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The RNAs bound to the genome and their binding sites are detected with GRID-seq. Higher eukaryotic genomes are bound by a large number of coding and non-coding RNAs, but approaches to comprehensively map the identity and binding sites of these RNAs are lacking. Here we report a method to capture in situ global RNA interactions with DNA by deep sequencing (GRID-seq), which enables the comprehensive identification of the entire repertoire of chromatin-interacting RNAs and their respective binding sites. In human, mouse, and Drosophila cells, we detected a large set of tissue-specific coding and non-coding RNAs that are bound to active promoters and enhancers, especially super-enhancers. Assuming that most mRNA–chromatin interactions indicate the physical proximity of a promoter and an enhancer, we constructed a three-dimensional global connectivity map of promoters and enhancers, revealing transcription-activity-linked genomic interactions in the nucleus.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome 1 . Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs. A combination of five assays is used to produce a catalogue of RNA elements to which RNA-binding proteins bind in human cells.

The lncRNA NEAT1 , a key component of paraspeckles, interacts with RNA-binding proteins, including NONO and PSF, and affects global pri-miRNA processing by recruiting the Drosha–DGCR8 Microprocessor. MicroRNA (miRNA) biogenesis is known to be modulated by a variety of RNA-binding proteins (RBPs), but in most cases, individual RBPs appear to influence the processing of a small subset of target miRNAs. Here, we report that the RNA-binding NONO–PSF heterodimer binds a large number of expressed pri-miRNAs in HeLa cells to globally enhance pri-miRNA processing by the Drosha–DGCR8 Microprocessor. NONO and PSF are key components of paraspeckles organized by the long noncoding RNA (lncRNA) NEAT1. We further demonstrate that NEAT1 also has a profound effect on global pri-miRNA processing. Mechanistic dissection reveals that NEAT1 broadly interacts with the NONO–PSF heterodimer as well as many other RBPs and that multiple RNA segments in NEAT1, including a 'pseudo pri-miRNA' near its 3′ end, help attract the Microprocessor. These findings suggest a 'bird nest' model in which an lncRNA orchestrates efficient processing of potentially an entire class of small noncoding RNAs in the nucleus.

Spermatogenesis in mammals is characterized by two waves of piRNA expression： one corresponds to classic piR- NAs responsible for silencing retrotransponsons and the second wave is predominantly derived from nontransposon intergenic regions in pachytene spermatocytes, but the function of these pachytene piRNAs is largely unknown. Here, we report the involvement of pachytene piRNAs in instructing massive mRNA elimination in mouse elongating spermatids （ES）. We demonstrate that a piRNA-induced silencing complex （pi-RISC） containing murine PIWI （MIWI） and deadenylase CAF1 is selectively assembled in ES, which is responsible for inducing mRNA deadenylation and decay via a mechanism that resembles the action of miRNAs in somatic cells. Such a highly orchestrated program appears to take full advantage of the enormous repertoire of diversified targeting capacity of pachytene piRNAs de rived from nontransposon intergenic regions. These findings suggest that pachytene piRNAs are responsible for inactivating vast cellular programs in preparation for sperm production from ES.

p63 and PAX6 act to specify limbal stem or progenitor cells (LSCs), and WNT7A controls corneal epithelium differentiation through PAX6; loss of WNT7A or PAX6 induces LSCs into epithelium, and transduction of PAX6 in skin epithelial stem cells converts them to LSC-like cells and transplantation in a rabbit corneal injury model can replenish corneal epithelial cells and repair damaged corneal surface. WNT7A and PAX6 signals in corneal pathogenesis The limbal stem cells (LSCs) in the basal limbal epithelium of the eye sustain corneal epithelial homeostasis and regeneration. Their loss due to injury or disease is one of the most common causes of blindness. Hong Ouyang et al . establish a method for culturing LSCs in a feeder-free medium. They identify the WNT7A–PAX6 signalling axis as a determinant of corneal lineage that has potential as a therapeutic target in corneal surface diseases. Transduction of PAX6 in skin epithelial stem cells converts them into LSC-like cells, and transplantation of the reprogrammed cells in a rabbit corneal injury model can repair damaged corneal surface. In a separate study in this issue of Nature , Bruce Ksander et al . identify a marker — the ABC transporter ABCB5 — that is functionally required for LSC maintenance, corneal development and repair, and can be used to identify LSCs in mouse and human eyes. LSC transplantation experiments suggest that ABCB5-expressing cells may have potential in the treatment of corneal disease, particularly corneal blindness due to LSC deficiency. The surface of the cornea consists of a unique type of non-keratinized epithelial cells arranged in an orderly fashion, and this is essential for vision by maintaining transparency for light transmission. Cornea epithelial cells (CECs) undergo continuous renewal from limbal stem or progenitor cells (LSCs) 1 , 2 , and deficiency in LSCs or corneal epithelium—which turns cornea into a non-transparent, keratinized skin-like epithelium—causes corneal surface disease that leads to blindness in millions of people worldwide 3 . How LSCs are maintained and differentiated into corneal epithelium in healthy individuals and which key molecular events are defective in patients have been largely unknown. Here we report establishment of an in vitro feeder-cell-free LSC expansion and three-dimensional corneal differentiation protocol in which we found that the transcription factors p63 (tumour protein 63) and PAX6 (paired box protein PAX6) act together to specify LSCs, and WNT7A controls corneal epithelium differentiation through PAX6. Loss of WNT7A or PAX6 induces LSCs into skin-like epithelium, a critical defect tightly linked to common human corneal diseases. Notably, transduction of PAX6 in skin epithelial stem cells is sufficient to convert them to LSC-like cells, and upon transplantation onto eyes in a rabbit corneal injury model, these reprogrammed cells are able to replenish CECs and repair damaged corneal surface. These findings suggest a central role of the WNT7A–PAX6 axis in corneal epithelial cell fate determination, and point to a new strategy for treating corneal surface diseases.

