Trends in Genetics
Volume 32, Issue 12, December 2016, Pages 828-838
Journal home page for Trends in Genetics

Review
Nascent Connections: R-Loops and Chromatin Patterning

https://doi.org/10.1016/j.tig.2016.10.002Get rights and content

Trends

R-loop structures are a type of non-B DNA structure that can form during transcription upon reannealing of the nascent RNA to the template DNA template. R-loops can now be profiled globally at high resolution in any genome.

R-loops in mammalian systems are abundant, covering over 100 Mb of DNA sequence, and form dynamically over conserved regions.

R-loops have important roles in chromosome dynamics, in particular because it concerns transcription termination and chromatin patterning.

Deregulation of R-loop metabolism is linked to an increasing number of human diseases, including cancers and several neurodegenerative disorders.

RNA molecules, such as long noncoding RNAs (lncRNAs), have critical roles in regulating gene expression, chromosome architecture, and the modification states of chromatin. Recent developments suggest that RNA also influences gene expression and chromatin patterns through the interaction of nascent transcripts with their DNA template via the formation of co-transcriptional R-loop structures. R-loop formation over specific, conserved, hotspots occurs at thousands of genes in mammalian genomes and represents an important and dynamic feature of mammalian chromatin. Here, focusing primarily on mammalian systems, I describe the accumulating connections and possible mechanisms linking R-loop formation and chromatin patterning. The possible contribution of aberrant R-loops to pathological conditions is also discussed.

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)

  • R. Pavri

    Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5

    Cell

    (2010)
  • B.B. Balter

    Mice lacking Smu tandem repeats maintain RNA polymerase patterns but exhibit histone modification pattern shifts linked to class switch site locations

    Mol. Immunol.

    (2012)
  • G. Li

    Combinatorial H3K9acS10 ph histone modification in IgH locus S regions targets 14-3-3 adaptors and AID to specify antibody class-switch DNA recombination

    Cell Rep.

    (2013)
  • I.X. Wang

    RNA-DNA differences are generated in human cells within seconds after RNA exits polymerase II

    Cell Rep.

    (2014)
  • P. Grzechnik

    Terminate and make a loop: regulation of transcriptional directionality

    Trends Biochem. Sci.

    (2014)
  • N.J. Krogan

    The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation

    Mol. Cell

    (2003)
  • J.H. Lee et al.

    CpG-binding protein (CXXC finger protein 1) is a component of the mammalian Set1 histone H3-Lys4 methyltransferase complex, the analogue of the yeast Set1/COMPASS complex

    J. Biol. Chem.

    (2005)
  • B.N. Tomson et al.

    The many roles of the conserved eukaryotic Paf1 complex in regulating transcription, histone modifications, and disease states

    Biochim. Biophys. Acta

    (2013)
  • R.N. de Jong

    Structure and DNA binding of the human Rtf1 Plus3 domain

    Structure

    (2008)
  • P. Huertas et al.

    Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination

    Mol. Cell

    (2003)
  • M. Castellano-Pozo

    R loops are linked to histone H3 S10 phosphorylation and chromatin condensation

    Mol. Cell

    (2013)
  • J.Y. Hsu

    Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes

    Cell

    (2000)
  • A.K. Sharma

    Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    Mutat Res.

    (2015)
  • A. Zippo

    Histone crosstalk between H3S10 ph and H4K16ac generates a histone code that mediates transcription elongation

    Cell

    (2009)
  • J. Sollier et al.

    Breaking bad: R-loops and genome integrity

    Trends Cell Biol.

    (2015)
  • O. Gursoy-Yuzugullu

    Patching broken DNA: nucleosome dynamics and the repair of DNA breaks

    J. Mol. Biol.

    (2016)
  • S. Hamperl et al.

    The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability

    DNA Repair (Amst)

    (2014)
  • A. Aguilera et al.

    R loops: from transcription byproducts to threats to genome stability

    Mol. Cell

    (2012)
  • L. Costantino et al.

    The Yin and Yang of R-loop biology

    Curr. Opin. Cell Biol.

    (2015)
  • Y.A. Chan

    Mechanisms of genome instability induced by RNA-processing defects

    Trends Genet.

    (2014)
  • E. Masse

    DNA topoisomerases regulate R-loop formation during transcription of the rrnB operon in Escherichia coli

    J. Biol. Chem.

    (1997)
  • Y. Yang

    Arginine methylation facilitates the recruitment of TOP3B to chromatin to prevent R loop accumulation

    Mol. Cell

    (2014)
  • J. Sollier

    Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability

    Mol. Cell

    (2014)
  • H.E. Mischo

    Yeast Sen1 helicase protects the genome from transcription-associated instability

    Mol. Cell

    (2011)
  • T.A. Brown

    Native R-loops persist throughout the mouse mitochondrial DNA genome

    J. Biol. Chem.

    (2008)
  • K.D. Westover

    Structural basis of transcription: separation of RNA from DNA by RNA polymerase II

    Science

    (2004)
  • G.A. Daniels et al.

    RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination

    Nucleic Acids Res.

    (1995)
  • D. Roy et al.

    G clustering is important for the initiation of transcription-induced R-loops in vitro, whereas high G density without clustering is sufficient thereafter

    Mol. Cell Biol.

    (2009)
  • D. Roy

    Competition between the RNA transcript and the nontemplate DNA strand during R-loop formation in vitro: a nick can serve as a strong R-loop initiation site

    Mol. Cell Biol.

    (2010)
  • L. Ratmeyer

    Sequence specific thermodynamic and structural properties for DNA.RNA duplexes

    Biochemistry

    (1994)
  • D. Roy

    Mechanism of R-loop formation at immunoglobulin class switch sequences

    Mol. Cell Biol.

    (2008)
  • B.P. Belotserkovskii

    Mechanisms and implications of transcription blockage by guanine-rich DNA sequences

    Proc. Natl. Acad. Sci. U.S.A.

    (2010)
  • K. Yu

    R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells

    Nat. Immunol.

    (2003)
  • D.D. Phillips

    The sub-nanomolar binding of DNA-RNA hybrids by the single-chain Fv fragment of antibody S9.6

    J. Mol. Recognit.

    (2013)
  • F.T. Huang

    Sequence dependence of chromosomal R-loops at the immunoglobulin heavy-chain Smu class switch region

    Mol. Cell Biol.

    (2007)
  • F.T. Huang

    Downstream boundary of chromosomal R-loops at murine switch regions: implications for the mechanism of class switch recombination

    Proc. Natl. Acad. Sci. U.S.A.

    (2006)
  • J. Nadel

    RNA: DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships

    Epigenetics Chromatin

    (2015)
  • P.A. Ginno

    GC skew at the 5′ and 3′ ends of human genes links R-loop formation to epigenetic regulation and transcription termination

    Genome Res.

    (2013)
  • Y.W. Lim

    Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutieres syndrome

    Elife

    (2015)
  • P.B. Chen

    R loops regulate promoter-proximal chromatin architecture and cellular differentiation

    Nat. Struct. Mol. Biol.

    (2015)
  • Cited by (0)

    View full text