Polycomb group proteins first announced themselves in fruit flies, where researchers screening for mutants noticed animals with an extra sex comb, a structure normally restricted to the front legs of males. At least sixteen of these genes were eventually identified as repressors of the homeotic, or Hox, genes, the ancient genetic address system that tells every cell where it sits along the body axis. The discovery revealed a profound biological principle: development depends not only on turning genes on, but on keeping the wrong genes firmly off. That principle has been conserved across hundreds of millions of years of evolution, and in vertebrates Polycomb proteins now appear central to cellular differentiation, proliferation, tissue integrity, homeostasis and stem cell renewal.

The molecular machinery is elegantly modular. Polycomb proteins assemble into two major multi-protein complexes, Polycomb Repressive Complex 1 and Polycomb Repressive Complex 2, each built around a catalytic core plus a variable set of accessory subunits. The catalytic heart of PRC1 is a heterodimer of RING1A or RING1B with one of six PCGF family members, and it stamps histone H2A with a single ubiquitin molecule at lysine 119, a mark abbreviated H2AK119ub. PRC2, whose catalytic engine is the methyltransferase EZH1 or EZH2 working alongside EED, SUZ12 and RBBP4/7, deposits di- and trimethyl marks on lysine 27 of histone H3, known as H3K27me2 and H3K27me3. The two complexes are deeply interdependent. The H3K27me3 mark laid down by PRC2 is recognised by the chromobox subunit of canonical PRC1, which is thereby recruited to the same target sites to spread H2AK119ub and lock genes into a repressed state. Variant PRC1 can operate in the opposite direction, ubiquitinating H2A first and pulling PRC2 in behind it, creating a self-reinforcing loop of silencing.

Studying these complexes in mammals is brutally difficult because embryos lacking Polycomb function typically die around gastrulation, long before organs form. This is precisely where the zebrafish, Danio rerio, earns its reputation as one of developmental biology’s most powerful model organisms. Introduced as a genetic system by George Streisinger in the 1970s, zebrafish combine optical clarity, rapid development, sexual maturity within twelve weeks of fertilisation, ex utero embryonic growth that allows manipulation from the single-cell stage, and clutches of hundreds of offspring per mating. Crucially, maternal gene products sustain the embryo up to roughly the 1000-cell stage, far longer than in mice, giving researchers an extended window to probe early gene regulation. Zebrafish embryos also survive early development even with severe cardiovascular defects, because they obtain oxygen by passive diffusion rather than relying solely on blood circulation, something no mammal can do.

The fish genome adds another layer of opportunity. Around 320 million years ago, the teleost lineage underwent a whole-genome duplication event, generating pairs of duplicate genes called ohnologs, of which roughly 15 to 20 percent were retained after rediploidization. In the zebrafish genome, thirteen Polycomb subunits exist as duplicated pairs, although some, including RING1A, PCGF2, PCGF3, RBBP7 and the accessory subunit HDAC2, were lost during rediploidization. Despite these losses, the diversity and complexity of Polycomb complexes remain intact, and researchers can now dissect their roles using an arsenal of techniques ranging from classical ENU mutagenesis and morpholino knockdowns to zinc-finger nucleases, TALENs and CRISPR/Cas9 gene editing.

When it comes to the heart, the review paints a picture of two complexes with distinct but complementary timing. Embryos lacking either PRC1 or PRC2 develop the striking ‘heart-strings’ phenotype: after 48 hours post-fertilisation, the heart fails to loop and instead remains a string-like tube rather than forming its two chambers, the atrium and ventricle. Loss of rnf2, the sole zebrafish homolog of PRC1’s enzymatic subunit, produces pericardial edema and this stringy heart. Yet early cardiac markers such as nkx2.5, tbx5 and hoxb5b appear at normal levels in the lateral plate mesoderm of rnf2 mutants, indicating that the heart field is specified correctly and that PRC1 acts later in the programme. RNA sequencing of individual mutant hearts by Chrispijn and colleagues showed that T-box transcription factors tbx2a, tbx2b, tbx3a and tbx5 become upregulated over time, while structural genes such as vmhc, myl7, myh6 and nppa are downregulated, pointing to a role for PRC1 in the structural maturation of the organ.