Structural and biochemical studies of RNA polymerase II (Pol II) assembled on DNA containing 5-carboxycytosine reveals that Pol II can sense the oxidized methylation state of DNA and transiently slows down during transcription. Pol II as a DNA methylation sensor Epigenetic DNA methylation — to produce 5-methylcytosine (5mC) residues — is an important gene transcription regulator recognized by various protein readers. 5mC can be oxidized by TET enzymes to produce 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). This study of the structure and biochemistry of RNA polymerase II (Pol II) assembled on DNA containing 5caC suggests that Pol II is able to function as an epigenetic DNA modification reader by specifically recognizing 5caC and 5fC during transcription elongation. Pol II can sense the oxidized methylation state of DNA and transiently slows down during transcription. The authors propose that Pol II may act as a direct sensor for a variety of DNA modification and damage events to instruct distinct downstream pathways. DNA methylation at selective cytosine residues (5-methylcytosine (5mC)) and their removal by TET-mediated DNA demethylation are critical for setting up pluripotent states in early embryonic development 1 , 2 . TET enzymes successively convert 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), with 5fC and 5caC subject to removal by thymine DNA glycosylase (TDG) in conjunction with base excision repair 1 , 2 , 3 , 4 , 5 , 6 . Early reports indicate that 5fC and 5caC could be stably detected on enhancers, promoters and gene bodies, with distinct effects on gene expression, but the mechanisms have remained elusive 7 , 8 . Here we determined the X-ray crystal structure of yeast elongating RNA polymerase II (Pol II) in complex with a DNA template containing oxidized 5mCs, revealing specific hydrogen bonds between the 5-carboxyl group of 5caC and the conserved epi-DNA recognition loop in the polymerase. This causes a positional shift for incoming nucleoside 5′-triphosphate (NTP), thus compromising nucleotide addition. To test the implication of this structural insight in vivo , we determined the global effect of increased 5fC/5caC levels on transcription, finding that such DNA modifications indeed retarded Pol II elongation on gene bodies. These results demonstrate the functional impact of oxidized 5mCs on gene expression and suggest a novel role for Pol II as a specific and direct epigenetic sensor during transcription elongation.

Resisting a dawn raid Circadian rhythms regulate a wide variety of developmental and metabolic processes resulting in enhanced fitness. This study of plant defence against the fungal pathogen Hyaloperonospora arabidopsidis , which causes downy mildew disease in Arabidopsis plants, reveals a link between the plant innate immune responses and the clock. The central circadian regulator CCA1 is shown to regulate a novel set of genes involved in R -gene-mediated defence. The mechanism allows plants to 'anticipate' infection at dawn when the pathogen normally disperses its spores. Circadian rhythms regulate a wide variety of developmental and metabolic processes resulting in enhanced fitness. In this study, a link is made between plant immune responses and the circadian clock. Plant defence against a fungal pathogen which causes downy mildew disease in Arabidopsis is studied, and it is shown that a novel set of defence genes are regulated by the circadian regulator CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1). The mechanism allows plants to 'anticipate' infection at dawn when the pathogen normally disperses its spores. Such a cross-talk mechanism reveals an important strategy for plants to time immune responses against pathogens. The principal immune mechanism against biotrophic pathogens in plants is the resistance ( R )-gene-mediated defence 1 . It was proposed to share components with the broad-spectrum basal defence machinery 2 . However, the underlying molecular mechanism is largely unknown. Here we report the identification of novel genes involved in R -gene-mediated resistance against downy mildew in Arabidopsis and their regulatory control by the circadian regulator, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1). Numerical clustering based on phenotypes of these gene mutants revealed that programmed cell death (PCD) is the major contributor to resistance. Mutants compromised in the R -gene-mediated PCD were also defective in basal resistance, establishing an interconnection between these two distinct defence mechanisms. Surprisingly, we found that these new defence genes are under circadian control by CCA1, allowing plants to ‘anticipate’ infection at dawn when the pathogen normally disperses the spores and time immune responses according to the perception of different pathogenic signals upon infection. Temporal control of the defence genes by CCA1 differentiates their involvement in basal and R -gene-mediated defence. Our study has revealed a key functional link between the circadian clock and plant immunity.

