Publications by authors named "Diego Pasini"

63 Publications

Coordinated maintenance of H3K36/K27 methylation by histone demethylases preserves germ cell identity and immortality.

Cell Rep 2021 Nov;37(8):110050

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes vej 5, Copenhagen DK-2200, Denmark. Electronic address:

Germ cells have evolved unique mechanisms to ensure the transmission of genetically and nongenetically encoded information, whose alteration compromises germ cell immortality. Chromatin factors play fundamental roles in these mechanisms. H3K36 and H3K27 methyltransferases shape and propagate a pattern of histone methylation essential for C. elegans germ cell maintenance, but the role of respective histone demethylases remains unexplored. Here, we show that jmjd-5 regulates H3K36me2 and H3K27me3 levels, preserves germline immortality, and protects germ cell identity by controlling gene expression. The transcriptional and biological effects of jmjd-5 loss can be hindered by the removal of H3K27demethylases, indicating that H3K36/K27 demethylases act in a transcriptional framework and promote the balance between H3K36 and H3K27 methylation required for germ cell immortality. Furthermore, we find that in wild-type, but not in jmjd-5 mutants, alterations of H3K36 methylation and transcription occur at high temperature, suggesting a role for jmjd-5 in adaptation to environmental changes.
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http://dx.doi.org/10.1016/j.celrep.2021.110050DOI Listing
November 2021

Polycomb-dependent histone H2A ubiquitination links developmental disorders with cancer.

Trends Genet 2021 Aug 20. Epub 2021 Aug 20.

European Institute of Oncology (IEO), Istituto Di Ricovero e Cura a Carattere Scientifico (IRCCS), Department of Experimental Oncology, Via Adamello 16, 20139 Milan, Italy; University of Milan, Department of Health Sciences, Via Antonio di Rudinì 8, 20142 Milan, Italy. Electronic address:

Cell identity is tightly controlled by specific transcriptional programs which require post-translational modifications of histones. These histone modifications allow the establishment and maintenance of active and repressed chromatin domains. Histone H2A lysine 119 ubiquitination (H2AK119ub1) has an essential role in building repressive chromatin domains during development. It is regulated by the counteracting activities of the Polycomb repressive complex 1 (PRC1) and the Polycomb repressive-deubiquitinase (PR-DUB) complexes, two multi-subunit ensembles that write and erase this modification, respectively. We have catalogued the recurrent genetic alterations in subunits of the PRC1 and PR-DUB complexes in both neurodevelopmental disorders and cancer. These genetic lesions are often shared across disorders, and we highlight common mechanisms of H2AK119ub1 dysregulation and how they affect development in multiple disease contexts.
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http://dx.doi.org/10.1016/j.tig.2021.07.011DOI Listing
August 2021

BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation.

Mol Cell 2021 09 28;81(17):3526-3541.e8. Epub 2021 Jun 28.

IEO, European Institute of Oncology IRCCS, Department of Experimental Oncology, Via Adamello 16, 20139 Milan, Italy; University of Milan, Via A. di Rudini 8, Department of Health Sciences, 20142 Milan, Italy. Electronic address:

BAP1 is mutated or deleted in many cancer types, including mesothelioma, uveal melanoma, and cholangiocarcinoma. It is the catalytic subunit of the PR-DUB complex, which removes PRC1-mediated H2AK119ub1, essential for maintaining transcriptional repression. However, the precise relationship between BAP1 and Polycombs remains elusive. Using embryonic stem cells, we show that BAP1 restricts H2AK119ub1 deposition to Polycomb target sites. This increases the stability of Polycomb with their targets and prevents diffuse accumulation of H2AK119ub1 and H3K27me3. Loss of BAP1 results in a broad increase in H2AK119ub1 levels that is primarily dependent on PCGF3/5-PRC1 complexes. This titrates PRC2 away from its targets and stimulates H3K27me3 accumulation across the genome, leading to a general chromatin compaction. This provides evidence for a unifying model that resolves the apparent contradiction between BAP1 catalytic activity and its role in vivo, uncovering molecular vulnerabilities that could be useful for BAP1-related pathologies.
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http://dx.doi.org/10.1016/j.molcel.2021.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428331PMC
September 2021

Prdm16-mediated H3K9 methylation controls fibro-adipogenic progenitors identity during skeletal muscle repair.

Sci Adv 2021 Jun 2;7(23). Epub 2021 Jun 2.

Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR) of Italy c/o Department of Biology and Biotechnology "C. Darwin," Sapienza University, 00185 Rome, Italy.

