Biology subjects

Gooding, A. R.

Publications and source records attributed to Gooding, A. R..

6 recordsLinked to original sources

bioRxiv2024

Diverse RNA Structures Induce PRC2 Dimerization and Inhibit Histone Methyltransferase Activity

Methyltransferase PRC2 (Polycomb Repressive Complex 2) deposits histone H3K27 trimethylation to establish and maintain epigenetic gene silencing. PRC2 is precisely regulated by accessory proteins, histone post-translational modifications and, particularly, 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, a mechanism explained by our recent cryo-EM structure showing G4 RNA-mediated PRC2 dimerization. However, PRC2 binds a wide variety of RNA sequences, and it remains unclear how diverse RNAs beyond G4 associate with and regulate PRC2. Here, we show that variations in RNA sequence elicit distinct effects on PRC2 function. A single-stranded G-rich RNA and an atypical G4 structure called a pUG-fold mediate PRC2 dimerization nearly identical to that induced by G4 RNA. In contrast, pyrimidine-rich RNAs, including a motif identified by CLIPseq in cells, do not induce PRC2 dimerization and instead bind PRC2 monomers with retention of methyltransferase activity. Only RNAs that dimerize PRC2 compete with nucleosome binding and inhibit PRC2 methyltransferase activity. CRISPR-dCas9 was adapted to localize different RNA elements onto a PRC2-targeted gene, revealing RNA sequence specificity for PRC2 regulation in cells. Thus, PRC2 binds many different RNAs with similar affinity, however, the functional effect on enzymatic activity depends entirely on the sequence of the bound RNA, a conclusion potentially applicable to any RNA- binding protein with a large transcriptome.

biochemistry↗

bioRxiv2023

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.

molecular biology↗

bioRxiv2023

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 3.3[A]-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 SummaryCryo-EM structure of RNA-bound PRC2 dimer elucidates an unexpected mechanism of PRC2 inhibition by RNA.

biochemistry↗

bioRxiv2022

PRC2 direct transfer from G-quadruplex RNA to dsDNA: 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 PRC2s RNA-binding activity is critical for facilitating its biological function(s). Here we use biochemical, biophysical, and computational approaches to interrogate PRC2s 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 ligands without a free-enzyme intermediate. Direct transfer explains the variation in dissociation kinetics reported previously, 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. SignificanceStudies of PRC2 in vitro indicate that RNA inhibits its histone methyltransferase (HMTase) activity through mutually antagonistic binding with nucleosomes, but some in vivo studies paradoxically suggest that RNA binding is necessary to facilitate chromatin occupancy and HMTase activity. Our findings unveil a protein-intrinsic mechanism for directly exchanging RNA and DNA/nucleosome in PRC2s binding site(s), which reconciles these prior findings by allowing antagonistic or synergistic RNA-mediated regulation dependent on RNA-nucleosome proximity. Furthermore, there is an increasing awareness that multiple chromatin-associated proteins exhibit regulatory RNA binding activity, and our findings indicate this "direct transfer" mechanism may be generally required for such regulation.

biophysics↗

bioRxiv2022

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 non-canonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicates that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA.

biochemistry↗

bioRxiv2018

Regulation of histone methylation by automethylation of PRC2

Polycomb Repressive Complex 2 (PRC2) is a histone methyltransferase whose function is critical for regulating transcriptional repression in many eukaryotes including humans. Its catalytic moiety EZH2 is responsible for the tri-methylation of H3K27 and also undergoes automethylation. Using mass spectroscopic analysis of recombinant human PRC2, we identified three methylated lysine residues (K510, K514, K515) on a disordered but highly conserved loop of EZH2. These lysines were mostly mono- and di-methylated. Either mutation of these lysines or their methylation increases PRC2 histone methyltransferase activity. In addition, mutation of these three lysines in HEK293T cells using CRISPR genome-editing increases global H3K27 methylation levels. EZH2 automethylation occurs intramolecularly (in cis) by methylation of a pseudosubstrate sequence on the flexible loop. This post-translational modification and cis-regulation of PRC2 are analogous to the activation of many protein kinases by autophosphorylation. We therefore propose that EZH2 automethylation provides a way for PRC2 to modulate its histone methyltransferase activity by sensing histone H3 tails, SAM concentration, and perhaps other effectors.

biochemistry↗