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

Beltran M., Tavares M., Justin N., Khandelwal G., Ambrose J., Foster B.M., Worlock K.B., Tvardovskiy A., Kunzelmann S., Herrero J., Bartke T., Gamblin S.J., Wilson J.R., Jenner R.G.

Polycomb repressive complex 2 (PRC2) maintains repression of cell-type-specific genes but also associates with genes ectopically in cancer. While it is currently unknown how PRC2 is removed from genes, such knowledge would be useful for the targeted reversal of deleterious PRC2 recruitment events. Here, we show that G-tract RNA specifically removes PRC2 from genes in human and mouse cells. PRC2 preferentially binds G tracts within nascent precursor mRNA (pre-mRNA), especially within predicted G-quadruplex structures. G-quadruplex RNA evicts the PRC2 catalytic core from the substrate nucleosome. In cells, PRC2 transfers from chromatin to pre-mRNA upon gene activation, and chromatin-associated G-tract RNA removes PRC2, leading to H3K27me3 depletion from genes. Targeting G-tract RNA to the tumor suppressor gene CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces senescence. These data support a model in which pre-mRNA evicts PRC2 during gene activation and provides the means to selectively remove PRC2 from specific genes. PRC2 preferentially binds G tracts within nascent pre-mRNAs, causing transfer of PRC2 from chromatin to RNA upon gene activation, thus providing a mechanism to selectively remove PRC2 from specific genes.

Wang X., Long Y., Paucek R.D., Gooding A.R., Lee T., Burdorf R.M., Cech T.R.

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.

Lee C., Yu J., Granat J., Saldaña-Meyer R., Andrade J., LeRoy G., Jin Y., Lund P., Stafford J.M., Garcia B.A., Ueberheide B., Reinberg D.

The histone methyltransferase activity of PRC2 is central to the formation of H3K27me3-decorated facultative heterochromatin and gene silencing. In addition, PRC2 has been shown to automethylate its core subunits, EZH1/EZH2 and SUZ12. Here, we identify the lysine residues at which EZH1/EZH2 are automethylated with EZH2-K510 and EZH2-K514 being the major such sites in vivo. Automethylated EZH2/PRC2 exhibits a higher level of histone methyltransferase activity and is required for attaining proper cellular levels of H3K27me3. While occurring independently of PRC2 recruitment to chromatin, automethylation promotes PRC2 accessibility to the histone H3 tail. Intriguingly, EZH2 automethylation is significantly reduced in diffuse intrinsic pontine glioma (DIPG) cells that carry a lysine-to-methionine substitution in histone H3 (H3K27M), but not in cells that carry either EZH2 or EED mutants that abrogate PRC2 allosteric activation, indicating that H3K27M impairs the intrinsic activity of PRC2. Our study demonstrates a PRC2 self-regulatory mechanism through its EZH1/2-mediated automethylation activity.

Barutcu A.R., Blencowe B.J., Rinn J.L.

Schertzer M.D., Braceros K.C., Starmer J., Cherney R.E., Lee D.M., Salazar G., Justice M., Bischoff S.R., Cowley D.O., Ariel P., Zylka M.J., Dowen J.M., Magnuson T., Calabrese J.M.

Long noncoding RNAs (lncRNAs) cause Polycomb repressive complexes (PRCs) to spread over broad regions of the mammalian genome. We report that in mouse trophoblast stem cells, the Airn and Kcnq1ot1 lncRNAs induce PRC-dependent chromatin modifications over multi-megabase domains. Throughout the Airn-targeted domain, the extent of PRC-dependent modification correlated with intra-nuclear distance to the Airn locus, preexisting genome architecture, and the abundance of Airn itself. Specific CpG islands (CGIs) displayed characteristics indicating that they nucleate the spread of PRCs upon exposure to Airn. Chromatin environments surrounding Xist, Airn, and Kcnq1ot1 suggest common mechanisms of PRC engagement and spreading. Our data indicate that lncRNA potency can be tightly linked to lncRNA abundance and that within lncRNA-targeted domains, PRCs are recruited to CGIs via lncRNA-independent mechanisms. We propose that CGIs that autonomously recruit PRCs interact with lncRNAs and their associated proteins through three-dimensional space to nucleate the spread of PRCs in lncRNA-targeted domains.

