Polycomb Group Complexes Research Articles

L3MBTL2 maintains MYCN-amplified neuroblastoma cell proliferation through silencing NRIP3 and BRME1 genes.

Epigenetic alterations critically affect tumor development. Polycomb-group complexes constitute an evolutionarily conserved epigenetic machinery that regulates stem cell fate and development. They are implicated in tumorigenesis, primarily via histone modification. Polycomb repressive complex 1 (PRC1) complexes 1-6 (PRC1.1-6) mediate the ubiquitination of histone H2A on lysine 119 (H2AK119ub). Here, we studied the functional roles of a PRC1.6 molecule, L3MBTL2, in neuroblastoma (NB) cells. L3MBTL2-knockout and knockdown revealed that L3MBTL2 depletion suppressed NB cell proliferation via cell-cycle arrest and gamma-H2A.X upregulation. L3MBTL2-knockout profoundly suppressed xenograft tumor formation. Transcriptome analysis revealed suppressed cell-cycle-related and activated differentiation-related pathways. Break repair meiotic recombinase recruitment factor 1 (BRME1) and nuclear receptor interacting protein 3 (NRIP3) were notably de-repressed by L3MBTL2-knockout. The deletion of L3MBTL2 reduced enrichment of H2AK119ub and PCGF6 at transcriptional start site proximal regions of the targets. Add-back studies unveiled the importance of L3MBTL2-BRME1 and -NRIP3 axes for NB cell proliferation. We further manifested the association of MYCN with de-repression of NRIP3 in an L3MBTL2-deficient context. Therefore, this study first revealed the significance of L3MBTL2-mediated gene silencing in MYCN-amplified NB cells.

Drosophila TET acts with PRC1 to activate gene expression independently of its catalytic activity.

Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS.

The TELOMERE REPEAT BINDING proteins TRB4 and TRB5 function as transcriptional activators of PRC2-controlled genes to regulate plant development

Plant-specific transcriptional regulators called TELOMERE REPEAT BINDING proteins (TRBs) combine two DNA-binding domains, the GH1 domain, which binds to linker DNA and is shared with H1 histones, and the Myb/SANT domain, which specifically recognizes the telobox DNA-binding site motif. TRB1, TRB2, and TRB3 proteins recruit Polycomb group complex 2 (PRC2) to deposit H3K27me3 and JMJ14 to remove H3K4me3 at gene promoters containing telobox motifs to repress transcription. Here, we demonstrate that TRB4 and TRB5, two related paralogs belonging to a separate TRB clade conserved in spermatophytes, regulate the transcription of several hundred genes involved in developmental responses to environmental cues. TRB4 binds to several thousand sites in the genome, mainly at transcription start sites and promoter regions of transcriptionally active and H3K4me3-marked genes, but, unlike TRB1, it is not enriched at H3K27me3-marked gene bodies. However, TRB4 can physically interact with the catalytic components of PRC2, SWINGER, and CURLY LEAF (CLF). Unexpectedly, we show that TRB4 and TRB5 are required for distinctive phenotypic traits observed in clf mutant plants and thus function as transcriptional activators of several hundred CLF-controlled genes, including key flowering genes. We further demonstrate that TRB4 shares multiple target genes with TRB1 and physically and genetically interacts with members of both TRB clades. Collectively, these results reveal that TRB proteins engage in both positive and negative interactions with other members of the family to regulate plant development through both PRC2-dependent and -independent mechanisms.

Research advances of polycomb group proteins in regulating mammalian development.

Polycomb group (PcG) proteins are a subset of epigenetic factors that are highly conserved throughout evolution. In mammals, PcG proteins can be classified into two muti-proteins complexes: Polycomb repressive complex 1 (PRC1) and PRC2. Increasing evidence has demonstrated that PcG complexes play critical roles in the regulation of gene expression, genomic imprinting, chromosome X-inactivation, and chromatin structure. Accordingly, the dysfunction of PcG proteins is tightly orchestrated with abnormal developmental processes. Here, we summarized and discussed the current knowledge of the biochemical and molecular functions of PcG complexes, especially the PRC1 and PRC2 in mammalian development including embryonic development and tissue development, which will shed further light on the deep understanding of the basic knowledge of PcGs and their functions for reproductive health and developmental disorders.