The finer details are remarkable. In homozygous rnf2(f5) mutants, genes involved in smooth muscle and skeletal muscle development, which normally participate in transforming the atrioventricular canal between the two chambers, become dysregulated. Transmission electron microscopy reveals misaligned cardiac sarcomeres, with closely packed myofibrils and abnormally spaced I-bands and Z-discs. Calcium imaging with the fluorescent dye fluo-4 AM shows a weakened calcium signal, and the result is a heart that contracts feebly. The expression of atrioventricular canal markers such as alcama, vcana and bmp4 becomes expanded and diffuse rather than tightly constricted. Together these data establish that PRC1 is essential for cardiac sarcomere assembly, the maintenance of contraction, and the proper constriction of the atrioventricular canal and its valves, largely by repressing inappropriate non-cardiac sarcomere genes. A second non-canonical PRC1 component, mga, adds another twist: morpholino knockdown of mgaa blocks heart tube looping, and the defect can be rescued by co-reducing gata4, whose transcript levels rise two- to four-fold in the mutant, showing that variant PRC1 fine-tunes this key regulator.

PRC2, by contrast, acts earlier. Maternal-zygotic ezh2 mutants, in which the methyltransferase is eliminated from both maternal and zygotic sources, show reduced numbers of nkx2.5-expressing cardiac progenitors, partially reduced chamber markers, and cardiomyocytes that wander away from the cardiac tube, leaving a small, tubular heart that never loops. The atrioventricular canal fails to form properly, possibly because of ectopic expression of has2, a gene that promotes cardiac cell migration. Chromatin immunoprecipitation sequencing shows that both ezh2 and the H3K27me3 mark are absent from heart-development genes such as tbx3a, tbx5 and isl1, and PRC1 peaks are missing from the same loci, consistent with PRC2 recruiting PRC1 to its targets. Intriguingly, ezh1 mutants show no heart defects despite a modest reduction in H3K27me3, and ezh1;ezh2 double mutants resemble ezh2 single mutants, demonstrating that ezh1 cannot compensate for ezh2 in the heart. Pharmacological inhibitors of EZH2, including GSK126, DZNep and PF-06726304 acetate, all induce heart edema, reinforcing the complex’s central role.

The pectoral fin story is equally compelling, and arguably more evolutionarily provocative. Zebrafish pectoral fins are the evolutionary relatives of tetrapod forelimbs, and although a fin and an arm look nothing alike, the underlying regulatory logic is deeply conserved. Retinoic acid, synthesised by the enzyme aldh1a2, initiates the programme by activating wnt2b and tbx5 in the lateral plate mesoderm; tbx5 then triggers the Fgf cascade that drives outgrowth, while shh, hox genes and the apical ectodermal ridge pattern the bud along its anterior-posterior, dorsal-ventral and proximal-distal axes. When PRC1 or PRC2 function is lost, pectoral fins simply fail to form. In rnf2 mutants, early fin markers such as tbx5 and hand2 appear on schedule but vanish from the fin mesenchyme by 40 hours post-fertilisation, the Fgf targets fgf24 and fgf10 shrink to a small patch, and apical ectodermal ridge markers such as dlx2a, fgf8 and versican are absent, so fin initiation occurs but outgrowth collapses.

The epigenetic mechanism behind the missing fins involves a runaway retinoic acid signal. In rnf2 mutants, aldh1a2 is overexpressed and spreads beyond its normal posterior domain, while cyp26a1, which degrades retinoic acid, is reduced, producing excess retinoic acid that the embryo tries to metabolise by upregulating genes such as dhrs3. Hox genes normally restricted to the anterior body, including hoxc6a and hoxc8a, are ectopically expressed in the posterior and throughout the brain, and fin-specific hox expression of hoxa9b, hoxc8a and hoxd9a is lost while their axial domains expand. Patterning markers such as shh, msxc, eng1a and wnt7a are absent or reduced. Even partially suppressing retinoic acid with DEAB restores tbx5 and hand2 only partially, insufficient to restart fin outgrowth. In maternal-zygotic ezh2 mutants the same picture emerges: tbx5 is entirely missing, the hoxab cluster expands beyond its normal domain, and shh is absent from the fin bud. Because PcG-deficient zebrafish survive gastrulation where mice do not, this model opens a unique window onto how epigenetic silencing sculpts organs, and the authors argue that understanding these molecular pathways could ultimately illuminate congenital heart defects and limb abnormalities in humans, and point toward new therapeutic targets in regenerative medicine.

