Mechanisms of Plant Epigenetic Regulation in Response to Plant Stress: Recent Discoveries and Implications.

Plant stress is a significant challenge that affects the development, growth, and productivity of plants and causes an adverse environmental condition that disrupts normal physiological processes and hampers plant survival. Epigenetic regulation is a crucial mechanism for plants to respond and adapt to stress. Several studies have investigated the role of DNA methylation (DM), non-coding RNAs, and histone modifications in plant stress responses. However, there are various limitations or challenges in translating the research findings into practical applications. Hence, this review delves into the recent recovery, implications, and applications of epigenetic regulation in response to plant stress. To better understand plant epigenetic regulation under stress, we reviewed recent studies published in the last 5-10 years that made significant contributions, and we analyzed the novel techniques and technologies that have advanced the field, such as next-generation sequencing and genome-wide profiling of epigenetic modifications. We emphasized the breakthrough findings that have uncovered specific genes or pathways and the potential implications of understanding plant epigenetic regulation in response to stress for agriculture, crop improvement, and environmental sustainability. Finally, we concluded that plant epigenetic regulation in response to stress holds immense significance in agriculture, and understanding its mechanisms in stress tolerance can revolutionize crop breeding and genetic engineering strategies, leading to the evolution of stress-tolerant crops and ensuring sustainable food production in the face of climate change and other environmental challenges. Future research in this field will continue to unveil the intricacies of epigenetic regulation and its potential applications in crop improvement.

Characterization of Gene Regulatory Networks in Plants Using New Methods and Data Types.

A major question in plant biology is to understand how plant growth, development, and environmental responses are controlled and coordinated by the activities of regulatory factors. Gene regulatory network (GRN) analyses require integrated approaches that combine experimental approaches with computational analyses. A wide range of experimental approaches and tools are now available, such as targeted perturbation of gene activities, quantitative and cell-type specific measurements of dynamic gene activities, and systematic analysis of the molecular 'hard-wiring' of the systems. At the computational level, different tools and databases are available to study regulatory sequences, including intuitive visualizations to explore data-driven gene regulatory networks in different plant species. Furthermore, advanced data integration approaches have recently been developed to efficiently leverage complementary regulatory data types and learn context-specific networks.

Genome-wide analysis of the Pleurotus eryngii laccase gene (PeLac) family and functional identification of PeLac5.

The laccase gene family encodes multiple isozymes that are crucial for the degradation of substrates and the regulation of developmental processes in fungi. Pleurotus eryngii is an important edible and medicinal fungus belonging to the Basidiomycota phylum and can grow on a variety of natural substrates. In the present study, genome-wide profiling of P. eryngii identified 10 genes encoding its laccase isoenzymes. Conservative sequence analysis demonstrated that all PeLacs possess classical laccase structural domains. Phylogenetic analysis yielded four major subgroups, the members of which are similar with respect to conserved gene organization, protein domain architecture, and consensus motifs. The 10 PeLacs formed three groups together with 12 PoLacs in Pleurotus ostreatus, indicating that they share a high level of evolutionary homology. Cis-responsive element analysis implied that PeLacs genes play a role in growth and development and lignocellulose degradation. Targeted overexpression of PeLac5 reduced the time to primordia formation and their development to fruiting bodies. Gene expression patterns in the presence of different lignocellulosic substrates indicate that three PeLacs genes (2, 4, and 9) are key to lignocellulose degradation. This work presents the first inventory of laccase genes in P. eryngii and preliminarily explores their functions, which may help to uncover the manner by which these proteins utilize substrates.

Comparative and integrative analysis of transcriptomic and epigenomic-wide DNA methylation changes in African American prostate cancer.

