Sites of transcription initiation drive mRNA isoform selection.

The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.

The histone variant H2A.W defines heterochromatin and promotes chromatin condensation in Arabidopsis.

Histone variants play crucial roles in gene expression, genome integrity, and chromosome segregation. We report that the four H2A variants in Arabidopsis define different genomic features, contributing to overall genomic organization. The histone variant H2A.W marks heterochromatin specifically and acts in synergy with heterochromatic marks H3K9me2 and DNA methylation to maintain transposon silencing. In vitro, H2A.W enhances chromatin condensation by promoting fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, H2A.W is required for heterochromatin condensation, demonstrating that H2A.W plays critical roles in heterochromatin organization. Similarities in conserved motifs between H2A.W and another H2A variant in metazoans suggest that plants and animals share common mechanisms for heterochromatin condensation.

Enterococcus faecalis Promotes Innate Immune Suppression and Polymicrobial Catheter-Associated Urinary Tract Infection.

Enterococcus faecalis, a member of the human gastrointestinal microbiota, is an opportunistic pathogen associated with hospital-acquired wound, bloodstream, and urinary tract infections. E. faecalis can subvert or evade immune-mediated clearance, although the mechanisms are poorly understood. In this study, we examined E. faecalis-mediated subversion of macrophage activation. We observed that E. faecalis actively prevents NF-κB signaling in mouse RAW264.7 macrophages in the presence of Toll-like receptor agonists and during polymicrobial infection with Escherichia coliE. faecalis and E. coli coinfection in a mouse model of catheter-associated urinary tract infection (CAUTI) resulted in a suppressed macrophage transcriptional response in the bladder compared to that with E. coli infection alone. Finally, we demonstrated that coinoculation of E. faecalis with a commensal strain of E. coli into catheterized bladders significantly augmented E. coli CAUTI. Taken together, these results support the hypothesis that E. faecalis suppression of NF-κB-driven responses in macrophages promotes polymicrobial CAUTI pathogenesis, especially during coinfection with less virulent or commensal E. coli strains.

Dynamic deposition of histone variant H3.3 accompanies developmental remodeling of the Arabidopsis transcriptome.

In animals, replication-coupled histone H3.1 can be distinguished from replication-independent histone H3.3. H3.3 variants are enriched at active genes and their promoters. Furthermore, H3.3 is specifically incorporated upon gene activation. Histone H3 variants evolved independently in plants and animals, and it is unclear whether different replication-independent H3.3 variants developed similar properties in both phyla. We studied Arabidopsis H3 variants in order to find core properties of this class of histones. Here we present genome-wide maps of H3.3 and H3.1 enrichment and the dynamic changes of their profiles upon cell division arrest. We find H3.3 enrichment to positively correlate with gene expression and to be biased towards the transcription termination site. In contrast with H3.1, heterochromatic regions are mostly depleted of H3.3. We report that, in planta, dynamic changes in H3.3 profiles are associated with the extensive remodeling of the transcriptome that occurs during cell differentiation. We propose that H3.3 dynamics are linked to transcription and are involved in resetting covalent histone marks at a genomic scale during plant development. Our study suggests that H3 variants properties likely result from functionally convergent evolution.

Coping with cryptic and defective transcripts in plant mitochondria.

Plant mitochondria are particularly prone to the production of both defective and cryptic transcripts as a result of the complex organisation and mode of expression of their genome. Cryptic transcripts are generated from intergenic regions due to a relaxed control of transcription. Certain intergenic regions are transcribed at higher rates than genuine genes and therefore, cryptic transcripts are abundantly produced in plant mitochondria. In addition, primary transcripts from genuine genes must go through complex post-transcriptional processes such as C to U editing and cis or trans splicing of group II introns. These post-transcriptional processes are rather inefficient and as a result, defective transcripts are constantly produced in plant mitochondria. In this review, we will describe the nature of cryptic and defective transcripts as well as their fate in plant mitochondria. Although RNA surveillance is crucial to establishing the final transcriptome by degrading cryptic transcripts, plant mitochondria are able to tolerate a surprising high level of defective transcripts.

Relaxed transcription in Arabidopsis mitochondria is counterbalanced by RNA stability control mediated by polyadenylation and polynucleotide phosphorylase.

Plant mitochondrial genomes are extraordinarily large and complex compared to their animal counterparts, due to the presence of large noncoding regions. Multiple promoters are common for plant mitochondrial genes, and transcription exhibits little or no modulation. Mature functional RNAs are produced through various posttranscriptional processes, and control of RNA stability has a major impact on RNA abundance. This control involves polyadenylation which targets RNA for degradation by polynucleotide phosphorylase (PNPase). Here, we have analyzed polyadenylated RNA fragments from Arabidopsis plants down-regulated for PNPase (PNP- plants). Because of their polyadenylated status and the accumulation of the corresponding RNA in PNP- versus wild-type plants, these sequences represent mitochondrial RNA degradation tags. Analysis of these tags revealed that PNPase is involved in degrading rRNA and tRNA maturation by-products but also RNA transcribed from regions that are in some cases highly expressed although lacking known functional genes. Some of these transcripts, such as RNA containing chimeric open reading frames created by recombination or antisense RNA transcribed on the opposite strand of a known gene, may present potential detrimental effects to mitochondrial function. Taken together, our data show that the relaxed transcription in Arabidopsis mitochondria is counterbalanced by RNA stability control mediated by polyadenylation and PNPase.

