SAFB restricts contact domain boundaries associated with L1 chimeric transcription.

Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.

Nuclear RNA homeostasis promotes systems-level coordination of cell fate and senescence.

Understanding cellular coordination remains a challenge despite knowledge of individual pathways. The RNA exosome, targeting a wide range of RNA substrates, is often downregulated in cellular senescence. Utilizing an auxin-inducible system, we observed that RNA exosome depletion in embryonic stem cells significantly affects the transcriptome and proteome, causing pluripotency loss and pre-senescence onset. Mechanistically, exosome depletion triggers acute nuclear RNA aggregation, disrupting nuclear RNA-protein equilibrium. This disturbance limits nuclear protein availability and hinders polymerase initiation and engagement, reducing gene transcription. Concurrently, it promptly disrupts nucleolar transcription, ribosomal processes, and nuclear exporting, resulting in a translational shutdown. Prolonged exosome depletion induces nuclear structural changes resembling senescent cells, including aberrant chromatin compaction, chromocenter disassembly, and intensified heterochromatic foci. These effects suggest that the dynamic turnover of nuclear RNA orchestrates crosstalk between essential processes to optimize cellular function. Disruptions in nuclear RNA homeostasis result in systemic functional decline, altering the cell state and promoting senescence.

A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency.

TET1 maintains hypomethylation at bivalent promoters through its catalytic activity in embryonic stem cells (ESCs). However, TET1 catalytic activity-independent function in regulating bivalent genes is not well understood. Using a proteomics approach, we map the TET1 interactome in ESCs and identify PSPC1 as a TET1 partner. Genome-wide location analysis reveals that PSPC1 functionally associates with TET1 and Polycomb repressive complex-2 (PRC2). We establish that PSPC1 and TET1 repress, and the lncRNA Neat1 activates, bivalent gene expression. In ESCs, Neat1 is preferentially bound to PSPC1 alongside its PRC2 association at bivalent promoters. During the ESC-to-epiblast-like stem cell (EpiLC) transition, PSPC1 and TET1 maintain PRC2 chromatin occupancy at bivalent gene promoters, while Neat1 facilitates the activation of certain bivalent genes by promoting PRC2 binding to their mRNAs. Our study demonstrates a TET1-PSPC1-Neat1 molecular axis that modulates PRC2-binding affinity to chromatin and bivalent gene transcripts in controlling stem cell bivalency.

Homotypic clustering of L1 and B1/Alu repeats compartmentalizes the 3D genome.

Organization of the genome into euchromatin and heterochromatin appears to be evolutionarily conserved and relatively stable during lineage differentiation. In an effort to unravel the basic principle underlying genome folding, here we focus on the genome itself and report a fundamental role for L1 (LINE1 or LINE-1) and B1/Alu retrotransposons, the most abundant subclasses of repetitive sequences, in chromatin compartmentalization. We find that homotypic clustering of L1 and B1/Alu demarcates the genome into grossly exclusive domains, and characterizes and predicts Hi-C compartments. Spatial segregation of L1-rich sequences in the nuclear and nucleolar peripheries and B1/Alu-rich sequences in the nuclear interior is conserved in mouse and human cells and occurs dynamically during the cell cycle. In addition, de novo establishment of L1 and B1 nuclear segregation is coincident with the formation of higher-order chromatin structures during early embryogenesis and appears to be critically regulated by L1 and B1 transcripts. Importantly, depletion of L1 transcripts in embryonic stem cells drastically weakens homotypic repeat contacts and compartmental strength, and disrupts the nuclear segregation of L1- or B1-rich chromosomal sequences at genome-wide and individual sites. Mechanistically, nuclear co-localization and liquid droplet formation of L1 repeat DNA and RNA with heterochromatin protein HP1α suggest a phase-separation mechanism by which L1 promotes heterochromatin compartmentalization. Taken together, we propose a genetically encoded model in which L1 and B1/Alu repeats blueprint chromatin macrostructure. Our model explains the robustness of genome folding into a common conserved core, on which dynamic gene regulation is overlaid across cells.

U1 snRNP regulates chromatin retention of noncoding RNAs.

