SART3 reads methylarginine-marked glycine- and arginine-rich motifs.

Glycine- and arginine-rich (GAR) motifs, commonly found in RNA-binding and -processing proteins, can be symmetrically (SDMA) or asymmetrically (ADMA) dimethylated at the arginine residue by protein arginine methyltransferases. Arginine-methylated protein motifs are usually read by Tudor domain-containing proteins. Here, using a GFP-Trap, we identify a non-Tudor domain protein, squamous cell carcinoma antigen recognized by T cells 3 (SART3), as a reader for SDMA-marked GAR motifs. Structural analysis and mutagenesis of SART3 show that aromatic residues lining a groove between two adjacent aromatic-rich half-a-tetratricopeptide (HAT) repeat domains are essential for SART3 to recognize and bind to SDMA-marked GAR motif peptides, as well as for the interaction between SART3 and the GAR-motif-containing proteins fibrillarin and coilin. Further, we show that the loss of this reader ability affects RNA splicing. Overall, our findings broaden the range of potential SDMA readers to include HAT domains.

Targeting of CYP2E1 by miRNAs in alcohol-induced intestine injury.

Although binge alcohol-induced gut leakage has been studied extensively in the context of reactive oxygen species-mediated signaling, it was recently revealed that post-transcriptional regulation plays an essential role as well. Ethanol (EtOH)-inducible cytochrome P450-2E1 (CYP2E1), a key enzyme in EtOH metabolism, promotes alcohol-induced hepatic steatosis and inflammatory liver disease, at least in part by mediating changes in intestinal permeability. For instance, gut leakage and elevated intestinal permeability to endotoxins have been shown to be regulated by enhancing CYP2E1 mRNA and CYP2E1 protein levels. Although it is understood that EtOH promotes CYP2E1 induction and activation, the mechanisms that regulate CYP2E1 expression in the context of intestinal damage remain poorly defined. Specific miRNAs, including miR-132, miR-212, miR-378, and miR-552, have been shown to repress the expression of CYP2E1, suggesting that these miRNAs contribute to EtOH-induced intestinal injury. Here, we have shown that CYP2E1 expression is regulated post-transcriptionally through miRNA-mediated degradation, as follows: (1) the RNA-binding protein AU-binding factor 1 (AUF1) binds mature miRNAs, including CYP2E1-targeting miRNAs, and this binding modulates the degradation of corresponding target mRNAs upon EtOH treatment; (2) the serine/threonine kinase mammalian Ste20-like kinase 1 (MST1) mediates oxidative stress-induced phosphorylation of AUF1. Those findings suggest that reactive oxygen species-mediated signaling modulates AUF1/miRNA interaction through MST1-mediated phosphorylation. Thus, our study demonstrates the critical functions of AUF1 phosphorylation by MST1 in the decay of miRNAs targeting CYP2E1, the stabilization of CYP2E1 mRNA in the presence of EtOH, and the relationship of this pathway to subsequent intestinal injury.

The intrinsic substrate specificity of the human tyrosine kinome.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

Genome-wide screening identifies Trim33 as an essential regulator of dendritic cell differentiation.

The development of dendritic cells (DCs), including antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs), is controlled by the growth factor Flt3 ligand (Flt3L) and its receptor Flt3. We genetically dissected Flt3L-driven DC differentiation using CRISPR-Cas9-based screening. Genome-wide screening identified multiple regulators of DC differentiation including subunits of TSC and GATOR1 complexes, which restricted progenitor growth but enabled DC differentiation by inhibiting mTOR signaling. An orthogonal screen identified the transcriptional repressor Trim33 (TIF-1γ) as a regulator of DC differentiation. Conditional targeting in vivo revealed an essential role of Trim33 in the development of all DCs, but not of monocytes or granulocytes. In particular, deletion of Trim33 caused rapid loss of DC progenitors, pDCs, and the cross-presenting cDC1 subset. Trim33-deficient Flt3+ progenitors up-regulated pro-inflammatory and macrophage-specific genes but failed to induce the DC differentiation program. Collectively, these data elucidate mechanisms that control Flt3L-driven differentiation of the entire DC lineage and identify Trim33 as its essential regulator.