Background Self-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is also regulated by OCT4. p21, a gene negatively associated with self-renewal and a senescence marker, is a member of the universal cyclin-dependent kinase inhibitors (CDKIs) and plays critical roles in the regulation of the G1/S transition. The expression of p21 can be regulated by OCT4-targeted DNA methyltransferases (DNMTs), which play distinct roles in gene regulation and maintaining pluripotency properties. The aim of this study was to determine the role of OCT4 in the regulation of self-renewal and senescence in human hair follicle mesenchymal stem cells (hHFMSCs) and to characterize the molecular mechanisms involved. Methods A lentiviral vector was used to ectopically express OCT4. The influences of OCT4 on the self-renewal and senescence of hHFMSCs were investigated. Next-generation sequencing (NGS) was performed to identify the downstream genes of OCT4 in this process. Methylation-specific PCR (MSP) analysis was performed to measure the methylation level of the p21 promoter region. p21 was overexpressed in hHFMSCs OCT4 to test its downstream effect on OCT4. The regulatory effect of OCT4 on DNMTs was examined by ChIP assay. 5-aza-dC/zebularine was used to inhibit the expression of DNMTs, and then self-renewal properties and senescence in hHFMSCs were detected. Results The overexpression of OCT4 promoted proliferation, cell cycle progression, and osteogenic differentiation capacity of hHFMSCs. The cell senescence of hHFMSCs was markedly suppressed due to the ectopic expression of OCT4. Through NGS, we identified 2466 differentially expressed genes (DEGs) between hHFMSCs OCT4 and hHFMSCs EGFP , including p21, which was downregulated. The overexpression of p21 abrogated the proliferation and osteogenic differentiation capacity of hHFMSCs OCT4 and promoted cell senescence. OCT4 enhanced the transcription of DNMT genes, leading to an elevation in the methylation of the p21 promoter. The inhibition of DNMTs reversed the OCT4-induced p21 reduction, depleted the self-renewal of hHFMSCs OCT4 , and triggered cell senescence. Conclusions OCT4 maintains the self-renewal ability of hHFMSCs and reverses senescence by suppressing the expression of p21 through the upregulation of DNMTs.

Substance abuse and addiction represent a significant public health problem that impacts multiple dimensions of society, including healthcare, the economy, and the workforce. In 2021, over 100,000 drug overdose deaths were reported in the US, with an alarming increase in fatalities related to opioids and psychostimulants. Understanding the fundamental gene regulatory mechanisms underlying addiction and related behaviors could facilitate more effective treatments. To explore how repeated drug exposure alters gene regulatory networks in the brain, we combined capped small (cs)RNA-seq, which accurately captures nascent-like initiating transcripts from total RNA, with Hi-C and single nuclei (sn)ATAC-seq. We profiled initiating transcripts in two addiction-related brain regions, the prefrontal cortex (PFC) and the nucleus accumbens (NAc), from rats that were never exposed to drugs or were subjected to prolonged abstinence after oxycodone or cocaine intravenous self-administration (IVSA). Interrogating over 100,000 active transcription start regions (TSRs) revealed that most TSRs had hallmarks of bonafide enhancers and highlighted the KLF/SP1, RFX, and AP1 transcription factors families as central to establishing brain-specific gene regulatory programs. Analysis of rats with addiction-like behaviors versus controls identified addiction-associated repression of transcription at regulatory enhancers recognized by nuclear receptor subfamily 3 group C (NR3C) factors, including glucocorticoid receptors. Cell-type deconvolution analysis using snATAC-seq uncovered a potential role of glial cells in driving the gene regulatory programs associated with addiction-related phenotypes. These findings highlight the power of advanced transcriptomics methods to provide insight into how addiction perturbs gene regulatory programs in the brain.

The downregulation of tight junction protein CLDN6 promotes breast cancer cell migration and invasion; however, the exact mechanism underlying CLDN6 downregulation remains unclear. CLDN6 silence is associated with DNA methyltransferase 1 (DNMT1) mediated DNA methylation, and DNMT1 is regulated by the transforming growth factor beta (TGFβ)/SMAD pathway. Therefore, we hypothesized that TGFβ/SMAD pathway, specifically SMAD2, may play a critical role for CLDN6 downregulation through DNA methyltransferase 1 (DNMT1) mediated DNA methylation. To test this hypothesis, we blocked the SMAD2 pathway with SB431542 in two human breast cancer cell lines (MCF-7 and SKBR-3). Our results showed that treatment with SB431542 led to a decrease of DNMT1 expression and the binding activity for CLDN6 promoter. The methylation level of CLDN6 promoter was decreased, and simultaneously CLDN6 protein expression increased. Upregulation of CLDN6 inhibited epithelial to mesenchymal transition (EMT) and reduced the migration and invasion ability of both MCF-7 and SKBR-3 cells. Furthermore, knocked down of CLDN6 abolished SB431542 effects on suppression of EMT associated gene expression and inhibition of migration and invasion. Thus, we demonstrated that the downregulation of CLDN6 is regulated through promoter methylation by DNMT1, which depends on the SMAD2 pathway, and that CLDN6 is a key regulator in the SMAD2/DNMT1/CLDN6 pathway to inhibit EMT, migration and invasion of breast cancer cells.