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1. Local Rather than Global H3K27me3 Dynamics Are Associated with Differential Gene Expression in Verticillium dahliae

   https://doi.org/10.1128/mbio.03566-21  

   Kramer, H. Martin ; Seidl, Michael F. ; Thomma, Bart P. ; Cook, David E. ( February 2022 , mBio)

   Fudal, Isabelle ; Di Pietro, Antonio (Ed.)

   ABSTRACT Differential growth conditions typically trigger global transcriptional responses in filamentous fungi. Such fungal responses to environmental cues involve epigenetic regulation, including chemical histone modifications. It has been proposed that conditionally expressed genes, such as those that encode secondary metabolites but also effectors in pathogenic species, are often associated with a specific histone modification, lysine27 methylation of H3 (H3K27me3). However, thus far, no analyses on the global H3K27me3 profiles have been reported under differential growth conditions in order to assess if H3K27me3 dynamics govern differential transcription. Using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing data from the plant-pathogenic fungus Verticillium dahliae grown in three in vitro cultivation media, we now show that a substantial number of the identified H3K27me3 domains globally display stable profiles among these growth conditions. However, we observe local quantitative differences in H3K27me3 ChIP-seq signals that are associated with a subset of differentially transcribed genes between media. Comparing the in vitro results to expression during plant infection suggests that in planta -induced genes may require chromatin remodeling to achieve expression. Overall, our results demonstrate that some loci display H3K27me3 dynamics associated with concomitant transcriptional variation, but many differentially expressed genes are associated with stable H3K27me3 domains. Thus,more »we conclude that while H3K27me3 is required for transcriptional repression, it does not appear that transcriptional activation requires the global erasure of H3K27me3. We propose that the H3K27me3 domains that do not undergo dynamic methylation may contribute to transcription through other mechanisms or may serve additional genomic regulatory functions. IMPORTANCE In many organisms, including filamentous fungi, epigenetic mechanisms that involve chemical and physical modifications of DNA without changing the genetic sequence have been implicated in transcriptional responses upon developmental or environmental cues. In fungi, facultative heterochromatin that can decondense to allow transcription in response to developmental changes or environmental stimuli is characterized by the trimethylation of lysine 27 on histone H3 (H3K27me3), and H3K27me3 has been implicated in transcriptional regulation, although the precise mechanisms and functions remain enigmatic. Based on ChIP and RNA sequencing data, we show for the soilborne broad-host-range vascular wilt plant-pathogenic fungus Verticillium dahliae that although some loci display H3K27me3 dynamics that can contribute to transcriptional variation, other loci do not show such a dependence. Thus, although we recognize that H3K27me3 is required for transcriptional repression, we also conclude that this mark is not a conditionally responsive global regulator of differential transcription upon responses to environmental cues.« less
2. Decoding co-/post-transcriptional complexities of plant transcriptomes and epitranscriptome using next-generation sequencing technologies

   https://doi.org/10.1042/BST20190492  

   Reddy, Anireddy S.N. ; Huang, Jie ; Syed, Naeem H. ; Ben-Hur, Asa ; Dong, Suomeng ; Gu, Lianfeng ( November 2020 , Biochemical Society Transactions)

   Next-generation sequencing (NGS) technologies - Illumina RNA-seq, Pacific Biosciences isoform sequencing (PacBio Iso-seq), and Oxford Nanopore direct RNA sequencing (DRS) - have revealed the complexity of plant transcriptomes and their regulation at the co-/post-transcriptional level. Global analysis of mature mRNAs, transcripts from nuclear run-on assays, and nascent chromatin-bound mRNAs using short as well as full-length and single-molecule DRS reads have uncovered potential roles of different forms of RNA polymerase II during the transcription process, and the extent of co-transcriptional pre-mRNA splicing and polyadenylation. These tools have also allowed mapping of transcriptome-wide start sites in cap-containing RNAs, poly(A) site choice, poly(A) tail length, and RNA base modifications. The emerging theme from recent studies is that reprogramming of gene expression in response to developmental cues and stresses at the co-/post-transcriptional level likely plays a crucial role in eliciting appropriate responses for optimal growth and plant survival under adverse conditions. Although the mechanisms by which developmental cues and different stresses regulate co-/post-transcriptional splicing are largely unknown, a few recent studies indicate that the external cues target spliceosomal and splicing regulatory proteins to modulate alternative splicing. In this review, we provide an overview of recent discoveries on the dynamics and complexities of plant transcriptomes,more »mechanistic insights into splicing regulation, and discuss critical gaps in co-/post-transcriptional research that need to be addressed using diverse genomic and biochemical approaches.« less
3. IMITATION SWITCH is required for normal chromatin structure and gene repression in PRC2 target domains

   https://doi.org/10.1073/pnas.2010003118  

   Kamei, Masayuki ; Ameri, Abigail J. ; Ferraro, Aileen R. ; Bar-Peled, Yael ; Zhao, Fangzhou ; Ethridge, Christina L. ; Lail, Kathleen ; Amirebrahimi, Mojgan ; Lipzen, Anna ; Ng, Vivian ; et al ( January 2021 , Proceedings of the National Academy of Sciences)