Functional characterization of QT interval associated SCN5A enhancer variants identify combined additive effects.

Several empirical and theoretical studies suggest presence of multiple enhancers per gene that collectively regulate gene expression, and that common sequence variation impacting on the activities of these enhancers is a major source of inter-individual variability in gene expression. However, for vast majority of genes, enhancers and the underlying regulatory variation remains unknown. Even for the genes with well-characterized enhancers, the nature of the combined effects from multiple enhancers and their variants, when known, on gene expression regulation remains unexplored. Here, we have evaluated the combined effects from five SCN5A enhancers and their regulatory variants that are known to collectively correlate with SCN5A cardiac expression and underlie QT interval association in the general population. Using small deletions centered at the regulatory variants in episomal reporter assays in a mouse cardiomyocyte cell line we demonstrate that the variants and their flanking sequences play critical role in individual enhancer activities, likely being a transcription factor (TF) binding site. By performing oligonucleotide-based pulldown assays on predicted TFs we identify the TFs likely driving allele-specific enhancer activities. Using all 32 possible allelic synthetic constructs in reporter assays, representing the five biallelic enhancers in tandem in their genomic order, we demonstrate combined additive effects on overall enhancer activities. Using transient enhancer assays in developing zebrafish embryos we demonstrate the four out the five enhancer elements act as enhancers in vivo . Together, these studies extend the previous findings to uncover the TFs driving the enhancer activities of QT interval associated SCN5A regulatory variants, reveal the additive effects from allelic combinations of these regulatory variants, and prove their potential to act as enhancers in vivo .

Molecular Basis for SPINDOC-Spindlin1 Engagement and Its Role in Transcriptional Attenuation.

Spindlin1 is a histone reader with three Tudor-like domains and its transcriptional co-activator activity could be attenuated by SPINDOC. The first two Tudors are involved in histone methylation readout, while the function of Tudor 3 is largely unknown. Here our structural and binding studies revealed an engagement mode of SPINDOC-Spindlin1, in which a hydrophobic motif of SPINDOC, DOCpep3, stably interacts with Spindlin1 Tudor 3, and two neighboring K/R-rich motifs, DOCpep1 and DOCpep2, bind to the acidic surface of Spindlin1 Tudor 2. Although DOCpep3-Spindlin1 engagement is compatible with histone readout, an extended SPINDOC fragment containing the K/R-rich region attenuates histone or TCF4 binding by Spindlin1 due to introduced competition. This inhibitory effect is more pronounced for weaker binding targets but not for strong ones such as H3 "K4me3-K9me3" bivalent mark. Further ChIP-seq and RT-qPCR indicated that SPINDOC could promote genomic relocation of Spindlin1, thus modulate downstream gene transcription. Collectively, we revealed multivalent engagement between SPINDOC and Spindlin1, in which a hydrophobic motif acts as the primary binding site for stable SPINDOC-Spindlin1 association, while K/R-rich region modulates the target selectivity of Spindlin1 via competitive inhibition, therefore attenuating the transcriptional co-activator activity of Spindlin1.

PRMT blockade induces defective DNA replication stress response and synergizes with PARP inhibition.

Multiple cancers exhibit aberrant protein arginine methylation by both type I arginine methyltransferases, predominately protein arginine methyltransferase 1 (PRMT1) and to a lesser extent PRMT4, and by type II PRMTs, predominately PRMT5. Here, we perform targeted proteomics following inhibition of PRMT1, PRMT4, and PRMT5 across 12 cancer cell lines. We find that inhibition of type I and II PRMTs suppresses phosphorylated and total ATR in cancer cells. Loss of ATR from PRMT inhibition results in defective DNA replication stress response activation, including from PARP inhibitors. Inhibition of type I and II PRMTs is synergistic with PARP inhibition regardless of homologous recombination function, but type I PRMT inhibition is more toxic to non-malignant cells. Finally, we demonstrate that the combination of PARP and PRMT5 inhibition improves survival in both BRCA-mutant and wild-type patient-derived xenografts without toxicity. Taken together, these results demonstrate that PRMT5 inhibition may be a well-tolerated approach to sensitize tumors to PARP inhibition.