Mask exhibits trxG-like behavior and associates with H3K27ac marked chromatin

The Trithorax group (trxG) proteins counteract the repressive effect of Polycomb group (PcG) complexes and maintain transcriptional memory of active states of key developmental genes. Although chromatin structure and modifications appear to play a fundamental role in this process, it is not clear how trxG prevents PcG-silencing and heritably maintains an active gene expression state. Here, we report a hitherto unknown role of Drosophila Multiple ankyrin repeats single KH domain (Mask), which emerged as one of the candidate trxG genes in our reverse genetic screen. The genome-wide binding profile of Mask correlates with known trxG binding sites across the Drosophila genome. In particular, the association of Mask at chromatin overlaps with CBP and H3K27ac, which are known hallmarks of actively transcribed genes by trxG. Importantly, Mask predominantly associates with actively transcribed genes in Drosophila. Depletion of Mask not only results in the downregulation of trxG targets but also correlates with diminished levels of H3K27ac. The fact that Mask positively regulates H3K27ac levels in flies was also found to be conserved in human cells. Strong suppression of Pc mutant phenotype by mutation in mask provides physiological relevance that Mask contributes to the anti-silencing effect of trxG, maintaining expression of key developmental genes. Since Mask is a downstream effector of multiple cell signaling pathways, we propose that Mask may connect cell signaling with chromatin mediated epigenetic cell memory governed by trxG.

Cell type-specific role of CBX2 and its disordered region in spermatogenesis.

Polycomb group (PcG) proteins maintain the repressed state of lineage-inappropriate genes and are therefore essential for embryonic development and adult tissue homeostasis. One critical function of PcG complexes is modulating chromatin structure. Canonical Polycomb repressive complex 1 (cPRC1), particularly its component CBX2, can compact chromatin and phase-separate in vitro. These activities are hypothesized to be critical for forming a repressed physical environment in cells. While much has been learned by studying these PcG activities in cell culture models, it is largely unexplored how cPRC1 regulates adult stem cells and their subsequent differentiation in living animals. Here, we show in vivo evidence of a critical nonenzymatic repressive function of cPRC1 component CBX2 in the male germline. CBX2 is up-regulated as spermatogonial stem cells differentiate and is required to repress genes that were active in stem cells. CBX2 forms condensates (similar to previously described Polycomb bodies) that colocalize with target genes bound by CBX2 in differentiating spermatogonia. Single-cell analyses of mosaic Cbx2 mutant testes show that CBX2 is specifically required to produce differentiating A1 spermatogonia. Furthermore, the region of CBX2 responsible for compaction and phase separation is needed for the long-term maintenance of male germ cells in the animal. These results emphasize that the regulation of chromatin structure by CBX2 at a specific stage of spermatogenesis is critical, which distinguishes this from a mechanism that is reliant on histone modification.

Abstract 3958: Transcriptome analysis of Small Cell Osteosarcoma reveals an embryonic stem cell-like phenotype