   Polycomb Group (PcG) proteins are part of an epigenetic cell memory system that plays essential roles in multicellular development, stem cell biology, X chromosome inactivation, and cancer. In animals, plants, and many fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to assemble transcriptionally repressed facultative heterochromatin. PRC2 is structurally and functionally conserved in the model fungusNeurospora crassa, and recent work in this organism has generated insights into PRC2 control and function. To identify components of the facultative heterochromatin pathway, we performed a targeted screen ofNeurosporadeletion strains lacking individual ATP-dependent chromatin remodeling enzymes. We found theNeurosporahomolog of IMITATION SWITCH (ISW) is critical for normal transcriptional repression, nucleosome organization, and establishment of typical histone methylation patterns in facultative heterochromatin domains. We also found that stable interaction between PRC2 and chromatin depends on ISW. A functional ISW ATPase domain is required for gene repression and normal H3K27 methylation. ISW homologs interact with accessory proteins to form multiple complexes with distinct functions. Using proteomics and molecular approaches, we identified three distinctNeurosporaISW-containing complexes. A triple mutant lacking three ISW accessory factors and disrupting multiple ISW complexes led to widespread up-regulation of PRC2 target genes and altered H3K27 methylation patterns,more »similar to an ISW-deficient strain. Taken together, our data show that ISW is a key component of the facultative heterochromatin pathway inNeurospora, and that distinct ISW complexes perform an apparently overlapping role to regulate chromatin structure and gene repression at PRC2 target domains.

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4. Chromatin accessibility profiling in Neurospora crassa reveals molecular features associated with accessible and inaccessible chromatin

   https://doi.org/10.1186/s12864-021-07774-0  

   Ferraro, Aileen R. ; Ameri, Abigail J. ; Lu, Zefu ; Kamei, Masayuki ; Schmitz, Robert J. ; Lewis, Zachary A. ( December 2021 , BMC Genomics)

   Abstract Background Regulation of chromatin accessibility and transcription are tightly coordinated processes. Studies in yeast and higher eukaryotes have described accessible chromatin regions, but little work has been done in filamentous fungi. Results Here we present a genome-scale characterization of accessible chromatin regions in Neurospora crassa , which revealed characteristic molecular features of accessible and inaccessible chromatin. We present experimental evidence of inaccessibility within heterochromatin regions in Neurospora, and we examine features of both accessible and inaccessible chromatin, including the presence of histone modifications, types of transcription, transcription factor binding, and relative nucleosome turnover rates. Chromatin accessibility is not strictly correlated with expression level. Accessible chromatin regions in the model filamentous fungus Neurospora are characterized the presence of H3K27 acetylation and commonly associated with pervasive non-coding transcription. Conversely, methylation of H3 lysine-36 catalyzed by ASH1 is correlated with inaccessible chromatin within promoter regions. Conclusions: In N. crassa, H3K27 acetylation is the most predictive histone modification for open chromatin. Conversely, our data show that H3K36 methylation is a key marker of inaccessible chromatin in gene-rich regions of the genome. Our data are consistent with an expanded role for H3K36 methylation in intergenic regions of filamentous fungi compared to the model yeasts,more »S. cerevisiae and S. pombe, which lack homologs of the ASH1 methyltransferase.« less
5. Intracellular Zn2+ transients modulate global gene expression in dissociated rat hippocampal neurons

   https://doi.org/10.1038/s41598-019-45844-2  

   Sanford, Lynn ; Carpenter, Margaret C. ; Palmer, Amy E. ( June 2019 , Scientific Reports)

   Zinc (Zn²⁺) is an integral component of many proteins and has been shown to act in a regulatory capacity in different mammalian systems, including as a neurotransmitter in neurons throughout the brain. While Zn²⁺plays an important role in modulating neuronal potentiation and synaptic plasticity, little is known about the signaling mechanisms of this regulation. In dissociated rat hippocampal neuron cultures, we used fluorescent Zn²⁺sensors to rigorously define resting Zn²⁺levels and stimulation-dependent intracellular Zn²⁺dynamics, and we performed RNA-Seq to characterize Zn²⁺-dependent transcriptional effects upon stimulation. We found that relatively small changes in cytosolic Zn²⁺during stimulation altered expression levels of 931 genes, and these Zn²⁺dynamics induced transcription of many genes implicated in neurite expansion and synaptic growth. Additionally, while we were unable to verify the presence of synaptic Zn²⁺in these cultures, we did detect the synaptic vesicle Zn²⁺transporter ZnT3 and found it to be substantially upregulated by cytosolic Zn²⁺increases. These results provide the first global sequencing-based examination of Zn²⁺-dependent changes in transcription and identify genes that may mediate Zn²⁺-dependent processes and functions.