Fang H., Bonora G., Lewandowski J.P., Thakur J., Filippova G.N., Henikoff S., Shendure J., Duan Z., Rinn J.L., Deng X., Noble W.S., Disteche C.M.

AbstractFirre encodes a lncRNA involved in nuclear organization in mammals. Here we find that Firre RNA is transcribed from the active X chromosome (Xa) and exerts trans-acting effects on the inactive X chromosome (Xi). Allelic deletion of Firre on the Xa in a mouse hybrid fibroblast cell line results in a dramatic loss of the histone modification H3K27me3 and of components of the PRC2 complex on the Xi as well as the disruption of the perinucleolar location of the Xi. These features are measurably rescued by ectopic expression of a mouse or human Firre/FIRRE cDNA transgene, strongly supporting a conserved trans-acting role of the Firre transcript in maintaining the Xi heterochromatin environment. Surprisingly, CTCF occupancy is decreased on the Xi upon loss of Firre RNA, but is partially recovered by ectopic transgene expression, suggesting a functional link between Firre RNA and CTCF in maintenance of epigenetic features and/or location of the Xi. Loss of Firre RNA results in dysregulation of genes implicated in cell division and development, but not in reactivation of genes on the Xi, which retains its bipartite structure despite some changes in chromatin contact distribution. Allelic deletion or inversion of Firre on the Xi causes localized redistribution of chromatin contacts, apparently dependent on the orientation of CTCF binding sites clustered at the locus. Thus, the Firre locus and its RNA have roles in the maintenance of epigenetic features and structure of the Xi.

Yu J., Lee C., Oksuz O., Stafford J.M., Reinberg D.

As the process that silences gene expression ensues during development, the stage is set for the activity of Polycomb-repressive complex 2 (PRC2) to maintain these repressed gene profiles. PRC2 catalyzes a specific histone posttranslational modification (hPTM) that fosters chromatin compaction. PRC2 also facilitates the inheritance of this hPTM through its self-contained “write and read” activities, key to preserving cellular identity during cell division. As these changes in gene expression occur without changes in DNA sequence and are inherited, the process is epigenetic in scope. Mutants of mammalian PRC2 or of its histone substrate contribute to the cancer process and other diseases, and research into these aberrant pathways is yielding viable candidates for therapeutic targeting. The effectiveness of PRC2 hinges on its being recruited to the proper chromatin sites; however, resolving the determinants to this process in the mammalian case was not straightforward and thus piqued the interest of many in the field. Here, we chronicle the latest advances toward exposing mammalian PRC2 and its high maintenance.

Laugesen A., Højfeldt J.W., Helin K.

The polycomb repressive complex 2 (PRC2) is a chromatin-associated methyltransferase catalyzing mono-, di-, and trimethylation of lysine 27 on histone H3 (H3K27). This activity is required for normal organismal development and maintenance of gene expression patterns to uphold cell identity. PRC2 function is often deregulated in disease and is a promising candidate for therapeutic targeting in cancer. In this review, we discuss the molecular mechanisms proposed to take part in modulating PRC2 recruitment and shaping H3K27 methylation patterns across the genome. This includes consideration of factors influencing PRC2 residence time on chromatin and PRC2 catalytic activity with a focus on the mechanisms giving rise to regional preferences and differential deposition of H3K27 methylation. We further discuss existing evidence for functional diversity between distinct subsets of PRC2 complexes with the aim of extracting key concepts and highlighting major open questions toward a more complete understanding of PRC2 function.

Casale A.M., Cappucci U., Fanti L., Piacentini L.