Abstract Introduction: Small round cell osteosarcoma (SCOS) is a very rare type of osteosarcoma, composed by small cells producing bone matrix, with limited studies that mainly focus on histological features. In this study we employed RNA sequencing to discover the transcriptional profile of SCOS in order to find a specific gene signatures useful to find new potential therapeutic targets. To date, the tumor is treated according to the osteosarcoma protocols. Material and Methods: We studied 15 primary tumor including 6 SCOS and 9 high grade Osteosarcoma (OS) by RNASeq. Pair-end libraries were synthesized using TruSeq RNA Library Prep Kit for Enrichment and loaded on NextSeq 500 platform (Illumina). After FastqQC quality control, fastq data were aligned to Homo_sapiens GRCh38 with STAR algorithm and counted by featureCounts. In Addition we randomly selected 30 muscle-skeletal normal tissues from 800 available samples from Genotype-Tissue Expression repository (GTEX). Using surrogate variables to remove batch effects and other unwanted variations, we performed differentially expression analysis employing DESeq2 (R package). We considered as differentially expressed genes those BH adj p-value <0.01 and absolute |log2FC|=2. We then performed preranked Gene set Enrichment analysis (GSEA) for biological function assessment Results: To elucidate the transcriptome landscape of SCOS we first performed differential gene expression analysis between SCOS and normal tissue identifying 5061 deregulated genes (2516 up-regulated and 2545 down-regulated in SCOS). Then, from this signature we filtered out the signature differentially expressed between OS and normal tissue, obtaining a specific SCOS signature of 1371 genes (1013 up-regulated and 358 down-regulated). To study the biological function behind SCOS we performed GSEA and we found that SCOS gene signature resulted enriched in epigenetics mechanisms as well as in embryonic stem cell (ES) gene sets. In particular, we found the overexpression of EZH2, ASXL1, HDAC9, MYCN, MYB genes involved in Polycomb group complex. This on one side confirms the highly undifferentiated nature of this tumor that could explain the more aggressive behaviour of SCOS in comparison to OS; on the other side, offers in epigenetic drugs a new therapeutic perspective. Validation studies are in progress. Conclusions: This work firstly profiled the transcriptome of SCOS and identifies new pathways that can be of interest for diagnostic and/or therapeutic purposes. This work was supported by Ministry of Health 5 × 1000, Anno 2020 Redditi 2019, contributions to the IRCCS, Istituto Ortopedico Rizzoli. Citation Format: Maria Antonella Laginestra, Alessandro Parra, Elisa Simonetti, Laura Formentini, Michela Pasello, Alberto Righi, Davide Maria Donati, Cristina Ferrari, Laura Pazzaglia, Katia Scotlandi. Transcriptome analysis of Small Cell Osteosarcoma reveals an embryonic stem cell-like phenotype. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3958.

Polycomb group genes are required for neuronal pruning in Drosophila

BackgroundPruning that selectively eliminates unnecessary or incorrect neurites is required for proper wiring of the mature nervous system. During Drosophila metamorphosis, dendritic arbourization sensory neurons (ddaCs) and mushroom body (MB) γ neurons can selectively prune their larval dendrites and/or axons in response to the steroid hormone ecdysone. An ecdysone-induced transcriptional cascade plays a key role in initiating neuronal pruning. However, how downstream components of ecdysone signalling are induced remains not entirely understood.ResultsHere, we identify that Scm, a component of Polycomb group (PcG) complexes, is required for dendrite pruning of ddaC neurons. We show that two PcG complexes, PRC1 and PRC2, are important for dendrite pruning. Interestingly, depletion of PRC1 strongly enhances ectopic expression of Abdominal B (Abd-B) and Sex combs reduced, whereas loss of PRC2 causes mild upregulation of Ultrabithorax and Abdominal A in ddaC neurons. Among these Hox genes, overexpression of Abd-B causes the most severe pruning defects, suggesting its dominant effect. Knockdown of the core PRC1 component Polyhomeotic (Ph) or Abd-B overexpression selectively downregulates Mical expression, thereby inhibiting ecdysone signalling. Finally, Ph is also required for axon pruning and Abd-B silencing in MB γ neurons, indicating a conserved function of PRC1 in two types of pruning.ConclusionsThis study demonstrates important roles of PcG and Hox genes in regulating ecdysone signalling and neuronal pruning in Drosophila. Moreover, our findings suggest a non-canonical and PRC2-independent role of PRC1 in Hox gene silencing during neuronal pruning.

