Biology subjects

Hsieh, T.-F.

Publications and source records attributed to Hsieh, T.-F..

10 recordsLinked to original sources

bioRxiv2025

Multi-Omics analyses reveal dynamic interactions between DNA methylation and transcriptional regulation during black raspberry ripening

We profiled developmental-series transcriptome, methylome, and metabolome profiling to reveal extensive epigenetic reprogramming during black raspberry (Rubus occidentalis) fruit ripening. Fruit tissues exhibit globally higher DNA methylation than leaves, particularly in CHG and CHH contexts. Local methylation in all cytosine contexts progressively decreases in promoter regions during ripening, whereas CHG and CHH methylation increase in transposon-rich regions. Two primary methylation transitions--promoter hypomethylation and CHH hypermethylation--govern transcriptional shifts in genes involved in ripening processes. Methyl-binding transcription factors with activation potential likely promote CHH hypermethylation-linked transcriptional activation. Multi-omics integration revealed coordinated anthocyanin accumulation parallels expression of biosynthetic and regulatory genes within coherent networks. Elevated non-CG methylation in heterochromatin coincides with increased transcription of histone variants mediating chromatin compaction, suggesting chromatin remodelers fine-tune accessibility for methyltransferases. Our study highlights both genome-wide and locus-specific epigenetic reprogramming and demonstrates a coordinated interplay between DNA methylation and transcriptional regulation during black raspberry fruit ripening.

genomics↗

bioRxiv2025

Integrated Genomic and Methylome Profiling Reveals Promoter Repression and Age-Linked CpGs in the California Mussel

While most knowledge of animal DNA methylation comes from vertebrates, this epigenetic mark remains poorly understood in invertebrates, which comprise the majority of animal diversity. For instance, how promoter and gene body methylation contribute to gene regulation, and how methylation relates to aging, are still relatively unknown in most invertebrates. Focusing on the California mussel (Mytilus californianus), we paired whole-genome resequencing and whole-genome bisulfite sequencing from the same individuals and evaluated relationships among promoter methylation, gene body methylation, gene expression, and age. Using seven individuals spanning a range of body sizes from the Santa Barbara Channel, California, we found standing genetic variation levels similar to related species and a relatively small effective population size. CpG methylation was enriched in gene bodies, and gene body methylation was positively associated with expression. Promoter methylation was less frequent but showed a strong negative association with expression and remained the best predictor of repression after accounting for gene body methylation, aligning with patterns widely documented in vertebrates and adding to the limited evidence in invertebrates that promoter methylation can be regulatory. We identified thousands of age-associated differentially methylated loci with directional changes across age classes, providing candidate sites for epigenetic clocks that could enable assessment of biological age, health, and stress resilience in wild and cultured populations.

evolutionary biology↗

bioRxiv2023

Temporal Transcriptome Analysis Uncovers Regulatory Modules Programming Embryo Development from Embryonic Morphogenesis to Post-Germination

We profiled the soybean seed embryo transcriptome across embryonic development to post-germinative development to understand gene activities and regulatory networks promoting these processes. Transcriptomic landscapes feature highly prevalent transcripts which are categorized into early and late groups with major functions of reserve accumulation and energy generation, respectively, and both functions are dominant during late reserve accumulation as the transitioning stage. During the mid-reserve accumulation, regulatory events simultaneously dominate at the transcriptional and chromatin levels, followed by the emergence of distinct mRNA populations during late reserve accumulation throughout germination. We identified diverse functions conducted by sequentially activated genes across developmental stages. Gene coexpression network analysis reveals modules associated with developmental stages, some of which are enriched in genes with functions involved in specific developmental processes. We identified an early-desiccation-associated gene module, containing most transcription factors responsive to abiotic stress, within which one transcription factor is functionally validated to demonstrate increased drought tolerance in Arabidopsis overexpression lines. Finally, we found that a subset of genes is under purifying selection, surpasses the number of their Arabidopsis germination-specific homologs and most are active before germination from embryonic morphogenesis through dormancy, suggesting a potential role in governing physical dormancy in soybean compared to physiological dormancy in Arabidopsis. Our data represent a step toward identifying genes and regulatory networks in the soybean genome facilitating developmental programs across transition phases to bridge embryonic and germinative development.

plant biology↗

bioRxiv2023

Embryo-specific epigenetic mechanisms reconstitute the CHH methylation landscape during Arabidopsis embryogenesis