A very important open question in stem cells regulation is how the fine balance between GSCs self-renewal and differentiation is orchestrated at the molecular level. In the past several years much progress has been made in understanding the molecular mechanisms underlying intrinsic and extrinsic controls of GSC regulation but the complex gene regulatory networks that regulate stem cell behavior are only partially understood. HP1 is a dynamic epigenetic determinant mainly involved in heterochromatin formation, epigenetic gene silencing and telomere maintenance. Furthermore, recent studies have revealed the importance of HP1 in DNA repair, sister chromatid cohesion and, surprisingly, in positive regulation of gene expression. Here, we show that HP1 plays a crucial role in the control of GSC homeostasis in Drosophila. Our findings demonstrate that HP1 is required intrinsically to promote GSC self-renewal and progeny differentiation by directly stabilizing the transcripts of key genes involved in GSCs maintenance.

The Gene Ontology resource (GO; http://geneontology.org) provides structured, computable knowledge regarding the functions of genes and gene products. Founded in 1998, GO has become widely adopted in the life sciences, and its contents are under continual improvement, both in quantity and in quality. Here, we report the major developments of the GO resource during the past two years. Each monthly release of the GO resource is now packaged and given a unique identifier (DOI), enabling GO-based analyses on a specific release to be reproduced in the future. The molecular function ontology has been refactored to better represent the overall activities of gene products, with a focus on transcription regulator activities. Quality assurance efforts have been ramped up to address potentially out-of-date or inaccurate annotations. New evidence codes for high-throughput experiments now enable users to filter out annotations obtained from these sources. GO-CAM, a new framework for representing gene function that is more expressive than standard GO annotations, has been released, and users can now explore the growing repository of these models. We also provide the 'GO ribbon' widget for visualizing GO annotations to a gene; the widget can be easily embedded in any web page.

Wang J., Luo J., Chen Q., Wang X., He J., Zhang W., Yin Z., Zheng F., Pan H., Li T., Lou Q., Wang B.

Atrial septal defect (ASD) is one of the most common cardiac malformations worldwide. Several genes have been identified so far, which can merely explain small proportion of all the cases, therefore, it is anticipated that there are additional genes causing ASD. The aims of this study were to identify the causal gene of ostium secundum atrial septal defect (ASDII) in a Chinese family.Whole exome sequencing was performed in three affected members and one control in the ASDII family. We screened mutations of LBX2 in 300 unrelated ASD patients and validated in 400 normal controls by Sanger sequencing. LBX2 knockout zebrafish was generated by CRISPR/Cas9 to detect whether lbx2 deficiency influenced cardiac development.A rare missense mutation in LBX2 (c.A403G: p.K135E) was identified as the pathogenic cause of ASD. Subsequent mutation screening revealed two missense variants in 3 of 300 sporadic patients. We observed expanded size of atrium and ventricle in LBX2 knockout zebrafish through hematoxylin-eosin staining, more incompact distribution of cardiac myocytes was also discovered in homozygote compared with in wildtype. Furthermore, we performed in situ hybridization of crip2 gene to trace the cardiac neural crest cells in the embryo stage and found that the migration of neural crest cells was obviously delayed in the homozygotes.We identified LBX2 for the first time as a pathogenic gene of ASDII. LBX2 deficiency may cause abnormal development of heart through influencing the migration of neural crest cells and affect the process of cardiac septation.

Oksuz O., Narendra V., Lee C., Descostes N., LeRoy G., Raviram R., Blumenberg L., Karch K., Rocha P.P., Garcia B.A., Skok J.A., Reinberg D.

