A New Strategy to Investigate RNA:DNA Triplex Using Atomic Force Microscopy.

Over the past decade, long non-coding RNAs (lncRNAs) have been recognized as key players in gene regulation, influencing genome organization and expression. The locus-specific binding of these non-coding RNAs (ncRNAs) to DNA involves either a non-covalent interaction with DNA-bound proteins or a direct sequence-specific interaction through the formation of RNA:DNA triplexes. In an effort to develop a novel strategy for characterizing a triple-helix formation, we employed atomic force microscopy (AFM) to visualize and study a regulatory RNA:DNA triplex formed between the Khps1 lncRNA and the enhancer of the proto-oncogene SPHK1. The analysis demonstrates the successful formation of RNA:DNA triplexes under various conditions of pH and temperature, indicating the effectiveness of the AFM strategy. Despite challenges in discriminating between the triple-helix and R-loop configurations, this approach opens new perspectives for investigating the role of lncRNAs in gene regulation at the single-molecule level.

TFIIIC as a Potential Epigenetic Modulator of Histone Acetylation in Human Stem Cells.

Regulation of histone acetylation dictates patterns of gene expression and hence cell identity. Due to their clinical relevance in cancer biology, understanding how human embryonic stem cells (hESCs) regulate their genomic patterns of histone acetylation is critical, but it remains largely to be investigated. Here, we provide evidence that acetylation of histone H3 lysine-18 (H3K18ac) and lysine-27 (H3K27ac) is only partially established by p300 in stem cells, while it represents the main histone acetyltransferase (HAT) for these marks in somatic cells. Our analysis reveals that whereas p300 marginally associated with H3K18ac and H3K27ac in hESCs, it largely overlapped with these histone marks upon differentiation. Interestingly, we show that H3K18ac is found at "stemness" genes enriched in RNA polymerase III transcription factor C (TFIIIC) in hESCs, whilst lacking p300. Moreover, TFIIIC was also found in the vicinity of genes involved in neuronal biology, although devoid of H3K18ac. Our data suggest a more complex pattern of HATs responsible for histone acetylations in hESCs than previously considered, suggesting a putative role for H3K18ac and TFIIIC in regulating "stemness" genes as well as genes associated with neuronal differentiation of hESCs. The results break ground for possible new paradigms for genome acetylation in hESCs that could lead to new avenues for therapeutic intervention in cancer and developmental diseases.

TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation.

How repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.

Alu RNA Modulates the Expression of Cell Cycle Genes in Human Fibroblasts.

Alu retroelements, whose retrotransposition requires prior transcription by RNA polymerase III to generate Alu RNAs, represent the most numerous non-coding RNA (ncRNA) gene family in the human genome. Alu transcription is generally kept to extremely low levels by tight epigenetic silencing, but it has been reported to increase under different types of cell perturbation, such as viral infection and cancer. Alu RNAs, being able to act as gene expression modulators, may be directly involved in the mechanisms determining cellular behavior in such perturbed states. To directly address the regulatory potential of Alu RNAs, we generated IMR90 fibroblasts and HeLa cell lines stably overexpressing two slightly different Alu RNAs, and analyzed genome-wide the expression changes of protein-coding genes through RNA-sequencing. Among the genes that were upregulated or downregulated in response to Alu overexpression in IMR90, but not in HeLa cells, we found a highly significant enrichment of pathways involved in cell cycle progression and mitotic entry. Accordingly, Alu overexpression was found to promote transition from G1 to S phase, as revealed by flow cytometry. Therefore, increased Alu RNA may contribute to sustained cell proliferation, which is an important factor of cancer development and progression.

Interpreting and integrating big data in non-coding RNA research.

In the last two decades, we have witnessed an impressive crescendo of non-coding RNA studies, due to both the development of high-throughput RNA-sequencing strategies and an ever-increasing awareness of the involvement of newly discovered ncRNA classes in complex regulatory networks. Together with excitement for the possibility to explore previously unknown layers of gene regulation, these advancements led to the realization of the need for shared criteria of data collection and analysis and for novel integrative perspectives and tools aimed at making biological sense of very large bodies of molecular information. In the last few years, efforts to respond to this need have been devoted mainly to the regulatory interactions involving ncRNAs as direct or indirect regulators of protein-coding mRNAs. Such efforts resulted in the development of new computational tools, allowing the exploitation of the information spread in numerous different ncRNA data sets to interpret transcriptome changes under physiological and pathological cell responses. While experimental validation remains essential to identify key RNA regulatory interactions, the integration of ncRNA big data, in combination with systematic literature mining, is proving to be invaluable in identifying potential new players, biomarkers and therapeutic targets in cancer and other diseases.