African American (AA) men have the highest incidence and mortality rate from Prostate cancer (PCa) than any other racial/ethnic group. To date, PCa genomic studies have largely under-represented tumour samples from AA men. We measured genome-wide DNA methylation in benign and tumor prostate tissues from AA men using the Illumina Infunium 850 K EPIC array. mRNA expression database from a subset of the AA biospecimen were used to assess correlation of transcriptome and methylation datasets. Genome-wide methylation analysis identified 11,460 probes that were significant (p < 0.01) and differentially methylated in AA PCa compared to normal prostate tissues and showed significant (p < 0.01) inverse-correlation with mRNA expression. Ingenuity pathway analysis and Gene Ontology analysis in our AA dataset compared with TCGA dataset showed similarities in methylation patterns: top candidate genes with significant hypermethylation and corresponding down-regulated gene expression were associated with biological pathways in hemidesmosome assembly, mammary gland development, epidermis development, hormone biosynthesis, and cell communication. In addition, top candidate genes with significant hypomethylation and corresponding up-regulated gene expression were associated with biological pathways in macrophage differentiation, cAMP-dependent protein kinase activity, protein destabilization, transcription co-repression, and fatty acid biosynthesis. In contrast, differences in genome-wide methylation in our AA dataset compared with TCGA dataset were enriched for genes in steroid signalling, immune signalling, chromatin structure remodelling and RNA processing. Overall, differential methylation of AMIGO3, IER3, UPB1, GRM7, TFAP2C, TOX2, PLSCR2, ZNF292, ESR2, MIXL1, BOLL, and FGF6 were significant and uniquely associated with PCa progression in our AA cohort.

A transcriptomic signature that predicts cancer recurrence after hepatectomy in patients with colorectal liver metastases.

BACKGROUND: Cancer recurrence is an important predictor of survival outcomes in patients with colorectal cancer-associated liver metastasis (CRLM), who undergo radical hepatectomy. Therefore, identification of patients with the greatest risk of recurrence is critical for developing a precision oncology strategy that might include frequent surveillance (in low-risk patients) or a more aggressive treatment approach (in high-risk patients). We performed genome-wide expression profiling, to identify and develop a transcriptomic signature for predicting recurrence in patients with CRLM. METHODS: We analysed a total of 383 patients with CRLM, including 63 patients from a publicly available data set (the NCBI's Gene Expression Omnibus with accession number GSE81423). and 320 patients from whom surgical specimens were collected for independent training (n = 169) and validation (n = 151) of identified biomarkers. Using Cox's proportional hazard regression analysis, we evaluated the clinical significance of the identified gene signature by comparing its performance with several key clinical factors. RESULTS: We identified a six-gene panel that robustly categorised patients with recurrence in the discovery (area under the curve (AUC) = 0.90). We showed that the panel was a significant predictor of recurrence in the clinical training (AUC = 0.83) and validation cohorts (AUC = 0.81). By combining our panel with key clinical factors, we established a risk-stratification model that emerged as an independent predictor of recurrence (AUC = 0.85; univariate: hazard ratio (HR) = 4.34, 95% confidence interval (CI) = 2.71-6.93, P < 0.001; multivariate: HR = 3.40, 95% CI = 1.76-6.56, P < 0.001). The stratification model revealed recurrence prediction in 89% of high-risk group and non-recurrence in 62% of low-risk group. CONCLUSIONS: We established a novel transcriptomic signature that robustly predicts recurrence, which has significant implications for the management of patients with CRLM.

Obese status is associated with accelerated DNA methylation change in peripheral blood of senior dogs.

Obesity and its associated comorbidities constitute a major and growing health problem worldwide not only involved with people but also dogs and cats. Although few genetic mutations have been associated with obesity in dogs, molecular mechanism remains to be clearly understood. Given the fact that DNA methylation leads to gene expression variability and has plasticity affected by metabolic phenotypes such as obesity in human, the objective of this study is to identify obesity-associated differentially methylated cytosine-phosphate-guanine (CpG) dinucleotide sites in dogs. With genome-wide DNA methylation analysis using next-generation sequencing for blood samples from fourteen Miniature dachshunds with body condition score (BCS) 4-5 and BCS ≥6, over 100,000 sites could be analysed to identify genomic locations of differentially methylated CpG sites. As a result, 191 differentially methylated CpG sites (89 CpG sites were hypermethylated in BCS ≥6 and 102 were hypermethylated in BCS 4-5) were identified. These sites included promoter regions of Kisspeptin receptor (KISS1R) and Calcyphosine 2 (CAPS2) genes which were subsequently validated by bisulfite-pyrosequencing for another set of 157 dog blood samples. KISS1R methylation levels were found to be higher in BCS ≥6 group than BCS 4-5 in senior (>84 months) dogs. Especially male dogs but not female dogs as well as uncastrated male dogs but not castrated male dogs showed this trend. DNA methylation of KISS1R gene will be useful for understanding of comprehensive epigenetic change in obese dogs.

