Chromatin Isolation Research Articles

Master and servant: LINC00152 \u2013 a STAT3-induced long noncoding RNA regulates STAT3 in a positive feedback in human multiple myeloma

BackgroundThe survival of INA-6 human multiple myeloma cells is strictly dependent upon the Interleukin-6-activated transcription factor STAT3. Although transcriptional analyses have revealed many genes regulated by STAT3, to date no protein-coding STAT3 target gene is known to mediate survival in INA-6 cells. Therefore, the aim here was to identify and analyze non-protein-coding STAT3 target genes. In addition to the oncogenic microRNA-21, we previously described five long noncoding RNAs (lncRNAs) induced by STAT3, named STAiRs. Here, we focus on STAT3-induced RNA 18 (STAiR18), an mRNA-like, long ncRNA that is duplicated in the human lineage. One STAiR18 locus is annotated as the already well described LINC00152/CYTOR, however, the other harbors the MIR4435-2HG gene and is, up to now, barely described.MethodsCAPTURE-RNA-sequencing was used to analyze STAiR18 transcript architecture. To identify the STAiR18 and STAT3 phenotype, siRNA-based knockdowns were performed and microarrays were applied to identify their target genes. RNA-binding partners of STAiR18 were determined by Chromatin-Isolation-by-RNA-Purification (ChIRP) and subsequent sequencing. STAT3 expression in dependence of STAiR18 was investigated by immunoblots, chromatin- and RNA-immunoprecipitations.ResultsAs identified by CAPTURE-RNA sequencing, a complex splice pattern originates from both STAiR18 loci, generating different transcripts. Knockdown of the most abundant STAiR18 isoforms dramatically decreased INA-6 cell vitality, suggesting a functional role in myeloma cells. Additionally, STAiR18 and STAT3 knockdowns yielded overlapping changes of transcription patterns in INA-6 cells, suggesting a close functional interplay between the two factors. Moreover, Chromatin isolation by RNA purification (ChIRP), followed by genome-wide RNA sequencing showed that STAiR18 associates specifically with the STAT3 primary transcript. Furthermore, the knockdown of STAiR18 reduced STAT3 levels on both the RNA and protein levels, suggesting a positive feedback between both molecules. Furthermore, STAiR18 knockdown changes the histone methylation status of the STAT3 locus, which explains the positive feedback and indicates that STAiR18 is an epigenetic modulator.ConclusionHence, STAiR18 is an important regulator of myeloma cell survival and is strongly associated with the oncogenic function of STAT3. The close functional interplay between STAT3 and STAiR18 suggests a novel principle of regulatory interactions between long ncRNAs and signaling pathways.

Ppp1r1b-lncRNA inhibits PRC2 at myogenic regulatory genes to promote cardiac and skeletal muscle development in mouse and human

Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators and play important roles in cardiac development and congenital heart disease. In a previous study, we identified a novel lncRNA, Ppp1r1b, with expression highly correlated with myogenesis. However, the molecular mechanism that underlies Ppp1r1b-lncRNA function in myogenic regulation is unknown. By silencing Ppp1r1b-lncRNA, mouse C2C12 and human skeletal myoblasts failed to develop fully differentiated myotubes. Myogenic differentiation was also impaired in PPP1R1B-lncRNA deficient human-induced pluripotent stem cell-derived cardiomyocytes (hiPSCs-CMs). The expression of myogenic transcription factors, including MyoD, Myogenin, and Tbx5, as well as sarcomere proteins, was significantly suppressed in Ppp1r1b-lncRNA inhibited myoblast cells and neonatal mouse heart. Histone modification analysis revealed increased H3K27 tri-methylation at MyoD1 and Myogenin promoters in GapmeR treated C2C12 cells. Furthermore, Ppp1r1b-lncRNA was found to bind to Ezh2, and chromatin isolation by RNA purification (ChIRP) assay revealed enriched interaction of Ppp1r1b-lncRNA with Myod1 and Tbx5 promoters, suggesting that Ppp1r1b-lncRNA induces transcription of myogenic transcription factors by interacting with the polycomb repressive complex 2 (PRC2) at the chromatin interface. Correspondingly, the silencing of Ppp1r1b-lncRNA increased EZH2 binding at promoter regions of myogenic transcription factors. Therefore, our results suggest that Ppp1r1b-lncRNA promotes myogenic differentiation through competing for PRC2 binding with chromatin of myogenic master regulators during heart and skeletal muscle development.

Chromatin Immunoprecipitation (ChIP) to Assess Histone Marks in Auxin-treated Arabidopsis thaliana Inflorescence Tissue.

