ABSTRACT
RNA can bind within the major groove of purine-rich DNA via Hoogsteen base pairing and form a triple helical RNA-DNA structure that anchors the RNA to specific DNA sequences, thereby targeting RNA-associated regulatory proteins to distinct genomic sites. Here we present methods to analyze the potential of a given RNA to form triplexes in vitro and to validate these structures in vivo.
Subject(s)
DNA/chemistry , Electrophoretic Mobility Shift Assay/methods , RNA/chemistry , HeLa Cells , Humans , Nucleic Acid ConformationABSTRACT
Transcription of the proto-oncogene SPHK1 is regulated by KHPS1, an antisense RNA that activates SPHK1 expression by forming a triple-helical RNA-DNA-DNA structure at the SPHK1 enhancer. Triplex-mediated tethering of KHPS1 to its target gene is required for recruitment of E2F1 and p300 and transcription of the RNA derived from the SPHK1 enhancer (eRNA-Sphk1). eRNA-Sphk1 evicts CTCF, which insulates the enhancer from the SPHK1 promoter, thus facilitating SPHK1 expression. Genomic deletion of the triplex-forming sequence attenuates SPHK1 expression, leading to decreased cell migration and invasion. Replacement of the triplex-forming region (TFR) of KHPS1 by the TFR of the lncRNA MEG3 tethers KHPS1 to the MEG3 target gene TGFBR1, underscoring the interchangeability and anchoring function of sequences involved in triplex formation. Altogether, the results reveal a triplex-driven feedforward mechanism involving lncRNA-dependent induction of eRNA, which enhances expression of specific target genes.
Subject(s)
Enhancer Elements, Genetic , Epigenesis, Genetic , RNA, Long Noncoding/metabolism , 3T3 Cells , Animals , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , Mice , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Proto-Oncogene Mas , RNA, Long Noncoding/genetics , Receptor, Transforming Growth Factor-beta Type I/genetics , Receptor, Transforming Growth Factor-beta Type I/metabolismABSTRACT
Although thousands of long noncoding RNAs (lncRNAs) have been discovered, very little is known about their mode of action. Here we functionally characterize an E2F1-regulated lncRNA named Khps1, which is transcribed in antisense orientation to the proto-oncogene SPHK1. Khps1 activates SPHK1 expression by recruiting the histone acetyltransferase p300/CBP to the SPHK1 promoter, which leads to local changes of the chromatin structure that ensures E2F1 binding and enhances transcription. Mechanistically, this is achieved by direct association of Khps1 with a homopurine stretch upstream of the transcription start site of SPHK1, which forms a DNA-RNA triplex that anchors the lncRNA and associated effector proteins to the gene promoter. The results reveal an lncRNA- and E2F1-driven regulatory loop in which E2F1-dependent induction of antisense RNA leads to changes in chromatin structure, facilitating E2F1-dependent expression of SPHK1 and restriction of E2F1-induced apoptosis.
Subject(s)
Chromatin Assembly and Disassembly , E2F1 Transcription Factor/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , RNA, Long Noncoding/metabolism , Apoptosis , Cell Proliferation , Gene Expression Regulation , Hep G2 Cells , Humans , Nucleic Acid Conformation , Promoter Regions, Genetic , Proto-Oncogene Mas , p300-CBP Transcription Factors/metabolismABSTRACT
A significant fraction of eukaryotic genomes comprises repetitive sequences, including rRNA genes, centromeres, telomeres, and retrotransposons. Repetitive elements are hotspots for recombination and represent a serious challenge for genome integrity. Maintaining these repeated elements in a compact heterochromatic structure suppresses recombination and unwanted mutagenic transposition, and is therefore indispensable for genomic stability. Paradoxically, repetitive elements are not transcriptionally inert, but produce RNA that has important functions in regulating and reinforcing the heterochromatic state. Here, we review the role of non-coding RNA (ncRNA) in recruiting chromatin-modifying enzymes to repetitive genomic loci to establish a repressive chromatin structure that safeguards chromosome integrity and genome stability.
Subject(s)
Heterochromatin/metabolism , RNA, Untranslated/metabolism , Repetitive Sequences, Nucleic Acid , Animals , Centromere/genetics , Chromatin Assembly and Disassembly , Epigenesis, Genetic , Genes, rRNA , Genomic Instability , Heterochromatin/genetics , Humans , RNA, Untranslated/genetics , Retroelements , Telomere/geneticsABSTRACT
Constitutive heterochromatin is crucial for the integrity of chromosomes and genomic stability. Here, we show that the chromatin remodelling complex NoRC, known to silence a fraction of rRNA genes, also establishes a repressive heterochromatic structure at centromeres and telomeres, preserving the structural integrity of these repetitive loci. Knockdown of NoRC leads to relaxation of centromeric and telomeric heterochromatin, abnormalities in mitotic spindle assembly, impaired chromosome segregation and enhanced chromosomal instability. The results demonstrate that NoRC safeguards genomic stability by coordinating enzymatic activities that establish features of repressive chromatin at centromeric and telomeric regions, and this heterochromatic structure is required for sustaining genomic integrity.
