Liqi Yao

Methyl-CpG binding protein 2 (MeCP2) is a chromatin-associated regulator essential for neuronal gene regulation, and pathogenic mutations in MeCP2 cause Rett syndrome (RTT). However, the mechanisms governing MeCP2 engagement on chromatin and the effects of RTT mutations on nucleosome interactions remain poorly understood. We determined cryo-EM structures of MeCP2 bound to mono-nucleosomes with or without linker DNA methylation. In the absence of linker DNA methylation, the methyl-CpG binding domain (MBD) engages nucleosomal DNA near superhelical location +/- 7, whereas a methylated linker CpG redirects MBD to the linker methylation site. Quantitative EMSA, MNase footprinting, and fluorescence polarization show that both MBD and AT-hook regions cooperate to stabilize nucleosome binding, and that six common RTT variants (R133C, T158M, R306C, R168X, R255X, R270X) fall into four mechanistic classes. Loss-of-function truncations progressively weaken binding, whereas missense variants show near-WT DNA affinity. Among the variants, R133C shows near wild-type affinity for naked DNA but loses methylation-directed nucleosome engagement. Our analysis of human neuronal transcriptomic and chromatin occupancy datasets showed that RTT variants R133C and R168X lose CpG-island specificity and redistribute in the genome through distinct biochemical routes. Together, this work establishes a framework linking variant-specific defects in nucleosome binding to chromatin-targeting failure and transcriptional dysregulation in RTT.

Summary: The restoration of chromatin in the wake of a DNA or RNA polymerase is essential to maintain the integrity of eukaryotic genomes. Human HIRA is a 1.8-megadalton, three-subunit histone chaperone that mediates all replication-independent deposition of the histone variant H3.3 at active chromatin regions ^1–6 . Disruption of HIRA perturbs active-chromatin organization and has wide-ranging consequences for development, cellular senescence, and genome integrity ^7–11 . Despite its central biological role in reassembling nucleosomes post-transcription, the structure of native human HIRA and the mechanism by which it organizes histones and DNA during nucleosome assembly remain unknown. In particular, the function of the largest HIRA subunit CABIN1 is enigmatic. Here, we show that HIRA is not simply a passive histone hand-off factor but remains engaged across multiple stages of nucleosome assembly, including a close interaction with the nucleosome. Cryo-EM structures reveal that HIRA forms an extended arch-like structure that binds the nucleosome primarily through extensive CABIN1 contacts with histones, histone tails, nucleosomal DNA, and linker DNA, during the final stage of nucleosome assembly. Together, our results suggest a testable mechanism for HIRA-mediated nucleosome assembly and product release and provide the basis for elucidating the molecular details of this fundamental biological process.

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 remained unclear how diverse RNAs beyond G4 associate with and regulate PRC2. Here, we show that variations in RNA sequence elicit disparate effects on PRC2 function. A G-rich RNA lacking consecutive G’s 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 CLIP-seq 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. 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.

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 remained unclear how diverse RNAs beyond G4 associate with and regulate PRC2. Here, we show that variations in RNA sequence elicit disparate effects on PRC2 function. A G-rich RNA lacking consecutive G’s 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.

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.

Polycomb repressive complexes are a family of chromatin modifier enzymes which are critical for regulating gene expression and maintaining cell-type identity. The reversible chemical modifications of histone H3 and H2A by the Polycomb proteins are central to its ability to function as a gene silencer. PRC2 is both a reader and writer of the tri-methylation of histone H3 lysine 27 (H3K27me3) which serves as a marker for transcription repression, and heterochromatin boundaries. Over the last few years, several studies have provided key insights into the mechanisms regulating the recruitment and activation of PRC2 at Polycomb target genes. In this review, we highlight the recent structural studies which have elucidated the roles played by Polycomb cofactor proteins in mediating crosstalk between histone post-translational modifications and the recruitment of PRC2 and the stimulation of PRC2 methyltransferase activity.