The modification of flowering plant DNA by CHH methylation acts primarily to silence transposable elements, of which many active copies are present in Arabidopsis thaliana. During embryogenesis, the CHH methylation landscape is dramatically reprogrammed, resulting in exceedingly high levels of this modification upon mature embryo formation. The mechanisms constituting the remodeling process, and its function in embryos, are unclear. Here, we isolate embryos from Arabidopsis plants harboring mutations for key components of the pathways that confer CHH methylation, namely RNA-directed DNA methylation (RdDM) and the Chromomethylase 2 (CMT2) pathways. We reveal that embryos are more methylated than leaves at shared CMT2 and RdDM targeting loci, accounting for most embryonic CHH hypermethylation. While the majority of embryo CHH methylated loci overlap with those in somatic tissues, a subset of conventional pericentric CMT2-methylated loci are instead targeted by RdDM in embryos. These loci, termed embRdDM exhibit intermediate H3K9me2 levels, associated with increased chromatin accessibility. Strikingly, more than 50% of the embRdDM loci in pollen vegetative (nurse) cells and ddm1 mutant somatic tissues are also targeted by RdDM, and these tissues were also reported to exhibit increased chromatin accessibility in pericentric heterochromatin. Furthermore, the root columella stem cell niche also displays CHH hypermethylation and an enriched presence of small RNAs at embRdDM loci. Finally, we observe a significant overlap of CHH hypermethylated loci with endosperm DEMETER targeting sites, suggesting that non-cell autonomous communication within the seed may contribute to the epigenetic landscape of the embryo. However, similar overlap with vegetative cell DEMETER targets indicates that the chromatin landscape that allows DEMETER access is mirrored in developing embryos, permitting CHH methylation catalysis at the same loci. Our findings demonstrate that both conserved and embryo-specific epigenetic mechanisms reshape CHH methylation profiles in the dynamic chromatin environment of embryogenesis.

plant biology↗

bioRxiv2023

H2A.X mutants exhibit enhanced DNA demethylation in Arabidopsis thaliana

H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction, though it is not known how FACT targets DME sites. Here, we investigated whether H2AX is involved in DME- and FACT-mediated DNA demethylation during Arabidopsis reproduction. We show that h2a.x mutants are more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to the Green Fluorescent Protein (GFP) gene under the H2A.X promoter was highly expressed in newly developing Arabidopsis tissues, including in male and female gametophytes. We examined DNA methylation in h2a.x developing seeds using whole genome bisulfite sequencing, and found that CG DNA methylation in the developing endosperm, but not the embryo, is decreased genome-wide in h2a.x mutants, predominately in transposons and intergenic DNA. Hypomethylated sites overlapped 62 % with canonical DME loci. These data indicate that H2A.X is not required for DME function, but is important for DNA methylation homeostasis in the unique chromatin environment of Arabidopsis endosperm.

plant biology↗

bioRxiv2022

Loss of Linker Histone H1 in the Maternal Genome Influences DEMETER-Mediated Demethylation and Affects the Endosperm DNA Methylation Landscape

The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the central cell genome prior to fertilization. This epigenetic reconfiguration of the female gamete companion cell establishes gene imprinting in the endosperm and is essential for seed viability. DME demethylates small and genic-flanking transposons as well as intergenic and heterochromatin sequences, but how DME is recruited to these target loci remains unknown. H1.2 was identified as a DME-interacting protein in a yeast two-hybrid screen, and maternal genome H1 loss affects DNA methylation and expression of selected imprinted genes in the endosperm. Yet, the extent to which how H1 influences DME demethylation and gene imprinting in the Arabidopsis endosperm has not been investigated. Here, we showed that unlike in the vegetative cell, both canonical histone H1 variants are present in the central cell. Our endosperm methylome analysis revealed that without the maternal linker histones, DME-mediated demethylation is facilitated, particularly in the heterochromatin regions, indicating that H1-containing nucleosomes are barriers for DME demethylation. Loss of H1 in the maternal genome has a very limited effect on gene transcription or gene imprinting regulation in the endosperm; however, it variably influences euchromatin TE methylation and causes a slight hypermethylation and a reduced expression in selected imprinted genes. We conclude that loss of maternal H1 indirectly influences DME-mediated demethylation and endosperm DNA methylation landscape but does not appear to affect endosperm gene transcription and overall imprinting regulation.

plant biology↗

bioRxiv2021

A merger between compatible but divergent genomes supports allopolyploidization in the Brassicaceae family

Hybridization and polyploidization are pivotal to plant evolution. Genetic crosses between distantly related species rarely occur in nature mainly due to reproductive barriers but how such hurdles can be overcome is largely unknown. xBrassicoraphanus is a fertile intergeneric allopolyploid synthesized between Brassica rapa and Raphanus sativus in the Brassicaceae family. Genomes of B. rapa and R. sativus are diverged enough to suppress synapsis formation between non-homologous progenitor chromosomes during meiosis, and we found that both genomes reside in the single nucleus of xBrassicoraphanus without genome loss or rearrangement. Expressions of syntenic orthologs identified in B. rapa and R. sativus were adjusted to a hybrid nuclear environment of xBrassicoraphanus, which necessitates reconfiguration of transcription network by rewiring cis-trans interactions. B. rapa coding sequences have a higher level of gene-body methylation than R. sativus, and such methylation asymmetry is maintained in xBrassicoraphanus. B. rapa-originated transposable elements were transcriptionally silenced in xBrassicoraphanus, rendered by gain of CHG methylation in trans via small RNAs derived from the same sequences of R. sativus subgenome. Our work proposes that not only transcription compatibility but also a certain extent of genome divergence supports hybrid genome stabilization, which may explain great diversification and expansion of angiosperms during evolution.

