Biology subjects

Pikaard, C.

Publications and source records attributed to Pikaard, C..

9 recordsLinked to original sources

bioRxiv2026

Epi-PoreC: a nanopore-based method for simultaneous profiling of chromatin conformation, DNA methylation, and chromatin accessibility

Understanding how the three-dimensional (3D) organization of the genome relates to chromatin and epigenetic landscapes requires methods capable of measuring chromosomal DNA interactions and chromatin modifications in an integrated manner. Current approaches typically use separate assays to assess chromatin conformation and epigenetic state, thus these features are not assessed on the same DNA molecules. Here, we describe Epi-PoreC, an Oxford Nanopore long-read sequencing-based method that combines the chromosome conformation capture method, PoreC with Fiber-seq, a method for detecting accessible chromatin regions based on their ability to be labeled using an exogenous adenosine methyltransferase. The positions of chromosome contacts, 6-methyladenosines, and endogenous 5-methylcytosines are then detected by nanopore sequencing, all on the same DNA molecules. Using Arabidopsis thaliana nuclei, we demonstrate that Epi-PoreC yields genome-wide chromatin contact maps comparable to those obtained by standard PoreC and DNA methylation and accessibility profiles comparable to standard Fiber-seq experiments. Epi-PoreC is thus an efficient means for conducting epigenetic and 3D genome profiling on individual DNA sequences in a single assay.

genomics↗

bioRxiv2023

Sequence and epigenetic landscapes of active and silenced nucleolus organizers in Arabidopsis

Arabidopsis thaliana has two ribosomal RNA gene loci, nucleolus organizer regions NOR2 and NOR4, whose complete sequences remain undefined. Ultra-long DNA sequences assembled using an unconventional approach yielded 5.5 and 3.9 Mbp sequences for NOR2 and NOR4 (in the reference strain, Col-0), revealing their distinct gene subtype compositions. RNA sequencing and identification of genes associated with flow-sorted nucleoli of wild-type or silencing-defective mutant plants shows that most of NOR4 is comprised of active genes whereas most, but not all, NOR2 genes are epigenetically silenced. Long intervals of low CG and CHG methylation overlap regions of gene activity and gene subtype homogenization. Collectively, the data reveal the genetic and epigenetic landscapes of the NORs and implicate transcription in rRNA gene concerted evolution. One Sentence SummaryNOR2 and NOR4 sequences fill genome gaps and enable megabase-scale analyses of rRNA gene regulation and concerted evolution.

genomics↗

bioRxiv2023

A Narrow Range of Transcript-error Rates Across the Tree of Life

The expression of genomically-encoded information is not error-free. Transcript-error rates are dramatically higher than DNA-level mutation rates, and despite their transient nature, the steady-state load of such errors must impose some burden on cellular performance. However, a broad perspective on the degree to which transcript-error rates are constrained by natural selection and diverge among lineages remains to be developed. Here, we present a genome-wide analysis of transcript-error rates across the Tree of Life using a modified rolling-circle sequencing method, revealing that the range in error rates is remarkably narrow across diverse species. Transcript errors tend to be randomly distributed, with little evidence supporting local control of error rates associated with gene-expression levels. A majority of transcript errors result in missense errors if translated, and as with a fraction of nonsense transcript errors, these are underrepresented relative to random expectations, suggesting the existence of mechanisms for purging some such errors. To quantitatively understand how natural selection and random genetic drift might shape transcript-error rates across species, we present a model based on cell biology and population genetics, incorporating information on cell volume, proteome size, average degree of exposure of individual errors, and effective population size. However, while this model provides a framework for understanding the evolution of this highly conserved trait, as currently structured it explains only 20% of the variation in the data, suggesting a need for further theoretical work in this area.

evolutionary biology↗

bioRxiv2023

Multimegabase-scale DNA hypermethylation and condensed chromatin correlate with chromosome-specific rRNA gene silencing in Arabidopsis

