Chromatin Isolation Research Articles

The long non-coding RNA PFI protects against pulmonary fibrosis by interacting with splicing regulator SRSF1.

Pulmonary fibrosis (PF) is a type of interstitial pneumonia with complex etiology and high mortality, characterized by progressive scarring of the alveolar interstitium and myofibroblastic lesions. Recently, there has been growing appreciation of the importance of long non-coding RNAs (lncRNAs) in organ fibrosis. The aim of this study was to investigate the role of lncRNAs in lung fibrosis. We used a qRT-PCR assay to identify dysregulated lncRNAs in the lungs of mice with experimental, bleomycin (BLM)-induced pulmonary fibrosis, and a series of molecular assays to assess the role of the novel lncRNA NONMMUT060091, designated as pulmonary fibrosis inhibitor (PFI), which was significantly downregulated in lung fibrosis. Functionally, knockdown of endogenous PFI by smart silencer promoted proliferation, differentiation, and extracellular matrix (ECM) deposition in primary mouse lung fibroblasts (MLFs). In contrast, overexpression of PFI partially abrogated TGF-β1-induced fibrogenesis both in MLFs and in the human fetal lung fibroblast MRC-5 cells. Similarly, PFI overexpression attenuated BLM-induced pulmonary fibrosis compared with wild type (WT) mice. Mechanistically, using chromatin isolation by RNA purification-mass spectrometry (ChIRP-MS) and an RNA pull-down assay, PFI was found to directly bind Serine/arginine-rich splicing factor 1 (SRSF1), and to repress its expression and pro-fibrotic activity. Furthermore, silencing of SRSF1 inhibited TGF-β1-induced proliferation, differentiation, and ECM deposition in MRC-5 cells by limiting the formation of the EDA+Fn1 splicing isoform; whereas forced expression of SRSF1 by intratracheal injection of adeno-associated virus 5 (AAV5) ablated the anti-fibrotic effect of PFI in BLM-treated mice. Overall, these data reveal that PFI mitigated pulmonary fibrosis through negative regulation of the expression and activity of SRSF1 to decrease the formation of the EDA+Fn1 splicing isoform, and suggest that PFI and SRSF1 may serve as potential targets for the treatment of lung fibrosis.

Neofunctionalization of a polyploidization-activated cotton long intergenic non-coding RNA DAN1 during drought stress regulation.

The genomic shock of whole-genome duplication (WGD) and hybridization introduces great variation into transcriptomes, for both coding and noncoding genes. An altered transcriptome provides a molecular basis for improving adaptation during the evolution of new species. The allotetraploid cotton, together with the putative diploid ancestor species compose a fine model for study the rapid gene neofunctionalization over the genome shock. Here we report on Drought-Associated Non-coding gene 1 (DAN1), a long intergenic noncoding RNA (lincRNA) that arose from the cotton progenitor A-diploid genome after hybridization and WGD events during cotton evolution. DAN1 in allotetraploid upland cotton (Gossypium hirsutum) is a drought-responsive lincRNA predominantly expressed in the nucleoplasm. Chromatin isolation by RNA purification profiling and electrophoretic mobility shift assay analysis demonstrated that GhDAN1 RNA can bind with DNA fragments containing AAAG motifs, similar to DNA binding with one zinc finger transcription factor binding sequences. The suppression of GhDAN1 mainly regulates genes with AAAG motifs in auxin-response pathways, which are associated with drought stress regulation. As a result, GhDAN1-silenced plants exhibit improved tolerance to drought stress. This phenotype resembles the drought-tolerant phenotype of the A-diploid cotton ancestor species, which has an undetectable expression of DAN1. The role of DAN1 in cotton evolution and drought tolerance regulation suggests that the genomic shock of interspecific hybridization and WGD stimulated neofunctionalization of non-coding genes during the natural evolutionary process.

Long noncoding RNA regulation of spermatogenesis via the spectrin cytoskeleton in Drosophila.