Cerebellar granule neurons induce Cyclin D1 before the onset of motor symptoms in Huntington’s disease mice

Although Huntington’s disease (HD) is classically defined by the selective vulnerability of striatal projection neurons, there is increasing evidence that cerebellar degeneration modulates clinical symptoms. However, little is known about cell type-specific responses of cerebellar neurons in HD. To dissect early disease mechanisms in the cerebellum and cerebrum, we analyzed translatomes of neuronal cell types from both regions in a new HD mouse model. For this, HdhQ200 knock-in mice were backcrossed with the calm 129S4 strain, to constrain experimental noise caused by variable hyperactivity of mice in a C57BL/6 background. Behavioral and neuropathological characterization showed that these S4-HdhQ200 mice had very mild behavioral abnormalities starting around 12 months of age that remained mild up to 18 months. By 9 months, we observed abundant Huntingtin-positive neuronal intranuclear inclusions (NIIs) in the striatum and cerebellum. The translatome analysis of GABAergic cells of the cerebrum further confirmed changes typical of HD-induced striatal pathology. Surprisingly, we observed the strongest response with 626 differentially expressed genes in glutamatergic neurons of the cerebellum, a population consisting primarily of granule cells, commonly considered disease resistant. Our findings suggest vesicular fusion and exocytosis, as well as differentiation-related pathways are affected in these neurons. Furthermore, increased expression of cyclin D1 (Ccnd1) in the granular layer and upregulated expression of polycomb group complex protein genes and cell cycle regulators Cbx2, Cbx4 and Cbx8 point to a putative role of aberrant cell cycle regulation in cerebellar granule cells in early disease.

Cbx2 is a functional target of the let-7 family in the gonad of Japanese flounder (Paralichthys olivaceus).

As a key member of the miRNA family, the role and target gene of the let-7 family in the gonad of Japanese flounder (Paralichthys olivaceus) is unclear. Chromobox homolog 2 (CBX2) is one of the core components of the polycomb group complex (PcG) and significantly influences gonadal development. The deletion of CBX2 can lead to sex reversal in mammals. Therefore, exploring the relationship between the let-7 family and cbx2 is crucial to clarify the role played by the let-7 family in the gonad of Japanese flounder. We predicted and verified the target interaction between the let-7 family and cbx2. The results showed that cbx2 was a direct target of let-7d, let-7e, let-7g, let-7j, and let-7b. Among them, let-7d, let-7e, let-7g, and let-7j exhibited an extremely significant targeting relationship with cbx2 (p<0.001). Taking let-7g as an example, we further investigated the regulatory role between let-7g and cbx2 in the gonad by miRNA overexpression and inhibition experiments in primary testis cells. The results revealed that let-7g could negatively regulate cbx2 at the level of primary testis cells. And the expression of sf1 (steroidogenic factor 1) was also significantly decreased after the interference of cbx2 siRNA. This suggests that the let-7 family may be involved in the Japanese flounder gonadal development via targeting cbx2.

Quantitative Analysis of PcG-Associated Condensates by Stochastic Optical Reconstruction Microscopy (STORM).

The polycomb group (PcG) proteins play a central role in the maintenance of a repressive state of gene expression. Recent findings demonstrate that PcG components are organized into nuclear condensates, contributing to the reshaping of chromatin architecture in physiological and pathological conditions, thus affecting the nuclear mechanics. In this context, direct stochastic optical reconstruction microscopy (dSTORM) provides an effective tool to achieve a detailed characterization of PcG condensates by visualizing them at a nanometric level. Furthermore, by analyzing dSTORM datasets with cluster analysis algorithms, quantitative information can be yielded regarding protein numbers, grouping, and spatial organization. Here, we describe how to set up a dSTORM experiment and perform the data analysis to study PcG complexes' components in adhesion cells quantitatively.

Roles of Polycomb Complexes in the Reconstruction of 3D Genome Architecture during Preimplantation Embryonic Development.