Genetic alterations associated with multiple primary malignancies.

Breast cancer (BC) patients are frequently at risk of developing other malignancies following treatment. Although studies have been conducted to elucidate the etiology of multiple primary malignancies (MPM) after a BC diagnosis, few studies have investigated other previously diagnosed primary malignancies (OPPM) before BC. Here, genome-wide profiling was used to identify potential driver DNA copy number alterations and somatic mutations that promote the development of MPMs. To compare the genomic profiles for two primary tumors (BC and OPPM) from the same patient, tumor pairs from 26 young women (≤50 years) diagnosed with one or more primary malignancies before breast cancer were analyzed. Malignant melanoma was the most frequent OPPM, followed by gynecologic- and hematologic malignancies. However, significantly more genetic alterations were detected in BC compared to the OPPM. BC also showed more genetic similarity as a group than the tumor pairs. Clonality testing showed that genetic alterations on chromosomes 1, 3, 16, and 19 were concordant in both tumors in 13 patients. TP53 mutations were also found to be prevalent in BC, MM, and HM. Although all samples were classified as genetically unstable, chromothripsis-like patterns were primarily observed in BC. Taken together, few recurrent genetic alterations were identified in both tumor pairs that can explain the development of MPMs in the same patient. However, larger studies are warranted to further investigate key driver mutations associated with MPMs.

Genome-Wide Circular RNA Expression Patterns Reflect Resistance to Immunomodulatory Drugs in Multiple Myeloma Cells.

Immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, may induce significant remissions in multiple myeloma (MM) patients, but relapses are frequently observed and the underlying molecular mechanisms for this are not completely understood. Circular RNAs (circRNAs) constitute an emerging class of non-coding RNAs with important roles in cancer. Here, we profiled genome-wide expression patterns of circRNAs in IMiD-sensitive MM cells and their resistant counterparts as well as in IMiD-resistant cells treated with specific epigenetic drugs alone or in combination. We found that genome-wide circRNA expression patterns reflect IMiD sensitivity and ciRS-7 (also known as CDR1as) was the most downregulated circRNA upon acquired resistance. The depletion of ciRS-7 correlated with increased methylation levels of the promoter CpG island of its host gene, LINC00632. Expression of LINC00632 and ciRS-7 was partly restored by treatment with a combination of an EZH2 inhibitor (EPZ-6438) and a DNA methyl transferase inhibitor (5-azacytidine), which also restores the IMiD sensitivity of the cells. However, knockdown of ciRS-7 did not affect IMiD sensitivity and we found that ciRS-7 also becomes epigenetically silenced after prolonged cell culture without drug-exposure. In conclusion, we found that genome-wide circRNA expression patterns reflect IMiD sensitivity in an in vitro model of acquired resistance.

Identification and requirements of enhancers that direct gene expression during zebrafish fin regeneration.

To identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified tens of thousands of DNA regions from each sample type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding expression changes by RNA-seq. To determine whether these profiles reveal bona fide TREEs, we tested the sufficiency and requirements of several sequences in stable transgenic lines and mutant lines with homozygous deletions. These experiments validated new non-coding regulatory sequences near induced and/or essential genes during fin regeneration, including fgf20a, mdka and cx43, identifying distinct domains of directed expression for each confirmed TREE. Whereas deletion of the previously identified LEN enhancer abolished detectable induction of the nearby leptin b gene during regeneration, deletions of enhancers linked to fgf20a, mdka and cx43 had no effect or partially reduced gene expression. Our study generates a new resource for dissecting the regulatory mechanisms of appendage generation and reveals a range of requirements for individual TREEs in control of regeneration programs.