LHP1 Interacts with ATRX through Plant-Specific Domains at Specific Loci Targeted by PRC2.

Heterochromatin Protein 1 (HP1) is a major regulator of chromatin structure and function. In animals, the network of proteins interacting with HP1 is mainly associated with constitutive heterochromatin marked by H3K9me3. HP1 physically interacts with the putative ortholog of the SNF2 chromatin remodeler ATRX, which controls deposition of histone variant H3.3 in mammals. In this study, we show that the Arabidopsis thaliana ortholog of ATRX participates in H3.3 deposition and possesses specific conserved domains in plants. We found that plant Like HP1 (LHP1) protein interacts with ATRX through domains that evolved specifically in land plant ancestors. Loss of ATRX function in Arabidopsis affects the expression of a limited subset of genes controlled by PRC2 (POLYCOMB REPRESSIVE COMPLEX 2), including the flowering time regulator FLC. The function of ATRX in regulation of flowering time requires novel LHP1-interacting domain and ATPase activity of the ATRX SNF2 helicase domain. Taken together, these results suggest that distinct evolutionary pathways led to the interaction between ATRX and HP1 in mammals and its counterpart LHP1 in plants, resulting in distinct modes of transcriptional regulation.

The histone H3 variant H3.3 regulates gene body DNA methylation in Arabidopsis thaliana.

BACKGROUND: Gene bodies of vertebrates and flowering plants are occupied by the histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. DNA methylation and H3.3 enrichment profiles over gene bodies are correlated and both have a similar dependence on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation. RESULTS: We engineered an H3.3 knockdown in Arabidopsis thaliana and observed transcription reduction that predominantly affects genes responsive to environmental cues. When H3.3 levels are reduced, gene bodies show a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome-wide distributions of several histone H3 marks, H2A.Z, and linker histone H1. We report that in the absence of H3.3, H1 distribution increases in gene bodies in a transcription-dependent manner. CONCLUSIONS: We propose that H3.3 prevents recruitment of H1, inhibiting H1's promotion of chromatin folding that restricts access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in conjunction with transcriptional activity.

Bladder catheterization increases susceptibility to infection that can be prevented by prophylactic antibiotic treatment.

Catheter-associated urinary tract infections (CAUTI) are the most common hospital-associated infections. Here, we report that bladder catheterization initiated a persistent sterile inflammatory reaction within minutes of catheter implantation. Catheterization resulted in increased expression of genes associated with defense responses and cellular migration, with ensuing rapid and sustained innate immune cell infiltration into the bladder. Catheterization also resulted in hypersensitivity to Enterococcus faecalis and uropathogenic Escherichia coli (UPEC) infection, in which colonization was achieved using an inoculum 100-fold lower than the ID90 for infection of an undamaged urothelium with the same uropathogens. As the time of catheterization increased, however, colonization by the Gram-positive uropathogen E. faecalis was reduced, whereas catheterization created a sustained window of vulnerability to infection for Gram-negative UPEC over time. As CAUTI contributes to poorer patient outcomes and increased health care expenditures, we tested whether a single prophylactic antibiotic treatment, concurrent with catheterization, would prevent infection. We observed that antibiotic treatment protected against UPEC and E. faecalis bladder and catheter colonization as late as 6 hours after implantation. Thus, our study has revealed a simple, safe, and immediately employable intervention, with the potential to decrease one of the most costly hospital-incurred infections, thereby improving patient and health care economic outcome.

The HIRA complex that deposits the histone H3.3 is conserved in Arabidopsis and facilitates transcriptional dynamics.

In animals, replication-independent incorporation of nucleosomes containing the histone variant H3.3 enables global reprogramming of histone modifications and transcriptional profiles. H3.3 enrichment over gene bodies correlates with gene transcription in animals and plants. In animals, H3.3 is deposited into chromatin by specific protein complexes, including the HIRA complex. H3.3 variants evolved independently and acquired similar properties in animals and plants, questioning how the H3.3 deposition machinery evolved in plants and what are its biological functions. We performed phylogenetic analyses in the plant kingdom and identified in Arabidopsis all orthologs of human genes encoding members of the HIRA complex. Genetic analyses, biochemical data and protein localisation suggest that these proteins form a complex able to interact with H3.3 in Arabidopsis in a manner similar to that described in mammals. In contrast to animals, where HIRA is required for fertilization and early development, loss of function of HIRA in Arabidopsis causes mild phenotypes in the adult plant and does not perturb sexual reproduction and embryogenesis. Rather, HIRA function is required for transcriptional reprogramming during dedifferentiation of plant cells that precedes vegetative propagation and for the appropriate transcription of genes responsive to biotic and abiotic factors. We conclude that the molecular function of the HIRA complex is conserved between plants and animals. Yet plants diversified HIRA functions to enable asexual reproduction and responsiveness to the environment in response to the plant sessile lifestyle.