Subject of Research: Polycomb group protein-mediated epigenetic regulation of heart and pectoral fin development in zebrafish

Article Title: Epigenetic regulation in zebrafish development: the roles of polycomb group proteins in heart and pectoral fin development

Article References: Kavade, P. S., Parab, S. S., Capone, V., Carannante, D., Ambrosino, C., Altucci, L., & Carafa, V. (2024). Epigenetic regulation in zebrafish development: the roles of polycomb group proteins in heart and pectoral fin development. Epigenetics Communications, 4(1), Article 7. https://doi.org/10.1186/s43682-024-00030-y

Image Credits: AI Generated

DOI: 10.1186/s43682-024-00030-y

Keywords: Polycomb group proteins, PRC1, PRC2, zebrafish, epigenetics, heart development, pectoral fin development, Hox genes, histone modification, retinoic acid signaling, EZH2, developmental biology

The review centers on CBX7, a member of the Chromobox protein family and a core subunit of Polycomb Repressive Complex 1, one of the two major Polycomb complexes that shape the epigenetic landscape of mammalian cells. Epigenetics, a field now seventy years old, concerns heritable changes in gene expression that occur without altering the underlying DNA sequence, chiefly through DNA methylation and histone modifications. Polycomb group proteins sit at the heart of this regulatory architecture, controlling cell fate decisions by modulating the expression of genes responsible for development, differentiation and, when things go wrong, cancer. Genome-wide studies have established that Polycomb Repressive Complex 1 and Polycomb Repressive Complex 2 function as crucial transcriptional regulators targeting a wide range of genes involved in tumor development, and the chromodomain-containing CBX proteins are the molecular anchors that tether Polycomb activity to specific chromatin regions.

Structurally, CBX7 is a study in molecular precision. Its chromodomain, spanning amino acids 9 to 48, descends from a conserved 37-amino acid sequence first identified in the Polycomb and heterochromatin protein 1 proteins of the fruit fly Drosophila melanogaster. Phylogenetic analyses show that the CBX7 chromodomain is more closely related to other Polycomb group proteins such as CBX2, CBX4, CBX6 and CBX8 than to the HP1 family members CBX1, CBX3 and CBX5, and unlike HP1 proteins, CBX7 lacks a chromoshadow domain. Instead, its chromodomain carries unique conserved residues exclusive to Polycomb proteins that are necessary for Polycomb dimer formation and for recognizing the tri-methylated lysine 27 mark on histone H3, known as H3K27me3. Thanks to the stable geometry of this domain, CBX7 preferentially binds H3K27me3, a mark laid down by the PRC2 complex through its core subunits EZH2, SUZ12 and EED. Once anchored, CBX7 recruits the canonical PRC1 complex, whose catalytic core ubiquitinates lysine 119 on histone H2A, compacting chromatin and silencing target genes. In this way, CBX7 acts as a functional bridge between PRC2 activity and stable transcriptional repression.

The protein also exists in two isoforms with strikingly divergent behavior. The 36-kilodalton nuclear form, p36CBX7, is expressed in proliferating cells and drives gene regulation, while the 22-kilodalton cytoplasmic form, p22CBX7, is induced by serum deprivation and restrains cell proliferation. This context- and tissue-specific duality foreshadows the protein’s complex role in cancer. Across human tumors, CBX7 behaves as either an oncogene or a tumor suppressor depending on the dominant signaling pathways and the genetic and epigenetic makeup of each tumor type. In lymphomas and gastric cancer, overexpressed CBX7 represses tumor-suppressive genes such as Ink4a/Arf and Trail, allowing malignant cells to evade senescence and apoptosis. In contrast, cancers of the breast, pancreas, lung, thyroid, colon, bladder and brain, including glioblastoma, consistently show reduced CBX7 expression compared with normal tissue, pointing to a tumor-suppressive function in these settings.

In glioblastoma specifically, the evidence for CBX7 as a tumor suppressor is compelling. High-grade gliomas exhibit significantly lower CBX7 levels than low-grade gliomas, and this downregulation correlates with aggressive disease and poor prognosis. When lost CBX7 is restored in glioma cells, tumor growth is inhibited and the cell cycle arrests at the G0/G1 phase. One well-characterized mechanism involves competition with the architectural transcription factor HMGA1 for control of the CCNE1 promoter, which encodes cyclin E1, a key driver of the G1/S transition. Under normal conditions, CBX7 partners with histone deacetylase 2 to form a repressive complex on the CCNE1 promoter. When CBX7 is lost, HMGA1 evicts this complex, cyclin E1 expression surges, and glioblastoma cells proliferate uncontrollably. HMGA1 also directly binds the CBX7 promoter itself, adding a further layer of antagonism.

The tumor-suppressive reach of CBX7 extends well beyond cell-cycle control into the machinery of invasion and stemness. CBX7 directly binds the promoter of CDH1, the gene encoding the adhesion molecule E-cadherin, where it restrains HDAC2 activity and promotes activating acetylation and methylation marks on histones H3 and H4, stabilizing the epithelial phenotype. Loss of CBX7 silences CDH1, promotes epithelial-to-mesenchymal transition and drives the invasiveness that typifies glioblastoma progression. CBX7 also prevents HMGA1 from transcriptionally activating SPP1, the gene encoding osteopontin, another potent EMT stimulator. Perhaps most strikingly, recent work shows that CBX7 engages PRC1 to facilitate ubiquitin-proteasome degradation of myosin heavy chain 9, thereby suppressing NF-kappaB signaling and stripping glioblastoma cells of their stem-like characteristics. Restoring MYH9 reverses this effect, re-establishing stemness, proliferation and invasion, and defining a CBX7-MYH9-NF-kappaB regulatory axis that researchers regard as a desirable therapeutic target.

