Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are transcriptional repressor complexes that play a fundamental role in epigenomic regulation and the cell-fate decision; these complexes are widely conserved in multicellular organisms. PRC1 is an E3 ubiquitin (ub) ligase that generates histone H2A ubiquitinated at lysine (K) 119 (H2AK119ub1), whereas PRC2 is a histone methyltransferase that specifically catalyzes tri-methylation of histone H3K27 (H3K27me3). Genome-wide analyses have confirmed that these two key epigenetic marks highly overlap across the genome and contribute to gene repression. We are now beginning to understand the molecular mechanisms that enable PRC1 and PRC2 to identify their target sites in the genome and communicate through feedback mechanisms to create Polycomb chromatin domains. Recently, it has become apparent that PRC1-induced H2AK119ub1 not only serves as a docking site for PRC2 but also affects the dynamics of the H3 tail, both of which enhance PRC2 activity, suggesting that trans-tail communication between H2A and H3 facilitates the formation of the Polycomb chromatin domain. In this review, we discuss the emerging principles that define how PRC1 and PRC2 establish the Polycomb chromatin domain and regulate gene expression in mammals.

Eukaryotic gene expression is regulated through chromatin conformation, in which enhancers and promoters physically interact (E–P interactions). How such chromatin-mediated E–P interactions affect gene expression is not yet fully understood, but the roles of histone acetylation and methylation, pioneer transcription factors, and architectural proteins such as CCCTC binding factor (CTCF) and cohesin have recently attracted attention. Moreover, accumulated data suggest that E–P interactions are mechanistically involved in biophysical events, including liquid–liquid phase separation, and in biological events, including cancers. In this review, we discuss various mechanisms that regulate eukaryotic gene expression, focusing on emerging views regarding chromatin conformations that are involved in E–P interactions and factors that establish and maintain them.

