Chromatin-associated RNA Research Articles

The lncRNA VELUCT strongly regulates viability of lung cancer cells despite its extremely low abundance.

Little is known about the function of most non-coding RNAs (ncRNAs). The majority of long ncRNAs (lncRNAs) is expressed at very low levels and it is a matter of intense debate whether these can be of functional relevance. Here, we identified lncRNAs regulating the viability of lung cancer cells in a high-throughput RNA interference screen. Based on our previous expression profiling, we designed an siRNA library targeting 638 lncRNAs upregulated in human cancer. In a functional siRNA screen analyzing the viability of lung cancer cells, the most prominent hit was a novel lncRNA which we called Viability Enhancing LUng Cancer Transcript (VELUCT). In silico analyses confirmed the non-coding properties of the transcript. Surprisingly, VELUCT was below the detection limit in total RNA from NCI-H460 cells by RT-qPCR as well as RNA-Seq, but was robustly detected in the chromatin-associated RNA fraction. It is an extremely low abundant lncRNA with an RNA copy number of less than one copy per cell. Blocking transcription with actinomycin D revealed that VELUCT RNA was highly unstable which may partially explain its low steady-state concentration. Despite its extremely low abundance, loss-of-function of VELUCT with three independent experimental approaches in three different lung cancer cell lines led to a significant reduction of cell viability: Next to four individual siRNAs, also two complex siPOOLs as well as two antisense oligonucleotides confirmed the strong and specific phenotype. In summary, the extremely low abundant lncRNA VELUCT is essential for regulation of cell viability in several lung cancer cell lines. Hence, VELUCT is the first example for a lncRNA that is expressed at a very low level, but has a strong loss-of-function phenotype. Thus, our study proves that at least individual low-abundant lncRNAs can play an important functional role.

Cellular Fractionation and Isolation of Chromatin-Associated RNA.

In eukaryotic cells, the synthesis, processing, and functions of RNA molecules are confined to distinct subcellular compartments. Biochemical fractionation of cells prior to RNA isolation thus enables the analysis of distinct steps in the lifetime of individual RNA molecules that would be masked in bulk RNA preparations from whole cells. Here, we describe a simple two-step differential centrifugation protocol for the isolation of cytoplasmic, nucleoplasmic, and chromatin-associated RNA that can be used in downstream applications such as qPCR or deep sequencing. We discuss various aspects of this fractionation protocol, which can be readily applied to many mammalian cell types. For the study of long noncoding RNAs and enhancer RNAs in regulation of transcription especially the preparation of chromatin-associated RNA can contribute significantly to further developments.

Detection and Analysis of Long Noncoding RNAs.

Recent genome-wide studies have yielded new insights into the biological function of long noncoding RNAs (lncRNAs), predominantly through analysis of their genomic addresses. These studies have revealed that a large number of lncRNAs map to regulatory elements in eukaryotic genome regions known as promoter and enhancer elements. Here, we review the principles of current methodologies for analyzing lncRNAs with high-throughput sequencing approaches. These include (1) direct RNA sequencing, (2) sequencing coupled with transcription, and (3) isolation of protein complexes associated with lncRNAs followed by high-throughput sequencing. Within these categories, we also describe detailed protocols for chromatin-associated RNA sequencing, nascent transcript Global run-on sequencing, and photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation.

Abstract 4881: Analysis of long-noncoding RNA interaction at chromatin by chromatin isolation by RNA purification (ChIRP)

