TRANSCRIPTION 2016, VOL. 7, NO. 4, 121–126 http://dx.doi.org/10.1080/21541264.2016.1198298
POINT-OF-VIEW
Regulation of chromatin structure and cell fate by R-loops Thomas G. Fazzioa,b a Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA; bProgram in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
ABSTRACT
ARTICLE HISTORY
Hybridization of RNA to its template DNA strand during transcription induces formation of R-loops— RNA:DNA hybrids with unpaired non-template DNA strands. Although unresolved R-loops can be detrimental, some R-loops contribute to regulation of chromatin structure. Consequently, R-loops help regulate gene expression and play important roles in numerous cellular processes.
Received 29 April 2016 Revised 30 May 2016 Accepted 1 June 2016 KEYWORDS
chromatin; embryonic stem cells; nucleosome; pluripotency; R-loops
During transcription, nascent RNAs are found in close proximity to unwound, complementary DNA, permitting hybridization of transcripts to their DNA templates at the expense of the non-template DNA strand. RNA:DNA hybrids are thermodynamically very stable in vitro, likely due to their intermediate conformation that resides in between the typical B-form of DNA: DNA duplexes and the more stable A-form of RNA: RNA duplexes.1 Studies have shown that R-loop accumulation in vivo is favored by G-rich transcripts.2,3 At least two structural features of R-loops may contribute to this observation. First, purine-rich RNAs hybridized to pyrimidine-rich DNA strands (rR:dY) adopt a conformation close to the highly stable A-form structure of RNA:RNA hybrids and are much more thermodynamically stable than rY:dR hybrids or DNA duplexes of the same sequence.4,5 Second, G-rich sequences on the displaced non-template DNA strand may be stabilized by formation of G-quadruplex structures.6 Therefore, given the access of nascent transcripts to their unwound, complementary templates during transcription, and the favorable energetics of RNA:DNA hybrid formation, it is perhaps not surprising that transcription-associated R-loops can be observed in eukaryotes from yeast to humans, as well as prokaryotes.7 Unresolved R-loops induce DNA single- and double-strand breaks, higher mutation rates, and genomic instability.7-10 Consequently, cells have evolved
CONTACT Thomas G. Fazzio [email protected] Medical School, Worcester, MA 01605, USA. © 2016 Taylor & Francis
multiple mechanisms for regulating the accumulation of R-loops that help prevent mutations, DNA damage, and genomic instability. With the exception of the CRISPR/Cas system,11 all regulators of R-loops that have been identified appear to act negatively—removing R-loops that have formed or preventing R-loop formation.7 One prominent example includes the DNA:RNA helicase SETX (Sen1 in yeast). SETX melts the RNA:DNA duplex of R-loops,12 which can be accompanied by degradation of the transcript by the RNA exonuclease, Xrn2.13 A second method of Rloop removal is through the actions of RNase H enzymes (RNase H1 and RNase H2 in eukaryotes), which degrade the RNA strand of the duplex through their endonuclease activities.14 It is not known whether additional proteins exist that specifically promote R-loop stability, either directly or by counteracting the functions of proteins that destabilize R-loops. Despite these mechanisms of R-loop removal, Rloops accumulate at multiple regions of the genome, particularly near the transcription start sites (TSSs) and transcription termination sites (TTSs) of transcribed genes.2,3,13,15 Steady-state R-loop levels are therefore likely a function of the combined effects of biophysical properties of the particular RNA:DNA hybrid, the level of transcription of the RNA component of the R-loop, and the presence or absence of proteins that inhibit R-loop formation or disrupt R-loops
Department of Molecular, Cell, and Cancer Biology, LRB 519, University of Massachusetts
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that have formed. Several functions have been described for R-loops in specific genomic contexts. For example, RNA:DNA hybrids function prominently in DNA replication, where thousands to millions of short RNA primers contribute to lagging strand DNA synthesis in eukaryotes, with a periodicity of approximately one RNA primer per nucleosome.16 Similarly, replication of mitochondrial DNA is primed from a transcript expressed from the mtDNA replication origin.17,18 In addition, R-loop formation at the immunoglobulin switch (S) regions allows access of the cytidine deaminase AID to single-stranded DNA, promoting DNA breakage and class-switch recombination.19 Besides these functions in DNA replication and recombination, recent studies from several labs have uncovered functions for R-loops in several different aspects of transcription.3,13,15,20,21 While R-loops appear to regulate expression of genes by several different mechanisms, one common theme appears to be their ability to alter local chromatin structure.