Chromatin Immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) or high-throughput sequencing (ChIP-seq) has become the gold standard for the identification of binding sites of DNA binding proteins and the localization of histone modification on a locus-specific or genome-wide scale, respectively. ChIP experiments can be divided into seven critical steps: (A) sample collection, (B) crosslinking of proteins to DNA, (C) nuclear extraction, (D) chromatin isolation and fragmentation by sonication, (E) immunoprecipitation of histone marks by appropriate antibodies, (F) DNA recovery, and (G) identification of precipitated protein-associated DNA by qPCR or high-throughput sequencing. Here, we describe a time-efficient protocol that can be used for ChIP-qPCR experiments to study the localization of histone modifications in young inflorescences of the model plants Arabidopsis thaliana.

Long Non-coding RNA LINC-PINT Suppresses Cell Proliferation and Migration of Melanoma via Recruiting EZH2.

Long non-coding RNAs (lncRNAs) have been identified as crucial regulators in many human cancers. Many lncRNAs show aberrant expression in cancer, and some of them play critical roles in tumor proliferation, invasion, and metastasis. However, the regulatory functions of lncRNAs in melanoma progression remain to be elucidated. We utilized the Real-time PCR methodology to determine the expression of LINC-PINT in melanoma cell lines. To evaluate the effect of LINC-PINT on tumorigenesis of melanoma, we used Cell Counting Kit-8 (CCK8) and colony formation assay. Flow cytometry assay was used to detect the function of LINC-PINT on cell cycle status. PINT-interacting proteins were identified by chromatin isolation using RNA purification (ChIRP). Microarray assay and bioinformatics analysis were used to find the potential target genes of LINC-PINT and the status of LINC-PINT target gene candidate was verified using chromatin immunoprecipitation assay (ChIP). LINC-PINT plays a role in suppressing the tumorigenicity of melanoma, which was further determined by xenograft model assay. LINC-PINT was significantly downregulated in melanoma tissues and cell lines. The overexpression of LINC-PINT in tumor cells resulted in significant tumor growth reduction and migration inhibition in A375, Mum2B and CRMM1 cells. Results based on the in vivo xenograft model were further consistent with the in vitro findings that LINC-PINT impeded growth and metastasis of melanoma cells. Microarray assay and bioinformatics analysis indicated that CDK1, CCNA2, AURKA, and PCNA were potential targets of LINC-PINT. In conclusion, LINC-PINT inhibits the tumorigenicity of melanoma through recruiting EZH2 to the promoter of its target genes, leading to H3K27 trimethylation and epigenetic silencing of target genes. LINC-PINT may serve as a novel diagnostic and therapeutic target for melanoma.

LINC02273 drives breast cancer metastasis by epigenetically increasing AGR2 transcription

BackgroundThe majority of breast cancer patients die of metastasis rather than primary tumors, whereas the molecular mechanisms orchestrating cancer metastasis remains poorly understood. Long noncoding RNAs (lncRNA) have been shown to regulate cancer occurrence and progression. However, the lncRNAs that drive metastasis in cancer patients and their underlying mechanisms are still largely unknown.MethodslncRNAs highly expressed in metastatic lymph nodes were identified by microarray. Survival analysis were made by Kaplan-Meier method. Cell proliferation, migration, and invasion assay was performed to confirm the phenotype of LINC02273. Tail vein model and mammary fat pad model were used for in vivo study. RNA pull-down and RIP assay were used to confirm the interaction of hnRNPL and LINC02273. Chromatin isolation by RNA purification followed by sequencing (ChIRP-seq), RNA-seq, ChIP-seq, and luciferase reporter assay reveal hnRNPL-LINC02273 regulates AGR2. Antisense oligonucleotides were used for in vivo treatment.ResultsWe identified a novel long noncoding RNA LINC02273, whose expression was significantly elevated in metastatic lesions compared to the primary tumors, by genetic screen of matched tumor samples. Increased LINC02273 promoted breast cancer metastasis in vitro and in vivo. We further showed that LINC02273 was stabilized by hnRNPL, a protein increased in metastatic lesions, in breast cancer cells. Mechanistically, hnRNPL-LINC02273 formed a complex which activated AGR2 transcription and promoted cancer metastasis. The recruitment of hnRNPL-LINC02273 complex to AGR2 promoter region epigenetically upregulated AGR2 by augmenting local H3K4me3 and H3K27ac levels. Combination of AGR2 and LINC02273 was an independent prognostic factor for predicting breast cancer patient survival. Moreover, our data revealed that LINC02273-targeting antisense oligonucleotides (ASO) substantially inhibited breast cancer metastasis in vivo.ConclusionsOur findings uncover a key role of LINC02273-hnRNPL-AGR2 axis in breast cancer metastasis and provide potential novel therapeutic targets for metastatic breast cancer intervention.