Loss of the methylarginine reader function of SND1 confers resistance to hepatocellular carcinoma.

Staphylococcal nuclease Tudor domain containing 1 (SND1) protein is an oncogene that 'reads' methylarginine marks through its Tudor domain. Specifically, it recognizes methylation marks deposited by protein arginine methyltransferase 5 (PRMT5), which is also known to promote tumorigenesis. Although SND1 can drive hepatocellular carcinoma (HCC), it is unclear whether the SND1 Tudor domain is needed to promote HCC. We sought to identify the biological role of the SND1 Tudor domain in normal and tumorigenic settings by developing two genetically engineered SND1 mouse models, an Snd1 knockout (Snd1 KO) and an Snd1 Tudor domain-mutated (Snd1 KI) mouse, whose mutant SND1 can no longer recognize PRMT5-catalyzed methylarginine marks. Quantitative PCR analysis of normal, KO, and KI liver samples revealed a role for the SND1 Tudor domain in regulating the expression of genes encoding major acute phase proteins, which could provide mechanistic insight into SND1 function in a tumor setting. Prior studies indicated that ectopic overexpression of SND1 in the mouse liver dramatically accelerates the development of diethylnitrosamine (DEN)-induced HCC. Thus, we tested the combined effects of DEN and SND1 loss or mutation on the development of HCC. We found that both Snd1 KO and Snd1 KI mice were partially protected against malignant tumor development following exposure to DEN. These results support the development of small molecule inhibitors that target the SND1 Tudor domain or the use of upstream PRMT5 inhibitors, as novel treatments for HCC.

CARM1 arginine methyltransferase as a therapeutic target for cancer.

Coactivator-associated arginine methyltransferase 1 (CARM1) is an arginine methyltransferase that posttranslationally modifies proteins that regulate multiple levels of RNA production and processing. Its substrates include histones, transcription factors, coregulators of transcription, and splicing factors. CARM1 is overexpressed in many different cancer types, and often promotes transcription factor programs that are co-opted as drivers of the transformed cell state, a process known as transcription factor addiction. Targeting these oncogenic transcription factor pathways is difficult but could be addressed by removing the activity of the key coactivators on which they rely. CARM1 is ubiquitously expressed, and its KO is less detrimental in embryonic development than deletion of the arginine methyltransferases protein arginine methyltransferase 1 and protein arginine methyltransferase 5, suggesting that therapeutic targeting of CARM1 may be well tolerated. Here, we will summarize the normal in vivo functions of CARM1 that have been gleaned from mouse studies, expand on the transcriptional pathways that are regulated by CARM1, and finally highlight recent studies that have identified oncogenic properties of CARM1 in different biological settings. This review is meant to kindle an interest in the development of human drug therapies targeting CARM1, as there are currently no CARM1 inhibitors available for use in clinical trials.

Effectors and effects of arginine methylation.

Arginine methylation is a ubiquitous and relatively stable post-translational modification (PTM) that occurs in three types: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Methylarginine marks are catalyzed by members of the protein arginine methyltransferases (PRMTs) family of enzymes. Substrates for arginine methylation are found in most cellular compartments, with RNA-binding proteins forming the majority of PRMT targets. Arginine methylation often occurs in intrinsically disordered regions of proteins, which impacts biological processes like protein-protein interactions and phase separation, to modulate gene transcription, mRNA splicing and signal transduction. With regards to protein-protein interactions, the major 'readers' of methylarginine marks are Tudor domain-containing proteins, although additional domain types and unique protein folds have also recently been identified as methylarginine readers. Here, we will assess the current 'state-of-the-art' in the arginine methylation reader field. We will focus on the biological functions of the Tudor domain-containing methylarginine readers and address other domains and complexes that sense methylarginine marks.