H3K9 methylation maintains cell identity orchestrating stable silencing and anchoring of alternate fate genes within the heterochromatic compartment underneath the nuclear lamina (NL). However, how cell type-specific genomic regions are specifically targeted to the NL is still elusive. Using fibro-adipogenic progenitors (FAPs) as a model, we identified Prdm16 as a nuclear envelope protein that anchors H3K9-methylated chromatin in a cell-specific manner. We show that Prdm16 mediates FAP developmental capacities by orchestrating lamina-associated domain organization and heterochromatin sequestration at the nuclear periphery. We found that Prdm16 localizes at the NL where it cooperates with the H3K9 methyltransferases G9a/GLP to mediate tethering and silencing of myogenic genes, thus repressing an alternative myogenic fate in FAPs. Genetic and pharmacological disruption of this repressive pathway confers to FAP myogenic competence, preventing fibro-adipogenic degeneration of dystrophic muscles. In summary, we reveal a druggable mechanism of heterochromatin perinuclear sequestration exploitable to reprogram FAPs in vivo.
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http://dx.doi.org/10.1126/sciadv.abd9371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8172132PMC
June 2021

Intestinal differentiation involves cleavage of histone H3 N-terminal tails by multiple proteases.

Nucleic Acids Res 2021 01;49(2):791-804

IEO European Institute of Oncology IRCCS, Department of Experimental Oncology, Via Adamello 16, 20139 Milan, Italy.

The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.
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http://dx.doi.org/10.1093/nar/gkaa1228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826276PMC
January 2021

Colorectal cancer residual disease at maximal response to EGFR blockade displays a druggable Paneth cell-like phenotype.

Sci Transl Med 2020 08;12(555)

Department of Oncology, University of Torino, 10060 Candiolo, Torino, Italy.

Blockade of epidermal growth factor receptor (EGFR) causes tumor regression in some patients with metastatic colorectal cancer (mCRC). However, residual disease reservoirs typically remain even after maximal response to therapy, leading to relapse. Using patient-derived xenografts (PDXs), we observed that mCRC cells surviving EGFR inhibition exhibited gene expression patterns similar to those of a quiescent subpopulation of normal intestinal secretory precursors with Paneth cell characteristics. Compared with untreated tumors, these pseudodifferentiated tumor remnants had reduced expression of genes encoding EGFR-activating ligands, enhanced activity of human epidermal growth factor receptor 2 (HER2) and HER3, and persistent signaling along the phosphatidylinositol 3-kinase (PI3K) pathway. Clinically, properties of residual disease cells from the PDX models were detected in lingering tumors of responsive patients and in tumors of individuals who had experienced early recurrence. Mechanistically, residual tumor reprogramming after EGFR neutralization was mediated by inactivation of Yes-associated protein (YAP), a master regulator of intestinal epithelium recovery from injury. In preclinical trials, Pan-HER antibodies minimized residual disease, blunted PI3K signaling, and induced long-term tumor control after treatment discontinuation. We found that tolerance to EGFR inhibition is characterized by inactivation of an intrinsic lineage program that drives both regenerative signaling during intestinal repair and EGFR-dependent tumorigenesis. Thus, our results shed light on CRC lineage plasticity as an adaptive escape mechanism from EGFR-targeted therapy and suggest opportunities to preemptively target residual disease.
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http://dx.doi.org/10.1126/scitranslmed.aax8313DOI Listing
August 2020

Dysfunctional polycomb transcriptional repression contributes to lamin A/C-dependent muscular dystrophy.

J Clin Invest 2020 05;130(5):2408-2421

Santa Lucia Foundation, Scientific Institute for Research, Hospitalization and Healthcare (IRCCS), Rome, Italy.

Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the lamin A/C gene cause several diseases belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of lamin A-dependent dystrophies are still largely unknown. The polycomb group (PcG) of proteins are epigenetic repressors and lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss muscular dystrophy (EDMD), we show here that lamin A loss deregulated PcG positioning in muscle satellite stem cells, leading to derepression of non-muscle-specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional program caused impairment in self-renewal, loss of cell identity, and premature exhaustion of the quiescent satellite cell pool. Genetic ablation of the Cdkn2a locus restored muscle stem cell properties in lamin A/C-null dystrophic mice. Our findings establish a direct link between lamin A and PcG epigenetic silencing and indicate that lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.
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http://dx.doi.org/10.1172/JCI128161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190994PMC
May 2020

PRMT1 Is Recruited via DNA-PK to Chromatin Where It Sustains the Senescence-Associated Secretory Phenotype in Response to Cisplatin.