The spectrin cytoskeleton has been shown to be critical in diverse processes such as axon development and degeneration, myoblast fusion, and spermatogenesis. Spectrin can be modulated in a tissue specific manner through junctional protein complexes, however, it has not been shown that long noncoding RNAs (lncRNAs) interact with and modulate spectrin. Here, we provide evidence of a lncRNA CR45362 that interacts with α-Spectrin, is required for spermatid nuclear bundling during Drosophila spermatogenesis. We observed that CR45362 showed high expression in the cyst cells at the basal testis, and CRISPR-mediated knockout of CR45362 led to sterile male, unbundled spermatid nuclei, and disrupted actin cones. Through chromatin isolation by RNA precipitation—mass spectrometry (ChIRP-MS), we identified actin-spectrin cytoskeletal components physically interact with the lncRNA CR45362. Genetic screening on identified cytoskeletal factors revealed that cyst cell-specific knockdown of α-Spectrin phenocopied CR45362 mutants and resulted in spermatid nuclear bundle defects. Consistently, CR45362 knockout disrupted the co-localization of α-Spectrin and spermatid nuclear bundles in the head cyst cells at the basal testis. Thus, we uncovered a novel lncRNA CR45362 that interacts with α-Spectrin to stabilize spermatid nuclear bundles during spermatid maturation.

A novel long intergenic non-coding RNA, Nostrill, regulates iNOS gene transcription and neurotoxicity in microglia

BackgroundMicroglia are resident immunocompetent and phagocytic cells in the CNS. Pro-inflammatory microglia, stimulated by microbial signals such as bacterial lipopolysaccharide (LPS), viral RNAs, or inflammatory cytokines, are neurotoxic and associated with pathogenesis of several neurodegenerative diseases. Long non-coding RNAs (lncRNA) are emerging as important tissue-specific regulatory molecules directing cell differentiation and functional states and may help direct proinflammatory responses of microglia. Characterization of lncRNAs upregulated in proinflammatory microglia, such as NR_126553 or 2500002B13Rik, now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus) increases our understanding of molecular mechanisms in CNS innate immunity.MethodsMicroglial gene expression array analyses and qRT-PCR were used to identify a novel long intergenic non-coding RNA, Nostrill, upregulated in LPS-stimulated microglial cell lines, LPS-stimulated primary microglia, and LPS-injected mouse cortical tissue. Silencing and overexpression studies, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification assays, and qRT-PCR were used to study the function of this long non-coding RNA in microglia. In vitro assays were used to examine the effects of silencing the novel long non-coding RNA in LPS-stimulated microglia on neurotoxicity.ResultsWe report here characterization of intergenic lncRNA, NR_126553, or 2500002B13Rik now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus). Nostrill is induced by LPS stimulation in BV2 cells, primary murine microglia, and in cortical tissue of LPS-injected mice. Induction of Nostrill is NF-κB dependent and silencing of Nostrill decreased inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in BV2 and primary microglial cells. Overexpression of Nostrill increased iNOS expression and NO production. RNA immunoprecipitation assays demonstrated that Nostrill is physically associated with NF-κB subunit p65 following LPS stimulation. Silencing of Nostrill significantly reduced NF-κB p65 and RNA polymerase II recruitment to the iNOS promoter and decreased H3K4me3 activating histone modifications at iNOS gene loci. In vitro studies demonstrated that silencing of Nostrill in microglia reduced LPS-stimulated microglial neurotoxicity.ConclusionsOur data indicate a new regulatory role of the NF-κB-induced Nostrill and suggest that Nostrill acts as a co-activator of transcription of iNOS resulting in the production of nitric oxide by microglia through modulation of epigenetic chromatin remodeling. Nostrill may be a target for reducing the neurotoxicity associated with iNOS-mediated inflammatory processes in microglia during neurodegeneration.

Study of Genome-Wide Occupancy of Long Non-Coding RNAs Using Chromatin Isolation by RNA Purification (ChIRP).

Long noncoding RNAs (lncRNAs) have recently emerged as masters of gene expression regulation by exerting their functions in all cell compartments through a wide repertoire of mechanisms. A high portion of lncRNAs are robustly enriched in the chromatin fraction suggesting a broad regulatory role in the nuclear compartment. Despite the advances in this field, the interaction between lncRNAs and the chromatin is still poorly understood. This led to the emergence of numerous hybridization capture assays such as the Chromatin Isolation by RNA Purification (ChIRP) which revealed at high resolution the genomic binding sites of several nuclear lncRNAs. In this chapter, we describe the ChIRP protocol that was successfully applied to the lncRNA ANRIL. We also provide a user-friendly bioinformatic pipeline for ChIRP-seq data analysis.

LncRNA PRADX-mediated recruitment of PRC2/DDX5 complex suppresses UBXN1 expression and activates NF-κB activity, promoting tumorigenesis.