CDK5-PRMT1-WDR24 signaling cascade promotes mTORC1 signaling and tumor growth.

The mammalian target of rapamycin complex1 (mTORC1) is a central regulator of metabolism and cell growth by sensing diverse environmental signals, including amino acids. The GATOR2 complex is a key component linking amino acid signals to mTORC1. Here, we identify protein arginine methyltransferase 1 (PRMT1) as a critical regulator of GATOR2. In response to amino acids, cyclin-dependent kinase 5 (CDK5) phosphorylates PRMT1 at S307 to promote PRMT1 translocation from nucleus to cytoplasm and lysosome, which in turn methylates WDR24, an essential component of GATOR2, to activate the mTORC1 pathway. Disruption of the CDK5-PRMT1-WDR24 axis suppresses hepatocellular carcinoma (HCC) cell proliferation and xenograft tumor growth. High PRMT1 protein expression is associated with elevated mTORC1 signaling in patients with HCC. Thus, our study dissects a phosphorylation- and arginine methylation-dependent regulatory mechanism of mTORC1 activation and tumor growth and provides a molecular basis to target this pathway for cancer therapy.

A complete methyl-lysine binding aromatic cage constructed by two domains of PHF2.

The N-terminal half of PHF2 harbors both a plant homeodomain (PHD) and a Jumonji domain. The PHD recognizes both histone H3 trimethylated at lysine 4 and methylated nonhistone proteins including vaccinia-related kinase 1 (VRK1). The Jumonji domain erases the repressive dimethylation mark from histone H3 lysine 9 (H3K9me2) at select promoters. The N-terminal amino acid sequences of H3 (AR2TK4) and VRK1 (PR2VK4) bear an arginine at position 2 and lysine at position 4. Here, we show that the PHF2 N-terminal half binds to H3 and VRK1 peptides containing K4me3, with dissociation constants (KD values) of 160 nM and 42 nM, respectively, which are 4 × and 21 × lower (and higher affinities) than for the isolated PHD domain of PHF2. X-ray crystallography revealed that the K4me3-containing peptide is positioned within the PHD and Jumonji interface, with the positively charged R2 residue engaging acidic residues of the PHD and Jumonji domains and with the K4me3 moiety encircled by aromatic residues from both domains. We suggest that the micromolar binding affinities commonly observed for isolated methyl-lysine reader domains could be improved via additional functional interactions within the same polypeptide or its binding partners.

The NFIB/CARM1 partnership is a driver in preclinical models of small cell lung cancer.

The coactivator associated arginine methyltransferase (CARM1) promotes transcription, as its name implies. It does so by modifying histones and chromatin bound proteins. We identified nuclear factor I B (NFIB) as a CARM1 substrate and show that this transcription factor utilizes CARM1 as a coactivator. Biochemical studies reveal that tripartite motif 29 (TRIM29) is an effector molecule for methylated NFIB. Importantly, NFIB harbors both oncogenic and metastatic activities, and is often overexpressed in small cell lung cancer (SCLC). Here, we explore the possibility that CARM1 methylation of NFIB is important for its transforming activity. Using a SCLC mouse model, we show that both CARM1 and the CARM1 methylation site on NFIB are critical for the rapid onset of SCLC. Furthermore, CARM1 and methylated NFIB are responsible for maintaining similar open chromatin states in tumors. Together, these findings suggest that CARM1 might be a therapeutic target for SCLC.

A small molecule antagonist of SMN disrupts the interaction between SMN and RNAP II.