plant biology↗

bioRxiv2021

Comparative phylogenomic analysis reveals evolutionary genomic changes and novel toxin families in endophytic Liberibacter pathogens

Liberibacter pathogens are the causative agents of several severe crop diseases worldwide, including citrus Huanglongbing and potato Zebra Chip. These bacteria are endophytic and non-culturable, which makes experimental approaches challenging and highlights the need for bioinformatic analysis in advancing our understanding about Liberibacter pathogenesis. Here, we performed an in-depth comparative phylogenomic analysis of the Liberibacter pathogens and their free-living, nonpathogenic, ancestral species, aiming to identify the major genomic changes and determinants associated with their evolutionary transitions in living habitats and pathogenicity. We found that prophage loci represent the most variable regions among Liberibacter genomes. Using gene neighborhood analysis and phylogenetic classification, we systematically recovered, annotated, and classified all prophage loci into four types, including one previously unrecognized group. We showed that these prophages originated through independent gene transfers at different evolutionary stages of Liberibacter and only the SC-type prophage was associated with the emergence of the pathogens. Using ortholog clustering, we vigorously identified two additional sets of genomic genes, which were either lost or gained in the ancestor of the pathogens. Consistent with the habitat change, the lost genes were enriched for biosynthesis of cellular building blocks. Importantly, among the gained genes, we uncovered several previously unrecognized toxins, including a novel class of polymorphic toxins, a YdjM phospholipase toxin, and a secreted EEP protein. Our results substantially extend the knowledge on the evolutionary events and potential determinants leading to the emergence of endophytic, pathogenic Liberibacter species and will facilitate the design of functional experiments and the development of new detection and blockage methods of these pathogens. ImportanceLiberibacter pathogens are associated with several severe crop diseases, including citrus Huanglongbing, the most destructive disease to the citrus industry. Currently, no effective cure or treatments are available, and no resistant citrus variety has been found. The fact that these obligate endophytic pathogens are not culturable has made it extremely challenging to experimentally uncover from the whole genome the genes/proteins important to Liberibacter pathogenesis. Further, earlier bioinformatics studies failed to identify the key genomic determinants, such as toxins and effector proteins, that underlie the pathogenicity of the bacteria. In this study, an in-depth comparative genomic analysis of Liberibacter pathogens together with their ancestral non-pathogenic species identified the prophage loci and several novel toxins that are evolutionarily associated with the emergence of the pathogens. These results shed new lights on the disease mechanism of Liberibacter pathogens and will facilitate the development of new detection and blockage methods targeting the toxins.

microbiology↗

bioRxiv2019

The Catalytic Core of DEMETER Guides Active DNA Demethylation in Arabidopsis

The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization, and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, and how it is recruited to these differing chromatin landscapes is unknown. The C-terminal DME catalytic core consists of three conserved regions required for catalysis in vitro. We show that the catalytic core of DME guides active demethylation at endogenous targets, rescuing the developmental and genomic hypermethylation phenotypes of DME mutants. However, without the N-terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. We used comparative analysis to reveal that the conserved DME N-terminal domains are only present in the flowering plants, whereas the domain architecture of DME-like proteins in non-vascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in all plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N-terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.

plant biology↗

bioRxiv2017

The roles of Conserved Domains in DEMETER-Mediated Active DNA Demethylation in planta

DNA methylation plays critical roles in maintaining genome stability, genomic imprinting, transposon silencing, and development. In Arabidopsis genomic imprinting is established in the central cell by DEMETER (DME)-mediated active DNA demethylation, and is essential for seed viability. DME is a large polypeptide with multiple poorly characterized conserved domains. Here we show that the C-terminal enzymatic core of DME is sufficient to complement dme associated developmental defects. When targeted by a native DME promoter, nuclear-localized DME C-terminal region rescues dme seed abortion and pollen germination defects, and ameliorates CG hypermethylation phenotype in dme-2 endosperm. Furthermore, targeted expression of the DME N-terminal region in wild-type central cell induces dme-like seed abortion phenotype. Our results support a bipartite organization for DME protein, and suggest that the N-terminal region might have regulatory function such as assisting in DNA binding and enhancing the processivity of active DNA demethylation in heterochromatin targets.

plant biology↗