Trends in Genetics
ReviewNascent Connections: R-Loops and Chromatin Patterning
Section snippets
Basic Determinants of Co-Transcriptional R-Loop Formation
During transcription, one strand of the DNA double helix is copied into a complementary RNA transcript. RNA synthesis occurs within the context of a moving transcription bubble where the two strands of DNA are physically separated and the nascent transcript is held through a transient 8-base pair (bp) RNA:DNA hybrid. The transcription elongation complex architecture ensures that the outgoing nascent RNA does not become entangled with the DNA helix as the RNA polymerase (RNAP) moves forward [1] (
Prevalent and Conserved R-Loop Formation in Mammalian Genomes
R-loops can now be effectively mapped at endogenous loci in genomic DNA using a variety of methods, including low-throughput, single-molecule approaches based on nondenaturing sodium bisulfite footprinting [12] and high-throughput, population average techniques based on the S9.6 anti-RNA:DNA hybrid antibody 13, 14 (Box 1). Initial evidence for endogenous R-loop formation came from footprinting analysis of murine switch regions, where R-loops were detected upon induction of the corresponding
Dynamic Turnover of R-Loop Structures
While genomic studies provide a wealth of information on the range of sequences that can form R-loops, they provide no information on two key parameters: the frequency at which R-loops form and the turnover rate of an R-loop once formed. DRIP-qPCR approaches suggest that R-loop formation frequencies range from 1% to 10% of input genomic DNA, depending on the locus 19, 29, 32. By contrast, negative loci (untranscribed and/or intergenic regions) range from 0.01% to 0.1% of input. Thus, while
Heightened Accessibility and RNAP Stalling Are Common Features of R-Loop Chromatin
The formation of long, stable R-loops likely alters local chromatin, and numerous studies have focused on defining these effects. Consistent with observations that RNA:DNA hybrids adopt a rigid A form-like conformation [33] and prevent nucleosome wrapping in vitro [34], R-loops associate with DNase I hyperaccessibility at all genic positions, as per DRIPc-seq [19]. This association could be explained by interference with nucleosome redeposition behind the advancing RNAP [35]. In support of
Defining Histone Modification Signatures of R-Loop Chromatin
In addition to chromatin accessibility and RNAP stalling, high-resolution R-loop maps have enabled the identification of a set of histone modifications that associate with R-loop chromatin under normal conditions 19, 22. Histone modifications that characterize ‘aberrant’ R-loops formed under altered conditions are described separately below. The existence of such signatures suggests that the transient formation of RNA:DNA hybrids is sensed by chromatin-modifying enzymes and translated into
Mechanisms of R-Loop-Mediated Chromatin Patterning
Now that a coherent set of R-loop chromatin signatures is emerging, the task is to decipher which of these marks can be causally linked to R-loop formation and to provide a mechanistic understanding of these relations. One way that R-loops may affect the chromatin landscape is by favoring the recruitment of chromatin-modifying complexes that are normally recruited co-transcriptionally. Trimethylation of histone H3 at lysine 36 (H3K36me3) is mediated by the SETD2 histone methyltransferase upon
Aberrant R-Loops and Chromatin Condensation
Defects in the THO mRNA export complex in yeast triggers marked genomic instability and increased R-loop formation [64]. This increased R-loop load is accompanied by higher levels of H3S10P [65], a mark typically associated with condensed chromosomes during mitosis [66]. Increased H3S10P and condensation were observed outside of mitosis and, while most visible over pericentromeric regions, they were also observed over coding genic regions. Since H3S10P levels could be suppressed by RNASEH1
Defining Aberrant R-Loop Formation under Pathological Conditions
R-loops are well known for their association with genomic instability, a topic extensively covered by several excellent recent reviews 75, 78, 79, 80, 81, 82. This association is clearest in the context of defects in a variety of factors involved in co-transcriptional processes, such as splicing, mRNA export, 3′-RNA processing, and transcription elongation, among others. Defects in other factors, such as topoisomerases, which are thought to prevent R-loop formation [83], or in enzymes such as
Concluding Remarks
The field of R-loop biology has made rapid progress in recent years thanks in part to technological advances in the detection of endogenous R-loops. While we now understand that R-loops are more abundant than previously thought, many questions remain (see Outstanding Questions). Efforts should focus in particular on identifying the protein complexes that maintain R-loop homeostasis in normal cells and how breakdown of this equilibrium is linked to disease. These studies will undoubtedly provide
Acknowledgments
Research in my laboratory is supported by NIH grants GM094299, GM113929, and GM120607.
Glossary
- CpG island
- a stretch of DNA sequence characterized by high GC content and high CG dinucleotide density compared with the rest of the human genome. CpG islands often map to the beginning of mammalian genes, where they serve as promoters. Nearly 60% of human genes have a CpG island promoter.
- GC skew
- a sequence property that describes the strand asymmetry in the distribution of guanine versus cytosine residues.
- R-loop structure
- a type of three-stranded non-B DNA structure in which the nontemplate DNA
References (109)
Transcription induces the formation of a stable RNA.DNA hybrid in the immunoglobulin alpha switch region
J. Biol. Chem.
(1994)R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters
Mol. Cell
(2012)Hypernegative supercoiling of the DNA template during transcription elongation in vitro
J. Biol. Chem.
(1994)Roles of DNA topoisomerases in the regulation of R-loop formation in vitro
J. Biol. Chem.
(1997)Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids
J. Immunol. Methods
(1986)Detection and characterization of R-loops at the murine immunoglobulin Salpha region
Mol. Immunol.
(2013)Prevalent, dynamic, and conserved R-loop structures associate with specific epigenomic signatures in mammals
Mol. Cell
(2016)RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability
Mol. Cell
(2011)Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination
Mol. Cell
(2011)- et al.
Nuclear architecture by RNA
Curr. Opin. Genet. Dev.
(2012)