Survival of motor neuron (SMN) functions in diverse biological pathways via recognition of symmetric dimethylarginine (Rme2s) on proteins by its Tudor domain, and deficiency of SMN leads to spinal muscular atrophy. Here we report a potent and selective antagonist with a 4-iminopyridine scaffold targeting the Tudor domain of SMN. Our structural and mutagenesis studies indicate that both the aromatic ring and imino groups of compound 1 contribute to its selective binding to SMN. Various on-target engagement assays support that compound 1 specifically recognizes SMN in a cellular context and prevents the interaction of SMN with the R1810me2s of RNA polymerase II subunit POLR2A, resulting in transcription termination and R-loop accumulation mimicking SMN depletion. Thus, in addition to the antisense, RNAi and CRISPR/Cas9 techniques, potent SMN antagonists could be used as an efficient tool to understand the biological functions of SMN.

TWIST1 methylation by SETD6 selectively antagonizes LINC-PINT expression in glioma.

Gliomas are one of the most common and lethal brain tumors among adults. One process that contributes to glioma progression and recurrence is the epithelial to mesenchymal transition (EMT). EMT is regulated by a set of defined transcription factors which tightly regulate this process, among them is the basic helix-loop-helix family member, TWIST1. Here we show that TWIST1 is methylated on lysine-33 at chromatin by SETD6, a methyltransferase with expression levels correlating with poor survival in glioma patients. RNA-seq analysis in U251 glioma cells suggested that both SETD6 and TWIST1 regulate cell adhesion and migration processes. We further show that TWIST1 methylation attenuates the expression of the long-non-coding RNA, LINC-PINT, thereby promoting EMT in glioma. Mechanistically, TWIST1 methylation represses the transcription of LINC-PINT by increasing the occupancy of EZH2 and the catalysis of the repressive H3K27me3 mark at the LINC-PINT locus. Under un-methylated conditions, TWIST1 dissociates from the LINC-PINT locus, allowing the expression of LINC-PINT which leads to increased cell adhesion and decreased cell migration. Together, our findings unravel a new mechanistic dimension for selective expression of LINC-PINT mediated by TWIST1 methylation.

Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction-Reconstruction and Fragment-Growing Approach.

Protein arginine methyltransferases (PRMTs) are important therapeutic targets, playing a crucial role in the regulation of many cellular processes and being linked to many diseases. Yet, there is still much to be understood regarding their functions and the biological pathways in which they are involved, as well as on the structural requirements that could drive the development of selective modulators of PRMT activity. Here we report a deconstruction-reconstruction approach that, starting from a series of type I PRMT inhibitors previously identified by us, allowed for the identification of potent and selective inhibitors of PRMT4, which regardless of the low cell permeability show an evident reduction of arginine methylation levels in MCF7 cells and a marked reduction of proliferation. We also report crystal structures with various PRMTs supporting the observed specificity and selectivity.

Inhibiting Type I Arginine Methyltransferase Activity Promotes T Cell-Mediated Antitumor Immune Responses.

Protein arginine methyltransferases (PRMT) are a widely expressed class of enzymes responsible for catalyzing arginine methylation on numerous protein substrates. Among them, type I PRMTs are responsible for generating asymmetric dimethylarginine. By controlling multiple basic cellular processes, such as DNA damage responses, transcriptional regulation, and mRNA splicing, type I PRMTs contribute to cancer initiation and progression. A type I PRMT inhibitor, GSK3368715, has been developed and has entered clinical trials for solid and hematologic malignancies. Although type I PRMTs have been reported to play roles in modulating immune cell function, the immunologic role of tumor-intrinsic pathways controlled by type I PRMTs remains uncharacterized. Here, our The Cancer Genome Atlas dataset analysis revealed that expression of type I PRMTs associated with poor clinical response and decreased immune infiltration in patients with melanoma. In cancer cell lines, inhibition of type I PRMTs induced an IFN gene signature, amplified responses to IFN and innate immune signaling, and decreased expression of the immunosuppressive cytokine VEGF. In immunocompetent mouse tumor models, including a model of T-cell exclusion that represents a common mechanism of anti-programmed cell death protein 1 (PD-1) resistance in humans, type I PRMT inhibition increased T-cell infiltration, produced durable responses dependent on CD8+ T cells, and enhanced efficacy of anti-PD-1 therapy. These data indicate that type I PRMT inhibition exhibits immunomodulatory properties and synergizes with immune checkpoint blockade (ICB) to induce durable antitumor responses in a T cell-dependent manner, suggesting that type I PRMT inhibition can potentiate an antitumor immunity in refractory settings.