Inhibition of protein-DNA interactions by R-loops Formation of an R-loop locks DNA in a locally unwound configuration and creates a three-stranded nucleic acid structure that is likely unsuitable for binding of many chromatin proteins. For example, due to structural constraints, it is unlikely that R-loops are compatible with the tight wrapping of DNA that occurs during formation of nucleosomes, raising the possibility that Rloops may locally regulate nucleosome positioning or occupancy by creating metastable, nucleosome-free “boundary” regions. While this possibility has not been tested genome-wide, R-loops appear to locally repress nucleosome formation near the regulatory regions of the vimentin gene in human colon cancer cells.22 At this locus, disruption of R-loop formation led to both decreased local chromatin accessibility and decreased binding of the transcription factor NF-kB, suggesting Rloops regulate NF-kB binding at this site by controlling nucleosome positioning or occupancy (Fig. 1). It remains to be determined how broadly this regulatory paradigm operates; however, R-loops could potentially regulate binding of many additional transcription factors at binding sites within or near transcribed regions. Loss of the histone chaperone complex FACT, which functions in chromatin re-assembly after passage of RNA Polymerase II (RNAPII), leads to R-loop accumulation,23 suggesting
Figure 1. R-loops both promote and inhibit binding of proteins with important regulatory functions. Shown is a model R-loop formed when a G-rich nascent RNA transcript hybridizes with its template DNA sequence. R-loops inhibit the binding and function of epigenetic regulators PRC2 and DNMT3B. In addition, R-loops may inhibit wrapping of DNA around histones to form nucleosomes (histone octamers depicted as dark unlabeled spheres divided into individual histone components). Conversely, R-loops promote binding of Tip60-p400 complex and the transcription factor NF-kB at some sites. See text for details.
that not only are R-loops inhibitory for nucleosome formation, but, conversely, nucleosomes also help prevent R-loop accumulation. Assuming R-loops are similarly inhibitory for nucleosome formation throughout the genome, R-loop formation likely alters the kinetics of nucleosome turnover, and consequently contributes to regulation of transcription.24 R-loops have also been implicated in the regulation of DNA methylation in mammals. Many CpG islands (CGIs) are unmethylated or lowly methylated relative to CpG dinucleotides elsewhere in the genome.25 CGIs often serve as promoters in mammalian cells, with more than 50% of human genes transcribed from CGI elements.26 Previously, Chedin and colleagues demonstrated that CGI-containing promoters frequently exhibit strand-biased distributions of G and C nucleotides downstream of TSSs, whereby G is overrepresented in the RNA and non-template DNA strand relative to the template strand.2,3,27 To assess the impact of GC skew on R-loop formation, the authors mapped the positions of R-loops throughout the genome by two independent techniques: DRIPseq, in which the S9.6 monoclonal antibody is used to immunoprecipitate RNA:DNA hybrids; and DRIVEseq, in which RNA:DNA hybrids are enriched by binding to catalytically-inactive RNase H1.3 Using
TRANSCRIPTION
both methods to sequence genomic fragments harboring R-loops, the authors showed that genes with skewed 50 GC distributions are most frequently associated with promoter-proximal R-loops and that R-loop formation plays an important role in maintaining these promoters in a lowly methylated state. Interestingly, R-loops appear to directly inhibit the functions of the de novo DNA methyltransferase Dnmt3b1, suggesting a mechanism by which R-loops negatively regulate DNA methylation3 (Fig. 1). R-loops also alter the chromatin landscape in cells by inhibiting the binding of chromatin regulatory factors. Studies over the past several years have shown that the Polycomb repressive complex 2 (PRC2), a key regulator of chromatin structure and development, binds to nascent transcripts near the promoter-proximal regions of many active genes in embryonic stem cells (ESCs).28 However, binding of RNA to PRC2 inhibits its ability to methylate histone H3 on lysine 27 to produce H3K27me3, a modification associated with repressive chromatin structure. Consequently, RNA binding has been proposed to allow sampling of the promoter-proximal regions of active genes by PRC2 without causing H3K27me3-mediated gene silencing. Given that PRC2 binds nascent RNAs near their 50 ends (corresponding to the regions where nascent RNAs frequently contribute to R-loops), we recently tested the possibility that R-loops might promote chromatin interaction by this complex. However, we observed the opposite result—upon reduction of R-loops by over-expression of the Rnaseh1 gene to specifically degrade RNAs within RNA:DNA hybrids, PRC2 binding to most of its normal targets is enhanced, along with ectopic binding to many genes not normally targeted by the complex.20 Combined with earlier findings,29,30 these data suggest PRC2 binds to nascent RNAs that do not favor R-loop formation and that disruption of normal promoter-proximal chromatin structure by R-loops inhibits PRC2 binding (Fig. 1). Consequently, RNA may have both positive and negative effects on PRC2 localization in ESCs, and the effects at each gene may vary according to the extent to which R-loops accumulate.
