtype Cas{Phi} enzyme, and yielded more offspring plants with inherited edits. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that Cas{Phi} is highly specific. The hypercompact size, T-rich minimal PAM and wide range of working temperatures make Cas{Phi} an excellent supplement to existing plant genome editing systems. Significance StatementPlant genome engineering with CRISPR-Cas systems is frequently used in both research and agriculture. Here, we demonstrate that the hypercompact Cas{Phi}-2 nuclease is able to generate heritable gene edits in Arabidopsis. Two Cas{Phi} protein variants vCas{Phi} and nCas{Phi} increased the editing efficiency in plants. Cas{Phi} also has a wide range of working temperatures and the editing by Cas{Phi} is highly specific. We also observed that editing by Cas{Phi} is sensitive to chromatin environment. The hypercompact size, T-rich minimal PAM and wide range of working temperatures make Cas{Phi} an excellent supplement to existing plant genome editing systems.

plant biology↗

bioRxiv2022

Single-nucleus RNA-seq reveals that MBD5, MBD6, and SILENZIO maintain silencing during epigenetic reprogramming in pollen

Silencing of transposable elements (TEs) drove the evolution of numerous redundant mechanisms of transcriptional regulation. Arabidopsis MBD5, MBD6, and SILENZIO act as TE repressors downstream of DNA methylation. Here we show via single-nucleus RNA-seq of developing male gametophytes that these repressors are critical for TE silencing in the pollen vegetative cell, which undergoes epigenetic reprogramming causing chromatin decompaction to support fertilization by sperm cells. Instead, other silencing mutants (met1, ddm1, mom1, morc) show loss of silencing in all pollen nucleus types and somatic cells. We found that TEs repressed by MBD5/6 gain accessibility in wild-type vegetative nuclei despite remaining silent, suggesting that loss of DNA compaction makes them sensitive to loss of MBD5/6. Consistently, crossing mbd5/6 to histone 1 mutants, which have decondensed chromatin in leaves, reveals derepression of MBD5/6-dependent TEs in leaves. MBD5/6 and SILENZIO thus act as a silencing system especially important when chromatin compaction is compromised.

molecular biology↗

bioRxiv2022

Histone chaperone ASF1 mediates H3.3-H4 deposition in Arabidopsis

Histone chaperones and chromatin remodelers control nucleosome dynamics, which are essential for transcription, replication, and DNA repair. The histone chaperone Anti-Silencing Factor 1 (ASF1) plays a central role in facilitating CAF-1-mediated replication-dependent H3.1 deposition and HIRA-mediated replication-independent H3.3 deposition in yeast and metazoans. Whether ASF1 function is evolutionarily conserved in plants is unknown. Here, we show that Arabidopsis ASF1 proteins display a preference for the HIRA complex. Simultaneous mutation of both Arabidopsis ASF1 genes caused a decrease in chromatin density and ectopic H3.1 occupancy at loci typically enriched with H3.3. Genetic, transcriptomic, and proteomic data indicate that ASF1 proteins strongly prefers the HIRA complex over CAF-1. asf1 mutants also displayed an increase in spurious Pol II transcriptional initiation and showed defects in the maintenance of gene body CG DNA methylation and in the distribution of histone modifications. Furthermore, ectopic targeting of ASF1 caused excessive histone deposition, less accessible chromatin, and gene silencing. These findings reveal the importance of ASF1-mediated histone deposition for proper epigenetic regulation of the genome.