Rationale: Accumulating evidence indicates that long noncoding RNAs (lncRNAs) play crucial roles in cancer progression; however, only few have been characterized in detail. The current study aimed to identify a novel cancer driver lncRNA in glioblastoma and colon adenocarcinoma.Methods: We performed whole transcriptome analysis of TCGA pan-cancer datasets to compare the lncRNA expression profiles of tumor and paired normal tissues. In situ hybridization of tissue sections was performed to validate the expression data and determine the localization of lncRNAs that may be linked to glioblastoma and colon adenocarcinoma. Chromatin isolation by RNA purification (ChIRP), chromatin immunoprecipitation (ChIP), and Co-immunoprecipitation (Co-IP) assays were performed to assess the interaction between lncRNA, proteins, and chromatin. The functional significance of the identified lncRNAs was verified in vitro and in vivo by knockdown or exogenous expression experiments.Results: We found a lncRNA ENST00000449248.1 termed PRC2 and DDX5 associated lncRNA (PRADX) that is highly expressed in glioblastoma and colon adenocarcinoma cells and tissues. PRADX, mainly located in the nucleus of tumor cells, could bind to EZH2 protein via the 5' terminal sequence. Moreover, PRADX increased the trimethylation of H3K27 in the UBXN1 gene promoter via PRC2/DDX5 complex recruitment and promoted NF-κB activity through UBXN1 suppression. Knockdown of PRADX significantly inhibited tumor cell viability and clonogenic growth in vitro. In xenograft models, PRADX knockdown suppressed tumor growth and tumorigenesis and prolonged the survival of tumor-bearing mice.Conclusions: PRADX acts as a cancer driver and may serve as a potential therapeutic target for glioblastoma and colon adenocarcinoma.

TINC— A Method to Dissect Regulatory Complexes at Single-Locus Resolution— Reveals an Extensive Protein Complex at the Nanog Promoter

SummaryCellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general.

Full-length annotation with multistrategy RNA-seq uncovers transcriptional regulation of lncRNAs in cotton.

Long noncoding RNAs (lncRNAs) are crucial factors during plant development and environmental responses. To build an accurate atlas of lncRNAs in the diploid cotton Gossypium arboreum, we combined Isoform-sequencing, strand-specific RNA-seq (ssRNA-seq), and cap analysis gene expression (CAGE-seq) with PolyA-seq and compiled a pipeline named plant full-length lncRNA to integrate multi-strategy RNA-seq data. In total, 9,240 lncRNAs from 21 tissue samples were identified. 4,405 and 4,805 lncRNA transcripts were supported by CAGE-seq and PolyA-seq, respectively, among which 6.7% and 7.2% had multiple transcription start sites (TSSs) and transcription termination sites (TTSs). We revealed that alternative usage of TSS and TTS of lncRNAs occurs pervasively during plant growth. Besides, we uncovered that many lncRNAs act in cis to regulate adjacent protein-coding genes (PCGs). It was especially interesting to observe 64 cases wherein the lncRNAs were involved in the TSS alternative usage of PCGs. We identified lncRNAs that are coexpressed with ovule- and fiber development-associated PCGs, or linked to GWAS single-nucleotide polymorphisms. We mapped the genome-wide binding sites of two lncRNAs with chromatin isolation by RNA purification sequencing. We also validated the transcriptional regulatory role of lnc-Ga13g0352 via virus-induced gene suppression assay, indicating that this lncRNA might act as a dual-functional regulator that either activates or inhibits the transcription of target genes.

Human shelterin protein POT1 prevents severe telomere instability induced by homology-directed DNA repair.

The evolutionarily conserved POT1 protein binds single-stranded G-rich telomeric DNA and has been implicated in contributing to telomeric DNA maintenance and the suppression of DNA damage checkpoint signaling. Here, we explore human POT1 function through genetics and proteomics, discovering that a complete absence of POT1 leads to severe telomere maintenance defects that had not been anticipated from previous depletion studies in human cells. Conditional deletion of POT1 in HEK293E cells gives rise to rapid telomere elongation and length heterogeneity, branched telomeric DNA structures, telomeric R-loops, and telomere fragility. We determine the telomeric proteome upon POT1-loss, implementing an improved telomeric chromatin isolation protocol. We identify a large set of proteins involved in nucleic acid metabolism that engage with telomeres upon POT1-loss. Inactivation of the homology-directed repair machinery suppresses POT1-loss-mediated telomeric DNA defects. Our results unravel as major function of human POT1 the suppression of telomere instability induced by homology-directed repair.