High-throughput RNA sequencing from paired lesional- and non-lesional skin reveals major alterations in the psoriasis circRNAome.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. It is one of the most prevalent chronic inflammatory skin conditions in adults worldwide, with a considerable negative impact on quality of life. Circular RNAs (circRNAs) are a recently identified type of non-coding RNA with diverse cellular functions related to their exceptional stability. In particular, some circRNAs can bind and regulate microRNAs (miRNAs), a group of RNAs that play a role in the pathogenesis of psoriasis. The aim of this study was to characterize the circRNAome in psoriasis and to assess potential correlations to miRNA expression patterns. METHODS: We used high-throughput RNA-sequencing (RNA-seq), NanoString nCounter technology and RNA chromogenic in situ hybridization (CISH) to profile the circRNA expression in paired lesional and non-lesional psoriatic skin from patients with psoriasis vulgaris. In addition, 799 miRNAs were profiled using NanoString nCounter technology and laser capture microdissection was used to study the dermis and epidermis separately. RESULTS: We found a substantial down-regulation of circRNA expression in lesional skin compared to non-lesional skin. We observed that this mainly applies to the epidermis by analyzing laser capture microdissected tissues. We also found that the majority of the circRNAs were downregulated independently of their corresponding linear host genes. The observed downregulation of circRNAs in psoriasis was neither due to altered expression levels of factors known to affect circRNA biogenesis, nor because lesional skin contained an increased number of inflammatory cells such as lymphocytes. Finally, we observed that the overall differences in available miRNA binding sites on the circRNAs between lesional and non-lesional skin did not correlate with differences in miRNA expression patterns. CONCLUSIONS: We have performed the first genome-wide circRNA profiling of paired lesional and non-lesional skin from patients with psoriasis and revealed that circRNAs are much less abundant in the lesional samples. Whether this is a cause or a consequence of the disease remains to be revealed, however, we found no evidence that the loss of miRNA binding sites on the circRNAs could explain differences in miRNA expression between lesional and non-lesional skin.

Epigenomic profiling of DNA methylation for hepatocellular carcinoma diagnosis and prognosis prediction.

BACKGROUND AND AIM: DNA hypermethylation has emerged as a novel molecular biomarker for the diagnosis and prognosis prediction of many cancers. We aimed to identify clinically useful biomarkers regulated by DNA methylation in hepatocellular carcinoma (HCC). METHODS: Genome-wide methylation analysis in HCCs and paired noncancerous tissues was performed using an Illumina Infinium HumanMethylation 450K BeadChip array. Methylation-specific polymerase chain reaction and pyrosequencing were used to validate the methylation status of selected genes in 100 paired HCCs and noncancerous samples. RESULTS: A total of 97 027 (20.0%) out of 485 577 CpG sites significantly were differed between HCC and noncancerous tissues. Among all the significant CpG sites, 48.8% are hypermethylated and 51.2% are hypomethylated in HCCs. Multiple signaling pathways (AMP-activated protein kinase, estrogen, and adipocytokine) involved in gene methylation were identified in HCC. FES was selected for further analysis based on its high level of methylation confirmed by polymerase chain reaction and pyrosequencing. The result showed that FES hypermethylation was correlated with tumor size (0.001), serum alpha fetoprotein (0.023), and tumor differentiation (0.006). FES protein was significantly downregulated in 51/100 (51%) HCCs, and 94.12% (48/51) of them were due to promoter hypermethylation. Both FES hypermethylation and protein downregulation were associated with the progression-free survival and overall survival of HCC patients. Overexpressed and knockdown of FES confirmed its inhibitory effect on the proliferation and migration of HCC cells. CONCLUSIONS: We identified many new differentially methylated CpGs in HCCs and demonstrate that FES functions as a tumor suppressor gene in HCC and its methylation status could be used as an indicator for prognosis of HCC.

Genome-wide probing RNA structure with the modified DMS-MaPseq in Arabidopsis.

Transcripts have intrinsic propensity to form stable secondary structure that is fundamental to regulate RNA transcription, splicing, translation, RNA localization and turnover. Numerous methods that integrate chemical reactions with next-generation sequencing (NGS) have been applied to study in vivo RNA structure, providing new insights into RNA biology. Dimethyl sulfate (DMS) probing coupled with mutational profiling through NGS (DMS-MaPseq) is a newly developed method for revealing genome-wide or target-specific RNA structure. Herein, we present our experimental protocol of a modified DMS-MaPseq method for plant materials. The DMS treatment condition was optimized, and library preparation procedures were simplified. We also provided custom scripts for bioinformatic analysis of genome-wide DMS-MaPseq data. Bioinformatic results showed that our method could generate high-quality and reproducible data. Further, we assessed sequencing depth and coverage for genome-wide RNA structure profiling in Arabidopsis, and provided two examples of in vivo structure of mobile RNAs. We hope that our modified DMS-MaPseq method will serve as a powerful tool for analyzing in vivo RNA structurome in plants.