Polycomb repressive complex 2 (PRC2) maintains gene silencing by catalyzing methylation of histone H3 at lysine 27 (H3K27me2/3) within chromatin. By designing a system whereby PRC2-mediated repressive domains were collapsed and then reconstructed in an inducible fashion in vivo, a two-step mechanism of H3K27me2/3 domain formation became evident. First, PRC2 is stably recruited by the actions of JARID2 and MTF2 to a limited number of spatially interacting "nucleation sites," creating H3K27me3-forming Polycomb foci within the nucleus. Second, PRC2 is allosterically activated via its binding to H3K27me3 and rapidly spreads H3K27me2/3 both in cis and in far-cis via long-range contacts. As PRC2 proceeds further from the nucleation sites, its stability on chromatin decreases such that domains of H3K27me3 remain proximal, and those of H3K27me2 distal, to the nucleation sites. This study demonstrates the principles of de novo establishment of PRC2-mediated repressive domains across the genome.

Youmans D.T., Schmidt J.C., Cech T.R.

Polycomb-repressive complex 2 (PRC2) is a histone methyltransferase that promotes epigenetic gene silencing, but the dynamics of its interactions with chromatin are largely unknown. Here we quantitatively measured the binding of PRC2 to chromatin in human cancer cells. Genome editing of a HaloTag into the endogenous EZH2 and SUZ12 loci and single-particle tracking revealed that ∼80% of PRC2 rapidly diffuses through the nucleus, while ∼20% is chromatin-bound. Short-term treatment with a small molecule inhibitor of the EED–H3K27me3 interaction had no immediate effect on the chromatin residence time of PRC2. In contrast, separation-of-function mutants of SUZ12, which still form the core PRC2 complex but cannot bind accessory proteins, revealed a major contribution of AEBP2 and PCL homolog proteins to chromatin binding. We therefore quantified the dynamics of this chromatin-modifying complex in living cells and separated the contributions of H3K27me3 histone marks and various PRC2 subunits to recruitment of PRC2 to chromatin.

Lee C., Yu J., Kumar S., Jin Y., LeRoy G., Bhanu N., Kaneko S., Garcia B.A., Hamilton A.D., Reinberg D.

PRC2 is a therapeutic target for several types of cancers currently undergoing clinical trials. Its activity is regulated by a positive feedback loop whereby its terminal enzymatic product, H3K27me3, is specifically recognized and bound by an aromatic cage present in its EED subunit. The ensuing allosteric activation of the complex stimulates H3K27me3 deposition on chromatin. Here we report a stepwise feedback mechanism entailing key residues within distinctive interfacing motifs of EZH2 or EED that are found to be mutated in cancers and/or Weaver syndrome. PRC2 harboring these EZH2 or EED mutants manifested little activity in vivo but, unexpectedly, exhibited similar chromatin association as wild-type PRC2, indicating an uncoupling of PRC2 activity and recruitment. With genetic and chemical tools, we demonstrated that targeting allosteric activation overrode the gain-of-function effect of EZH2Y646X oncogenic mutations. These results revealed critical implications for the regulation and biology of PRC2 and a vulnerability in tackling PRC2-addicted cancers.

Long Y., Bolanos B., Gong L., Liu W., Goodrich K.J., Yang X., Chen S., Gooding A.R., Maegley K.A., Gajiwala K.S., Brooun A., Cech T.R., Liu X.

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.

Lucero K., Han S., Huang P., Qiu X., Mazzoni E.O., Reinberg D.

SUMMARYCCCTC-binding factor (CTCF) is essential for chromatin organization. CTCF interacts with endogenous RNAs, and deletion of its ZF1 RNA-binding region (ΔZF1) disrupts chromatin loops in mouse embryonic stem cells (ESCs). However, the functional significance of CTCF-ZF1 RNA interactions during cell differentiation is unknown. Using an ESC-to-neural progenitor cell (NPC) differentiation model, we show that CTCF-ZF1 is crucial for maintaining cell-type-specific chromatin loops. Expression of CTCF-ΔZF1 leads to disrupted loops and dysregulation of genes within these loops, particularly those involved in neuronal development and function. We identified NPC-specific, CTCF-ZF1 interacting RNAs. Truncation of two such coding RNAs,PodxlandGrb10, disrupted chromatin loopsin cis, similar to the disruption seen in CTCF-ΔZF1 expressing NPCs. These findings underscore the inherent importance of CTCF-ZF1 RNA interactions in preserving cell-specific genome structure and cellular identity.HIGHLIGHTSCTCF loop anchors induced after differentiation are disrupted in the ΔZF1 RNA-binding mutant.Loop loss in the ΔZF1 mutant is independent of its DNA binding and protein interactions.Chromatin loop loss is associated with gene dysregulation.Truncation of cell-specific, CTCF-ZF1-interacting RNAs disrupts chromatin loopsin cis.GRAPHICAL ABSTRACT