Epigenetic and Transcriptional Modifications in Repetitive Elements in Petrol Station Workers Exposed to Benzene and MTBE.

Benzene, a known human carcinogen, and methyl tert-butyl ether (MTBE), not classifiable as to its carcinogenicity, are fuel-related pollutants. This study investigated the effect of these chemicals on epigenetic and transcriptional alterations in DNA repetitive elements. In 89 petrol station workers and 90 non-occupationally exposed subjects the transcriptional activity of retrotransposons (LINE-1, Alu), the methylation on repeated-element DNA, and of H3K9 histone, were investigated in peripheral blood lymphocytes. Median work shift exposure to benzene and MTBE was 59 and 408 µg/m³ in petrol station workers, and 4 and 3.5 µg/m³, in controls. Urinary benzene (BEN-U), S-phenylmercapturic acid, and MTBE were significantly higher in workers than in controls, while trans,trans-muconic acid (tt-MA) was comparable between the two groups. Increased BEN-U was associated with increased Alu-Y and Alu-J expression; moreover, increased tt-MA was associated with increased Alu-Y and Alu-J and LINE-1 (L1)-5'UTR expression. Among repetitive element methylation, only L1-Pa5 was hypomethylated in petrol station workers compared to controls. While L1-Ta and Alu-YD6 methylation was not associated with benzene exposure, a negative association with urinary MTBE was observed. The methylation status of histone H3K9 was not associated with either benzene or MTBE exposure. Overall, these findings only partially support previous observations linking benzene exposure with global DNA hypomethylation.

Hydroquinone induces DNA hypomethylation-independent overexpression of retroelements in human leukemia and hematopoietic stem cells.

Hydroquinone (HQ) is an important benzene-derived metabolite associated with acute myelogenous leukemia risk. Although altered DNA methylation has been reported in both benzene-exposed human subjects and HQ-exposed cultured cells, the inventory of benzene metabolite effects on the epigenome is only starting to be established. In this study, we used a monocytic leukemia cell line (THP-1) and hematopoietic stem cells (HSCs) from cord blood to investigate the effects of HQ treatment on the expression of the three most important families of retrotransposons in the human genome: LINE-1, Alu and Endogenous retroviruses (HERVs), that are normally subjected to tight epigenetic silencing. We found a clear tendency towards increased retrotransposon expression in response to HQ exposure, more pronounced in the case of LINE-1 and HERV. Such a partial loss of silencing, however, was generally not associated with HQ-induced DNA hypomethylation. On the other hand, retroelement derepression was also observed in the same cells in response to the hypomethylating agent decitabine. These observations suggest the existence of different types of epigenetic switches operating at human retroelements, and point to retroelement activation in response to benzene-derived metabolites as a novel factor deserving attention in benzene carcinogenesis studies.

A novel snRNA-like transcript affects amyloidogenesis and cell cycle progression through perturbation of Fe65L1 (APBB2) alternative splicing.

FE65 proteins constitute a family of adaptors which modulates the processing of amyloid precursor protein and the consequent amyloid ß production. Thus, they have been involved in the complex and partially unknown cascade of reactions at the base of Alzheimer's disease etiology. However, FE65 and FE65-like proteins may be linked to neurodegeneration through the regulation of cell cycle in post-mitotic neurons. In this work we disclose novel molecular mechanisms by which APBB2 can modulate APP processing. We show that APBB2 mRNA splicing, driven by the over-expression of a novel non-coding RNA named 45A, allow the generation of alternative protein forms endowed with differential effects on Aß production, cell cycle control, and DNA damage response. 45A overexpression also favors cell transformation and tumorigenesis leading to a marked increase of malignancy of neuroblastoma cells. Therefore, our results highlight a novel regulatory pathway of considerable interest linking APP processing with cell cycle regulation and DNA-surveillance systems, that may represent a molecular mechanism to induce neurodegeneration in post-mitotic neurons.