Differential genome-wide profiling of alternative polyadenylation sites in nasopharyngeal carcinoma by high-throughput sequencing.

BACKGROUND: Alternative polyadenylation (APA) is a widespread phenomenon in the posttranscriptional regulation of gene expression that generates mRNAs with alternative 3'-untranslated regions (3'UTRs). APA contributes to the pathogenesis of various diseases, including cancer. However, the potential role of APA in the development of nasopharyngeal carcinoma (NPC) remains largely unknown. METHODS: A strategy of sequencing APA sites (SAPAS) based on second-generation sequencing technology was carried out to explore the global patterns of APA sites and identify genes with tandem 3'UTRs in samples from 6 NPC and 6 normal nasopharyngeal epithelial tissue (NNET). Sequencing results were then validated using quantitative RT-PCR in a larger cohort of 16 NPC and 16 NNET samples. RESULTS: The sequencing data showed that the use of tandem APA sites was prevalent in NPC, and numerous genes with APA-switching events were discovered. In total, we identified 195 genes with significant differences in the tandem 3'UTR length between NPC and NNET; including 119 genes switching to distal poly (A) sites and 76 genes switching to proximal poly (A) sites. Several gene ontology (GO) terms were enriched in the list of genes with switched APA sites, including regulation of cell migration, macromolecule catabolic process, protein catabolic process, proteolysis, small conjugating protein ligase activity, and ubiquitin-protein ligase activity. CONCLUSIONS: APA site-switching events are prevalent in NPC. APA-mediated regulation of gene expression may play an important role in the development of NPC, and more detailed studies targeting genes with APA-switching events may contribute to the development of novel future therapeutic strategies for NPC.

CircRNAs in Plants.

Circular RNAs (circRNAs) are covalently closed, single-stranded transcripts that are ubiquitously expressed in all eukaryotes and even prokaryotic archaea. Although once regarded as splicing artifacts, circRNAs are a novel class of regulatory molecules with diverse biological functions, including regulation of transcription, modulation of alternative splicing, and binding of miRNAs and proteins. The majority of studies of circRNAs have been performed in animals with a focus on the biogenesis, function, and mechanistic characterization of these molecules. In contrast, the study of circRNAs in plants is just emerging. Interestingly, recent circRNA profiling studies in model plant systems show distinct features of plant circRNAs compared with those from animals, including putative roles in stress response, differences in expression patterns, and novel biogenesis mechanisms. This provides a great opportunity to broaden our knowledge of circRNAs using plant model systems, such as Arabidopsis and rice, which are ideal for phenotypic characterization and genetic studies. In this review, we summarize current knowledge of plant circRNAs, discuss their identification and biogenesis, describe potential functions, and propose future perspectives for plant circRNA study.

Nuclear Receptor Nur77 Limits the Macrophage Inflammatory Response through Transcriptional Reprogramming of Mitochondrial Metabolism.

Activation of macrophages by inflammatory stimuli induces reprogramming of mitochondrial metabolism to support the production of pro-inflammatory cytokines and nitric oxide. Hallmarks of this metabolic rewiring are downregulation of α-ketoglutarate formation by isocitrate dehydrogenase (IDH) and accumulation of glutamine-derived succinate, which enhances the inflammatory response via the activity of succinate dehydrogenase (SDH). Here, we identify the nuclear receptor Nur77 (Nr4a1) as a key upstream transcriptional regulator of this pro-inflammatory metabolic switch in macrophages. Nur77-deficient macrophages fail to downregulate IDH expression and accumulate higher levels of succinate and other TCA cycle-derived metabolites in response to inflammatory stimulation in a glutamine-independent manner. Consequently, these macrophages produce more nitric oxide and pro-inflammatory cytokines in an SDH-dependent manner. In vivo, bone marrow Nur77 deficiency exacerbates atherosclerosis development and leads to increased circulating succinate levels. In summary, Nur77 induces an anti-inflammatory metabolic state in macrophages that protects against chronic inflammatory diseases such as atherosclerosis.