Identification of ncRNA-Mediated Functions of Nucleus-Localized miR-320 in Cardiomyocytes

In recent years, systematic analyses of the subcellular distribution of microRNAs (miRNAs) suggest that the majority of miRNAs are present in both nuclear and cytoplasmic compartments. However, the full extent of nuclear miRNA function in cardiomyocytes is currently unknown. Here, subcellular fractionation, followed by the miRNA microarray, revealed that most miRNAs were detectable in both nuclear and cytoplasmic fractions of cardiomyocytes. We employed miR-320 as an example to explore the function of nucleus-localized miRNAs, finding that CRISPR-Cas9-mediated Ago2 knockdown abolished miR-320-induced transcriptional remodeling. Furthermore, nuclear Ago2 re-expression restored the effects of miR-320 in the nucleus. Moreover, liquid chromatography-mass spectrometry (LC-MS) analysis revealed the association of nuclear Ago2 with transcription factors YLP motif-containing protein 1 (Ylpm1) and single-stranded DNA binding protein 1 (Ssbp1). Intersection of the data of transcriptome-sequencing (seq) with Ago2-chromatin immunoprecipitation (ChIP)-seq revealed that the binding of Ago2 with the target promoter DNA may require promoter RNAs. Specifically, Cep57 was upregulated, whereas Fscn2 was downregulated by miR-320, and a similar effect was also observed by knockdown of their promoter RNA, respectively. Chromatin isolation by RNA purification (ChIRP) analysis showed decreased binding of the Cep57 and Fscn2 promoter RNA on their promoter DNA by miR-320 overexpression.Our work provided a preliminary idea that promoter RNA transcripts act as “pioneers” to disrupt chromatin that permits Ago2/miR-320 complexes to target Cep57 or Fscn2 promoter DNA for transcriptional regulation. miRNAs are naturally located in both cytoplasm and nucleus; however, their pathophysiological functions are largely unknown. Our work provided a theoretical basis for developing nuclear miRNA-based therapeutics against various diseases in the future.

Hottip Lncrna Reinforces CTCF Defined Chromatin Boundaries and Drives Wnt Target Gene Expression in AML Leukemogenesis

We reported recently that HOXA locus associated lncRNA, HOTTIP, is highly expressed in AML patients carrying MLL rearrangement and NPM1c+ mutations. The expression of HOTTIP positively correlates with posterior HOXA gene expression and poor patient survival. We further demonstrated that HOTTIP acts as an epigenetic regulator to define oncogenic HOXA topologically associated domain (TAD) and drive HOXA associated leukemic transcription program. However, it remains unclear whether and how HOTTIP lncRNA is involved in remodeling leukemic genome to facilitate AML leukemogenesis. Here, we showed that HOTTIP regulates a fraction of CTCF binding sites (CBSs) in the AML genome by directly interacting with CTCF and its binding motifs. We carried out CTCF ChIP-seq and HOTTIP ChIRP (chromatin isolation by RNA purification)-seq comparing WT and HOTTIP knockout (KO) MOLM13 cells. KO of HOTTIP in MLL-rearranged MOLM13 AML cells specifically impaired CTCF binding sites that were co-occupied by HOTTIP lncRNA, whereas loss of HOTTIP did not affect global CTCF binding. These target genes include posterior HOXA genes and Wnt target genes such as C-MYC, EVI1, AXIN, and TWIST1. Furthermore, we found that HOTTIP interacts with its putative target sites by formation of DNA: RNA hybridization structure triple helix and R-loop in vivo and in vitro. We then carried out DRIP (DNA-RNA immunoprecipitation)-seq and DRIPc(DNA-RNA immunoprecipitation followed by cDNA conversion)-Seq, which utilize a sequence independent but structure-specific S9.6 antibody for DRIP to capture global R-loops, by comparing WT and HOTTIP KO MOLM13 cells. The obtained DRIP-seq and DRIPc-seq data were then incorporated and integrated with the HOTTIP ChIRP-seq and CTCF ChIP-seq data to explore global collaboration between R-loop and HOTTIP associated CTCF binding sites. We found that HOTTIP interacts with CTCF binding motif that defines the TADs and the promoters of the HOTTIP target genes by formation of R-loop or triple helix structure. Loss of HOTTIP disrupted the R-loop formation at promoters and enhancers of the HOTTIP target genes to inhibit their expression. In MLL-rearranged AML genome, in addition to the HOXA locus, CTCF forms leukemic specific TADs that protect aberrant Wnt target genes. Depletion of HOTTIP lncRNA impaired CTCF defined TADs in the Wnt target gene loci and reduced Wnt target gene expression. In contrast, overexpression of Hottip lncRNA (Hottip-Tg) in the mice bone marrow hematopoietic compartment perturbs hematopoietic stem cell (HSC) self-renewal and differentiation leading to AML like disease by reinforcing CTCF defined TADs, enhancing chromatin accessibility within TADs, and upregulating gene transcription in the Wnt target loci. Finally, when we treated HOTTIP expressed primary patient AML cells carrying MLL-rearrangement and their derived PDX mouse model with a canonical Wnt inhibitor, ICG-001, ICG-001 inhibited AML LSC self-renewal in in vitro by LTC-IC assays and in vivo leukemogenesis in the PDX mouse models with an aberrant HOTTIP lncRNA expression, but not in HOTTIP negative/low non-MLL AML samples. Thus, HOTTIP lncRNA and CTCF cooperate to specifically reinforce CTCF defined WNT target locus TADs and drive Wnt target gene expression in the HOTTIP expressed AML. Disclosures No relevant conflicts of interest to declare.