The ERG-Regulated LINC00920 Promotes Prostate Cancer Cell Survival via the 14-3-3ε-FOXO Pathway.

Numerous noncoding transcripts have been reported to correlate with cancer development and progression. Nevertheless, there remains a paucity of long noncoding RNAs (lncRNA) with well-elucidated functional roles. Here, we leverage the International Cancer Genome Consortium-Early Onset Prostate Cancer transcriptome and identify the previously uncharacterized lncRNA LINC00920 to be upregulated in prostate tumors. Phenotypic characterization of LINC00920 revealed its positive impact on cellular proliferation, colony formation, and migration. We demonstrate that LINC00920 transcription is directly activated by ERG, an oncogenic transcription factor overexpressed in 50% of prostate cancers. Chromatin isolation by RNA purification-mass spectrometry revealed the interaction of LINC00920 with the 14-3-3ε protein, leading to enhanced sequestration of tumor suppressive FOXO1. Altogether, our results provide a rationale on how ERG overexpression, partly by driving LINC00920 transcription, could confer survival advantage to prostate cancer cells and potentially prime PTEN-intact prostate cells for cellular transformation through FOXO inactivation. IMPLICATIONS: The study describes a novel lncRNA-mediated mechanism of regulating the FOXO signaling pathway and provides additional insight into the role of ERG in prostate cancer cells.

The long noncoding RNA LUCAT1 promotes colorectal cancer cell proliferation by antagonizing Nucleolin to regulate MYC expression

The long noncoding RNA (lncRNA) LUCAT1 was recently reported to be upregulated and to play an essential role in multiple cancer types, especially colorectal cancer (CRC), but the molecular mechanisms of LUCAT1 in CRC are mostly unreported. Here, a systematic analysis of LUACT1 expression is performed with data from TCGA database and clinic CRC samples. LUCAT1 is identified as a putative oncogene, which is significantly upregulated in CRC and is associated with poor prognosis. Loss of LUCAT1 restricts CRC proliferative capacities in vitro and in vivo. Mechanically, NCL is identified as the protein binding partner of LUCAT1 by using chromatin isolation by RNA purification coupled with mass spectrometry (ChIRP-MS) and RNA immunoprecipitation assays. We also show that NCL directly binds to LUCAT1 via its putative G-quadruplex-forming regions from nucleotides 717 to 746. The interaction between LUCAT1 and NCL interferes NCL-mediated inhibition of MYC and promote the expression of MYC. Cells lacking LUCAT1 show a decreased MYC expression, and NCL knockdown rescue LUCAT1 depletion-induced inhibition of CRC cell proliferation and MYC expression. Our results suggest that LUCAT1 plays a critical role in CRC cell proliferation by inhibiting the function of NCL via its G-quadruplex structure and may serve as a new prognostic biomarker and effective therapeutic target for CRC.

Long Noncoding RNA AW112010 Promotes the Differentiation of Inflammatory T Cells by Suppressing IL-10 Expression through Histone Demethylation.

Long noncoding RNAs (lncRNAs) have been demonstrated to play important regulatory roles in gene expression, from histone modification to protein stability. However, the functions of most identified lncRNAs are not known. In this study, we investigated the role of an lncRNA called AW112010. The expression of AW112010 was significantly increased in CD4+ T cells from C57BL/6J mice activated in vivo with myelin oligodendrocyte glycoprotein, Staphylococcal enterotoxin B, or in vitro with anti-CD3 anti-CD28 mAbs, thereby demonstrating that activation of T cells leads to induction of AW112010. In contrast, anti-inflammatory cannabinoids such as cannabidiol or δ-9-tetrahydrocannabinol decreased the expression of AW112010 in T cells. Interestingly, the expression of AW112010 was high in in vitro-polarized Th1 and Th17 cells but low in Th2 cells, suggesting that this lncRNA may regulate inflammation. To identify genes that might be regulated by AW112010, we used chromatin isolation by RNA purification, followed by sequencing. This approach demonstrated that AW112010 regulated the transcription of IL-10. Additionally, the level of IL-10 in activated T cells was low when the expression of AW112010 was increased. Use of small interfering RNA to knock down AW112010 expression in activated T cells led to increased IL-10 expression and a decrease in the expression of IFN-γ. Further studies showed that AW112010 interacted with histone demethylase KDM5A, which led to decreased H3K4 methylation in IL-10 gene locus. Together, these studies demonstrate that lncRNA AW112010 promotes the differentiation of inflammatory T cells by suppressing IL-10 expression through histone demethylation.