R-loops promote the activities of some chromatin regulatory factors Since most chromatin-binding proteins interact with histones, DNA, and/or other chromatin proteins, it is
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not surprising the presence of R-loops would impair the association of some factors with chromatin. However, recent findings show R-loops also promote the binding and functions of a subset of chromatin regulatory factors, revealing an additional (and still poorly understood) role for R-loops in regulation of chromatin structure at key regulatory regions. Despite the possibility that promoter-proximal R-loops inhibit nucleosome assembly at some regulatory regions of genes, R-loops were shown to positively correlate with H3S10P, a histone modification associated mainly with mitotic chromatin condensation, in yeast, nematodes, and mammalian cells.31 Yeast mutants with increased accumulation of R-loops exhibited increased H3S10P on chromatin not only during mitosis, but also in G1 cells, and disruption of R-loops reversed this phenomenon. Interestingly, the increase in H3S10P in cells with higher levels of Rloops coincided with reduced chromatin accessibility at some loci. Therefore, R-loops can either increase or decrease chromatin accessibility, depending on the context. The molecular mechanisms underlying the correlation between R-loops and H3S10P remain to be determined. Along with promoter-proximal regions of transcribed genes, R-loops also accumulate near the 30 ends of genes, where RNAPII pauses prior to transcription termination.13 A recent study from Proudfoot and colleagues found that R-loops that form as a result of RNAPII pausing near the TTSs of genes induce antisense transcription that produces double-stranded RNA (dsRNA).32 Interestingly, the accumulation of dsRNA at these sites leads to recruitment of components of the RNAi machinery— DICER, AGO1, and AGO2, which in turn leads to recruitment of the histone H3K9 methyltransferase, G9a. The authors observed accumulation of H3K9me2 near TTSs, consistent with the actions of G9a, and showed that H3K9me2 accumulation was dependent on R-loops and AGO2. Finally, the authors showed that HP1g, a known “reader” of H3K9 methyl marks, binds, and helps promote RNAPII pausing-dependent transcription termination. Consistent with this latter finding, triplet repeat expansions associated with Friedreich ataxia and Fragile X syndrome induce formation of R-loops, which in turn induce spread of H3K9me2 and repression of transcription.33 These latter findings suggest the R-loopdependent transcription termination process described above can be ectopically triggered by some types of repeat expansions and contribute to disease.
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These studies show R-loops can induce antisense transcription, but how? Simply by maintaining the non-template DNA strand in an unpaired state, it is possible that promiscuous binding and initiation of RNAPII could produce antisense transcripts at this site. Another question is how the antisense RNA transcript then swaps positions with the template DNA strand in the R-loop to form dsRNA. One possibility is that the actions of a helicase that unwinds R-loops, such as SETX, may play a role in this process. Most interestingly, do R-loops elsewhere (such as near TSSs) also induce antisense transcription? Many promoters produce divergent antisense transcripts,34 but these tend to arise from independent RNAPII binding sites upstream of the coding gene promoter, and most do not overlap with the coding gene transcript. Nonetheless, it remains possible that promoter-proximal R-loops could induce antisense transcription and dsRNA production at some coding gene promoters. Besides PRC2, a large number of other chromatin regulatory complexes have been shown to bind RNA and could therefore be regulated—either positively or negatively—by R-loops. In a 2011 study from Guttman, Lander, and colleagues, 12 regulators of chromatin structure with important roles in ESC pluripotency were shown to bind to some fraction of 74 lncRNAs tested.35 We recently showed that one of these complexes, the Tip60-p400 histone acetyltransferase and H2A.Z deposition complex, binds not only to lncRNAs, but also binds to approximately 2000 nascent transcripts from coding genes.20 Interestingly, while many of the transcripts bound by Tip60-p400 were expressed at moderate to high levels, a portion of them were expressed at relatively low levels, suggesting the complex does not simply bind transcripts in proportion to their expression level. When we compared the genome-wide binding profiles of Tip60-p400 complex (which binds most prominently to promoter-proximal regions) to the presence of R-loops, we observed a strong association between the two. However, unlike PRC2, we found that upon reduction in R-loop levels by Rnaseh1 over-expression, Tip60-p400 binding to its target promoters was substantially reduced. These data reveal that normal binding of Tip60-p400 to most target genes depends on R-loops.20 Together with our findings that Tip60-p400 binds to nascent RNAs, these studies suggest Tip60-p400 may be recruited to genes in part via direct interaction with the RNA component of promoter-proximal R-loops (Fig. 1). Several questions arise from these findings. First, how does Tip60-p400 interact with RNA within R-
loops or otherwise? One possibility is that the complex interacts with RNA indirectly, through interaction with an RNA-binding protein. A second question arising from these studies is whether other RNAbinding chromatin-remodeling complexes are also recruited to their genomic targets by R-loops. Finally, since R-loops are tightly regulated and likely to be transient (in order to avoid mutagenesis and genomic instability), are there continuous cycles of R-loop formation and Tip60-p400 recruitment followed by R-loop dissolution and Tip60-p400 eviction? Sophisticated studies that can capture these potential dynamic behaviors will be required to address this issue.