Long noncoding RNAs (lncRNAs) and promoter- or enhancer-associated unstable transcripts locate preferentially to chromatin, where some regulate chromatin structure, transcription and RNA processing1-13. Although several RNA sequences responsible for nuclear localization have been identified-such as repeats in the lncRNA Xist and Alu-like elements in long RNAs14-16-how lncRNAs as a class are enriched at chromatin remains unknown. Here we describe a random, mutagenesis-coupled, high-throughput method that we name 'RNA elements for subcellular localization by sequencing' (mutREL-seq). Using this method, we discovered an RNA motif that recognizes the U1 small nuclear ribonucleoprotein (snRNP) and is essential for the localization of reporter RNAs to chromatin. Across the genome, chromatin-bound lncRNAs are enriched with 5' splice sites and depleted of 3' splice sites, and exhibit high levels of U1 snRNA binding compared with cytoplasm-localized messenger RNAs. Acute depletion of U1 snRNA or of the U1 snRNP protein component SNRNP70 markedly reduces the chromatin association of hundreds of lncRNAs and unstable transcripts, without altering the overall transcription rate in cells. In addition, rapid degradation of SNRNP70 reduces the localization of both nascent and polyadenylated lncRNA transcripts to chromatin, and disrupts the nuclear and genome-wide localization of the lncRNA Malat1. Moreover, U1 snRNP interacts with transcriptionally engaged RNA polymerase II. These results show that U1 snRNP acts widely to tether and mobilize lncRNAs to chromatin in a transcription-dependent manner. Our findings have uncovered a previously unknown role of U1 snRNP beyond the processing of precursor mRNA, and provide molecular insight into how lncRNAs are recruited to regulatory sites to carry out chromatin-associated functions.

Genomic Repeats Categorize Genes with Distinct Functions for Orchestrated Regulation.

Repetitive elements are abundantly distributed in mammalian genomes. Here, we reveal a striking association between repeat subtypes and gene function. SINE, L1, and low-complexity repeats demarcate distinct functional categories of genes and may dictate the time and level of gene expression by providing binding sites for different regulatory proteins. Importantly, imaging and sequencing analysis show that L1 repeats sequester a large set of genes with specialized functions in nucleolus- and lamina-associated inactive domains that are depleted of SINE repeats. In addition, L1 transcripts bind extensively to its DNA in embryonic stem cells (ESCs). Depletion of L1 RNA in ESCs leads to relocation of L1-enriched chromosomal segments from inactive domains to the nuclear interior and de-repression of L1-associated genes. These results demonstrate a role of L1 DNA and RNA in gene silencing and suggest a general theme of genomic repeats in orchestrating the function, regulation, and expression of their host genes.

RNase H1 Cooperates with DNA Gyrases to Restrict R-Loops and Maintain Genome Integrity in Arabidopsis Chloroplasts.

Maintaining organellar genome integrity is essential for eukaryotic cells, and many factors can threaten genome integrity. R-loops are DNA:RNA duplexes produced during transcription, with the nontemplated DNA forming a single-stranded region. R-loops function in the regulation of transcription, DNA replication, and DNA repair, but can also be susceptible to lesions that form double-stranded breaks and thus induce genome instability. From investigating the function of a plant chloroplast-localized R-loop removing enzyme AtRNH1C, we have found that it is responsible for plastid R-loop homeostasis, chloroplast genome instability, and development. Interactome analysis revealed that AtRNH1C associates with multiple chloroplast-localized DNA and RNA metabolism-related proteins, including the core DNA gyrases complex. The interaction between AtRNH1C and AtGyrases was critical for R-loop homeostasis in chloroplast and important to release the transcription-replication conflicts in the highly transcribed and replication originated cp-rDNA regions and thus to reduce the DNA damage. Our results reveal the plastid R-loop accumulation leads to chloroplast DNA instability and provide insight into the maintenance of genome integrity in chloroplasts, in which the evolutionarily conserved RNase H1 and DNA gyrase proteins are involved.

TaPIMP2, a pathogen-induced MYB protein in wheat, contributes to host resistance to common root rot caused by Bipolaris sorokiniana.