264The long noncoding RNA H19 modulates cardiac remodeling after infarction

Abstract Noncoding RNAs account for 80% of human transcripts, but functional studies on noncoding RNAs are relatively few and limited. Long noncoding RNAs (lncRNAs) are known to have an important role in cardiac development, and lately, high-throughput RNA sequencing has been extensively utilized to profile and explore the transcriptome landscape of lncRNAs in failing hearts. These studies have revealed that lncRNAs are mostly dysregulated in failing hearts and their expression signature can discriminate failing hearts of different etiologies. H19 is abundantly expressed in failing human hearts and its polymorphism was shown to possess a significant correlation with the risk of coronary artery diseases. In our study using murine hearts, we discovered that H19 was significantly up regulated in the heart after ischemic injury, with predominant expression in cardiac fibroblasts. This finding piqued our interest to further investigate the function of H19 in the heart. We demonstrated that ectopic overexpression of H19 using the AAV approach led to severe cardiac fibrosis in mouse hearts following myocardial infarction. In light of this finding, we generated H19 knockout mice to further investigate the functionality of H19 and we found that cardiac fibrosis was attenuated in these mice. Altogether, these findings suggested that H19 is a fibrosis regulator during cardiac remodeling process after infarction. Due to the multiple regulatory roles of lncRNAs, we then took advantage of chromatin isolation by RNA purification (ChIRP) to identify the H19-interacting protein, YB-1. Surprisingly, mice with YB-1 knockdown displayed severe cardiac fibrosis even without injury. Furthermore, we demonstrated that YB-1 is a transcriptional suppressor of collagen 1A1. Knockout of H19 in YB-1 knockdown partially suppressed Col1a1 expression, which suggests a negative regulatory role of H19 on YB-1 towards the expression of Col1a1. Taking into account all of these findings, we concluded that H19 mediates collagen expression in fibroblasts through the inhibition of YB-1 activity during cardiac remodeling.

LncRNA SATB2-AS1 inhibits tumor metastasis and affects the tumor immune cell microenvironment in colorectal cancer by regulating SATB2

BackgroundEmerging studies suggest that long non-coding RNAs (lncRNAs) play crucial roles in colorectal cancer (CRC). Here, we report a lncRNA, SATB2-AS1, which is specifically expressed in colorectal tissue and is significantly reduced in CRC. We systematically elucidated its functions and possible molecular mechanisms in CRC.MethodsLncRNA expression in CRC was analyzed by RNA-sequencing and RNA microarrays. The expression level of SATB2-AS1 in tissues was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH). The functional role of SATB2-AS1 in CRC was investigated by a series of in vivo and in vitro assays. RNA pull-down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), chromatin isolation by RNA purification (ChIRP), Bisulfite Sequencing PCR (BSP) and bioinformatics analysis were utilized to explore the potential mechanisms of SATB2-AS1.ResultsSATB2-AS1 is specifically expressed in colorectal tissues and downregulated in CRC. Survival analysis indicates that decreased SATB2-AS1 expression is associated with poor survival. Functional experiments and bioinformatics analysis revealed that SATB2-AS1 inhibits CRC cell metastasis and regulates TH1-type chemokines expression and immune cell density in CRC. Mechanistically, SATB2-AS1 directly binds to WDR5 and GADD45A, cis-activating SATB2 (Special AT-rich binding protein 2) transcription via mediating histone H3 lysine 4 tri-methylation (H3K4me3) deposition and DNA demethylation of the promoter region of SATB2.ConclusionsThis study reveals the functions of SATB2-AS1 in CRC tumorigenesis and progression, suggesting new biomarkers and therapeutic targets in CRC.

