Diverse RNA Structures Induce PRC2 Dimerization and Inhibit Histone Methyltransferase Activity.

Methyltransferase PRC2 (Polycomb Repressive Complex 2) introduces histone H3K27 trimethylation, a repressive chromatin mark, to tune the differential expression of genes. PRC2 is precisely regulated by accessory proteins, histone post-translational modifications and, notably, RNA. Research on PRC2-associated RNA has mostly focused on the tight-binding G-quadruplex (G4) RNAs, which inhibit PRC2 enzymatic activity in vitro and in cells. Our recent cryo-EM structure provided a molecular mechanism for G4 RNA inactivating PRC2 via dimerization, but it remained unclear how diverse RNAs associate with and regulate PRC2. Here, we show that a single-stranded G-rich RNA and an atypical G4 structure called pUG-fold unexpectedly also mediate near-identical PRC2 dimerization resulting in inhibition of PRC2 methyltransferase activity. The conformational flexibility of arginine-rich loops within subunits EZH2 and AEBP2 of PRC2 can accommodate diverse RNA secondary structures, resulting in protein-RNA and protein-protein interfaces similar to those observed previously with G4 RNA. Furthermore, we address a recent report that failed to detect PRC2-associated RNAs in living cells by demonstrating the insensitivity of PRC2-RNA interaction to photochemical crosslinking. Our results support the significance of RNA-mediated PRC2 regulation by showing that this interaction is not limited to a single RNA secondary structure, consistent with the broad PRC2 transcriptome containing many G-tract RNAs incapable of folding into G4 structures.

Structural basis for inactivation of PRC2 by G-quadruplex RNA.

Polycomb repressive complex 2 (PRC2) silences genes through trimethylation of histone H3K27. PRC2 associates with numerous precursor messenger RNAs (pre-mRNAs) and long noncoding RNAs (lncRNAs) with a binding preference for G-quadruplex RNA. In this work, we present a 3.3-Å-resolution cryo-electron microscopy structure of PRC2 bound to a G-quadruplex RNA. Notably, RNA mediates the dimerization of PRC2 by binding both protomers and inducing a protein interface composed of two copies of the catalytic subunit EZH2, thereby blocking nucleosome DNA interaction and histone H3 tail accessibility. Furthermore, an RNA-binding loop of EZH2 facilitates the handoff between RNA and DNA, another activity implicated in PRC2 regulation by RNA. We identified a gain-of-function mutation in this loop that activates PRC2 in zebrafish. Our results reveal mechanisms for RNA-mediated regulation of a chromatin-modifying enzyme.

Evaluation of the RNA-dependence of PRC2 binding to chromatin in human pluripotent stem cells.

Polycomb Repressive Complex 2 (PRC2), an important histone modifier and epigenetic repressor, has been known to interact with RNA for almost two decades. In our previous publication (Long, Hwang et al. 2020), we presented data supporting the functional importance of RNA interaction in maintaining PRC2 occupancy on chromatin, using comprehensive approaches including an RNA-binding mutant of PRC2 and an rChIP-seq assay. Recently, concerns have been expressed regarding whether the RNA-binding mutant has impaired histone methyltransferase activity and whether the rChIP-seq assay can potentially generate artifacts. Here we provide new data that support a number of our original findings. First, we found the RNA-binding mutant to be fully capable of maintaining H3K27me3 levels in human induced pluripotent stem cells. The mutant had reduced methyltransferase activity in vitro, but only on some substrates at early time points. Second, we found that our rChIP-seq method gave consistent data across antibodies and cell lines. Third, we further optimized rChIP-seq by using lower concentrations of RNase A and incorporating a catalytically inactive mutant RNase A as a control, as well as using an alternative RNase (RNase T1). The EZH2 rChIP-seq results using the optimized protocols supported our original finding that RNA interaction contributes to the chromatin occupancy of PRC2.

PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.

The chromatin-modifying enzyme, Polycomb Repressive Complex 2 (PRC2), deposits the H3K27me3 epigenetic mark to negatively regulate expression at numerous target genes, and this activity has been implicated in embryonic development, cell differentiation, and various cancers. A biological role for RNA binding in regulating PRC2 histone methyltransferase activity is generally accepted, but the nature and mechanism of this relationship remains an area of active investigation. Notably, many in vitro studies demonstrate that RNA inhibits PRC2 activity on nucleosomes through mutually antagonistic binding, while some in vivo studies indicate that PRC2's RNA-binding activity is critical for facilitating its biological function(s). Here we use biochemical, biophysical, and computational approaches to interrogate PRC2's RNA and DNA-binding kinetics. Our findings demonstrate that PRC2-polynucleotide dissociation rates are dependent on the concentration of free ligand, indicating the potential for direct transfer between nucleic acid ligands without a free-enzyme intermediate. Direct transfer explains the variation in previously reported dissociation kinetics, allows reconciliation of prior in vitro and in vivo studies, and expands the potential mechanisms of RNA-mediated PRC2 regulation. Moreover, simulations indicate that such a direct transfer mechanism could be obligatory for RNA to recruit proteins to chromatin.