Two further oncogenic pathways fall under CBX7’s control. The Wnt/beta-catenin pathway, aberrantly activated in many glioblastomas, is dampened when CBX7 binds the promoter of DKK1, a Wnt inhibitor, upregulating its expression and simultaneously reducing ZEB1, a transcription factor that promotes invasion, metastasis and epithelial-to-mesenchymal transition. Restoring CBX7 in glioblastoma cells suppresses both Wnt signaling and ZEB1, curtailing tumor growth and metastatic potential. CBX7 likewise modulates the Hippo pathway through connective tissue growth factor, encoded by CTGF, a key component of the YAP/TAZ signaling axis. When CBX7 is lost, PRC1-mediated gene suppression fails, YAP/TAZ signaling becomes hyperactive, TEAD-dependent transcription rises and CTGF is overexpressed, activating kinases such as SAPK/JNK and fueling glioma cell invasion and migration. Ectopic CBX7 expression additionally reduces the matrix metalloproteinases MMP2 and MMP9, major markers of glioblastoma invasiveness, angiogenesis and immune modulation.

How does such a critical tumor suppressor get silenced in the first place? The review details two principal mechanisms. The first is epigenetic: hypermethylation of the CBX7 promoter, confirmed by bisulfite sequencing in glioblastoma tissues, glioma-derived cell lines and normal brain, with clear enrichment of methylation in tumor samples. Knockdown studies identified the DNA methyltransferases DNMT1 and DNMT3A, but not DNMT3B, as the enzymes responsible for this silencing, and treatment with a methylation inhibitor restored CBX7 mRNA expression in glioma cell lines. The second mechanism is post-transcriptional, mediated by microRNAs. The MYC-responsive microRNA miR-9 lowers CBX7 protein levels, while CBX7 in turn binds the miR-9 promoter and represses its expression, creating an autoregulatory loop that also feeds into the control of p16-INK4a, a key effector of replicative senescence. Separately, the oncogenic miR-18a, highly expressed in glioblastoma, directly targets CBX7 mRNA, and silencing miR-18a in animal models markedly slows tumor growth and prolongs survival.

The translational implications are substantial, though tempered by caution. No CBX7-targeted therapy is yet in clinical use for glioblastoma, and the review’s authors stress that the protein’s role in normal cells must be fully understood before therapeutic modulation is attempted. Nevertheless, pharmacological proof of concept already exists in other malignancies: chromodomain-targeting inhibitors such as UNC3866, UNC4976, MS452, EC-134 and BDA-41 have dislodged overexpressed CBX7 from chromatin in lymphoid leukemia models, inducing differentiation and growth arrest. For glioblastoma, the therapeutic logic runs in the opposite direction, toward restoring rather than inhibiting CBX7 function, using epigenetic drugs, miRNA-based approaches or targeted modulation of CBX7-associated complexes and downstream pathways. Beyond therapy, CBX7’s consistent downregulation and correlation with tumor aggressiveness make it a promising prognostic biomarker, measurable through immunohistochemistry, methylation profiling or miRNA expression analysis to classify patients and guide treatment decisions. As the authors conclude, CBX7 represents a particularly promising starting point for developing more accurate and potent targeted treatments, and for deepening our understanding of glioblastoma biology in a disease that urgently needs both.

Subject of Research: The role of the Polycomb protein CBX7 as an epigenetic tumor suppressor in glioblastoma and its therapeutic potential.

Article Title: Epigenetic regulation by CBX7 in glioblastoma: molecular mechanisms and translational perspectives

Article References: Fayyaz, F., Favale, G., Capasso, L., Casalino, R., Mele, D., Verrilli, G., Conte, M., Carafa, V., Nebbioso, A., & Altucci, L. (2025). Epigenetic regulation by CBX7 in glioblastoma: molecular mechanisms and translational perspectives. Epigenetics Communications, 6(1), Article 2. https://doi.org/10.1186/s43682-025-00043-1

Image Credits: AI Generated

DOI: 10.1186/s43682-025-00043-1

Keywords: CBX7, glioblastoma, epigenetics, Polycomb, PRC1, PRC2, chromodomain, DNA methylation, microRNA, Wnt signaling, Hippo pathway, tumor suppressor