Abstract MP140: Epigenetic Silencing of SERCA2a by Long Noncoding RNA Lnc-SYNPO Contributes to Impaired Ca 2+ Homeostasis and Contractility in the Failing Heart

Abnormal sarcolemmal Ca 2+ handling and impaired contractile function owing to sarcoplasmic reticulum/endoplasmic reticulum ATPase 2a (SERCA2a, encoded by ATP2A2 ) downregulation and/or dysfunction are pathognomonic hallmarks of heart failure (HF). There is, however, no clinically proven HF therapy that targets SERCA2a/ ATP2A2 dysregulation directly. Exploiting gene co-expression network analysis using an RNA sequencing dataset generated from human failing and non-failing left ventricular (LV) tissues, we identified long noncoding RNA lnc-SYNPO , transcribed from the 3’ untranslated region of SYNPO2 , as a potential regulator of SERCA2a/ ATP2A2 . Myocardial lnc-SYNPO was markedly upregulated during HF and its expression level showed a strong negative correlation (Pearson correlation coefficient=-0.60) with that of SERCA2a/ ATP2A2 in both mouse and human LV. Knockdown of lnc-SYNPO in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) increased, whereas overexpression of lnc-SYNPO reduced, SERCA2a/ ATP2A2 expression. In addition, lnc-SYNPO knockdown reversed endothelin-1 (ET-1) treatment-induced SERCA2a/ ATP2A2 downregulation, impaired Ca 2+ homeostasis and contractile dysfunction in hiPSC-CM. Mechanistically, the depletion of lnc-SYNPO, a chromatin-associated nuclear lncRNA, led to increased H3K4me3 and reduced H3K27me3 occupancy at SERCA2a/ ATP2A2 promoter. Chromatin isolation by RNA purification showed direct physical binding between lnc-SYNPO and the promoter of SERCA2a/ ATP2A2 . In addition, RNA pull-down assays showed that lnc-SYNPO binds to EZH2 and SUZ12, two core components of the polycomb repressor complex 2 (PRC2), suggesting that lnc-SYNPO represses SERCA2a/ ATP2A2 expression by recruiting PRC2 complex to the promoter of SERCA2a/ ATP2A2 , leading to epigenetic silencing of SERCA2a/ ATP2A2 . Taken together, these results unveiled a previously undiscovered epigenetic regulatory mechanism of SERCA2a/ ATP2A2 by lnc-SYNPO , contributing to impaired myocardial Ca 2+ homeostasis and contractile dysfunction observed with HF. Targeting lnc-SYNPO , therefore, could be a novel therapeutic approach to improve Ca 2+ homeostasis and contractile function in the failing human heart.

A novel LncRNA transcript, RBAT1, accelerates tumorigenesis through interacting with HNRNPL and cis-activating E2F3

BackgroundLong non-coding RNAs (lncRNAs) have been identified as important epigenetic regulators that play critical roles in human cancers. However, the regulatory functions of lncRNAs in tumorigenesis remains to be elucidated. Here, we aimed to investigate the molecular mechanisms and potential clinical application of a novel lncRNA, retinoblastoma associated transcript-1 (RBAT1), in tumorigenesis.MethodsRBAT1 expression was determined by real-time PCR in both retinoblastoma (Rb) and bladder cancer (BCa) cell lines and clinical tissues. Chromatin isolation using RNA purification (ChIRP) assays were performed to identify RBAT1-interacting proteins. Patient-derived xenograft (PDX) retinoblastoma models were established to test the therapeutic potential of RBAT1-targeting GapmeRs.ResultsHere, we found that RBAT1 expression was significantly higher in Rb and BCa tissues than that in adjacent tissues. Functional assays revealed that RBAT1 accelerated tumorigenesis both in vitro and in vivo. Mechanistically, RBAT1 recruited HNRNPL protein to E2F3 promoter, thereby activating E2F3 transcription. Therapeutically, GapmeR-mediated RBAT1 silencing significantly inhibited tumorigenesis in orthotopic xenograft retinoblastoma models derived from Rb cell lines and Rb primary cells.ConclusionsRBAT1 overexpression upregulates a known oncogene, E2F3, via directly recruiting HNPNPL to its promoter and cis-activating its expression. Our finding provides a novel mechanism of lncRNA biology and provides potential targets for diagnosis and treatment of Rb and BCa.