Structural basis for inactivation of PRC2 by G-quadruplex RNA.

The histone methyltransferase PRC2 (Polycomb Repressive Complex 2) silences genes via successively attaching three methyl groups to lysine 27 of histone H3. PRC2 associates with numerous pre-mRNA and lncRNA transcripts with a binding preference for G-quadruplex RNA. Here, we present a 3.3Å-resolution cryo-EM structure of PRC2 bound to a G-quadruplex RNA. Notably, RNA mediates the dimerization of PRC2 by binding both protomers and inducing a protein interface comprised of two copies of the catalytic subunit EZH2, which limits nucleosome DNA interaction and occludes H3 tail accessibility to the active site. Our results reveal an unexpected mechanism for RNA-mediated inactivation of a chromatin-modifying enzyme. Furthermore, the flexible loop of EZH2 that helps stabilize RNA binding also facilitates the handoff between RNA and DNA, an activity implicated in PRC2 regulation by RNA. One-Sentence Summary: Cryo-EM structure of RNA-bound PRC2 dimer elucidates an unexpected mechanism of PRC2 inhibition by RNA.

DNMT1 inhibition by pUG-fold quadruplex RNA.

Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation remains poorly understood. A link between RNA and chromatin-associated proteins has recently emerged, and several studies have shown that DNMT1 can be regulated by a variety of RNAs. In this study, we have confirmed that human DNMT1 indeed interacts with multiple RNAs, including its own nuclear mRNA. Unexpectedly, we found that DNMT1 exhibits a strong and specific affinity for GU-rich RNAs that form a pUG-fold, a noncanonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and we additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicate that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA.

Competition between PRC2.1 and 2.2 subcomplexes regulates PRC2 chromatin occupancy in human stem cells.

Polycomb repressive complex 2 (PRC2) silences expression of developmental transcription factors in pluripotent stem cells by methylating lysine 27 on histone H3. Two mutually exclusive subcomplexes, PRC2.1 and PRC2.2, are defined by the set of accessory proteins bound to the core PRC2 subunits. Here we introduce separation-of-function mutations into the SUZ12 subunit of PRC2 to drive it into a PRC2.1 or 2.2 subcomplex in human induced pluripotent stem cells (iPSCs). We find that PRC2.2 occupies polycomb target genes at low levels and that homeobox transcription factors are upregulated when this complex is exclusively present. In contrast with previous studies, we find that chromatin occupancy of PRC2 increases drastically when it is forced to form PRC2.1. Additionally, several cancer-associated mutations also coerce formation of PRC2.1. We suggest that PRC2 chromatin occupancy can be altered in the context of disease or development by tuning the ratio of PRC2.1 to PRC2.2.

RNA is essential for PRC2 chromatin occupancy and function in human pluripotent stem cells.

Many chromatin-binding proteins and protein complexes that regulate transcription also bind RNA. One of these, Polycomb repressive complex 2 (PRC2), deposits the H3K27me3 mark of facultative heterochromatin and is required for stem cell differentiation. PRC2 binds RNAs broadly in vivo and in vitro. Yet, the biological importance of this RNA binding remains unsettled. Here, we tackle this question in human induced pluripotent stem cells by using multiple complementary approaches. Perturbation of RNA-PRC2 interaction by RNase A, by a chemical inhibitor of transcription or by an RNA-binding-defective mutant all disrupted PRC2 chromatin occupancy and localization genome wide. The physiological relevance of PRC2-RNA interactions is further underscored by a cardiomyocyte differentiation defect upon genetic disruption. We conclude that PRC2 requires RNA binding for chromatin localization in human pluripotent stem cells and in turn for defining cellular state.

Bending and looping of long DNA by Polycomb repressive complex 2 revealed by AFM imaging in liquid.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that methylates histone H3 at Lysine 27. PRC2 is critical for epigenetic gene silencing, cellular differentiation and the formation of facultative heterochromatin. It can also promote or inhibit oncogenesis. Despite this importance, the molecular mechanisms by which PRC2 compacts chromatin are relatively understudied. Here, we visualized the binding of PRC2 to naked DNA in liquid at the single-molecule level using atomic force microscopy. Analysis of the resulting images showed PRC2, consisting of five subunits (EZH2, EED, SUZ12, AEBP2 and RBBP4), bound to a 2.5-kb DNA with an apparent dissociation constant ($K_{\rm{D}}^{{\rm{app}}}$) of 150 ± 12 nM. PRC2 did not show sequence-specific binding to a region of high GC content (76%) derived from a CpG island embedded in such a long DNA substrate. At higher concentrations, PRC2 compacted DNA by forming DNA loops typically anchored by two or more PRC2 molecules. Additionally, PRC2 binding led to a 3-fold increase in the local bending of DNA's helical backbone without evidence of DNA wrapping around the protein. We suggest that the bending and looping of DNA by PRC2, independent of PRC2's methylation activity, may contribute to heterochromatin formation and therefore epigenetic gene silencing.