Role of R-loops in regulation of the ESC state The Tip60-p400 and PRC2 chromatin remodeling complexes play important roles in ESC pluripotency. For example, mutations in genes encoding the Suz12, Eed, and Ezh2 subunits of PRC2 complex cause defects in ESC differentiation,36,37 whereby mesendoderm-specific genes are poorly induced when ESCs are grown in differentiation-promoting medium. The phenotypes of Tip60p400 mutants or RNAi-mediated knockdowns (KDs) are more complicated. KD of most subunits results in a partial defect in self-renewal, whereby expression of some markers of pluripotency, such as alkaline phosphatase, are reduced, while other markers (Oct4, Nanog) are maintained.38,39 Perhaps not surprisingly, given this altered ESC state, Tip60-p400 KD ESCs also have defects in normal differentiation.20,38,39 Partial loss of Tip60p400 function, as observed in the case of KD or mutation of the gene encoding the Tip60-p400 interacting protein Hdac6, result in differentiation defects without any significant effect on self-renewal.39 Since R-loops were found to be necessary to restrict PRC2 targets and activity while simultaneously promoting Tip60-p400 binding, one might expect reduction of R-loops to result in defects in self-renewal or differentiation. While self-renewal was not affected in Rnaseh1 over-expressing cells (in which R-loops were reduced, but not completely eliminated), differentiation was significantly impaired.20 Future work will be necessary to understand whether this differentiation defect can be attributed to enhanced PRC2 binding and H3K27 methylation, loss of Tip60-p400 function, or both. In addition, R-loops may also affect other gene regulators necessary for ESC pluripotency beyond Tip60-p400 and PRC2.
TRANSCRIPTION
Conclusions and perspectives The discoveries that R-loops regulate the binding and activities of multiple gene regulatory factors suggest that the presence or absence of R-loops must be determined in order to understand how each specific gene is regulated. Since R-loops require transcription to generate the RNA component of the RNA:DNA hybrid, the mechanisms by which R-loops regulate gene expression may take the form of positive or negative feedback loops. For example, low levels of transcription may induce R-loop formation at some genes, causing displacement of PRC2 and recruitment of Tip60-p400, potentially facilitating increased transcription. In addition to R-loop formation by nascent transcripts at their genes of origin, to what extent do R-loops form in trans? While technical limitations make trans-R-loops difficult to identify, there are a few examples in the literature,40,41 raising the intriguing possibility that hundreds or thousands of trans-Rloops result from the actions of thousands of lncRNAs expressed in mammals and other eukaryotes. The finding that R-loops near mammalian TTSs can induce the formation of dsRNA, followed by recruitment of the RNAi machinery and the G9a histone methyltransferase raises two related questions. What portion of Rloops elsewhere in the genome induces antisense RNA expression? Furthermore, what determines which Rloops are transcribed in the antisense orientation to produce dsRNA? Whether or not the sequence of the nontemplate DNA strand is conducive to transcription presumably plays a role, as does the local chromatin context. In order to accurately predict the functions that R-loops might play on a gene-by-gene basis, we must gain a better understanding of their apparent context-specific effects on chromatin structure. Similarly, a better understanding of the factors controlling the balance between R-loop stabilization and destabilization will facilitate our understanding of how these structures are employed to regulate transcription.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Acknowledgments I thank J. Benanti, S. Hainer, L. Ee, and K. McCannell for critical reading of the manuscript.