Rrp6 Regulates Heterochromatic Gene Silencing via ncRNA RUF6 Decay in Malaria Parasites.

The heterochromatin environment plays a central role in silencing genes associated with the malaria parasite's development, survival in the host, and transmission to the mosquito vector. However, the underlying mechanism regulating the dynamic chromatin structure is not understood yet. Here, we have uncovered that Plasmodium falciparum Rrp6, an orthologue of eukaryotic RNA exosome-associated RNase, controls the silencing of heterochromatic genes. PfRrp6 knockdown disrupted the singular expression of the GC-rich ncRNA RUF6 family, a known critical regulator of virulence gene expression, through the stabilization of the nascent transcripts. Mechanistic investigation showed that the accumulation of the multiple RUF6 ncRNAs triggered local chromatin remodeling in situ, which activated their adjacent var genes. Strikingly, chromatin isolation by RNA purification analysis (ChIRP-seq) revealed that a remarkable RUF6 ncRNA had interacted with distal heterochromatin regions directly and stimulated a global derepression effect on heterochromatic genes, including all variant gene families and the sexual commitment-associated regulator ap2-g gene. Collectively, Rrp6 appears to conduct the epigenetic surveillance of heterochromatic gene expression through controlling RUF6 levels, thereby securing antigenic variation and sexual commitment of malaria parasites during the infection of the host.IMPORTANCE Malaria remains a major public health and economic burden. The heterochromatin environment controls the silencing of genes associated with the fate of malaria parasites. Previous studies have demonstrated that a group of GC-rich ncRNAs (RUF6) is associated with the mutually exclusive expression of var genes, but the underlying mechanisms remain elusive. Here, through a series of genetic manipulation and genome-wide multiomics analysis, we have identified the plasmodial orthologue of RNA exosome-associated Rrp6 as an upstream regulator of RUF6 expression and revealed that the dysregulation of RUF6 upon Rrp6 knockdown triggered local chromatin alteration, thereby activating most heterochromatic genes via direct interaction of RUF6 and distal gene loci. This finding not only uncovered the in-depth mechanism of RUF6-mediated regulation of heterochromatic genes but also identified Rrp6 as a novel regulator of gene expression in human malaria parasites, which provides a new target for developing intervention strategies against malaria.

574-P: Antioxidant Defence and Long Noncoding RNA in Diabetic Retinopathy

Retinal oxidative stress is increased and the antioxidant defence system is compromised in diabetic retinopathy. Expression of many key antioxidant genes is regulated by a master regulator, nuclear factor erythroid 2-related factor (Nrf2). The transcriptional activity of Nrf2 is mediated by an intracellular inhibitor Kelch-like ECH-associated protein 1 (Keap1), which regulates its nuclear movement. In diabetes, Nrf2 transcriptional activity is decreased and Keap1 levels are increased. A long noncoding RNA (IncRNA), Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), plays a role in regulating Keap1 expression, and retinal MALAT1 levels are upregulated in diabetes. Our aim is to investigate the role of MALAT1 in maintaining the antioxidant defence system in diabetic retinopathy. Human retinal endothelial cells (untransfected or transfected with MALAT1-siRNA) were incubated in 20mM glucose for 96 hours. MALAT1 expression was analysed by q-RTPCR and RNA sequencing, and role of MALAT1 in Keap1 transcription was quantified using MALAT1 RNA pull down assay. Simultaneous occupancy of MALAT1 and transcription factor Sp1 on Keap1 promoter was quantified by Chromatin Isolation by RNA Purification (ChIRP)-reChIP technique, nuclear translocation of Nrf2 by immunofluorescence imaging, and Nrf2-responsive genes superoxide dismutase 2 (Sod2) and heme oxygenase 1 (HO1) by q-RTPCR. Compared to cells in 5mM glucose, high glucose increased co-occupancy of MALAT1 and Sp1 at Keap1 promoter. MALAT1-siRNA ameliorated glucose-induced increase in co-occupancy of MALAT1-Sp1 at its promoter and decreased Keap1 expression. MALAT1-siRNA also restored nuclear localization of Nrf2 and the transcription of Sod2 and HO1. Thus, IncRNA MALAT1, via Keap1, modulates the transcriptional activity of Nrf2 and the antioxidant defence system in diabetic retinopathy. Regulation of MALAT1 can protect the retina from oxidative damage, and prevent/slow down the development of diabetic retinopathy. Disclosure R. Kowluru: None. R. Radhakrishnan: None. Funding EY014370, EY017313, EY022230

Repeat RNAs associate with replication forks and post-replicative DNA.