Abstract Gene regulation plays a critical role during development and progression of Cancer. While the regulation of gene expression by transcription factors and epigenetic influences has been well studied over time, pervasive genomic transcription and the role of non-coding RNAs in this process is a rapidly evolving field that remains to be thoroughly explored. Chromatin Isolation by RNA Purification (ChIRP) is one of the methods, which allows discovery of the sites of interaction of chromatin-associated RNAs (e.g. lncRNAs) with genomic DNA sequences by using probe-based hybridization to target RNA molecules in chromatin. To perform ChIRP multiple biotinylated oligonucleotide probes complementary to the RNA of interest are used. To help eliminate non-specific signals two different pools of probes are used (even and odd probe sets). These probe sets are combined with chromatin and hybridized to the chromatin-associated RNA. Complexes containing biotinylated-probes bound to the chromatin-associated RNA are then isolated using streptavidin magnetic beads. DNA can then be recovered and analyzed by quantitative PCR or next generation sequencing (ChIRP-seq). Alternatively, RNA may also be isolated from an aliquot of the recovered chromatin to detect other RNA molecules that may be associated with the RNA of interest. To enable the exploration of these RNA interactions in chromatin regulation, we have optimized the methods and developed ChIRP reagents. Using these reagents ChIRP experiments can be performed with reliable recovery of chromatin using lncRNA or other chromatin associated RNA as targets. Additionally, negative and positive control probe sets, and detection primers were developed to ensure first time success. We have performed ChIRP experiments with a HeLa cell lysate and capture oligos for the NEAT1 lncRNA. Isolated DNA was subjected to NGS library construction and sequenced on an Illumina HiSeq™ instrument. Sequences were aligned to the reference genome (hg19). The peaks were called separately for odd and even reactions, and only common peaks were selected. We successfully identified several NEAT1 binding sites in the genomic DNA sequence. In summary the methods developed allow unbiased discovery of RNA-associated genomic DNA sequences, RNA sequences, and potentially proteins. Citation Format: Kan Saito, Konstantin Taganov, John M. Rosenfeld, Nick Asbrock, Vi Chu. Analysis of long-noncoding RNA interaction at chromatin by chromatin isolation by RNA purification (ChIRP). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4881. doi:10.1158/1538-7445.AM2015-4881

RNA-mediated epigenetic regulation of gene expression.

Diverse classes of RNA, ranging from small to long non-coding RNAs, have emerged as key regulators of gene expression, genome stability and defence against foreign genetic elements. Small RNAs modify chromatin structure and silence transcription by guiding Argonaute-containing complexes to complementary nascent RNA scaffolds and then mediating the recruitment of histone and DNA methyltransferases. In addition, recent advances suggest that chromatin-associated long non-coding RNA scaffolds also recruit chromatin-modifying complexes independently of small RNAs. These co-transcriptional silencing mechanisms form powerful RNA surveillance systems that detect and silence inappropriate transcription events, and provide a memory of these events via self-reinforcing epigenetic loops.

Integrated analysis of long noncoding RNAs and mRNAs reveals their potential roles in the pathogenesis of uterine leiomyomas

Global expression profiling studies showed that miRNAs are aberrantly expressed in uterine leiomyomas (ULMs) and are involved in ULM pathogenesis. Long noncoding RNAs (lncRNAs) are another group of regulatory RNA whose expression and roles in ULMs have not been explored. In this study, we examined the global expressions of lncRNAs and mRNAs in ULMs using microarray and interrogated their interrelationship through co-expression analysis. We found that lncRNAs and mRNAs were dysregulated in ULMs and the degree of dysregulation was positively correlated with tumor size. Further analysis showed that lncRNAs correlate to their cis mRNAs in expression levels depending on genomic locations and orientations. Moreover, we identified several dysregulated pathways that were correlated to dysregulated lncRNAs. We validated several aberrantly expressed lncRNAs in extended samples and confirmed that AK023096 was down-regulated and chromatin-associated RNA (CAR) Intergenic 10 was up-regulated in the majority of leiomyomas. Knockdown of Intergenic 10 inhibited the proliferation of leiomyoma cells in vitro, indicating its functional importance in ULM pathogenesis. The neighboring protein-coding gene ADAM12 was also downregulated in Intergenic 10 knockdown leiomyoma cells. We showed for the first time that lncRNAs were dysregulated in uterine leiomyomas. Aberrantly expressed lncRNAs may contribute to the pathogenesis of uterine leiomyomas.

Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs.