BAT Hi-C maps global chromatin interactions in an efficient and economical way.

Chromosome Conformation Capture (3C)-based technologies, such as Hi-C, have represented a significant breakthrough in investigating the structure and function of higher-order genome architecture. However, the mapping of global chromatin interactions remains challenging across many biological conditions due to high background noise and financial constraints, especially for small laboratories. Here, we describe the Bridge linker-Alul-Tn5 Hi-C (BAT Hi-C) method, which is a simple and efficient method for delineating chromatin conformational features of mouse embryonic stem (mES) cells and uncover DNA loops. This protocol combines Alul fragmentation and biotinylated linker-mediated proximity ligation to obtain kilobase (kb) resolution with a marked increase in the amount of unique read pairs. The protocol also includes chromatin isolation to reduce background noise and Tn5 tagmentation to cut down on preparation time. Importantly, with only one-third sequencing depth, our method revealed the same spectrum of chromatin contacts as in situ Hi-C. BAT Hi-C is an economical (i.e., approximately $40 for library preparation) and straightforward (total hands-on time of 3 days) tool that is ideal for the in-depth analysis of long-range chromatin looping events in a genome-wide fashion.

Abstract 657: Long Noncoding RNA Ppp1r1b Regulates Myogenic Differentiation Through Modulating Histone 3 Methylation

Introduction: Emerged as critical epigenetic regulators of the transcriptome, the long noncoding RNAs (lncRNAs) play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction in congenital heart disease. In our work, we identified a novel lncRNA that regulates myogenic differentiation. Methods: Antisense oligonucleotides (GapmeR) were used to suppress Ppp1r1b-lncRNA. Chromatin immunoprecipitation (CHIP) and chromatin isolation by RNA purification (CHIRP) were used to analyze gene specific histone modification level and lncRNA:chromatin interaction, respectively. RNA pull-down and RNA immunoprecipitation (RIP) were used to identify the protein molecules that interact with Ppp1r1b-lncRNA. Results: By silencing Ppp1r1b-lncRNA, C2C12, a skeletal myoblast cell line, and human-induced pluripotent stem cell derived cardiomyocytes failed to develop fully differentiated myotubes. Analysis of GapmeR injected neonatal mouse heart ( in vivo ) and siRNA silenced human myoblasts ( in vitro ) further confirmed the role of Ppp1r1b-lncRNA in normal myogenesis. Members of the MyoD family of muscle-specific transcription factors were not induced during myogenic differentiation in C2C12 cells treated with Ppp1r1b-lncRNA specific GapmeR. Proteins essential for striated muscle development were also suppressed in Ppp1r1b-lncRNA- deficient cells. Histone modification analysis revealed enrichment of histone tri-methylation at Myogenin and MyoD1 promoters in GapmeR treated C2C12 cells. Subsequently, lncRNA- protein complex isolation and lncRNA-DNA interaction assays confirmed Ppp1r1b-lncRNA function. Conclusions: Our findings support important role of Ppp1r1b-lncRNA in promoting myogenic differentiation. Ppp1r1b-lncRNA function is mediated by inhibiting histone methylation on promoters of key myogenic genes. Particularly, the identification of EZH2 in pulled Pp1r1b-lncRNA: protein complex supports that Polycomb repressive complex 2 is involved in Ppp1r1b-lncRNA modulated myoblast differentiation. The recovery of MyoD1 promoter DNA in isolated lncRNA: chromatin DNA interactome implies that Ppp1r1b-lncRNA regulates transcription by recruiting PRC2 complex to its target genes.