Noncoding RNA has a proven ability to direct and regulate chromatin modifications by acting as scaffolds between DNA and histone-modifying complexes. However, it is unknown if ncRNA plays any role in DNA replication and epigenome maintenance, including histone eviction and reinstallment of histone modifications after genome duplication. Isolation of nascent chromatin has identified a large number of RNA-binding proteins in addition to unknown components of the replication and epigenetic maintenance machinery. Here, we isolated and characterized long and short RNAs associated with nascent chromatin at active replication forks and track RNA composition during chromatin maturation across the cell cycle. Shortly after fork passage, GA-rich-, alpha- and TElomeric Repeat-containing RNAs (TERRA) are associated with replicated DNA. These repeat containing RNAs arise from loci undergoing replication, suggesting an interaction in cis. Post-replication during chromatin maturation, and even after mitosis in G1, the repeats remain enriched on DNA. This suggests that specific types of repeat RNAs are transcribed shortly after DNA replication and stably associate with their loci of origin throughout the cell cycle. The presented method and data enable studies of RNA interactions with replication forks and post-replicative chromatin and provide insights into how repeat RNAs and their engagement with chromatin are regulated with respect to DNA replication and across the cell cycle.

Epigenetic Mechanisms Involved in Anti‐Inflammatory Actions of a Diabetes Regulated lncRNA DRAIR in Monocytes

Type 2 diabetes (T2D) is associated with monocyte activation and chronic inflammation implicated in accelerated cardiovascular disease. Evidence shows that epigenetic mechanisms and long non‐coding RNAs (lncRNA)s can regulate inflammatory phenotype of monocytes, but their role in T2D induced monocyte dysfunction is unclear. Here, we describe the regulation and functions of a novel lncRNA Diabetes Regulated anti‐ inflammatory RNA (DRAIR) in monocytes. Using RNA‐seq we identified downregulation of several anti‐inflammatory genes and lncRNAs including DRAIR in peripheral blood CD14+ monocytes isolated from subjects with T2D versus healthy controls. Further studies showed that high glucose and palmitic acid significantly downregulated DRAIR expression in CD14+ monocytes and THP1 human monocytes. Conversely, DRAIR was upregulated by anti‐inflammatory cytokines and by PMA (inducer of macrophage differentiation). ChIP assays showed that KLF4 transcription factor binds at KLF4 binding sites in the DRAIR promoter and KLF4 overexpression upregulates DRAIR expression in monocytes. Loss and gain of function approaches revealed that DRAIR regulates anti‐inflammatory phenotype in monocytes and macrophages. Mouse orthologous Drair was also similarly downregulated in macrophages from T2D db/db mice and its knockdown with LNA‐GapmeRs increased expression of pro‐inflammatory genes. Furthermore, we found that DRAIR is a chromatin associated lncRNA. Chromatin isolation by RNA purification and RNA‐immunoprecipitation revealed DRAIR interaction with chromatin near target genes and with a histone methyl transferase G9a. Mechanistically, DRAIR overexpression inhibited G9a recruitment and enrichment of the repressive histone modification H3K9me2 at anti‐inflammatory genes. Together, these results demonstrate that DRAIR can mediate anti‐inflammatory phenotype in monocytes/macrophages via epigenetic mechanisms, and its downregulation in diabetes might contribute to mechanisms promoting chronic inflammation associated with diabetes complications.Support or Funding InformationThis research work was funded by National Institutes of Health grant NIH R01 DK 065073.

LncRNA-PLACT1 sustains activation of NF-κB pathway through a positive feedback loop with IκBα/E2F1 axis in pancreatic cancer