A unified phylogeny-based nomenclature for histone variants.

Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

Zygotic resetting of the HISTONE 3 variant repertoire participates in epigenetic reprogramming in Arabidopsis.

In most eukaryotes, the HISTONE 3 family comprises several variants distinguished by their amino acid sequence, localization, and correlation with transcriptional activity. Transgenerational inheritance of epigenetic information carried by histones is still unclear. In addition to covalent histone modifications, the mosaic distribution of H3 variants onto chromatin has been proposed to provide a new level of epigenetic information. To study the transmission of patterns of H3 variants through generations, we combined transcriptional profiling and live imaging of the 13 H3 variants encoded by the Arabidopsis plant genome. In comparison with somatic cells, only a restricted number of H3 variants are present in male and female gametes. Upon fertilization, H3 variants contributed by both gametes are actively removed from the zygote chromatin. The somatic H3 composition is restored in the embryo by de novo synthesis of H3 variants. A survey of Arabidopsis homologs of animal H3 chaperones suggests that removal of parental H3 from the zygote nucleus relies on a new mechanism. Our results suggest that reprogramming of parental genomes in the zygote limits the inheritance of epigenetic information carried by H3 variants across generations.

Ribonucleotide reductase regulation in response to genotoxic stress in Arabidopsis.

Ribonucleotide reductase (RNR) is an essential enzyme that provides dNTPs for DNA replication and repair. Arabidopsis (Arabidopsis thaliana) encodes three AtRNR2-like catalytic subunit genes (AtTSO2, AtRNR2A, and AtRNR2B). However, it is currently unclear what role, if any, each gene contributes to the DNA damage response, and in particular how each gene is transcriptionally regulated in response to replication blocks and DNA damage. To address this, we investigated transcriptional changes of 17-d-old Arabidopsis plants (which are enriched in S-phase cells over younger seedlings) in response to the replication-blocking agent hydroxyurea (HU) and to the DNA double-strand break inducer bleomycin (BLM). Here we show that AtRNR2A and AtRNR2B are specifically induced by HU but not by BLM. Early AtRNR2A induction is decreased in an atr mutant, and this induction is likely required for the replicative stress checkpoint since rnr2a mutants are hypersensitive to HU, whereas AtRNR2B induction is abolished in the rad9-rad17 double mutant. In contrast, AtTSO2 transcription is only activated in response to double-strand breaks (BLM), and this activation is dependent upon AtE2Fa. Both TSO2 and E2Fa are likely required for the DNA damage response since tso2 and e2fa mutants are hypersensitive to BLM. Interestingly, TSO2 gene expression is increased in atr versus wild type, possibly due to higher ATM expression in atr. On the other hand, a transient ATR-dependent H4 up-regulation was observed in wild type in response to HU and BLM, perhaps linked to a transient S-phase arrest. Our results therefore suggest that individual RNR2-like catalytic subunit genes participate in unique aspects of the cellular response to DNA damage in Arabidopsis.

Polyadenylation-mediated RNA degradation in plant mitochondria.

In plant mitochondria, polyadenylation-mediated RNA degradation is involved in several key aspects of genome expression, including RNA maturation, RNA turnover, and RNA surveillance. We describe here a combination of in vivo, in vitro, and in organello methods that have been developed or optimized to characterize this RNA degradation pathway. These approaches include several PCR-based methods designed to identify polyadenylated RNA substrates, as well as in vitro and in organello systems, to study functional aspects of the RNA degradation processes. Taken together, identification of RNA substrates combined with information from degradation assays are invaluable tools to dissect the mechanisms and roles of RNA degradation in plant mitochondrial genome expression.

Degradation of a polyadenylated rRNA maturation by-product involves one of the three RRP6-like proteins in Arabidopsis thaliana.

Yeast Rrp6p and its human counterpart, PM/Scl100, are exosome-associated proteins involved in the degradation of aberrant transcripts and processing of precursors to stable RNAs, such as the 5.8S rRNA, snRNAs, and snoRNAs. The activity of yeast Rrp6p is stimulated by the polyadenylation of its RNA substrates. We identified three RRP6-like proteins in Arabidopsis thaliana: AtRRP6L3 is restricted to the cytoplasm, whereas AtRRP6L1 and -2 have different intranuclear localizations. Both nuclear RRP6L proteins are functional, since AtRRP6L1 complements the temperature-sensitive phenotype of a yeast rrp6Delta strain and mutation of AtRRP6L2 leads to accumulation of an rRNA maturation by-product. This by-product corresponds to the excised 5' part of the 18S-5.8S-25S rRNA precursor and accumulates as a polyadenylated transcript, suggesting that RRP6L2 is involved in poly(A)-mediated RNA degradation in plant nuclei. Interestingly, the rRNA maturation by-product is a substrate of AtRRP6L2 but not of AtRRP6L1. This result and the distinctive subcellular distribution of AtRRP6L1 to -3 indicate a specialization of RRP6-like proteins in Arabidopsis.