Heterochromatin protein 1 (HP1a) has conserved roles in gene silencing and heterochromatin and is also implicated in transcription, DNA replication, and repair. Here we identify chromatin-associated protein and RNA interactions of HP1a by BioTAP-XL mass spectrometry and sequencing from Drosophila S2 cells, embryos, larvae, and adults. Our results reveal an extensive list of known and novel HP1a-interacting proteins, of which we selected three for validation. A strong novel interactor, dADD1 (Drosophila ADD1) (CG8290), is highly enriched in heterochromatin, harbors an ADD domain similar to human ATRX, displays selective binding to H3K9me2 and H3K9me3, and is a classic genetic suppressor of position-effect variegation. Unexpectedly, a second hit, HIPP1 (HP1 and insulator partner protein-1) (CG3680), is strongly connected to CP190-related complexes localized at putative insulator sequences throughout the genome in addition to its colocalization with HP1a in heterochromatin. A third interactor, the histone methyltransferase MES-4, is also enriched in heterochromatin. In addition to these protein-protein interactions, we found that HP1a selectively associated with a broad set of RNAs transcribed from repetitive regions. We propose that this rich network of previously undiscovered interactions will define how HP1a complexes perform their diverse functions in cells and developing organisms.

Inhibition of U4 snRNA in human cells causes the stable retention of polyadenylated pre-mRNA in the nucleus.

Most human pre-mRNAs contain introns that are removed by splicing. Such a complex process needs strict control and regulation in order to prevent the expression of aberrant or unprocessed transcripts. To analyse the fate of pre-mRNAs that cannot be spliced, we inhibited splicing using an anti-sense morpholino (AMO) against U4 snRNA. As a consequence, splicing of several selected transcripts was strongly inhibited. This was accompanied by the formation of enlarged nuclear speckles containing polyadenylated RNA, splicing factors and the nuclear poly(A) binding protein. Consistently, more polyadenylated pre-mRNA could be isolated from nucleoplasmic as well as chromatin-associated RNA fractions following U4 inhibition. Further analysis demonstrated that accumulated pre-mRNAs were stable in the nucleus and that nuclear RNA degradation factors did not re-localise to nuclear speckles following splicing inhibition. The accumulation of pre-mRNA and the formation of enlarged speckles were sensitive to depletion of the 3′ end processing factor, CPSF73, suggesting a requirement for poly(A) site processing in this mechanism. Finally, we provide evidence that the pre-mRNAs produced following U4 snRNA inhibition remain competent for splicing, perhaps providing a biological explanation for their stability. These data further characterise processes ensuring the nuclear retention of pre-mRNA that cannot be spliced and suggest that, in some cases, unspliced transcripts can complete splicing sometime after their initial synthesis.

Df31 Protein and snoRNAs Maintain Accessible Higher-Order Structures of Chromatin

Packaging of DNA into nucleosomes and the formation of higher-order chromatin structures determine DNA accessibility and activity of genome domains. We identified an RNA-dependent mechanism maintaining the open chromatin structure within euchromatic regions in Drosophila cells. The mechanism of reversible chromatin opening, reconstituted in vitro, depends on the Drosophila decondensation factor 31 (Df31) that specifically binds to RNA and localizes to euchromatic regions. Df31 is capable to tether a heterogeneous pool of short, single-stranded RNAs to chromatin. This class of chromatin-associated RNA (caRNA) is stably linked to chromatin and is largely composed of snoRNAs, which are preferentially bound by Df31. We suggest that the Df31-mediated linkage of snoRNAs and chromatin, forms a RNA-chromatin network resulting in the establishment of open chromatin domains. Analysis of caRNAs in human cells also reveals a strong enrichment of snoRNAs, implying a conserved role for these molecules in higher-order structures of chromatin.

Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs

Splicing remains an incompletely understood process. Recent findings suggest that chromatin structure participates in its regulation. Here, we analyze the RNA from subcellular fractions obtained through RNA-seq in the cell line K562. We show that in the human genome, splicing occurs predominantly during transcription. We introduce the coSI measure, based on RNA-seq reads mapping to exon junctions and borders, to assess the degree of splicing completion around internal exons. We show that, as expected, splicing is almost fully completed in cytosolic polyA+ RNA. In chromatin-associated RNA (which includes the RNA that is being transcribed), for 5.6% of exons, the removal of the surrounding introns is fully completed, compared with 0.3% of exons for which no intron-removal has occurred. The remaining exons exist as a mixture of spliced and fewer unspliced molecules, with a median coSI of 0.75. Thus, most RNAs undergo splicing while being transcribed: “co-transcriptional splicing.” Consistent with co-transcriptional spliceosome assembly and splicing, we have found significant enrichment of spliceosomal snRNAs in chromatin-associated RNA compared with other cellular RNA fractions and other nonspliceosomal snRNAs. CoSI scores decrease along the gene, pointing to a “first transcribed, first spliced” rule, yet more downstream exons carry other characteristics, favoring rapid, co-transcriptional intron removal. Exons with low coSI values, that is, in the process of being spliced, are enriched with chromatin marks, consistent with a role for chromatin in splicing during transcription. For alternative exons and long noncoding RNAs, splicing tends to occur later, and the latter might remain unspliced in some cases.

Transcription, Splicing, and Release: Are We There Yet?

A systematic analysis of LPS-induced gene expression in macrophages by Bhatt et al. demonstrates that inflammatory responses are governed primarily at the level of transcription initiation. Unexpectedly, full-length nascent RNAs that contain introns appear to accumulate on chromatin, presumably to complete processing, prior to release of functional mRNA for export to the cytoplasm.

A Long ncRNA Links Copy Number Variation to a Polycomb/Trithorax Epigenetic Switch in FSHD Muscular Dystrophy

SummaryRepetitive sequences account for more than 50% of the human genome. Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease associated with reduction in the copy number of the D4Z4 repeat mapping to 4q35. By an unknown mechanism, D4Z4 deletion causes an epigenetic switch leading to de-repression of 4q35 genes. Here we show that the Polycomb group of epigenetic repressors targets D4Z4 in healthy subjects and that D4Z4 deletion is associated with reduced Polycomb silencing in FSHD patients. We identify DBE-T, a chromatin-associated noncoding RNA produced selectively in FSHD patients that coordinates de-repression of 4q35 genes. DBE-T recruits the Trithorax group protein Ash1L to the FSHD locus, driving histone H3 lysine 36 dimethylation, chromatin remodeling, and 4q35 gene transcription. This study provides insights into the biological function of repetitive sequences in regulating gene expression and shows how mutations of such elements can influence the progression of a human genetic disease.

Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila

RNAi pathways have evolved as important modulators of gene expression that act in the cytoplasm by degrading RNA target molecules via the activity of short (21-30nt) RNAs1-6 RNAi components have been reported to play a role in the nucleus as they are involved in epigenetic regulation and heterochromatin formation7-10. However, although RNAi-mediated post-transcriptional silencing (PTGS) is well documented, mechanisms of RNAi-mediated transcriptional gene silencing (TGS) and in particular the role of RNAi components in chromatin, especially in higher eukaryotes, are still elusive. Here we show that key RNAi components Dicer-2 (Dcr2) and and Argonaute-2 (AGO2) AGO2 associate with chromatin, with strong preference for euchromatic, transcriptionally active loci and interact with core transcription machinery. Notably Dcr2 and AGO2 loss of function show that transcriptional defects are accompanied by perturbation of Pol II positioning on promoters. Further, both Dcr2 and Ago2 null mutations as well as missense mutations compromising the RNAi activity impair global Pol II dynamics upon heat shock. Finally, AGO2 RIP-seq experiments reveal that, AGO2 is strongly enriched in small-RNAs encompassing promoter as well as other parts of heat shock and other gene loci on both sense and antisense, with a strong bias for antisense, particularly after heat shock. Taken together our results reveal a new scenario in which Dcr2 and AGO2 are globally associated with transcriptionally active loci and may play a pivotal role in shaping the transcriptome by controlling RNA Pol II processivity.