Regulation of histone methylation by automethylation of PRC2.

Polycomb-repressive complex 2 (PRC2) is a histone methyltransferase that is critical for regulating transcriptional repression in mammals. Its catalytic subunit, EZH2, is responsible for the trimethylation of H3K27 and also undergoes automethylation. Using mass spectrometry analysis of recombinant human PRC2, we identified three methylated lysine residues (K510, K514, and K515) on a disordered but highly conserved loop of EZH2. Methylation of these lysines increases PRC2 histone methyltransferase activity, whereas their mutation decreases activity in vitro. De novo histone methylation in an EZH2 knockout cell line is greatly impeded by mutation of the automethylation lysines. EZH2 automethylation occurs intramolecularly (in cis) by methylation of a pseudosubstrate sequence on a flexible loop. This posttranslational modification and cis regulation of PRC2 are analogous to the activation of many protein kinases by autophosphorylation. We propose that EZH2 automethylation allows PRC2 to modulate its histone methyltransferase activity by sensing histone H3 tails, SAM concentration, and perhaps other effectors.

Conserved RNA-binding specificity of polycomb repressive complex 2 is achieved by dispersed amino acid patches in EZH2.

Polycomb repressive complex 2 (PRC2) is a key chromatin modifier responsible for methylation of lysine 27 in histone H3. PRC2 has been shown to interact with thousands of RNA species in vivo, but understanding the physiological function of RNA binding has been hampered by the lack of separation-of-function mutants. Here, we use comprehensive mutagenesis and hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify critical residues for RNA interaction in PRC2 core complexes from Homo sapiens and Chaetomium thermophilum, for which crystal structures are known. Preferential binding of G-quadruplex RNA is conserved, surprisingly using different protein elements. Key RNA-binding residues are spread out along the surface of EZH2, with other subunits including EED also contributing, and missense mutations of some of these residues have been found in cancer patients. The unusual nature of this protein-RNA interaction provides a paradigm for other epigenetic modifiers that bind RNA without canonical RNA-binding motifs.

Molecular analysis of PRC2 recruitment to DNA in chromatin and its inhibition by RNA.

Many studies have revealed pathways of epigenetic gene silencing by Polycomb repressive complex 2 (PRC2) in vivo, but understanding the underlying molecular mechanisms requires biochemistry. Here we analyze interactions of reconstituted human PRC2 with nucleosome complexes. Histone modifications, the H3K27M cancer mutation, and inclusion of JARID2 or EZH1 in the PRC2 complex have unexpectedly minor effects on PRC2-nucleosome binding. Instead, protein-free linker DNA dominates the PRC2-nucleosome interaction. Specificity for CG-rich sequences is consistent with PRC2 occupying CG-rich DNA in vivo. PRC2 preferentially binds methylated DNA regulated by its AEBP2 subunit, suggesting how DNA and histone methylation collaborate to repress chromatin. We find that RNA, known to inhibit PRC2 activity, is not a methyltransferase inhibitor per se. Instead, RNA sequesters PRC2 from nucleosome substrates, because PRC2 binding requires linker DNA, and RNA and DNA binding are mutually exclusive. Together, we provide a model for PRC2 recruitment and an explanation for how actively transcribed genomic regions bind PRC2 but escape silencing.

Targeting of Polycomb Repressive Complex 2 to RNA by Short Repeats of Consecutive Guanines.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that trimethylates H3K27, a mark of repressed chromatin. Mammalian PRC2 binds RNA promiscuously, with thousands of target transcripts in vivo. But what does PRC2 recognize in these RNAs? Here we show that purified human PRC2 recognizes G > C,U ≫ A in single-stranded RNA and has a high affinity for folded guanine quadruplex (G4) structures but little binding to duplex RNAs. Importantly, G-tract motifs are significantly enriched among PRC2-binding transcripts in vivo. DNA sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on chromatin and H3K27me3-modified nucleosomes. Collectively, the abundance of PRC2-binding RNA motifs rationalizes the promiscuous RNA binding of PRC2, and their enrichment at Polycomb target genes provides a means for RNA-mediated regulation.

RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure.

RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.

Toward a consensus on the binding specificity and promiscuity of PRC2 for RNA.