Costa S., La Rocca G., Cavalieri V.

The bulk of RNA produced from the genome of complex organisms consists of a very large number of transcripts lacking protein translational potential and collectively known as noncoding RNAs (ncRNAs). Initially thought to be mere products of spurious transcriptional noise, ncRNAs are now universally recognized as pivotal players in cell regulatory networks across a broad spectrum of biological processes. Owing to their critical regulatory roles, ncRNA dysfunction is closely associated with the etiopathogenesis of various human malignancies, including cancer. As such, ncRNAs represent valuable diagnostic biomarkers as well as potential targets for innovative therapeutic intervention. In this review, we focus on microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), the two most extensively studied classes in the field of ncRNA biology. After outlining key concepts of miRNA and lncRNA biogenesis pathways, we examine their multiple roles in mediating epigenetic regulation of gene expression and chromatin organization. Finally, by providing numerous examples of specific miRNAs and lncRNAs, we discuss how dysregulation of these mechanisms contributes to the onset and/or progression of various human diseases.

Colemon A., Prioleau T., Rouse C., Pendergast A.M.

AbstractTriple-negative breast cancer (TNBC) remains a leading cause of cancer associated deaths in women owing to its highly metastatic potential and limited treatment options. Recent studies have shown that expression of proteins associated with epigenetic regulation of gene expression are associated with metastatic relapse, however targeting epigenetic regulatory proteins has not resulted in effective therapies for TNBC in the clinic. The ABL tyrosine kinases promote metastasis of breast cancer cells in mouse models. However, a role of ABL kinases in the regulation of epigenetic processes in solid tumor metastasis remains unexplored. Here we show that inactivation of ABL kinases in bone metastatic TNBC cells led to a significant enrichment in gene signatures associated with the PRC2 protein complex, revealing a functional link between ABL kinases and the PRC2 complex. ABL inactivation promotes EZH2-T487 phosphorylation through the regulation of a FAK-CDK1 signaling axis. We find that phosphorylated EZH2 T487 or a phosphomimic EZH2 T487D mutant exhibit increased binding to non-canonical binding partners of EZH2 including c-MYC and ZMYND8. Notably, we identify a therapeutic vulnerability in TNBC cells whereby combination treatment with ABL allosteric inhibitors and EZH2 inhibitors elicits a synergistic decrease in TNBC cell survival in vitro, and impairs TNBC metastasis, prolonging survival of tumor-bearing mice treated with the combination therapy.One Sentence SummaryABL Kinases indirectly impact EZH2 catalytic activity by blocking a signaling cascade that leads to changes in the phosphorylation, protein interactions, and function of the PRC2 catalytic component EZH2 in TNBC.

Ding C., Chen G., Luan S., Gao R., Fan Y., Zhang Y., Wang X., Li G., Foda M.F., Yan J., Li X.