YY1/LncRNA GAS5 complex aggravates cerebral ischemia/reperfusion injury through enhancing neuronal glycolysis

Yin-Yang 1 (YY1) has been identified as playing critical roles in multiple diseases. However, little is known regarding its roles and mechanisms in cerebral ischemia/reperfusion (I/R) injury. This study is aimed to explore the roles of YY1 in regulating neuronal apoptosis in cerebral I/R injury and its underlying mechanisms. Primary mouse cerebral cortical neurons were isolated and subjected to OGD/R to mimic cerebral I/R injury in vitro. The roles of YY1 on OGD/R-induced neuronal injury were investigated by performing western blotting, quantitative real-time polymerase chain reaction, TUNEL, RNA-binding protein immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification assay, glucose uptake assay, lactate production assay, and extracellular acidification rate assay. YY1-binding long non-coding RNAs (LncRNAs) in neurons subjected to OGD/R were identified by RIP and RNA sequencing. The roles of YY1 on cerebral I/R in vivo were detected by assessing neuronbehaviour, infarct size, and neuronal apoptosis. We found that YY1 expression is downregulated, and LncRNA GAS5 is upregulated in neurons subjected to OGD/R. OGD/R treatment promotes YY1 interacting with GAS5 in neurons, and YY1 negatively regulates GAS5 expression by binding to GAS5 promoter to repress its transcription. Besides, YY1 and GAS5 bind to the same region of PFKFB3 promoter to promote PFKFB3 expression and strengthen neuronal glycolysis, resulting in aggravating OGD/R-induced neuronal apoptosis. Knockdown of YY1 or GAS5 protects against I/R-induced ischemic brain damage and improves overall neurological functions in vivo. Overall, YY1 interacts with LncRNA GAS5 to promote PFKFB3 transcription to enhance neuronal glycolysis, resulting in aggravating cerebral I/R injury.

ALDH1A3-regulated long non-coding RNA NRAD1 is a potential novel target for triple-negative breast tumors and cancer stem cells

To discover novel therapeutic targets for triple-negative breast cancer (TNBC) and cancer stem cells (CSCs), we screened long non-coding RNAs (lncRNAs) most enriched in TNBCs for high expression in CSCs defined by high Aldefluor activity and associated with worse patient outcomes. This led to the identification of non-coding RNA in the aldehyde dehydrogenase 1 A pathway (NRAD1), also known as LINC00284. Targeting NRAD1 in TNBC tumors using antisense oligonucleotides reduced cell survival, tumor growth, and the number of cells with CSC characteristics. Expression of NRAD1 is regulated by an enzyme that causes Aldefluor activity in CSCs, aldehyde dehydrogenase 1A3 (ALDH1A3) and its product retinoic acid. Cellular fractionation revealed that NRAD1 is primarily nuclear localized, which suggested a potential function in gene regulation. This was confirmed by transcriptome profiling and chromatin isolation by RNA purification, followed by sequencing (ChIRP-seq), which demonstrated that NRAD1 has enriched chromatin interactions among the genes it regulates. Gene Ontology enrichment analysis revealed that NRAD1 regulates expression of genes involved in differentiation and catabolic processes. NRAD1 also contributes to gene expression changes induced by ALDH1A3; thereby, the induction of NRAD1 is a novel mechanism through which ALDH1A3 regulates gene expression. Together, these data identify lncRNA NRAD1 as a downstream effector of ALDH1A3, and a target for TNBCs and CSCs, with functions in cell survival and regulation of gene expression.

Interaction of Sox2 with RNA binding proteins in mouse embryonic stem cells

Sox2 is a master transcriptional regulator of embryonic development. In this study, we determined the protein interactome of Sox2 in the chromatin and nucleoplasm of mouse embryonic stem (mES) cells. Apart from canonical interactions with pluripotency-regulating transcription factors, we identified interactions with several chromatin modulators, including members of the heterochromatin protein 1 (HP1) family, suggesting a role for Sox2 in chromatin-mediated transcriptional repression. Sox2 was also found to interact with RNA binding proteins (RBPs), including proteins involved in RNA processing. RNA immunoprecipitation followed by sequencing revealed that Sox2 associates with different messenger RNAs, as well as small nucleolar RNA Snord34 and the non-coding RNA 7SK. 7SK has been shown to regulate transcription at gene regulatory regions, which could suggest a functional interaction with Sox2 for chromatin recruitment. Nevertheless, we found no evidence of Sox2 modulating recruitment of 7SK to chromatin when examining 7SK chromatin occupancy by Chromatin Isolation by RNA Purification (ChIRP) in Sox2 depleted mES cells. In addition, knockdown of 7SK in mES cells did not lead to any change in Sox2 occupancy at 7SK-regulated genes. Thus, our results show that Sox2 extensively interacts with RBPs, and suggest that Sox2 and 7SK co-exist in a ribonucleoprotein complex whose function is not to regulate chromatin recruitment, but could rather regulate other processes in the nucleoplasm.

Inhibition of the Hypoxia-Inducible Factor 1α-Induced Cardiospecific HERNA1 Enhance-Templated RNA Protects From Heart Disease.