Co-transcriptional splicing of constitutive and alternative exons

In metazoan organisms, pre-mRNA splicing is thought to occur during transcription, and it is postulated that these two processes are functionally coupled via still-unknown mechanisms. Current evidence supports co-transcriptional spliceosomal assembly, but there is little quantitative information on how much splicing is completed during RNA synthesis. Here we isolate nascent chromatin-associated RNA from free, nucleoplasmic RNA already released from the DNA template. Using a quantitative RT-PCR assay, we show that the majority of introns separating constitutive exons are already excised from the human c-Src and fibronectin pre-mRNAs that are still in the process of synthesis, and that these introns are removed in a general 5'-to-3' order. Introns flanking alternative exons in these transcripts are also removed during synthesis, but show differences in excision efficiency between cell lines with different regulatory conditions. Our data suggest that skipping of an exon can induce a lag in splicing compared to intron removal under conditions of exon inclusion. Nevertheless, excision of the long intron encompassing the skipped exon is still completed prior to transcript release into the nucleoplasm. Thus, we demonstrate that the decision to include or skip an alternative exon is made during transcription and not post-transcriptionally.

RNA Is an Integral Component of Chromatin that Contributes to Its Structural Organization

Chromatin structure is influenced by multiples factors, such as pH, temperature, nature and concentration of counterions, post-translational modifications of histones and binding of structural non-histone proteins. RNA is also known to contribute to the regulation of chromatin structure as chromatin-induced gene silencing was shown to depend on the RNAi machinery in S. pombe, plants and Drosophila. Moreover, both in Drosophila and mammals, dosage compensation requires the contribution of specific non-coding RNAs. However, whether RNA itself plays a direct structural role in chromatin is not known. Here, we report results that indicate a general structural role for RNA in eukaryotic chromatin. RNA is found associated to purified chromatin prepared from chicken liver, or cultured Drosophila S2 cells, and treatment with RNase A alters the structural properties of chromatin. Our results indicate that chromatin-associated RNAs, which account for 2%–5% of total chromatin-associated nucleic acids, are polyA− and show a size similar to that of the DNA contained in the corresponding chromatin fragments. Chromatin-associated RNA(s) are not likely to correspond to nascent transcripts as they are also found bound to chromatin when cells are treated with α-amanitin. After treatment with RNase A, chromatin fragments of molecular weight >3.000 bp of DNA showed reduced sedimentation through sucrose gradients and increased sensitivity to micrococcal nuclease digestion. This structural transition, which is observed both at euchromatic and heterochromatic regions, proceeds without loss of histone H1 or any significant change in core-histone composition and integrity.

Immunocytogenetics: localization of transcriptionally active rRNA genes in nucleoli and nucleolus organizer regions by use of human autoantibodies to RNA polymerase I.

Cytological staining with silver nitrate (AgNO3) has proved useful for the localization of nucleoli in interphase nuclei, as well as of nucleolus organizer regions (NORs) in metaphase chromosomes. The affinity of interphase nucleoli and chromosomal NORs to silver is a direct measure of the ongoing transcriptional activity of the rRNA genes or their activity during the preceding interphase, respectively. Correspondingly, human autoantibodies directed against chromatin-associated RNA polymerase I (RPI) should also be of value in the investigation of transcribed rRNA genes. Indirect immunofluorescence using the anti-RPI antibody as a probe has been employed successfully to visualize the chromosomal distribution of NORs in various mammalian species, as well as in human tumor cells. Immunofluorescence staining even permits the identification of heteromorphisms and small aberrations of the chromosomal NORs. The fluorescent intensity of interphase nucleoli is correlated with the different stages of nucleolar activation. In male gametogenesis, RPI-positive granules are present during meiotic prophase up to pachytene, as well as during the early and middle spermatid stages.