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Funding T.G.F. is supported by grants HD072122 from the NIH, RSG-14-220-01 from the American Cancer Society, and is a Leukemia and Lymphoma Society Scholar.
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POINT-OF-VIEW
Regulation of chromatin structure and cell fate by R-loops Thomas G. Fazzioa,b a Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA; bProgram in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
ABSTRACT
ARTICLE HISTORY
Hybridization of RNA to its template DNA strand during transcription induces formation of R-loops— RNA:DNA hybrids with unpaired non-template DNA strands. Although unresolved R-loops can be detrimental, some R-loops contribute to regulation of chromatin structure. Consequently, R-loops help regulate gene expression and play important roles in numerous cellular processes.
Received 29 April 2016 Revised 30 May 2016 Accepted 1 June 2016 KEYWORDS
chromatin; embryonic stem cells; nucleosome; pluripotency; R-loops
During transcription, nascent RNAs are found in close proximity to unwound, complementary DNA, permitting hybridization of transcripts to their DNA templates at the expense of the non-template DNA strand. RNA:DNA hybrids are thermodynamically very stable in vitro, likely due to their intermediate conformation that resides in between the typical B-form of DNA: DNA duplexes and the more stable A-form of RNA: RNA duplexes.1 Studies have shown that R-loop accumulation in vivo is favored by G-rich transcripts.2,3 At least two structural features of R-loops may contribute to this observation. First, purine-rich RNAs hybridized to pyrimidine-rich DNA strands (rR:dY) adopt a conformation close to the highly stable A-form structure of RNA:RNA hybrids and are much more thermodynamically stable than rY:dR hybrids or DNA duplexes of the same sequence.4,5 Second, G-rich sequences on the displaced non-template DNA strand may be stabilized by formation of G-quadruplex structures.6 Therefore, given the access of nascent transcripts to their unwound, complementary templates during transcription, and the favorable energetics of RNA:DNA hybrid formation, it is perhaps not surprising that transcription-associated R-loops can be observed in eukaryotes from yeast to humans, as well as prokaryotes.7 Unresolved R-loops induce DNA single- and double-strand breaks, higher mutation rates, and genomic instability.7-10 Consequently, cells have evolved
CONTACT Thomas G. Fazzio [email protected] Medical School, Worcester, MA 01605, USA. © 2016 Taylor & Francis
multiple mechanisms for regulating the accumulation of R-loops that help prevent mutations, DNA damage, and genomic instability. With the exception of the CRISPR/Cas system,11 all regulators of R-loops that have been identified appear to act negatively—removing R-loops that have formed or preventing R-loop formation.7 One prominent example includes the DNA:RNA helicase SETX (Sen1 in yeast). SETX melts the RNA:DNA duplex of R-loops,12 which can be accompanied by degradation of the transcript by the RNA exonuclease, Xrn2.13 A second method of Rloop removal is through the actions of RNase H enzymes (RNase H1 and RNase H2 in eukaryotes), which degrade the RNA strand of the duplex through their endonuclease activities.14 It is not known whether additional proteins exist that specifically promote R-loop stability, either directly or by counteracting the functions of proteins that destabilize R-loops. Despite these mechanisms of R-loop removal, Rloops accumulate at multiple regions of the genome, particularly near the transcription start sites (TSSs) and transcription termination sites (TTSs) of transcribed genes.2,3,13,15 Steady-state R-loop levels are therefore likely a function of the combined effects of biophysical properties of the particular RNA:DNA hybrid, the level of transcription of the RNA component of the R-loop, and the presence or absence of proteins that inhibit R-loop formation or disrupt R-loops
Department of Molecular, Cell, and Cancer Biology, LRB 519, University of Massachusetts
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that have formed. Several functions have been described for R-loops in specific genomic contexts. For example, RNA:DNA hybrids function prominently in DNA replication, where thousands to millions of short RNA primers contribute to lagging strand DNA synthesis in eukaryotes, with a periodicity of approximately one RNA primer per nucleosome.16 Similarly, replication of mitochondrial DNA is primed from a transcript expressed from the mtDNA replication origin.17,18 In addition, R-loop formation at the immunoglobulin switch (S) regions allows access of the cytidine deaminase AID to single-stranded DNA, promoting DNA breakage and class-switch recombination.19 Besides these functions in DNA replication and recombination, recent studies from several labs have uncovered functions for R-loops in several different aspects of transcription.3,13,15,20,21 While R-loops appear to regulate expression of genes by several different mechanisms, one common theme appears to be their ability to alter local chromatin structure.