The appropriate deployment of developmental programs depends on complex genetic information encoded by genomic DNA sequences and their positioning and contacts in the three-dimensional (3D) space within the nucleus. Current studies using novel techniques including, but not limited to, Hi-C, ChIA-PET, and Hi-ChIP reveal that regulatory elements (Res), such as enhancers and promoters, may participate in the precise regulation of expression of tissue-specific genes important for both embryogenesis and organogenesis by recruiting Polycomb Group (PcG) complexes. PcG complexes usually poise the transcription of developmental genes by forming Polycomb bodies to compact poised enhancers and promoters marked by H3K27me3 in the 3D space. Additionally, recent studies have also uncovered their roles in transcriptional activation. To better understand the full complexities in the mechanisms of how PcG complexes regulate transcription and long-range 3D contacts of enhancers and promoters during developmental programs, we outline novel insights regarding PcG-associated dramatic changes in the 3D chromatin conformation in developmental programs of early embryos and naïve-ground-state transitions of pluripotent embryonic stem cells (ESCs), and highlight the distinct roles of unique and common subunits of canonical and non-canonical PcG complexes in shaping genome architectures and transcriptional programs.

Identification of hepatocellular carcinoma subtypes based on PcG-related genes and biological relevance with cancer cells

BackgroundHepatocellular carcinoma (HCC) is an extensive heterogeneous disease where epigenetic factors contribute to its pathogenesis. Polycomb group (PcG) proteins are a group of subunits constituting various macro-molecular machines to regulate the epigenetic landscape, which contributes to cancer phenotype and has the potential to develop a molecular classification of HCC.ResultsHere, based on multi-omics data analysis of DNA methylation, mRNA expression, and copy number of PcG-related genes, we established an epigenetic classification system of HCC, which divides the HCC patients into two subgroups with significantly different outcomes. Comparing these two epigenetic subgroups, we identified different metabolic features, which were related to epigenetic regulation of polycomb-repressive complex 1/2 (PRC1/2). Furthermore, we experimentally proved that inhibition of PcG complexes enhanced the lipid metabolism and reduced the capacity of HCC cells against glucose shortage. In addition, we validated the low chemotherapy sensitivity of HCC in Group A and found inhibition of PRC1/2 promoted HCC cells’ sensitivity to oxaliplatin in vitro and in vivo. Finally, we found that aberrant upregulation of CBX2 in Group A and upregulation of CBX2 were associated with poor prognosis in HCC patients. Furthermore, we found that manipulation of CBX2 affected the levels of H3K27me3 and H2AK119ub.ContributionsOur study provided a novel molecular classification system based on PcG-related genes data and experimentally validated the biological features of HCC in two subgroups. Our founding supported the polycomb complex targeting strategy to inhibit HCC progression where CBX2 could be a feasible therapeutic target.

Regulation of Polyhomeotic Condensates by Intrinsically Disordered Sequences That Affect Chromatin Binding.

The Polycomb group (PcG) complex PRC1 localizes in the nucleus in condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains an oligomerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph) forms phase-separated condensates with DNA or chromatin in vitro, suggesting that PcG bodies may form through SAM-driven phase separation. In cells, Ph forms multiple small condensates, while mini-Ph typically forms a single large nuclear condensate. We therefore hypothesized that sequences outside of mini-Ph, which are predicted to be intrinsically disordered, are required for proper condensate formation. We identified three distinct low-complexity regions in Ph based on sequence composition. We systematically tested the role of each of these sequences in Ph condensates using live imaging of transfected Drosophila S2 cells. Each sequence uniquely affected Ph SAM-dependent condensate size, number, and morphology, but the most dramatic effects occurred when the central, glutamine-rich intrinsically disordered region (IDR) was removed, which resulted in large Ph condensates. Like mini-Ph condensates, condensates lacking the glutamine-rich IDR excluded chromatin. Chromatin fractionation experiments indicated that the removal of the glutamine-rich IDR reduced chromatin binding and that the removal of either of the other IDRs increased chromatin binding. Our data suggest that all three IDRs, and functional interactions among them, regulate Ph condensate size and number. Our results can be explained by a model in which tight chromatin binding by Ph IDRs antagonizes Ph SAM-driven phase separation. Our observations highlight the complexity of regulation of biological condensates housed in single proteins.