Eukaryotic genomes are extensively transcribed into various types of RNAs, many of which are physically associated with chromatin in cis at their transcription sites or in trans to other genomic loci. Emerging roles have been uncovered for these chromatin-associated RNAs (caRNAs) in gene regulation and genome organization, yet they remain challenging to interrogate. Here, we present TaDRIM-seq, a technique employing Protein G (PG)-Tn5-targeted DNA elements and in situ proximity ligation to concurrently probe caRNAs across diverse genomic regions as well as global RNA-RNA interactions within intact nuclei. Notably, this approach diminishes required cell inputs, minimizes hands-on time compared to established methodologies, and is compatible in both mammalian cells and plants. Using this technique, we identify extensive caRNAs at DNA anchor regions associated with chromatin loops and reveal diurnal variation in RNA-DNA and RNA-RNA connectivity networks within rice. Chromatin-associated RNAs play various roles in gene regulation and genome organization, yet they remain challenging to interrogate. Here the authors report a targeted DNA-associated RNA and RNA-RNA interaction mapping method and use it to identify caRNAs at DNA anchor regions associated with chromatin loops.

Li J., Xu S., Liu Z., Yang L., Ming Z., Zhang R., Zhao W., Peng H., Quinn J.J., Wu M., Geng Y., Zhang Y., He J., Chen M., Li N., et. al.

Long noncoding RNAs known as roX (RNA on the X) are crucial for male development in Drosophila, as their loss leads to male lethality from the late larval stages. While roX RNAs are recognized for their role in sex-chromosome dosage compensation, ensuring balanced expression of X-linked genes in both sexes, their potential influence on autosomal gene regulation remains unexplored. Here, using an integrative multi-omics approach, we show that roX RNAs not only govern the X chromosome but also target genes on autosomes that lack male-specific lethal (MSL) complex occupancy, together with Polycomb repressive complexes (PRCs). We observed that roX RNAs colocalize with MSL proteins on the X chromosome and PRC components on autosomes. Intriguingly, loss of roX function reduces X-chromosomal H4K16ac levels and autosomal H3K27me3 levels. Correspondingly, X-linked genes display reduced expression, whereas many autosomal genes exhibit elevated expression upon roX loss. Our findings propose a dual role for roX RNAs: activators of X-linked genes and repressors of autosomal genes, achieved through interactions with MSL and PRC complexes, respectively. This study uncovers the unconventional epigenetic repressive function of roX RNAs with PRC interaction. roX (RNA on the X) are crucial for male development in Drosophila. Here, the authors propose a role for roX RNAs in regulating gene expression at specific autosomal loci, working in coordination with Polycomb repressive complexes (PRCs).

Gao L., Li S., Chang H.S., Kim Y.J.

Fosseprez O., Cuvier O.

Over the past decade, regulatory non-coding RNAs (ncRNAs) produced by RNA Pol II have been revealed as meaningful players in various essential cellular functions. In particular, thousands of ncRNAs are produced at transcriptional regulatory elements such as enhancers and promoters, where they may exert multiple functions to regulate proper development, cellular programming, transcription or genomic stability. Here, we review the mechanisms involving these regulatory element-associated ncRNAs, and particularly enhancer RNAs (eRNAs) and PROMoter uPstream Transcripts (PROMPTs). We contextualize the mechanisms described to the processing and degradation of these short lived RNAs. We summarize recent findings explaining how ncRNAs operate locally at promoters and enhancers, or further away, either shortly after their production by RNA Pol II, or through post-transcriptional stabilization. Such discoveries lead to a converging model accounting for how ncRNAs influence cellular fate, by acting on transcription and chromatin structure, which may further involve factors participating to 3D nuclear organization.

Piscitelli S., Cascone E., D'Ambrosio C., Divisato G., De Lisio L., Leoni G., Matassa D.S., Lanzuolo C., Rosti V., Zizolfi M.C., Matuozzo M., di Patrizio Soldateschi E., Maiuri P., Scaloni A., Passaro F., et. al.