Inhibition of protein-DNA interactions by R-loops Formation of an R-loop locks DNA in a locally unwound configuration and creates a three-stranded nucleic acid structure that is likely unsuitable for binding of many chromatin proteins. For example, due to structural constraints, it is unlikely that R-loops are compatible with the tight wrapping of DNA that occurs during formation of nucleosomes, raising the possibility that Rloops may locally regulate nucleosome positioning or occupancy by creating metastable, nucleosome-free “boundary” regions. While this possibility has not been tested genome-wide, R-loops appear to locally repress nucleosome formation near the regulatory regions of the vimentin gene in human colon cancer cells.22 At this locus, disruption of R-loop formation led to both decreased local chromatin accessibility and decreased binding of the transcription factor NF-kB, suggesting Rloops regulate NF-kB binding at this site by controlling nucleosome positioning or occupancy (Fig. 1). It remains to be determined how broadly this regulatory paradigm operates; however, R-loops could potentially regulate binding of many additional transcription factors at binding sites within or near transcribed regions. Loss of the histone chaperone complex FACT, which functions in chromatin re-assembly after passage of RNA Polymerase II (RNAPII), leads to R-loop accumulation,23 suggesting
Figure 1. R-loops both promote and inhibit binding of proteins with important regulatory functions. Shown is a model R-loop formed when a G-rich nascent RNA transcript hybridizes with its template DNA sequence. R-loops inhibit the binding and function of epigenetic regulators PRC2 and DNMT3B. In addition, R-loops may inhibit wrapping of DNA around histones to form nucleosomes (histone octamers depicted as dark unlabeled spheres divided into individual histone components). Conversely, R-loops promote binding of Tip60-p400 complex and the transcription factor NF-kB at some sites. See text for details.
that not only are R-loops inhibitory for nucleosome formation, but, conversely, nucleosomes also help prevent R-loop accumulation. Assuming R-loops are similarly inhibitory for nucleosome formation throughout the genome, R-loop formation likely alters the kinetics of nucleosome turnover, and consequently contributes to regulation of transcription.24 R-loops have also been implicated in the regulation of DNA methylation in mammals. Many CpG islands (CGIs) are unmethylated or lowly methylated relative to CpG dinucleotides elsewhere in the genome.25 CGIs often serve as promoters in mammalian cells, with more than 50% of human genes transcribed from CGI elements.26 Previously, Chedin and colleagues demonstrated that CGI-containing promoters frequently exhibit strand-biased distributions of G and C nucleotides downstream of TSSs, whereby G is overrepresented in the RNA and non-template DNA strand relative to the template strand.2,3,27 To assess the impact of GC skew on R-loop formation, the authors mapped the positions of R-loops throughout the genome by two independent techniques: DRIPseq, in which the S9.6 monoclonal antibody is used to immunoprecipitate RNA:DNA hybrids; and DRIVEseq, in which RNA:DNA hybrids are enriched by binding to catalytically-inactive RNase H1.3 Using
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both methods to sequence genomic fragments harboring R-loops, the authors showed that genes with skewed 50 GC distributions are most frequently associated with promoter-proximal R-loops and that R-loop formation plays an important role in maintaining these promoters in a lowly methylated state. Interestingly, R-loops appear to directly inhibit the functions of the de novo DNA methyltransferase Dnmt3b1, suggesting a mechanism by which R-loops negatively regulate DNA methylation3 (Fig. 1). R-loops also alter the chromatin landscape in cells by inhibiting the binding of chromatin regulatory factors. Studies over the past several years have shown that the Polycomb repressive complex 2 (PRC2), a key regulator of chromatin structure and development, binds to nascent transcripts near the promoter-proximal regions of many active genes in embryonic stem cells (ESCs).28 However, binding of RNA to PRC2 inhibits its ability to methylate histone H3 on lysine 27 to produce H3K27me3, a modification associated with repressive chromatin structure. Consequently, RNA binding has been proposed to allow sampling of the promoter-proximal regions of active genes by PRC2 without causing H3K27me3-mediated gene silencing. Given that PRC2 binds nascent RNAs near their 50 ends (corresponding to the regions where nascent RNAs frequently contribute to R-loops), we recently tested the possibility that R-loops might promote chromatin interaction by this complex. However, we observed the opposite result—upon reduction of R-loops by over-expression of the Rnaseh1 gene to specifically degrade RNAs within RNA:DNA hybrids, PRC2 binding to most of its normal targets is enhanced, along with ectopic binding to many genes not normally targeted by the complex.20 Combined with earlier findings,29,30 these data suggest PRC2 binds to nascent RNAs that do not favor R-loop formation and that disruption of normal promoter-proximal chromatin structure by R-loops inhibits PRC2 binding (Fig. 1). Consequently, RNA may have both positive and negative effects on PRC2 localization in ESCs, and the effects at each gene may vary according to the extent to which R-loops accumulate.