Methyl-lysine readers PHF20 and PHF20L1 define two distinct gene expression-regulating NSL complexes.

The methyl-lysine readers plant homeodomain finger protein 20 (PHF20) and its homolog PHF20-like protein 1 (PHF20L1) are known components of the nonspecific lethal (NSL) complex that regulates gene expression through its histone acetyltransferase activity. In the current model, both PHF homologs coexist in the same NSL complex, although this was not formally tested; nor have the functions of PHF20 and PHF20L1 regarding NSL complex integrity and transcriptional regulation been investigated. Here, we perform an in-depth biochemical and functional characterization of PHF20 and PHF20L1 in the context of the NSL complex. Using mass spectrometry, genome-wide chromatin analysis, and protein-domain mapping, we identify the existence of two distinct NSL complexes that exclusively contain either PHF20 or PHF20L1. We show that the C-terminal domains of PHF20 and PHF20L1 are essential for complex formation with NSL, and the Tudor 2 domains are required for chromatin binding. The genome-wide chromatin landscape of PHF20-PHF20L1 shows that these proteins bind mostly to the same genomic regions, at promoters of highly expressed/housekeeping genes. Yet, deletion of PHF20 and PHF20L1 does not abrogate gene expression or impact the recruitment of the NSL complex to those target gene promoters, suggesting the existence of an alternative mechanism that compensates for the transcription of genes whose sustained expression is important for critical cellular functions. This work shifts the current paradigm and lays the foundation for studies on the differential roles of PHF20 and PHF20L1 in regulating NSL complex activity in physiological and diseases states.

Pan-methylarginine antibody generation using PEG linked GAR motifs as antigens.

Arginine methylation is a prevalent posttranslational modification which is deposited by a family of protein arginine methyltransferases (PRMTs), and is found in three different forms in mammalian cells: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Pan-methylarginine antibodies are critical for identifying proteins that are methylated on arginine residues, and are also used for evaluating signaling pathways that modulate this methyltransferase activity. Although good pan-MMA, -ADMA and -SDMA antibodies have been developed over the years, there is still room for improvement. Here we use a novel antigen approach, which involves the separation of short methylated motifs with inert polyethylene glycol (PEG) linkers, to generate a set of pan antibodies to the full range of methylarginine marks. Using these antibodies, we observed substrate scavenging by PRMT1, when PRMT5 activity is blocked. Specifically, we find that the splicing factor SmD1 displays increased ADMA levels upon PRMT5 inhibitor treatment. Furthermore, when the catalysis of both SDMA and ADMA is blocked with small molecule inhibitors, we demonstrate that SmD1 and SMN no longer interact. This could partially explain the synergistic effect of PRMT5 and type I PRMT inhibition on RNA splicing and cancer cell growth.

Reprogramming CBX8-PRC1 function with a positive allosteric modulator.

Canonical targeting of Polycomb repressive complex 1 (PRC1) to repress developmental genes is mediated by cell-type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7, and 8). Based on their central role in silencing and their dysregulation associated with human disease including cancer, CBX proteins are attractive targets for small-molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids. We show that treatment with UNC7040 leads to efficient and selective eviction of CBX8-containing PRC1 from chromatin, loss of silencing, and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only reveals the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of Polycomb regulation by non-specific CBX nucleotide binding activity.