Enhancers are genomic regulatory elements conferring spatiotemporal and signal-dependent control of gene expression. Recent evidence suggests that enhancers can generate noncoding enhancer RNAs, but their (patho)biological functions remain largely elusive. We performed chromatin immunoprecipitation-coupled sequencing of histone marks combined with RNA sequencing of left ventricular biopsies from experimental and genetic mouse models of human cardiac hypertrophy to identify transcripts revealing enhancer localization, conservation with the human genome, and hypoxia-inducible factor 1α dependence. The most promising candidate, hypoxia-inducible enhancer RNA ( HERNA)1, was further examined by investigating its capacity to modulate neighboring coding gene expression by binding to their gene promoters by using chromatin isolation by RNA purification and λN-BoxB tethering-based reporter assays. The role of HERNA1 and its neighboring genes for pathological stress-induced growth and contractile dysfunction, and the therapeutic potential of HERNA1 inhibition was studied in gapmer-mediated loss-of-function studies in vitro using human induced pluripotent stem cell-derived cardiomyocytes and various in vivo models of human pathological cardiac hypertrophy. HERNA1 is robustly induced on pathological stress. Production of HERNA1 is initiated by direct hypoxia-inducible factor 1α binding to a hypoxia-response element in the histoneH3-lysine27acetylation marks-enriched promoter of the enhancer and confers hypoxia responsiveness to nearby genes including synaptotagmin XVII, a member of the family of membrane-trafficking and Ca2+-sensing proteins and SMG1, encoding a phosphatidylinositol 3-kinase-related kinase. Consequently, a substrate of SMG1, ATP-dependent RNA helicase upframeshift 1, is hyperphoshorylated in a HERNA1- and SMG1-dependent manner. In vitro and in vivo inactivation of SMG1 and SYT17 revealed overlapping and distinct roles in modulating cardiac hypertrophy. Finally, in vivo administration of antisense oligonucleotides targeting HERNA1 protected mice from stress-induced pathological hypertrophy. The inhibition of HERNA1 postdisease development reversed left ventricular growth and dysfunction, resulting in increased overall survival. HERNA1 is a novel heart-specific noncoding RNA with key regulatory functions in modulating the growth, metabolic, and contractile gene program in disease, and reveals a molecular target amenable to therapeutic exploitation.

LUCAT1 promotes colorectal cancer tumorigenesis by targeting the ribosomal protein L40-MDM2-p53 pathway through binding with UBA52.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in both men and women in the USA. However, the underlying molecular mechanisms that drive CRC tumorigenesis are still not clear. Several studies have reported that long noncoding RNAs (lncRNAs) have important roles in tumor development. Here, we undertook a transcriptome microarray analysis in 6 pairs of CRC tissues and their corresponding adjacent normal tissues. A total of 1705 differentially expressed lncRNAs were detected in CRC tissues at stages I/II and III/IV (fold change greater than or equal to 2 or less than or equal to 0.5). Among them, we found that the lncRNA lung cancer‐associated transcript 1 (LUCAT1) was upregulated in CRC tissues and was closely associated with poor overall survival of CRC patients, through analysis of clinical data and The Cancer Genome Atlas. Functional studies indicated that LUCAT1 promoted CRC cell proliferation, apoptosis, migration, and invasion in vitro and in vivo. Furthermore, knockdown of LUCAT1 rendered CRC cells hypersensitive to oxaliplatin treatment. Mechanistically, bioinformatic analysis indicated that low expression of LUCAT1 was associated with the p53 signaling pathway. Chromatin isolation by RNA purification followed by mass spectrometry and RNA immunoprecipitation revealed that LUCAT1 bound with UBA52, which encodes ubiquitin and 60S ribosomal protein L40 (RPL40). We found that RPL40 functions in the ribosomal protein‐MDM2‐p53 pathway to regulate p53 expression. Taken together, our findings indicate that suppression of LUCAT1 induces CRC cell cycle arrest and apoptosis by binding UBA52 and activating the RPL40‐MDM2‐p53 pathway. These results implicate LUCAT1 as a potential prognostic biomarker and therapeutic target for CRC.