R-loops promote the activities of some chromatin regulatory factors Since most chromatin-binding proteins interact with histones, DNA, and/or other chromatin proteins, it is
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not surprising the presence of R-loops would impair the association of some factors with chromatin. However, recent findings show R-loops also promote the binding and functions of a subset of chromatin regulatory factors, revealing an additional (and still poorly understood) role for R-loops in regulation of chromatin structure at key regulatory regions. Despite the possibility that promoter-proximal R-loops inhibit nucleosome assembly at some regulatory regions of genes, R-loops were shown to positively correlate with H3S10P, a histone modification associated mainly with mitotic chromatin condensation, in yeast, nematodes, and mammalian cells.31 Yeast mutants with increased accumulation of R-loops exhibited increased H3S10P on chromatin not only during mitosis, but also in G1 cells, and disruption of R-loops reversed this phenomenon. Interestingly, the increase in H3S10P in cells with higher levels of Rloops coincided with reduced chromatin accessibility at some loci. Therefore, R-loops can either increase or decrease chromatin accessibility, depending on the context. The molecular mechanisms underlying the correlation between R-loops and H3S10P remain to be determined. Along with promoter-proximal regions of transcribed genes, R-loops also accumulate near the 30 ends of genes, where RNAPII pauses prior to transcription termination.13 A recent study from Proudfoot and colleagues found that R-loops that form as a result of RNAPII pausing near the TTSs of genes induce antisense transcription that produces double-stranded RNA (dsRNA).32 Interestingly, the accumulation of dsRNA at these sites leads to recruitment of components of the RNAi machinery— DICER, AGO1, and AGO2, which in turn leads to recruitment of the histone H3K9 methyltransferase, G9a. The authors observed accumulation of H3K9me2 near TTSs, consistent with the actions of G9a, and showed that H3K9me2 accumulation was dependent on R-loops and AGO2. Finally, the authors showed that HP1g, a known “reader” of H3K9 methyl marks, binds, and helps promote RNAPII pausing-dependent transcription termination. Consistent with this latter finding, triplet repeat expansions associated with Friedreich ataxia and Fragile X syndrome induce formation of R-loops, which in turn induce spread of H3K9me2 and repression of transcription.33 These latter findings suggest the R-loopdependent transcription termination process described above can be ectopically triggered by some types of repeat expansions and contribute to disease.
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These studies show R-loops can induce antisense transcription, but how? Simply by maintaining the non-template DNA strand in an unpaired state, it is possible that promiscuous binding and initiation of RNAPII could produce antisense transcripts at this site. Another question is how the antisense RNA transcript then swaps positions with the template DNA strand in the R-loop to form dsRNA. One possibility is that the actions of a helicase that unwinds R-loops, such as SETX, may play a role in this process. Most interestingly, do R-loops elsewhere (such as near TSSs) also induce antisense transcription? Many promoters produce divergent antisense transcripts,34 but these tend to arise from independent RNAPII binding sites upstream of the coding gene promoter, and most do not overlap with the coding gene transcript. Nonetheless, it remains possible that promoter-proximal R-loops could induce antisense transcription and dsRNA production at some coding gene promoters. Besides PRC2, a large number of other chromatin regulatory complexes have been shown to bind RNA and could therefore be regulated—either positively or negatively—by R-loops. In a 2011 study from Guttman, Lander, and colleagues, 12 regulators of chromatin structure with important roles in ESC pluripotency were shown to bind to some fraction of 74 lncRNAs tested.35 We recently showed that one of these complexes, the Tip60-p400 histone acetyltransferase and H2A.Z deposition complex, binds not only to lncRNAs, but also binds to approximately 2000 nascent transcripts from coding genes.20 Interestingly, while many of the transcripts bound by Tip60-p400 were expressed at moderate to high levels, a portion of them were expressed at relatively low levels, suggesting the complex does not simply bind transcripts in proportion to their expression level. When we compared the genome-wide binding profiles of Tip60-p400 complex (which binds most prominently to promoter-proximal regions) to the presence of R-loops, we observed a strong association between the two. However, unlike PRC2, we found that upon reduction in R-loop levels by Rnaseh1 over-expression, Tip60-p400 binding to its target promoters was substantially reduced. These data reveal that normal binding of Tip60-p400 to most target genes depends on R-loops.20 Together with our findings that Tip60-p400 binds to nascent RNAs, these studies suggest Tip60-p400 may be recruited to genes in part via direct interaction with the RNA component of promoter-proximal R-loops (Fig. 1). Several questions arise from these findings. First, how does Tip60-p400 interact with RNA within R-
loops or otherwise? One possibility is that the complex interacts with RNA indirectly, through interaction with an RNA-binding protein. A second question arising from these studies is whether other RNAbinding chromatin-remodeling complexes are also recruited to their genomic targets by R-loops. Finally, since R-loops are tightly regulated and likely to be transient (in order to avoid mutagenesis and genomic instability), are there continuous cycles of R-loop formation and Tip60-p400 recruitment followed by R-loop dissolution and Tip60-p400 eviction? Sophisticated studies that can capture these potential dynamic behaviors will be required to address this issue.