plant biology↗

bioRxiv2022

Haplotype resolved DNA methylome of African cassava genome

Cytosine DNA methylation is involved in biological processes such as transposable element (TE) silencing, imprinting, and X chromosome inactivation. Plant methylation is mediated by MET1 (mammalian DNMT1), DRM2 (mammalian DNMT3), and two plant-specific DNA methyltransferases, CMT2 and CMT3 (Law and Jacobsen, 2010). De novo DNA methylation in plants is established by DRM2 via the plant specific RNA-directed DNA methylation (RdDM) pathway that depends on two DNA-dependent RNA polymerases, Pol IV and Pol V (Gallego-Bartolome et al., 2019; Law and Jacobsen, 2010; Stroud et al., 2013). The DNA methylome of cassava has been previously documented based on its haploid collapsed genome (Wang et al., 2015). Since the cassava genome is highly heterozygous, DNA methylome analysis of the haplotype-collapsed genome misses many features of the methylome. With the development of long read sequencing and chromosomal conformation capture techniques, haplotype resolved genomes are available for highly heterozygous genomes (Mansfeld et al., 2021; Qi et al., 2022; Zhou et al., 2020), which provides high-quality reference genomes facilitating studies of haplotype resolved DNA methylomes.

plant biology↗

bioRxiv2022

Loss of H3.1K27me1 in Arabidopsis confers resistance to Geminivirus by sequestering DNA repair proteins onto rDNA and defense-related genes

The H3 methyltransferases ATXR5 and ATXR6 deposit H3.1K27me1 to heterochromatin to prevent genomic instability and transposon reactivation. Here, we report that atxr5 atxr6 mutants displayed robust resistance to Geminivirus. The viral resistance correlated with activation of DNA repair pathways, but not with transposon reactivation or heterochromatin amplification. We identified RAD51 and RPA1A as partners of virus-encoded Rep protein. The two DNA repair proteins showed increased binding to heterochromatic regions and defense-related genes in atxr5 atxr6 vs wild type plants. Consequently, the proteins had reduced interactions to viral DNA in the mutant, thus hampering viral replication. Additionally, RAD51 recruitment to the host genome arose via BRCA1, HOP2 and CYCB1, and this recruitment was essential for viral resistance in atxr5 atxr6. Thus, Geminiviruses adapt to healthy plants by hijacking its DNA repairing pathways for replication, but the host could retain DNA repairing proteins via sacrificing its genome stability to suppress viral infection.

genetics↗

bioRxiv2022

Drosophila Males Use 5'-to-3' Phased Biogenesis to Make Stellate-silencing piRNAs that Lack Homology to Maternally Deposited piRNA Guides

PIWI-interacting RNAs (piRNAs) direct PIWI proteins to silence complementary targets such as transposons. In animals with a maternally specified germline, e.g. Drosophila melanogaster, maternally deposited piRNAs initiate piRNA biogenesis in the progeny. Normal fertility in D. melanogaster males requires repression of tandemly repeated Stellate genes by piRNAs from Suppressor of Stellate [Su(Ste)]. Because the Su(Ste) loci are on the Y chromosome, Su(Ste) piRNAs are not deposited in oocytes. How the male germline produces Su(Ste) piRNAs in the absence of maternally deposited Su(Ste) piRNAs is unknown. Here, we show that Su(Ste) piRNAs are made in the early male germline via 5'-to-3' phased piRNA biogenesis triggered by maternally deposited 1360/Hoppel transposon piRNAs. Strikingly, deposition of Su(Ste) piRNAs from XXY mothers obviates the need for phased piRNA biogenesis in sons. Together, our study uncovers the developmentally programmed mechanism that allows fly mothers to protect their sons using a Y-linked piRNA locus.

developmental biology↗

bioRxiv2022

Mutations in DNA polymerase δ subunit 1 mediate CMD2-type resistance to Cassava Mosaic Geminiviruses

Cassava mosaic disease suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to the cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for loss of resistance, we performed whole genome sequencing and genetic variant analysis and fine-mapped the CMD2 locus to a 190 kilobase interval. Data suggest that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase {delta} subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a Cassava mosaic disease-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional resistance alleles within MePOLD1. MePOLD1 resistance alleles represent important genetic resources for resistance breeding or genome editing, and elucidating mechanisms of resistance to geminiviruses.

plant biology↗

bioRxiv2022

Improving disease resistance in plants by editing the epigenome

Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas phaseoli pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. In this work, we direct methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display decreased CBB symptoms while maintaining normal growth and development. This work therefore presents an epigenome editing approach useful for crop improvement.

plant biology↗