Cell Rep 2020 01;30(4):1208-1222.e9

Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan 20139, Italy. Electronic address:

Protein arginine methyltransferase 1 (PRMT1) is overexpressed in various human cancers and linked to poor response to chemotherapy. Various PRMT1 inhibitors are currently under development; yet, we do not fully understand the mechanisms underpinning PRMT1 involvement in tumorigenesis and chemoresistance. Using mass spectrometry-based proteomics, we identified PRMT1 as regulator of arginine methylation in ovarian cancer cells treated with cisplatin. We showed that DNA-dependent protein kinase (DNA-PK) binds to and phosphorylates PRMT1 in response to cisplatin, inducing its chromatin recruitment and redirecting its enzymatic activity toward Arg3 of histone H4 (H4R3). On chromatin, the DNA-PK/PRMT1 axis induces senescence-associated secretory phenotype through H4R3me2a deposition at pro-inflammatory gene promoters. Finally, PRMT1 inhibition reduces the clonogenic growth of cancer cells exposed to low doses of cisplatin, sensitizing them to apoptosis. While unravelling the role of PRMT1 in response to genotoxic agents, our findings indicate the possibility of targeting PRMT1 to overcome chemoresistance in cancer.
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http://dx.doi.org/10.1016/j.celrep.2019.12.061DOI Listing
January 2020

Cooperation Between MYC and β-Catenin in Liver Tumorigenesis Requires Yap/Taz.

Hepatology 2020 10 29;72(4):1430-1443. Epub 2020 Jul 29.

European Institute of Oncology (IEO)-IRCCS, Milan, Italy.

Background And Aims: Activation of MYC and catenin beta-1 (CTNNB1, encoding β-catenin) can co-occur in liver cancer, but how these oncogenes cooperate in tumorigenesis remains unclear.

Approach And Results: We generated a mouse model allowing conditional activation of MYC and WNT/β-catenin signaling (through either β-catenin activation or loss of APC - adenomatous polyposis coli) upon expression of CRE recombinase in the liver and monitored their effects on hepatocyte proliferation, apoptosis, gene expression profiles, and tumorigenesis. Activation of WNT/β-catenin signaling strongly accelerated MYC-driven carcinogenesis in the liver. Both pathways also cooperated in promoting cellular transformation in vitro, demonstrating their cell-autonomous action. Short-term induction of MYC and β-catenin in hepatocytes, followed by RNA-sequencing profiling, allowed the identification of a "Myc/β-catenin signature," composed of a discrete set of Myc-activated genes whose expression increased in the presence of active β-catenin. Notably, this signature enriched for targets of Yes-associated protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz), two transcriptional coactivators known to be activated by WNT/β-catenin signaling and to cooperate with MYC in mitogenic activation and liver transformation. Consistent with these regulatory connections, Yap/Taz accumulated upon Myc/β-catenin activation and were required not only for the ensuing proliferative response, but also for tumor cell growth and survival. Finally, the Myc/β-catenin signature was enriched in a subset of human hepatocellular carcinomas characterized by comparatively poor prognosis.

Conclusions: Myc and β-catenin show a strong cooperative action in liver carcinogenesis, with Yap and Taz serving as mediators of this effect. These findings warrant efforts toward therapeutic targeting of Yap/Taz in aggressive liver tumors marked by elevated Myc/β-catenin activity.
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http://dx.doi.org/10.1002/hep.31120DOI Listing
October 2020

Histone H2AK119 Mono-Ubiquitination Is Essential for Polycomb-Mediated Transcriptional Repression.

Mol Cell 2020 02 26;77(4):840-856.e5. Epub 2019 Dec 26.

Department of Experimental Oncology, IEO European Institute of Oncology IRCCS, Via Adamello 16, 20139 Milan, Italy; Department of Health Sciences, University of Milan, Via A. di Rudinì 8, 20142 Milan, Italy. Electronic address:

Polycomb group proteins (PcGs) maintain transcriptional repression to preserve cellular identity in two distinct repressive complexes, PRC1 and PRC2, that modify histones by depositing H2AK119ub1 and H3K27me3, respectively. PRC1 and PRC2 exist in different variants and show a complex regulatory cross-talk. However, the contribution that H2AK119ub1 plays in mediating PcG repressive functions remains largely controversial. Using a fully catalytic inactive RING1B mutant, we demonstrated that H2AK119ub1 deposition is essential to maintain PcG-target gene repression in embryonic stem cells (ESCs). Loss of H2AK119ub1 induced a rapid displacement of PRC2 activity and a loss of H3K27me3 deposition. This preferentially affected PRC2.2 variant with respect to PRC2.1, destabilizing canonical PRC1 activity. Finally, we found that variant PRC1 forms can sense H2AK119ub1 deposition, which contributes to their stabilization specifically at sites where this modification is highly enriched. Overall, our data place H2AK119ub1 deposition as a central hub that mounts PcG repressive machineries to preserve cell transcriptional identity.
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http://dx.doi.org/10.1016/j.molcel.2019.11.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033561PMC
February 2020

Loss of PRC1 activity in different stem cell compartments activates a common transcriptional program with cell type-dependent outcomes.