AbstractThe transition of embryonic stem cells (ESCs) from pluripotency to lineage commitment is regulated by multiple mechanisms, including chromatin dynamics and both transcriptional and post-transcriptional processes. Recent advances have highlighted that these mechanisms often interact, forming intricate multi-layered regulatory networks that require detailed characterization. In this study, we demonstrate that the RNA-binding protein LIN28A plays a pivotal role in neuronal differentiation by mediating RNA-dependent interactions with the Polycomb repressive complex 2 (PRC2). This interaction facilitates the eviction of PRC2 from chromatin, thereby activating a neuronal lineage-specific transcriptional program. Proteomic analyses revealed that the LIN28A interactome undergoes substantial remodeling during differentiation, corresponding to changes in LIN28A localization. In ESCs, LIN28A is predominantly nuclear and interacts with several components of the PRC2 complex in an RNA-dependent manner, assisting in chromatin dynamics. Our findings show that in the absence of LIN28A, PRC2 remains associated with chromatin, impairing the expression of genes critical for neuronal differentiation in ESCs. Chromatin immunoprecipitation sequencing (ChIP-seq) further confirmed that loss of LIN28A results in preferential PRC2 occupancy at the promoters of differentiation-associated genes. This study uncovers a novel role for LIN28A in epigenetic remodeling, which is essential for the proper differentiation of ESCs into the neuronal lineage.

Shu X., Kato M., Takizawa S., Suzuki Y., Carninci P.

Abstract Many RNAs associate with chromatin, either directly or indirectly. Several technologies for mapping regions where RNAs interact across the genome have been developed to investigate the function of these RNAs. Obtaining information on the proteins involved in these RNA–chromatin interactions is critical for further analysis. Here, we developed RADIP [RNA and DNA interacting complexes ligated and sequenced (RADICL-seq) with immunoprecipitation], a novel technology that combines RADICL-seq technology with chromatin immunoprecipitation to characterize RNA–chromatin interactions mediated by individual proteins. Building upon the foundational principles of RADICL-seq, RADIP extends its advantages by increasing genomic coverage and unique mapping rate efficiency compared to existing methods. To demonstrate its effectiveness, we applied an anti-H3K27me3 antibody to the RADIP technology and generated libraries from mouse embryonic stem cells (mESCs). We identified a multitude of RNAs, including RNAs from protein-coding genes and non-coding RNAs, that are associated with chromatin via H3K27me3 and that likely facilitate the spread of Polycomb repressive complexes over broad regions of the mammalian genome, thereby affecting gene expression, chromatin structures and pluripotency of mESCs. Our study demonstrates the applicability of RADIP to investigations of the functions of chromatin-associated RNAs.

Valyaeva A.A., Sheval E.V.

Eukaryotic cells are characterized by a high degree of compartmentalization of their internal contents, which ensures precise and controlled regulation of intracellular processes. During many processes, including different stages of transcription, dynamic membrane-free compartments called biomolecular condensates are formed. Transcription condensates contain various transcription factors and RNA polymerase and are formed by high and low specificity interactions between protein factors, DNA and nearby RNA. This review discusses recent data demonstrating the important role of nonspecific multivalent protein-protein and RNA-protein interactions in the organization and regulation of transcription.

Lu Y., Ruan X., Xiao G., Dai Y., Li G., Cai G., Zheng L., Guan Z., Sun W., Wang H.

ABSTRACTAimTo investigate the role of lncRNA Lockd in mandibular mesenchymal stem cell (M‐MSC) proliferation and osteogenic capability in the inflammatory microenvironment, focusing on its interaction with SUZ12.Materials and MethodsUsing lncR Lockd knockdown/overexpression cell models and a murine periodontitis model, we explored Lockd's effects on M‐MSC proliferation and osteogenic capability in the inflammatory microenvironment. Predictions from multiple databases and a series of rescue experiments revealed the regulatory role of the Lockd/SUZ12 signalling axis of M‐MSC in the inflammatory microenvironment.ResultsLockd was found to stimulate M‐MSC proliferation but impair osteogenic differentiation. The in vitro studies suggested that the activation of Lockd negatively inhibited the osteogenic differentiation process and may ultimately impact bone formation in periodontitis. Mechanistically, it was elucidated that Lockd interacts with SUZ12, a core component of the polycomb repressive complex 2 (PRC2), and may affect the PRC2 complex's role in osteogenic gene expression.ConclusionsLockd boosts the proliferation of M‐MSCs but inhibits their osteogenic differentiation by interacting with SUZ12, potentially inhibiting osteogenic capability in the inflammatory microenvironment.