Arabidopsis thaliana has two nucleolus organizer regions (NORs), consisting of long tandem arrays of 45S rRNA genes on chromosomes 2 (NOR2) and 4 (NOR4). The rRNA gene subtypes mapping to NOR2 are mostly silenced during development, whereas most of those mapping to NOR4 are active. Cytosine methylation of promoters plays a significant role in rRNA gene silencing, but recent findings demonstrate that gene body methylation also play a key role in gene silencing. Previous studies have demonstrated that CG methylation plays a role in rRNA gene silencing. However, it remains to be determined whether Chromomethylase 2 (CMT2)-mediated, RdDM-independent CHH methylation plays any role in rRNA gene silencing. To find out the relative importance of cytosine methylation in each of the sequence contexts (CG, CHG, and CHH, where H=A, T, or C), we studied multiple DNA methylation-deficient Arabidopsis mutants. Primarily, we show that the mutants that displayed higher losses of CG or CHH methylation at rDNA loci, display higher degrees of NOR2 rRNA gene silencing disruption. Using whole genome bisulfite sequencing data, we show that the loss of RdDM-independent CMT2, not the loss of RdDM-dependent DRM2, erases most of the CHH methylation at rDNA loci, while retaining CG methylation levels comparable to those of wild-type Col-0, demonstrating the sufficiency of CHH methylation loss for releasing rRNA gene silencing. Analysis of Arabidopsis and tomato NOR sequences revealed that in 45S rRNA gene sequences, cytosines mostly occur in the CHH context, followed by CG and the lowest being in CHG contexts. The predominance of CHH sites is even more pronounced when the rRNA gene promoter and spacer promoter(s) are considered (0-7% CG, 14-25% CHG, and 79-80% CHH), suggesting a mechanism to explain the stronger disruption of NOR2 gene silencing in mutants that display higher losses of CHH methylation. Our data also reveal a role for gene body methylation in rRNA gene silencing. We show that NOR2 genes are relatively hypermethylated compared to NOR4, consistent with findings from recent studies. By contrast, human rRNA genes have more CG sites than CHH and CHG, correlating with the evolutionary loss of CMTs in mammals. CMTs are plant-specific, which primarily methylate CHG and CHH sites. Our data define a critical role for CMT2-mediated RdDM-independent CHH methylation, in combination with MET1-mediated CG methylation, in rRNA gene silencing, which is entirely mediated by CG methylation in mammals.

plant biology↗

bioRxiv2021

Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest or termination, involving the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The dsRNAs are then released from the Pol IV-RDR2 complex and diced into siRNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 [A], determined by cryo-electron microscopy. The N-terminal region contains an RNA-recognition motif (RRM) adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1-2 nucleotides (nt) internal to the 3 ends of its templates and can transcribe the RNA of an RNA-DNA hybrid provided that 9 or more nucleotides at the RNAs 3 end is unpaired. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3 end occurs as the DNA template and non-template strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3 end as the DNA strands reanneal, allowing RDR2 to engage the RNA and transcribe the second strand. SignificanceRDR2 is critical for siRNA-directed DNA methylation in Arabidopsis, functioning in physical association with DNA-dependent Pol IV to synthesize the second strands of double-stranded siRNA precursors. Basepairing between the DNA template strand transcribed by Pol IV and the nontemplate DNA strand is known to induce Pol IV arrest and Pol IV-RDR2 transcriptional coupling, but how this occurs is unknown. We report the structure of RDR2 and experimental evidence for how RDR2 engages its RNA templates and initiates transcription. RDR2 engages the ends of RNAs displaced from RNA-DNA hybrids, suggesting a model in which Pol IV arrest and backtracking, accompanied by DNA strand reannealing, extrudes the 3 end of the Pol IV transcript, allowing RNA engagement and second-strand synthesis.

molecular biology↗

bioRxiv2020

Assembly of a double-stranded RNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 docks with NUCLEAR RNA POLYMERASE IV at the clamp domain