BackgroundThe activation of NF-κB signaling pathway is regarded as the dominant process that correlates with tumorigenesis. Recently, increasing evidence shows that long noncoding RNAs (lncRNAs) play crucial roles in sustaining the NF-κB signaling pathway. However, the underlying mechanisms have not yet been elucidated.MethodsThe expression and clinical features of PLACT1 were analyzed in a 166-case cohort of PDAC by qRT-PCR and in situ hybridization. The functional role of PLACT1 was evaluated by both in vitro and in vivo experiments. Chromatin isolation by RNA purification assays were utilized to examine the interaction of PLACT1 with IκBα promoter.ResultsWe identified a novel lncRNA-PLACT1, which was significantly upregulated in tumor tissues and correlated with progression and poor survival in PDAC patients. Moreover, PLACT1 promoted the proliferation and invasion of PDAC cells in vitro. Consistently, PLACT1 overexpression fostered the progression of PDAC both in orthotopic and lung metastasis mice models. Mechanistically, PLACT1 suppressed IκBα expression by recruiting hnRNPA1 to IκBα promoter, which led to increased H3K27me3 that decreased the transcriptional level of IκBα. Furthermore, E2F1-mediated overexpression of PLACT1 modulated the progression of PDAC by sustained activation of NF-κB signaling pathway through forming a positive feedback loop with IκBα. Importantly, administration of the NF-κB signaling pathway inhibitor significantly suppressed PLACT1-induced sustained activation of NF-κB signaling pathway, leading to reduced tumorigenesis in vivo.ConclusionsOur findings suggest that PLACT1 provides a novel epigenetic mechanism involved in constitutive activation of NF-κB signaling pathway and may represent a new therapeutic target of PDAC.

Abstract P4-04-05: circHEACA promotes anti-HER2 drug resistance by HEACA-GRB7/AKT axis in HER2+ breast cancer patients

Abstract Background: Some HER2-positive (HER2+) breast cancer patient are resistant to anti-HER2 therapy, and the potential reasons of anti-HER2 drug resistance remain unclear. It has been demonstrated that circRNAs play an important role in breast cancer progression, however, the role of circRNAs in anti-Her2 drug resistance remains unknown. In our study, we found and identified a HER2-related circRNA that is associated with malignant progression of HER2 positive breast cancer and preliminary explore their potential mechanisms and clinical significance. Materials and Methods: To identify the HER2-related circRNAs, the deep circRNAs RNA sequencing was performed between the HER2 positive and HER2 negative human breast cancer tissues, and then selected the candidate circRNAs using qPCR to verify the expression level. K-M model was used to define the prognostic performance of circRNA candidates in a breast cancer patient cohort with anti-HER2 drug treatment(N=120). Divergent primers assay and actinomycin D experiments were used to identify the circular features of circRNA candidates. To detect the biological function in breast cancer cells, we knocked-down or over-expressed the circular RNA by siRNA and plasmid transfection. We conducted CHiRP (Chromatin Isolation by RNA Purification) assay to pull down protein complex using biotinylated circRNA probe, and performed mass spectrometric to find out the potential binding protein. Western blot and immunofluorescence (IF) verified that circRNA interacted the protein candidate Results: In this study, we identified a HER2 -Associated CircRNA (named HEACA), and found that divergent primers were amplified in cDNA but not gDNA. Convergent primers amplified in the both cDNA and gDNA. As expected, HEACA was more stable than its linear mRNA when both of them were treated by actinomycin D. Clinical data indicated that HER2+ breast cancer patients with high expression level of HEACA was significantly associated with larger tumour size, more much lymphatic metastasis and higher ki67 index. Kaplan-Meier survival curves showed that HER2+ breast cancer patients with high HEACA who received anti-HER2 therapy has shorter disease-free survival (DFS) and overall survival (OS) than those with low HEACA. Next, we found that know-down HEACA significantly inhibited the proliferation of HER2 positive breast cancer cells through cell variability assay and colony formation, while over-express HEACA highly increased cell proliferation. However, neither know-down nor over-express resulted in affecting the biological function in MCF-7 cells. CircRNA pull down assay and mass spectrometric indicated HEACA may bind to Growth factor receptor-bound protein 7(GRB7). Western blot and IF showed that the protein expression of GRB7 increased or decreased by knocking down or over-express HEACA. Furthermore, we demonstrated that upregulated HEACA promoted breast cancer progression via GRB7/AKT axis in HER2+ cell lines, consequently resulted in a malignant progression of breast cancer cells in vitro and in vivo. Conclusions: HEACA-GRB7/AKT axis plays a critical role in HER2+ breast cancer progression and may be a predictive biomarker for anti-her2 therapy for HER2+ BC patients. Citation Format: Yun Ling, Liang Gehao, Zihao Liu, Zhenluan Tian, Wenjing Zhong, Wanyi Lin, Erwei Song, Gong Chang. circHEACA promotes anti-HER2 drug resistance by HEACA-GRB7/AKT axis in HER2+ breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-04-05.