Role of R-loops in regulation of the ESC state The Tip60-p400 and PRC2 chromatin remodeling complexes play important roles in ESC pluripotency. For example, mutations in genes encoding the Suz12, Eed, and Ezh2 subunits of PRC2 complex cause defects in ESC differentiation,36,37 whereby mesendoderm-specific genes are poorly induced when ESCs are grown in differentiation-promoting medium. The phenotypes of Tip60p400 mutants or RNAi-mediated knockdowns (KDs) are more complicated. KD of most subunits results in a partial defect in self-renewal, whereby expression of some markers of pluripotency, such as alkaline phosphatase, are reduced, while other markers (Oct4, Nanog) are maintained.38,39 Perhaps not surprisingly, given this altered ESC state, Tip60-p400 KD ESCs also have defects in normal differentiation.20,38,39 Partial loss of Tip60p400 function, as observed in the case of KD or mutation of the gene encoding the Tip60-p400 interacting protein Hdac6, result in differentiation defects without any significant effect on self-renewal.39 Since R-loops were found to be necessary to restrict PRC2 targets and activity while simultaneously promoting Tip60-p400 binding, one might expect reduction of R-loops to result in defects in self-renewal or differentiation. While self-renewal was not affected in Rnaseh1 over-expressing cells (in which R-loops were reduced, but not completely eliminated), differentiation was significantly impaired.20 Future work will be necessary to understand whether this differentiation defect can be attributed to enhanced PRC2 binding and H3K27 methylation, loss of Tip60-p400 function, or both. In addition, R-loops may also affect other gene regulators necessary for ESC pluripotency beyond Tip60-p400 and PRC2.
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Conclusions and perspectives The discoveries that R-loops regulate the binding and activities of multiple gene regulatory factors suggest that the presence or absence of R-loops must be determined in order to understand how each specific gene is regulated. Since R-loops require transcription to generate the RNA component of the RNA:DNA hybrid, the mechanisms by which R-loops regulate gene expression may take the form of positive or negative feedback loops. For example, low levels of transcription may induce R-loop formation at some genes, causing displacement of PRC2 and recruitment of Tip60-p400, potentially facilitating increased transcription. In addition to R-loop formation by nascent transcripts at their genes of origin, to what extent do R-loops form in trans? While technical limitations make trans-R-loops difficult to identify, there are a few examples in the literature,40,41 raising the intriguing possibility that hundreds or thousands of trans-Rloops result from the actions of thousands of lncRNAs expressed in mammals and other eukaryotes. The finding that R-loops near mammalian TTSs can induce the formation of dsRNA, followed by recruitment of the RNAi machinery and the G9a histone methyltransferase raises two related questions. What portion of Rloops elsewhere in the genome induces antisense RNA expression? Furthermore, what determines which Rloops are transcribed in the antisense orientation to produce dsRNA? Whether or not the sequence of the nontemplate DNA strand is conducive to transcription presumably plays a role, as does the local chromatin context. In order to accurately predict the functions that R-loops might play on a gene-by-gene basis, we must gain a better understanding of their apparent context-specific effects on chromatin structure. Similarly, a better understanding of the factors controlling the balance between R-loop stabilization and destabilization will facilitate our understanding of how these structures are employed to regulate transcription.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Acknowledgments I thank J. Benanti, S. Hainer, L. Ee, and K. McCannell for critical reading of the manuscript.
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Funding T.G.F. is supported by grants HD072122 from the NIH, RSG-14-220-01 from the American Cancer Society, and is a Leukemia and Lymphoma Society Scholar.
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