Chromatin Landscape Distinguishes the Genomic Loci of Hundreds of Androgen-Receptor-Associated LincRNAs From the Loci of Non-associated LincRNAs.

Cell signaling events triggered by androgen hormone in prostate cells is dependent on activation of the androgen receptor (AR) transcription factor. Androgen hormone binding to AR promotes its displacement from the cytoplasm to the nucleus and AR binding to DNA motifs, thus inducing activatory and inhibitory transcriptional programs through a complex regulatory mechanism not yet fully understood. In this work, we performed RNA-seq deep-sequencing of LNCaP prostate cancer cells and found over 7000 expressed long intergenic non-coding RNAs (lincRNAs), of which ∼4000 are novel lincRNAs, and 258 lincRNAs have their expression activated by androgen. Immunoprecipitation of AR, followed by large-scale sequencing of co-immunoprecipitated RNAs (RIP-Seq) has identified in the LNCaP cell line a total of 619 lincRNAs that were significantly enriched (FDR < 10%, DESeq2) in the anti-Androgen Receptor (antiAR) fraction in relation to the control fraction (non-specific IgG), and we named them Androgen-Receptor-Associated lincRNAs (ARA-lincRNAs). A genome-wide analysis showed that protein-coding gene neighbors to ARA-lincRNAs had a significantly higher androgen-induced change in expression than protein-coding genes neighboring lincRNAs not associated to AR. To find relevant epigenetic signatures enriched at the ARA-lincRNAs' transcription start sites (TSSs) we used a machine learning approach and identified that the ARA-lincRNA genomic loci in LNCaP cells are significantly enriched with epigenetic marks that are characteristic of in cis enhancer RNA regulators, and that the H3K27ac mark of active enhancers is conspicuously enriched at the TSS of ARA-lincRNAs adjacent to androgen-activated protein-coding genes. In addition, LNCaP topologically associating domains (TADs) that comprise chromatin regions with ARA-lincRNAs exhibit transcription factor contents, epigenetic marks and gene transcriptional activities that are significantly different from TADs not containing ARA-lincRNAs. This work highlights the possible involvement of hundreds of lincRNAs working in synergy with the AR on the genome-wide androgen-induced gene regulatory program in prostate cells.

Dynamic changes in DNA methylation patterns in canine lymphoma cell lines demonstrated by genome-wide quantitative DNA methylation analysis.

DNA methylation is the conversion of cytosine to 5-methylcytosine, leading to changes in the interactions between DNA and proteins. Methylation of cytosine-guanine (CpG) islands (CGIs) is associated with gene expression silencing of the involved promoter. Although studies focussing on global changes or a few single loci in DNA methylation have been performed in dogs with certain diseases, genome-wide analysis of DNA methylation is required to prospectively identify specific regions with DNA methylation change. The hypothesis of this study was that next-generation sequencing with methylation-specific signatures created by sequential digestion of genomic DNA with SmaI and XmaI enzymes can provide quantitative information on methylation levels. Using blood from healthy dogs and cells obtained from canine lymphoma cell lines, approximately 100,000CpG sites across the dog genome were analysed with the novel method established in this study. CpG sites in CGIs broadly were shown to be either methylated or unmethylated in normal blood, while CpG sites not within CpG islands (NCGIs) were largely methylated. Thousands of CpG sites in lymphoma cell lines were found to gain methylation at normally unmethylated CGI sites and lose methylation at normally methylated NCGI sites. These hypermethylated CpG sites are located at promoter regions of hundreds of genes, such as TWIST2 and TLX3. In addition, genes annotated with 'Homeobox' and 'DNA-binding' characteristics have hypermethylated CpG sites in their promoter CGIs. Genome-wide quantitative DNA methylation analysis is a sensitive method that is likely to be suitable for studies of DNA methylation changes in cancer, as well as other common diseases in dogs.