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Early works suggested binding specificity of PRC2 to certain long non-coding RNAs for recruitment to chromatin. More recent studies provided evidence both in favor and against this idea. Here, we bridge the two existing models of PRC2-RNA interaction. RepA RNA is a good binding partner for PRC2, while multiple non-relevant RNAs, including bacterial mRNAs, also bind PRC2; Kds depend to some extent on the experimental conditions. Human and mouse PRC2 have broadly similar RNA-binding properties in vitro. Examination of evidence supporting an existing model for site-specific recruitment of PRC2 by a well-defined RNA motif in cells reveals that results are PRC2 independent. We conclude that promiscuous and specific RNA-binding activities of PRC2 in vitro are not mutually exclusive, and that binding specificity in vivo remains to be demonstrated.

A dimeric state for PRC2.

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) can recruit PRC2 to chromatin. Previous studies identified PRC2 subunits in a complex with the apparent molecular weight of a dimer, which might be accounted for by the incorporation of additional protein subunits or RNA rather than PRC2 dimerization. Here we show that reconstituted human PRC2 is in fact a dimer, using multiple independent approaches including analytical size exclusion chromatography (SEC), SEC combined with multi-angle light scattering and co-immunoprecipitation of differentially tagged subunits. Even though it contains at least two RNA-binding subunits, each PRC2 dimer binds only one RNA molecule. Yet, multiple PRC2 dimers bind a single RNA molecule cooperatively. These observations suggest a model in which the first RNA binding event promotes the recruitment of multiple PRC2 complexes to chromatin, thereby nucleating repression.

Tetrahymena telomerase protein p65 induces conformational changes throughout telomerase RNA (TER) and rescues telomerase reverse transcriptase and TER assembly mutants.

The biogenesis of the Tetrahymena telomerase ribonucleoprotein particle (RNP) is enhanced by p65, a La family protein. Single-molecule and biochemical studies have uncovered a hierarchical assembly of the RNP, wherein the binding of p65 to stems I and IV of telomerase RNA (TER) causes a conformational change that facilitates the subsequent binding of telomerase reverse transcriptase (TERT) to TER. We used purified p65 and variants of TERT and TER to investigate the conformational rearrangements that occur during RNP assembly. Nuclease protection assays and mutational analysis revealed that p65 interacts with and stimulates conformational changes in regions of TER beyond stem IV. Several TER mutants exhibited telomerase activity only in the presence of p65, revealing the importance of p65 in promoting the correct RNP assembly pathway. In addition, p65 rescued TERT assembly mutants but not TERT activity mutants. Taken together, these results suggest that p65 stimulates telomerase assembly and activity in two ways. First, by sequestering stems I and IV, p65 limits the ensemble of structural conformations of TER, thereby presenting TERT with the active conformation of TER. Second, p65 acts as a molecular buttress within the assembled RNP, mutually stabilizing TER and TERT in catalytically active conformations.

Preparation of group I introns for biochemical studies and crystallization assays by native affinity purification.

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides -- some containing exons -- were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3' overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules.

The Cech Symposium: a celebration of 25 years of ribozymes, 10 years of TERT, and 60 years of Tom.

The Cech Symposium was held in Boulder, Colorado, on July 12-13, 2007, to celebrate a triple anniversary: 25 years since the first publication reporting RNA self-splicing, 10 years since the identification of reverse transcriptase motifs in the catalytic subunit of telomerase, and 60 years since the birth of Thomas R. Cech. Past and present members of the Cech laboratory presented on their current research, which branched into many categories of study including RNA-mediated catalysis, telomerase and telomeres, new frontiers in nucleic acids, alternative splicing, as well as scientific research with direct medical applications.

Local RNA structural changes induced by crystallization are revealed by SHAPE.

We present a simple approach to locate sites that undergo conformational changes upon crystallization by comparative structural mapping of the same RNA in three different environments. As a proof of principle, we probed the readily crystallized P4-P6DeltaC209 domain from the Tetrahymena thermophila group I intron in a native solution, in a solution mimicking the crystallization drop, and in crystals. We chose the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, which monitors the flexibility and the conformation of each nucleotide. First, SHAPE successfully revealed the structural changes that occur during the crystallization process. Specifically, 64% of the nucleotides implicated in packing contacts and present in the portion of the molecule analyzed were identified. Second, reactivity differences for some of these nucleotides were already observed in the crystallization solution, suggesting that the crystallization buffer locked down a particular structure that was favorable to crystal formation. Third, the probing of a known structure extends our understanding of the structural basis for the SHAPE reaction by suggesting that reactivity is enhanced by a C2'-endo sugar pucker. Furthermore, by identifying local conformational changes of the RNA that take place during crystallization, SHAPE could be combined with the in vitro selection of stable mutants to rationalize the design of RNA candidates for crystallization.