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24 nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 co-immunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that RDR2 stably associates with Pol IVs largest catalytic subunit, NRPD1 at three sites, all within the clamp module. The clamp is a ubiquitous feature of DNA-dependent RNA polymerases that opens to allow DNA template entry and closes to encase the DNA-RNA hybrid adjacent to the RNA exit channel. The clamp also provides binding sites for polymerase-specific subunits or regulatory proteins, thus RDR2 binding to the Pol IV clamp is consistent with this theme. Within RDR2, the site of interaction with NRPD1 is very near the catalytic center. The locations of the NRPD1-RDR2 contact sites suggest a model in which transcripts emanating from Pol IVs RNA exit channel align with the template cleft of RDR2, facilitating rapid conversion of terminated Pol IV transcripts into double-stranded RNAs. Significance StatementShort interfering RNAs (siRNAs) play important roles in gene regulation by inhibiting mRNA translation into proteins or by guiding chromatin modifications that inhibit gene transcription. In plants, transcriptional gene silencing is guided by siRNAs derived from double-stranded (ds) RNAs generated by coupling the activities of DNA-dependent NUCLEAR RNA POLYMERASE IV and RNA-DEPENDENT RNA POLYMERASE 2. We show that the physical basis for Pol IV-RDR2 coupling is RDR2 binding to the clamp domain of Pol IVs largest subunit. The positions of the protein docking sites suggest that nascent Pol IV transcripts are generated in close proximity to RDR2s catalytic site, enabling rapid conversion of Pol IV transcripts into dsRNAs.

plant biology↗

bioRxiv2019

Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway

In eukaryotes with multiple small RNA pathways the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the siRNA biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV) whose atypical termination mechanism, induced by nontemplate DNA basepairing, channels transcripts to the associated RNA-dependent RNA polymerase, RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs then typically adds an extra untemplated 3 terminal nucleotide to the second strands. The dicer endonuclease, DCL3 cuts resulting duplexes to generate 24 and 23nt siRNAs. The 23nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling and RNA channeling from template DNA transcription to siRNA guide strand/passenger strand discrimination.

biochemistry↗

bioRxiv2017

Catalytic properties of RNA polymerases IV and V: accuracy, nucleotide incorporation and rNTP/dNTP discrimination

Catalytic subunits of DNA-dependent RNA polymerases of bacteria, archaea and eukaryotes share hundreds of ultra-conserved amino acids. Remarkably, the plant-specific RNA silencing enzymes, Pol IV and Pol V differ from Pols I, II and III at ~140 of these positions, yet remain capable of RNA synthesis. Whether these amino acid changes in Pols IV and V alter their catalytic properties in comparison to Pol II, from which they evolved, is unknown. Here, we show that Pols IV and V differ from one another, and Pol II, in nucleotide incorporation rate, transcriptional accuracy and the ability to discriminate between ribonucleotides and deoxyribonucleotides. Pol IV transcription is notably error-prone, which may be tolerable, or even beneficial, for biosynthesis of siRNAs targeting transposon families in trans. By contrast, Pol V exhibits high fidelity transcription, suggesting a need for Pol V transcripts to faithfully reflect the DNA sequence of target loci in order to recruit siRNA-Argonaute protein silencing complexes.

biochemistry↗

bioRxiv2017

Subdomains of the Pol V largest subunit CTD mediate locus-specific RNA-directed DNA methylation

Plant multisubunit RNA Polymerase V transcription recruits Argonaute siRNA complexes that specify sites of RNA-directed DNA methylation (RdDM) for gene silencing. Pol V's largest subunit, NRPE1, evolved from the largest subunit of Pol II but has a distinctive carboxyl-terminal domain (CTD). We show that the Pol V CTD is dispensable for catalytic activity in vitro, yet essential in vivo. One CTD subdomain (DeCL), is required for Pol V function at virtually all loci. other CTD subdomains have locusspecific effects. In a yeast two-hybrid screen, the 3'->5' exoribonuclease, RRP6L1 was identified as an interactor with the DeCL subdomain and DeCL and glutamine-serine-rich (QS) subdomains, located downstream from an Argonaute-binding repeat subdomain. Experimental evidence indicates that RRP6L1 trims the 3 ends of Pol V transcripts sliced by ARGONAUTE 4 (AGO4), suggesting a model whereby the CTD enables the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.

molecular biology↗