Chromatin landscape distinguishes the genomic loci of hundreds of Androgen-Receptor-Associated LincRNAs from the loci of non-associated LincRNAs

Cell signaling events triggered by androgen hormone in prostate cells is dependent on activation of the androgen receptor (AR) transcription factor. Androgen hormone binding to AR promotes its displacement from the cytoplasm to the nucleus and AR binding to DNA motifs, thus inducing activatory and inhibitory transcriptional programs through a complex regulatory mechanism not yet fully understood. In this work, we performed RNA-seq deep-sequencing of LNCaP prostate cancer cells and found over 7000 expressed long intergenic non-coding RNAs (lincRNAs), of which similar to 4000 are novel lincRNAs, and 258 lincRNAs have their expression activated by androgen. Immunoprecipitation of AR, followed by large-scale sequencing of co-immunoprecipitated RNAs (RIP-Seq) has identified in the LNCaP cell line a total of 619 lincRNAs that were significantly enriched (FDR < 10%, DESeq2) in the anti-Androgen Receptor (antiAR) fraction in relation to the control fraction (non-specific IgG), and we named them Androgen-Receptor-Associated lincRNAs (ARA-lincRNAs). A genome-wide analysis showed that protein-coding gene neighbors to ARA-lincRNAs had a significantly higher androgen-induced change in expression than protein-coding genes neighboring lincRNAs not associated to AR. To find relevant epigenetic signatures enriched at the ARA-lincRNAs' transcription start sites (TSSs) we used a machine learning approach and identified that the ARA-lincRNA genomic loci in LNCaP cells are significantly enriched with epigenetic marks that are characteristic of in cis enhancer RNA regulators, and that the H3K27ac mark of active enhancers is conspicuously enriched at the TSS of ARA-lincRNAs adjacent to androgen-activated protein-coding genes. In addition, LNCaP topologically associating domains (TADs) that comprise chromatin regions with ARA-lincRNAs exhibit transcription factor contents, epigenetic marks and gene transcriptional activities that are significantly different from TADs not containing ARA-lincRNAs. This work highlights the possible involvement of hundreds of lincRNAs working in synergy with the AR on the genome-wide androgen-induced gene regulatory program in prostate cells.

Genome-wide profiling of the 3' ends of polyadenylated RNAs.

Alternative polyadenylation (APA) diversifies the 3' termini of a majority of mRNAs in most eukaryotes, and is consequently inferred to have substantial consequences for the utilization of post-transcriptional regulatory mechanisms. Since conventional RNA-sequencing methods do not accurately define mRNA termini, a number of protocols have been developed that permit sequencing of the 3' ends of polyadenylated transcripts (3'-seq). We present here our experimental protocol to generate 3'-seq libraries using a dT-priming approach, including extensive details on considerations that will enable successful library cloning. We pair this with a set of computational tools that allow the user to process the raw sequence data into a filtered set of clusters that represent high-confidence functional polyadenylation sites. The data are single-nucleotide resolution and quantitative, and can be used for downstream analyses of APA.

Systematic identification of Ctr9 regulome in ERα-positive breast cancer.

BACKGROUND: We had previously identified Ctr9, the key scaffold subunit of the human RNA polymerase II (RNAPII) associated factor complex (PAFc), as a key factor regulating a massive ERα target gene expression and ERα-positive breast cancer growth. Furthermore, we have shown that knockdown of Ctr9 reduces ERα protein stability and decreases the occupancy of ERα and RNAPII at a few ERα-target loci. However, it remains to be determined whether Ctr9 controls ERα-target gene expression by regulating the global chromatin occupancy of ERα and RNAPII in the presence of estrogen. RESULTS: In this study, we determined the genome-wide ERα and RNAPII occupancy in response to estrogen reatment and/or Ctr9 knockdown by performing chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq). We found that loss of Ctr9 dramatically decreases the global occupancy of ERα and RNAPII, highlighting the significance of Ctr9 in regulating estrogen signaling in ERα-positive breast cancer cells. Combining this resource with previously published genomic data sets, we identified a unique subset of ERα and Ctr9 target genes, and further delineated the independent function of Ctr9 from other subunits in PAFc when regulating transcription. CONCLUSIONS: Our data demonstrated that Ctr9, independent of other PAFc subunits, controls ERα-target gene expression by regulating global chromatin occupancies of ERα and RNAPII.