MYB transcription factors (TFs) have been implicated in various biology processes in model plants. However, functions of the great majority of MYB TFs in wheat (Triticum aestivum L.) have not been characterized. The soil-borne fungal pathogens Bipolaris sorokiniana and Rhizoctonia cerealis are the causal agents of important destructive diseases of wheat. Here, the TaPIMP2 gene, encoding a pathogen-induced MYB protein in wheat, was isolated through comparative transcriptomic analysis, and its defensive role was studied. TaPIMP2 was proved to localize in nuclei. TaPIMP2 responded in a different extent and speed upon infections of B. sorokiniana or R. cerealis. TaPIMP2 displayed different expression patterns after exogenous application of phytohormones, including abscisic acid, ethylene, and salicylic acid. Silencing of TaPIMP2 repressed resistance of wheat cultivar Yangmai 6 to B. sorokiniana, but did not alter resistance of wheat line CI12633 to R. cerealis. TaPIMP2 overexpression significantly improved resistance to B. sorokiniana rather than R. cerealis in transgenic wheat. Moreover, TaPIMP2 positively modulated the expression of pathogenesis-related genes, including PR1a, PR2, PR5, and PR10. Collectively, TaPIMP2 positively contributes to wheat resistance to B. sorokiniana possibly through regulating the expression of defense-related genes, and TaPIMP2 plays distinct roles in defense responses to different fungal infection.

The wheat calcium-dependent protein kinase TaCPK7-D positively regulates host resistance to sharp eyespot disease.

Sharp eyespot, caused mainly by the necrotrophic fungus Rhizoctonia cerealis, limits wheat production worldwide. Here, TaCPK7-D, encoding a subgroup III member of the calcium-dependent protein kinase (CPK) family, was identified from the sharp eyespot-resistant wheat line CI12633 through comparative transcriptomic analysis. Subsequently, the defence role of TaCPK7-D against R. cerealis infection was studied by the generation and characterization of TaCPK7-D-silenced and TaCPK7-D-overexpressing wheat plants. Rhizoctonia cerealis inoculation induced a higher transcriptional level of TaCPK7-D in the resistant wheat line CI12633 than in the susceptible cultivar Wenmai 6. The expression of TaCPK7-D was significantly induced after exogenous application of 1-aminocyclopropane-1-carboxylic acid (an ethylene biosynthesis precursor). The green fluorescent protein signal distribution assays indicated that TaCPK7-D localizes to the plasma membrane in both onion epidermal cells and wheat protoplasts. Following R. cerealis inoculation, TaCPK7-D-silenced wheat CI12633 plants displayed more severe sharp eyespot symptoms than control CI12633 plants. Four defence-associated genes (ß-1,3-glucanase, chitinase 1, defensin and TaPIE1) and an ethylene biosynthesis key gene, ACO2, were significantly suppressed in the TaCPK7-D-silenced wheat plants compared with control plants. Conversely, TaCPK7-D-overexpressing wheat lines showed increased resistance to sharp eyespot compared with untransformed recipient wheat Yangmai 16. Furthermore, the transcriptional levels of these four defence-related genes and ACO2 gene were significantly elevated in TaCPK7-D-overexpressing plants compared with untransformed recipient wheat plants. These results suggest that TaCPK7-D positively regulates the wheat resistance response to R. cerealis infection through the modulation of the expression of these defence-associated genes, and that TaCPK7-D is a candidate to improve sharp eyespot resistance in wheat.

Transcript suppression of TaGW2 increased grain width and weight in bread wheat.

Bread wheat (Triticum aestivum L.) is a major staple crop in the world. Grain weight is a major factor of grain yield in wheat, and the identification of candidate genes associated with grain weight is very important for high-yield breeding of wheat. TaGW2 is an orthologous gene of rice OsGW2 that negatively regulates the grain width and weight in rice. There are three TaGW2 homoeologs in bread wheat, TaGW2A, TaGW2B, and TaGW2D. In this study, a specific TaGW2-RNA interference (RNAi) cassette was constructed and transformed into a Chinese bread wheat variety 'Shi 4185' with small grain. The transcript levels of TaGW2A, TaGW2B, and TaGW2D were simultaneously downregulated in TaGW2-RNAi transgenic wheat lines. Compared with the controls, TaGW2-underexpressing transgenic lines displayed significantly increases in the grain width and weight, suggesting that TaGW2 negatively regulated the grain width and weight in bread wheat. Further transcript analysis showed that in different bread wheat accessions, the transcript abundance of TaGW2A was negatively associated with the grain width.