Sci Adv 2019 05 15;5(5):eaav1594. Epub 2019 May 15.

European Institute of Oncology-IRCCS, Department of Experimental Oncology, Milan, Italy.

Polycomb repressive complexes are evolutionarily conserved complexes that maintain transcriptional repression during development and differentiation to establish and preserve cell identity. We recently described the fundamental role of PRC1 in preserving intestinal stem cell identity through the inhibition of non-lineage-specific transcription factors. To further elucidate the role of PRC1 in adult stem cell maintenance, we now investigated its role in LGR5 hair follicle stem cells during regeneration. We show that PRC1 depletion severely affects hair regeneration and, different from intestinal stem cells, derepression of its targets induces the ectopic activation of an epidermal-specific program. Our data support a general role of PRC1 in preserving stem cell identity that is shared between different compartments. However, the final outcome of the ectopic activation of non-lineage-specific transcription factors observed upon loss of PRC1 is largely context-dependent and likely related to the transcription factors repertoire and specific epigenetic landscape of different cellular compartments.
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http://dx.doi.org/10.1126/sciadv.aav1594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520019PMC
May 2019

Functional Landscape of PCGF Proteins Reveals Both RING1A/B-Dependent-and RING1A/B-Independent-Specific Activities.

Mol Cell 2019 06 24;74(5):1037-1052.e7. Epub 2019 Apr 24.

IEO European Institute of Oncology IRCCS, Department of Experimental Oncology, Via Adamello 16, 20139 Milan, Italy; University of Milan, Department of Health Sciences, Via A. di Rudinì, 8, 20142 Milan, Italy. Electronic address:

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) control cell identity by establishing facultative heterochromatin repressive domains at common sets of target genes. PRC1, which deposits H2Aub1 through the E3 ligases RING1A/B, forms six biochemically distinct subcomplexes depending on the assembled PCGF protein (PCGF1-PCGF6); however, it is yet unclear whether these subcomplexes have also specific activities. Here we show that PCGF1 and PCGF2 largely compensate for each other, while other PCGF proteins have high levels of specificity for distinct target genes. PCGF2 associates with transcription repression, whereas PCGF3 and PCGF6 associate with actively transcribed genes. Notably, PCGF3 and PCGF6 complexes can assemble and be recruited to several active sites independently of RING1A/B activity (therefore, of PRC1). For chromatin recruitment, the PCGF6 complex requires the combinatorial activities of its MGA-MAX and E2F6-DP1 subunits, while PCGF3 requires an interaction with the USF1 DNA binding transcription factor.
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http://dx.doi.org/10.1016/j.molcel.2019.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561742PMC
June 2019

Dissecting the role of H3K27 acetylation and methylation in PRC2 mediated control of cellular identity.

Nat Commun 2019 04 11;10(1):1679. Epub 2019 Apr 11.

IEO European Institute of Oncology IRCCS, Department of Experimental Oncology, Via Adamello 16, 20139, Milan, Italy.

The Polycomb repressive complexes PRC1 and PRC2 act non-redundantly at target genes to maintain transcriptional programs and ensure cellular identity. PRC2 methylates lysine 27 on histone H3 (H3K27me), while PRC1 mono-ubiquitinates histone H2A at lysine 119 (H2Aub1). Here we present engineered mouse embryonic stem cells (ESCs) targeting the PRC2 subunits EZH1 and EZH2 to discriminate between contributions of distinct H3K27 methylation states and the presence of PRC2/1 at chromatin. We generate catalytically inactive EZH2 mutant ESCs, demonstrating that H3K27 methylation, but not recruitment to the chromatin, is essential for proper ESC differentiation. We further show that EZH1 activity is sufficient to maintain repression of Polycomb targets by depositing H3K27me2/3 and preserving PRC1 recruitment. This occurs in the presence of altered H3K27me1 deposition at actively transcribed genes and by a diffused hyperacetylation of chromatin that compromises ESC developmental potential. Overall, this work provides insights for the contribution of diffuse chromatin invasion by acetyltransferases in PRC2-dependent loss of developmental control.
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http://dx.doi.org/10.1038/s41467-019-09624-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459869PMC
April 2019

Increased Lactate Secretion by Cancer Cells Sustains Non-cell-autonomous Adaptive Resistance to MET and EGFR Targeted Therapies.