PBRM1, SETD2 and BAP1 - the trinity of 3p in clear cell renal cell carcinoma.

Biallelic inactivation of the tumour suppressor gene Von Hippel-Lindau (VHL) occurs in the vast majority of clear cell renal cell carcinoma (ccRCC) instances, disrupting cellular oxygen-sensing mechanisms to yield a state of persistent pseudo-hypoxia, defined as a continued hypoxic response despite the presence of adequate oxygen levels. However, loss of VHL alone is often insufficient to drive oncogenesis. Results from genomic studies have shown that co-deletions of VHL with one (or more) of three genes encoding proteins involved in chromatin modification and remodelling, polybromo-1 gene (PBRM1), BRCA1-associated protein 1 (BAP1) and SET domain-containing 2 (SETD2), are common and important co-drivers of tumorigenesis. These genes are all located near VHL on chromosome 3p and are often altered following cytogenetic rearrangements that lead to 3p loss and precede the establishment of ccRCC. These three proteins have multiple roles in the regulation of crucial cancer-related pathways, including protection of genomic stability, antagonism of polycomb group (PcG) complexes to maintain a permissive transcriptional landscape in physiological conditions, and regulation of genes that mediate responses to immune checkpoint inhibitor therapy. An improved understanding of these mechanisms will bring new insights regarding cellular drivers of ccRCC growth and therapy response and, ultimately, will support the development of novel translational therapeutics.

Variant Polycomb complexes in Drosophila consistent with ancient functional diversity.

Polycomb group (PcG) mutants were first identified in Drosophila on the basis of their failure to maintain proper Hox gene repression during development. The proteins encoded by the corresponding fly genes mainly assemble into one of two discrete Polycomb repressive complexes: PRC1 or PRC2. However, biochemical analyses in mammals have revealed alternative forms of PRC2 and multiple distinct types of noncanonical or variant PRC1. Through a series of proteomic analyses, we identify analogous PRC2 and variant PRC1 complexes in Drosophila, as well as a broader repertoire of interactions implicated in early development. Our data provide strong support for the ancient diversity of PcG complexes and a framework for future analysis in a longstanding and versatile genetic system.

Know when to fold 'em: Polycomb complexes in oncogenic 3D genome regulation.

Chromatin is spatially and temporally regulated through a series of orchestrated processes resulting in the formation of 3D chromatin structures such as topologically associating domains (TADs), loops and Polycomb Bodies. These structures are closely linked to transcriptional regulation, with loss of control of these processes a frequent feature of cancer and developmental syndromes. One such oncogenic disruption of the 3D genome is through recurrent dysregulation of Polycomb Group Complex (PcG) functions either through genetic mutations, amplification or deletion of genes that encode for PcG proteins. PcG complexes are evolutionarily conserved epigenetic complexes. They are key for early development and are essential transcriptional repressors. PcG complexes include PRC1, PRC2 and PR-DUB which are responsible for the control of the histone modifications H2AK119ub1 and H3K27me3. The spatial distribution of the complexes within the nuclear environment, and their associated modifications have profound effects on the regulation of gene transcription and the 3D genome. Nevertheless, how PcG complexes regulate 3D chromatin organization is still poorly understood. Here we glean insights into the role of PcG complexes in 3D genome regulation and compaction, how these processes go awry during tumorigenesis and the therapeutic implications that result from our insights into these mechanisms.

Context-specific Polycomb mechanisms in development.

Polycomb group (PcG) proteins are crucial chromatin regulators that maintain repression of lineage-inappropriate genes and are therefore required for stable cell fate. Recent advances show that PcG proteins form distinct multi-protein complexes in various cellular environments, such as in early development, adult tissue maintenance and cancer. This surprising compositional diversity provides the basis for mechanistic diversity. Understanding this complexity deepens and refines the principles of PcG complex recruitment, target-gene repression and inheritance of memory. We review how the core molecular mechanism of Polycomb complexes operates in diverse developmental settings and propose that context-dependent changes in composition and mechanism are essential for proper epigenetic regulation in development.