Effects of 3′-methyl-4-dimethylaminoazobenzene on RNA processing in rat liver nuclei

Treatment of rats with the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB) leads to the accumulation of polyadenylated RNA in rat liver nuclei. This polyadenylated nuclear RNA contains more double-stranded stretches than polyadenylated nuclear RNA isolated from liver of control animals. Treatment with 3'MeDAB also results in an increase of sequences homologous to small chromatin-associated RNA (fr 3-RNA) in the polyadenylated nuclear RNA and in the appearance of these sequences in polyadenylated nuclear RNA which contains some double-stranded stretches. The last mentioned fraction of nuclear RNA from liver of control animals did not hybridize with a DNA probe for fr 3-RNA. It is proposed that, perhaps as a result of inhibition of RNA processing by 3'MeDAB, the transient stages of RNA processing can be observed in animals treated with this compound.

Azocarcinogen-induced release of chromatin-associated RNA into liver cytoplasm: The possible role of reiterated RNA sequences in the control of RNA processing

In the liver of rats fed the azocarcinogen 3′-methyl-4-dimethylaminoazobenzene (3′MeDAB) reiterated RNA sequence transcribed from middle repetitive DNA are released into the cytoplasm. The same repetitive nucleotide sequences can be isolated from the chromatin of the liver of control animals in the form of metabolically highly active, 13 000 daltons RNA. This small, chromatin-associated RNA originates from nuclear RNA larger than 10 S. The discontinuation of the feeding of the azocarcinogen will not stop the release of the nuclear reiterated RNA sequences into the cytoplasm. The repetitive sequences of nuclear RNA which are released into the cytoplasm in animals fed the azocarcinogen can no longer be found in the chromatin in the form of small RNA molecules. The results can be explained by the assumption that the reiterated RNA sequences are involved in the upholding of RNA processing. A cell-specific processing of RNA will be maintained by the interaction of reiterated RNA fragments from already processed RNA with the reiterated complementary sequences on RNA yet to be processed. Existence of such a feed-back circuit would make it possible to explain how a temporary interference of the azocarcinogen with RNA processing will result in the disappearance of specific reiterated RNA sequences from the chromatin. It could also explain the continuation of the release of the same repeated RNA sequences into the cytoplasm as part of larger RNA molecules even after the removal of the carcinogen.

Nonhistone chromosomal proteins from gibberellic acid treated maize and pea plants, and their effect on transcription in vitro

1. 1. Nonhistone chromosomal proteins (NHP) isolated from gibberellin-(GA 3)-treated and untreated maize seedlings and pea embryos activate the rate of transcription of the calf thymus DNA- E. coli RNA polymerase system but not of the maize chromatin- E. coli RNA polymerase system. 2. 2. Studies of the effect of NHP on the chromatin-associated RNA polymerase activity showed that only NHP originating from the gibberellin-treated plants exhibited activation of transcription. 3. 3. Histone fractions isolated from this material exerted an inhibitory effect on the calf thymus DNA- E. coli RNA polymerase system but were without effect on the maize chromatin-chromatin-associated RNA polymerase system. 4. 4. Results were discussed with respect to the specific effect of GA 3 on the regulation of transcription with involvement of NHP.

Chromatin-associated RNA: Differential extraction and characterization

Mammalian cells in different states of cytodifferentiation exhibited different RNA-synthesizing and processing patterns that could be used as markers for phenotypic variability. Inherent in these patterns was an RNA class which was differentially extracted from the cellular homogenate by elevating the temperature and pH of the buffer used in the phenol procedure. This class of RNA was initially designated fraction B (chromatin-associated RNA). In the characterization of fraction B, human myeloma cells labeled for 3 and 24 h were fractionated into subcytoplasmic and subnuclear components and the [ 3H]-RNA was differentially extracted. After 3 and 24 h labeling 84% and 73%, respectively, of the labeled RNA in the chromatin was extracted in fraction B. Only 10–20% of the polysomal RNA was extracted in fraction B with little enrichment in poly(A) RNA. These and other observations suggested that fraction B was a subpopulation of heterogeneous nuclear RNA which was tightly bound to the chromatin complex.