RNA polymerase III drives alternative splicing of the potassium channel-interacting protein contributing to brain complexity and neurodegeneration.

Alternative splicing generates protein isoforms that are conditionally or differentially expressed in specific tissues. The discovery of factors that control alternative splicing might clarify the molecular basis of biological and pathological processes. We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4). The alternative KCNIP4 isoform cannot interact with the γ-secretase complex, resulting in modification of γ-secretase activity, amyloid precursor protein processing, and increased secretion of ß-amyloid enriched in the more toxic Aß x-42 species. Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels. This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.

17A, a novel non-coding RNA, regulates GABA B alternative splicing and signaling in response to inflammatory stimuli and in Alzheimer disease.

Alternative splicing is a central component of human brain complexity; nonetheless, its regulatory mechanisms are still largely unclear. In this work, we describe a novel non-coding (nc) RNA (named 17A) RNA polymerase (pol) III-dependent embedded in the human G-protein-coupled receptor 51 gene (GPR51, GABA B2 receptor). The stable expression of 17A in SHSY5Y neuroblastoma cells induces the synthesis of an alternative splicing isoform that abolish GABA B2 intracellular signaling (i.e., inhibition of cAMP accumulation and activation of K(+) channels). Indeed, 17A is expressed in human brain, and we report that it is upregulated in cerebral tissues derived from Alzheimer disease patients. We demonstrate that 17A expression in neuroblastoma cells enhances the secretion of amyloid ß peptide (Aß) and the Aß x-42/Αß x-40 peptide ratio and that its synthesis is induced in response to inflammatory stimuli. These data correlate, for the first time, the activity of a novel pol III-dependent ncRNA to alternative splicing events and, possibly, to neurodegeneration induced by abnormal GABA B function. We anticipate that further analysis of pol III-dependent regulation of alternative splicing will disclose novel regulatory pathways associated to brain physiology and/or pathology.

A common sequence motif involved in selection of transcription start sites of Arabidopsis and budding yeast tRNA genes.

The transcription start site (TSS) is useful to predict gene and to understand transcription initiation. Although vast data on mRNA TSSs are available, little is known about tRNA genes because of rapid processing. Using a tobacco in vitro transcription system under conditions of impaired 5' end processing, TSSs were determined for 64 Arabidopsis tRNA genes. This analysis revealed multiple TSSs distributed in a region from 10 to 2bp upstream of the mature tRNA coding sequence (-10 to -2). We also analyzed 31 Saccharomyces cerevisiae tRNA genes that showed a smaller number but a broader distribution (-13 to -1) of TSSs. In both cases, transcription was initiated preferentially at adenosine, and a common 'TCAACA' sequence was found spanning the TSSs. In plant, this motif caused multiple TSSs to converge at one site and enhanced transcription. The TATA-like sequence upstream of Arabidopsis tRNA genes also contributed to TSS selection.

An Alu-like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells.

Neuroblastoma (NB) is a pediatric cancer characterized by remarkable cell heterogeneity within the tumor nodules. Here, we demonstrate that the synthesis of a pol III-transcribed noncoding (nc) RNA (NDM29) strongly restricts NB development by promoting cell differentiation, a drop of malignancy processes, and a dramatic reduction of the tumor initiating cell (TIC) fraction in the NB cell population. Notably, the overexpression of NDM29 also confers to malignant NB cells an unpredicted susceptibility to the effects of antiblastic drugs used in NB therapy. Altogether, these results suggest the induction of NDM29 expression as possible treatment to increase cancer cells vulnerability to therapeutics and the measure of its synthesis in NB explants as prognostic factor of this cancer type.

New small nuclear RNA gene-like transcriptional units as sources of regulatory transcripts.

By means of a computer search for upstream promoter elements (distal sequence element and proximal sequence element) typical of small nuclear RNA genes, we have identified in the human genome a number of previously unrecognized, putative transcription units whose predicted products are novel noncoding RNAs with homology to protein-coding genes. By elucidating the function of one of them, we provide evidence for the existence of a sense/antisense-based gene-regulation network where part of the polymerase III transcriptome could control its polymerase II counterpart.