The role of RNA in recruitment of various chromatin factors is widely acknowledged to contribute to nuclear transcriptional regulation. For instance, in PIWI–piRNA-mediated nuclear co-transcriptional silencing, piRNAs act as guides for the PIWI–piRNA complex, facilitating its localization at target nascent RNAs through sequence complementarity (Iwasaki et al., 2015). This complex recruits silencing effectors, including chromatin modifiers, thereby initiating co-transcriptional silencing. Similarly, long non-coding RNAs (lncRNAs) have diverse roles in nuclear transcriptional regulation (Yao et al., 2019). Xist serves as a prime example, orchestrating the architecture of the inactive X chromosome (Xi) during X chromosome inactivation. Xist recruits HDAC1-associated repressor proteins, SMART, HDAC3 and Polycomb repressive complex 1 (PRC1) to silence Xi (McHugh et al, 2015; Chu et al., 2015). Another lncRNA, Mhrt, prevents SWI/SNF binding to corresponding DNA loci (Han et al., 2014). Khps1 enhances RNA polymerase II (Pol II) transcription by forming an R-loop that anchors Khps1-interacting p300/CBP to the SPHK1 promoter (Postepska-Igielska et al., 2015). Additionally, some lncRNAs, such as SLERT, promote RNA polymerase I (Pol I) transcription by binding to DDX21, altering its conformation, and releasing its inhibitory effect on Pol I (Xing et al., 2017). These examples underscore the significance of non-coding RNAs in collaborating with proteins for diverse nuclear regulatory and genome function aspects.Polycomb repressive complex 2 (PRC2) mediates gene silencing by associating with CpG islands (CGIs) by trimethylation of histone H3 at lysine 27 (H3K27me3). Recent reports indicate that its recruitment and catalytic activity are closely linked to recognition of H2A mono-ubiquitylation given by PRC1 and unmethylated CpG dyads in CGIs (Blackledge and Klose, 2021; Kasinath et al., 2021). Nonetheless, PRC2 is also known to interact with RNA through several mechanisms to exert precise control over gene expression (Almeida et al., 2020; Davidovich and Cech, 2015). Notably, there are some models suggesting that PRC2 interacts with non-coding RNAs, which can bind to specific genes, guiding PRC2 to their genomic locations. RNA, through binding to regulatory regions of particular genes, recruits PRC2 to those specific loci, initiating H3K27 methylation. This epigenetic modification promotes gene silencing. A prominent example occurs in the Kcnq1 domain of an imprinted allele, where it has been reported that Kcnq1ot1 lncRNA interacts with PRC2 and recruits it to the imprinted allele, increasing H3K27 methylation levels and ensuring gene silencing (Pandey et al., 2008). Although it was initially postulated that RNA facilitates the recruitment of PRC2 to chromatin, another line of reports have demonstrated that RNA inhibits the interaction of PRC2 with nucleosomes (Beltran et al., 2016) and inhibits its methyltransferase activity (Kaneko et al., 2014). Therefore, proposed models of RNA-mediated PRC2 regulation remain a controversial subject. Nevertheless, the complex interaction between RNA and PRC2 contributes to precise gene regulation through H3K27 methylation and affects various biological processes, and disruptions in this regulatory process can potentially lead to diverse diseases.Healy and colleagues (2023 preprint) and Hall Hickman and Jenner (2023 preprint) have raised concerns about a high-throughput methodology employed to investigate the requirement for RNA-PRC2 interaction in the localization of PRC2 components in their preprints. The methodology, known as RNase-ChIP (rChIP), uses RNase A treatment during chromatin immunoprecipitation to observe the reduction of PRC2 occupancy on chromatin upon RNA depletion, suggesting an ‘RNA bridge’ hypothesis between RNA molecules and chromatin modifiers (Long et al., 2020). However, a closer examination of this methodology has yielded unexpected findings. Instead of solely affecting PRC2, Healy and colleagues revealed that RNase A treatment during chromatin immunoprecipitation leads to the apparent loss of all facultative heterochromatin, including PRC2 and its associated H3K27me3 marks, across the entire genome. This phenomenon was consistent across various cell types, including mouse embryonic stem cells, human cancer cells and human induced pluripotent stem cells. The study suggests that this is linked to a global increase in pulled-down DNA, which artificially diminishes ChIP signals from facultative heterochromatin during data normalization. The second preprint from Hall Hickman and Jenner (2023 preprint) arrived at the same conclusion. In this case, the authors demonstrated that the reduction of the relative enrichment of PRC2-bound genomic regions upon RNase A treatment was caused by non-specific chromatin precipitation, which could be rescued by adding the anionic polymer poly-L-glutamic acid, which maintains chromatin solubility in RNase A-treated samples. Consequently, these two independent preprints collectively suggest that RNase A treatment may not be as reliable a method as previously thought for mapping RNA-dependent chromatin occupancy.Importantly, a recent preprint by Long and colleagues (2023 preprint) also addresses a similar issue in their original PRC2 RNase-ChIP (Long et al., 2020), that RNase A treatment caused a substantial increase in the DNA pulled down by magnetic antibody beads. Meanwhile, the authors re-verified that chromatin binding of, for example, TBP (TATA box-binding protein) is not affected by the treatment with RNase A, suggesting that the issue may be antibody specific. They suggested that with the use of lower RNase A concentrations or the use of the RNase T1 RNA can still be effectively digested while reducing excess DNA pull-down. rChIP with lower RNase A concentration resulted in fewer genes significantly affected, suggesting the involvement of an ‘RNA bridge’ between PRC2...

Understanding the relationship between viral proteins and host factors is essential for developing strategies to control virus infections. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells using angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) as viral receptor and priming protease during the viral entry phase. Here, we report that TMPRSS2 reduces the infectivity of SARS-CoV-2 and HIV-1 during the viral production phase. Treatment of virus-producing cells with a TMPRSS2 inhibitor increases the production of infectious virions. TMPRSS2 enzymatic activity specifically disrupts the trans-Golgi by phosphorylating Golgi stacking proteins through ERK activation, which disturbs virion spike incorporation and structural maturation of the viral envelope glycoproteins, causing lower viral infectivity. We find that SARS-CoV-2 envelope protein (E protein) counteracts this TMPRSS2 activity to rescue SARS-CoV-2-S incorporation. These results demonstrate negative regulation of viral envelope glycoprotein incorporation by TMPRSS2 and reveal that SARS-CoV-2 regulates the Golgi system to create an optimal viral replication environment.