LncRNA BCAR4 Increases Viability, Invasion, and Migration of Non-Small Cell Lung Cancer Cells by Targeting Glioma-Associated Oncogene 2 (GLI2)

This study aimed to explore the effects of lncRNA BCAR4 on the viability and aggressiveness of non-small cell lung cancer (NSCLC) cells. qRT-PCR was used to determine the expression of BCAR4 and GLI2 downstream genes in NSCLC tissues and cell lines. Chromatin isolation by RNA purification (CHIRP) and Western blot were employed to measure the expression of the GLI2 downstream proteins. Ki-67 expression in nude mice tumors was tested by immunohistochemistry. MTT assay, wound healing assay, and Transwell assay were used to assess NSCLC cell viability and aggressiveness, respectively. Tumor xenograft was conducted to determine the effects of BCAR4 and GLI2 on NSCLC tumorigenesis in vivo. The expression of BCAR4 in NSCLC tissues and cells was significantly higher than the normal level. The overexpression of BCAR4 promoted NSCLC cell viability, migration, and invasion. The suppression of BCAR4 and GLI2 showed the opposite effects. The overexpression of BCAR4 led to an increase in the expression of GLI2 downstream proteins, while the suppression of BCAR4 and GLI2 reduced their expression. In a tumor xenograft assay, the tumors in mice of the BCAR4 group showed the biggest volume, while those in mice of the si-GLI2 group showed the smallest volume. Ki-67 showed much higher levels in the BCAR4 overexpression group but much lower levels in the si-GLI2 group. In summary, the cooperative mechanism of lncRNA BCAR4 and GLI2 might provide a new opportunity for treating NSCLC.

Egg Lysis Buffer–Chromatin Isolation Buffer (ELB–CIB)

Egg Lysis Buffer–Chromatin Isolation Buffer (ELB–CIB)

Hypoxia-induced H19/YB-1 cascade modulates cardiac remodeling after infarction.

Rationale: Long non-coding RNA (lncRNAs) has been identified as a pivotal novel regulators in cardiac development as well as cardiac pathogenesis. lncRNA H19 is known as a fetal gene but it is exclusively abundant in the heart and skeletal muscles in adulthood, and is evolutionarily conserved in humans and mice. It has been reported to possess a significant correlation with the risk of coronary artery diseases. However, the function of H19 is not well characterized in heart.Methods: Loss-of-function and gain-of-function mouse models with left anterior descending coronary artery-ligation surgery were utilized to evaluate the functionality of H19 in vivo. For mechanistic studies, hypoxia condition were exerted in in vitro models to mimic cardiac ischemic injury. Chromatin isolation by RNA immunoprecipitation (ChIRP) was performed to reveal the interacting protein of lncRNA H19.Results: lncRNA H19 was significantly upregulated in the infarct area post-surgery day 4 in mouse model. Ectopic expression of H19 in the mouse heart resulted in severe cardiac dilation and fibrosis. Several extracellular matrix (ECM) genes were significantly upregulated. While genetic ablation of H19 by CRISPR-Cas9 ameliorated post-MI cardiac remodeling with reduced expression in ECM genes. Through chromatin isolation by RNA purification (ChIRP), we identified Y-box-binding protein (YB)-1, a suppressor of Collagen 1A1, as an interacting protein of H19. Furthermore, H19 acted to antagonize YB-1 through direct interaction under hypoxia, which resulted in de-repression of Collagen 1A1 expression and cardiac fibrosis.Conclusions: Together these results demonstrate that lncRNA H19 and its interacting protein YB-1 are crucial for ECM regulation during cardiac remodeling.

Reconstruction of the Regulatory Network in a Minimal Bacterium Reveals Extensive Non-Transcription Factor Dependent Regulation

Determining the gene regulatory network of an organism is fundamental for achieving a global understanding of cellular behaviour. Here, we determine the relative importance of different transcription regulators in the genome-reduced bacterium Mycoplasma pneumoniae, by integrating DNA affinity chromatography, chromatin isolation, ChIP-seq, transcriptomics, proteomics and growth curves experiments for 143 genes (20.8% of the bacteria´s NCBI annotated proteins). In 55% of the cases, gene overexpression or mutation does not result in transcriptional nor growth phenotypes, highlighting the robustness of even very simple cells. We identified nine TFs (besides the sigma factor) and their targets, representing 33% of the genome. Nevertheless, we find that the majority of transcription regulation in M. pneumoniae is not mediated by canonical TFs, but rather occurs at the level of supercoiling, metabolic control, RNA degradation, and chromosome topology. For the first time, we quantify the non-redundant contribution of each of these mechanisms in various environmental perturbations using a random forest model, showing that cells use different mechanisms to respond to distinct stresses. The gene changes predicted by our model correlate with the actual changes in 95% of the tested perturbations, and after accounting for experimental and gene expression noise, we can explain over 70% of the total variance. This comprehensive analysis highlights the importance of non-TF-mediated regulation in bacteria, an ancient type of regulation that allows adaptation to mild environmental changes.