Lee Y., Lee J.T.

Here, we expound on the view that Xist RNA directly controls Polycomb repressive complex 2 (PRC2) recruitment, off-loading to chromatin, catalytic activity, and eviction from chromatin. RNA-PRC2 interactions also control RNA polymerase II transcription pausing. Dynamic RNA folding determines PRC2 activity. Disparate studies and interpretations abound but can be reconciled.

Anastasakis D.G., Apostolidi M., Garman K.A., Polash A.H., Umar M.I., Meng Q., Scutenaire J., Jarvis J.E., Wang X., Haase A.D., Brownell I., Rinehart J., Hafner M.

Nuclear localization of the metabolic enzyme PKM2 is widely observed in various cancer types. We identify nuclear PKM2 as a non-canonical RNA-binding protein (RBP) that specifically interacts with folded RNA G-quadruplex (rG4) structures in precursor mRNAs (pre-mRNAs). PKM2 occupancy at rG4s prevents the binding of repressive RBPs, such as HNRNPF, and promotes the expression of rG4-containing pre-mRNAs (the "rG4ome"). We observe an upregulation of the rG4ome during epithelial-to-mesenchymal transition and a negative correlation of rG4 abundance with patient survival in different cancer types. By preventing the nuclear accumulation of PKM2, we could repress the rG4ome in triple-negative breast cancer cells and reduce migration and invasion of cancer cells in vitro and in xenograft mouse models. Our data suggest that the balance of folded and unfolded rG4s controlled by RBPs impacts gene expression during tumor progression.

Gallardo-Dodd C.J., Kutter C.

AbstractBiological systems encompass intricate networks governed by RNA-protein interactions that play pivotal roles in cellular functions. RNA and proteins constituting 1.1% and 18% of the mammalian cell weight, respectively, orchestrate vital processes from genome organization to translation. To date, disentangling the functional fraction of the human genome has presented a major challenge, particularly for noncoding regions, yet recent discoveries have started to unveil a host of regulatory functions for noncoding RNAs (ncRNAs). While ncRNAs exist at different sizes, structures, degrees of evolutionary conservation and abundances within the cell, they partake in diverse roles either alone or in combination. However, certain ncRNA subtypes, including those that have been described or remain to be discovered, are poorly characterized given their heterogeneous nature. RNA activity is in most cases coordinated through interactions with RNA-binding proteins (RBPs). Extensive efforts are being made to accurately reconstruct RNA-RBP regulatory networks, which have provided unprecedented insight into cellular physiology and human disease. In this review, we provide a comprehensive view of RNAs and RBPs, focusing on how their interactions generate functional signals in living cells, particularly in the context of post-transcriptional regulatory processes and cancer.

Lee Y., Blum R., Rosenberg M., Lee J.

SUMMARYUsing halo-tagged PRC2 and “CLAP” methodology, Guo et al. recently came to the conclusion that PRC2 is not an RNA binding protein (RBP). They suggested that previous findings are CLIP artifacts and argue that RNA cannot play a direct role in PRC2 regulation. Here, we perform a re-analysis of the authors’ raw datasets and come to contrary conclusions. First, CLAP demonstrates significant PRC2 enrichment throughout the transcriptome, including in XIST’s Repeat A (RepA) motif. Second, our re-analysis of the authors’ CLAP and CLIP datasets demonstrates that the two methods yield similar outcomes, with both showing PRC2 enrichment in the transcriptome. Furthermore, PRC2 demonstrates more RNA binding peaks than SAF-A and PTBP1. Additionally, re-analysis of CLAP contradicts the authors’ conclusion that CTCF and YY1 are not RBP. The discrepancies may be attributable to the authors’ unconventional data normalization, methods of determining significance, and lack of minus-tag and input controls in some experiments.