Significance of chromobox protein (CBX) expression in diffuse LBCL.

Diffuse large B-cell lymphoma (DLBCL) is the main pathological type of non-Hodgkin lymphoma (NHL). Chromobox (CBX) family proteins are classical components of polycomb group (PcG) complexes in many cancer types, resulting in accelerated carcinogenesis. Nevertheless, the prognostic, functional and expression significance of these CBX family members in DLBCL remain unclear and elusive. CBX transcriptional levels were confirmed using Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA) and Cancer Cell Line Encyclopedia (CCLE) databases. The protein levels of CBX family members were analysed using The Human Protein Atlas (HPA) database. Information on the PPI network, functional enrichment, drug sensitivity, prognostic value, miRNA network, protein structure, genetic alteration and immune cell infiltration were generated using the GeneMANIA, Metascape, GSCALite, GEPIA, PDB, cBioPortal, and TIMER databases, and the correlation of these factors with CBX expression levels in DLBCL was assessed. CBX1/2/3/5/6/8 mRNA levels were significantly enhanced in DLBCL tissues compared to corresponding normal tissues. CBX1/3/4/5/8 protein expression levels were obviously increased, whereas CBX7 was obviously decreased. This difference might be attributed to miRNA regulation based on the miRNA network. Overall survival (OS) analysis showed that CBX levels were not correlated with prognosis in DLBCL patients, indicating that CBXs are not good biomarkers for DLBCL patients. Furthermore, functional enrichment analyses indicated that CBXs were closely related to DNA duplex unwinding, covalent chromatin modification, and histone lysine methylation. The levels of CBXs were also significantly associated with diverse immune cell infiltration in DLBCL. This study reveals that dysregulated CBXs are involved in DLBCL development and might represent potential therapeutic targets for DLBCL.

Enhancer of trithorax/polycomb, Corto, regulates timing of hunchback gene relocation and competence in Drosophila neuroblasts

BackgroundNeural progenitors produce diverse cells in a stereotyped birth order, but can specify each cell type for only a limited duration. In the Drosophila embryo, neuroblasts (neural progenitors) specify multiple, distinct neurons by sequentially expressing a series of temporal identity transcription factors with each division. Hunchback (Hb), the first of the series, specifies early-born neuronal identity. Neuroblast competence to generate early-born neurons is terminated when the hb gene relocates to the neuroblast nuclear lamina, rendering it refractory to activation in descendent neurons. Mechanisms and trans-acting factors underlying this process are poorly understood. Here we identify Corto, an enhancer of Trithorax/Polycomb (ETP) protein, as a new regulator of neuroblast competence.MethodsWe used the GAL4/UAS system to drive persistent misexpression of Hb in neuroblast 7–1 (NB7-1), a model lineage for which the early competence window has been well characterized, to examine the role of Corto in neuroblast competence. We used immuno-DNA Fluorescence in situ hybridization (DNA FISH) in whole embryos to track the position of the hb gene locus specifically in neuroblasts across developmental time, comparing corto mutants to control embryos. Finally, we used immunostaining in whole embryos to examine Corto’s role in repression of Hb and a known target gene, Abdominal B (Abd-B).ResultsWe found that in corto mutants, the hb gene relocation to the neuroblast nuclear lamina is delayed and the early competence window is extended. The delay in gene relocation occurs after hb transcription is already terminated in the neuroblast and is not due to prolonged transcriptional activity. Further, we find that Corto genetically interacts with Posterior Sex Combs (Psc), a core subunit of polycomb group complex 1 (PRC1), to terminate early competence. Loss of Corto does not result in derepression of Hb or its Hox target, Abd-B, specifically in neuroblasts.ConclusionsThese results show that in neuroblasts, Corto genetically interacts with PRC1 to regulate timing of nuclear architecture reorganization and support the model that distinct mechanisms of silencing are implemented in a step-wise fashion during development to regulate cell fate gene expression in neuronal progeny.