Cell Metab 2018 12 30;28(6):848-865.e6. Epub 2018 Aug 30.

Candiolo Cancer Institute - FPO, IRCCS, Strada Provinciale 142, Torino, Candiolo 10060, Italy; University of Torino, Department of Oncology, 10060, Torino, Candiolo, Italy. Electronic address:

The microenvironment influences cancer drug response and sustains resistance to therapies targeting receptor-tyrosine kinases. However, if and how the tumor microenvironment can be altered during treatment, contributing to resistance onset, is not known. We show that, under prolonged treatment with tyrosine kinase inhibitors (TKIs), EGFR- or MET-addicted cancer cells displayed a metabolic shift toward increased glycolysis and lactate production. We identified secreted lactate as the key molecule instructing cancer-associated fibroblasts to produce hepatocyte growth factor (HGF) in a nuclear factor κB-dependent manner. Increased HGF, activating MET-dependent signaling in cancer cells, sustained resistance to TKIs. Functional or pharmacological targeting of molecules involved in the lactate axis abrogated in vivo resistance, demonstrating the crucial role of this metabolite in the adaptive process. This adaptive resistance mechanism was observed in lung cancer patients progressed on EGFR TKIs, demonstrating the clinical relevance of our findings and opening novel scenarios in the challenge to drug resistance.
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http://dx.doi.org/10.1016/j.cmet.2018.08.006DOI Listing
December 2018

The H3K36me2 Methyltransferase Nsd1 Demarcates PRC2-Mediated H3K27me2 and H3K27me3 Domains in Embryonic Stem Cells.

Mol Cell 2018 04 29;70(2):371-379.e5. Epub 2018 Mar 29.

School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Dublin 4, Ireland. Electronic address:

The Polycomb repressor complex 2 (PRC2) is composed of the core subunits Ezh1/2, Suz12, and Eed, and it mediates all di- and tri-methylation of histone H3 at lysine 27 in higher eukaryotes. However, little is known about how the catalytic activity of PRC2 is regulated to demarcate H3K27me2 and H3K27me3 domains across the genome. To address this, we mapped the endogenous interactomes of Ezh2 and Suz12 in embryonic stem cells (ESCs), and we combined this with a functional screen for H3K27 methylation marks. We found that Nsd1-mediated H3K36me2 co-locates with H3K27me2, and its loss leads to genome-wide expansion of H3K27me3. These increases in H3K27me3 occurred at PRC2/PRC1 target genes and as de novo accumulation within what were previously broad H3K27me2 domains. Our data support a model in which Nsd1 is a key modulator of PRC2 function required for regulating the demarcation of genome-wide H3K27me2 and H3K27me3 domains in ESCs.
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http://dx.doi.org/10.1016/j.molcel.2018.02.027DOI Listing
April 2018

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

Circ Res 2018 01 12;122(2):231-245. Epub 2017 Dec 12.

From the IFOM, FIRC Institute of Molecular Oncology, Milan, Italy (M.F.M., C.G., M.C., F.P., E.D., A.T.); Department of Biomedicine, University of Basel, Switzerland (M.F.M.); Laboratory of Thermodynamics in Emerging Technologies, Department of Mechanical and Process Engineering, ETH Zurich, Switzerland (C.G.); Cogentech, Milan, Italy (F.P.); Department of Experimental Oncology (E.L., D.P.) and Unit of Gynecological Oncology Research (M.L., U.C.), European Institute of Oncology, Milan, Italy; Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Sweden (S.I.C., L.L.C., E.D.); Peptide Chemistry (N.O., D.J.), Structural Biology (S.K., R.G.), Experimental Histopathology (E.N.), Bioinformatics & Biostatistics Department (R.M.), and Immunity and Cancer Laboratory (D.P.C., A.T.), The Francis Crick Institute, London, United Kingdom; Center for Chemical Biology and Drug Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY (A.M., J.J.); and Department of Oncology and Hemato-Oncology, University of Milan, Italy (E.D.).

Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability.

Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization.

Methods And Results: We compared a VEC-null cell line with the same line reconstituted with wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including , vascular endothelial-protein tyrosine phosphatase (), and von Willebrand factor (). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of , , and . VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to , , and promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP.

Conclusions: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.312392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771688PMC
January 2018

Fam60a defines a variant Sin3a-Hdac complex in embryonic stem cells required for self-renewal.

EMBO J 2017 08 29;36(15):2216-2232. Epub 2017 May 29.

Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland

Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a-Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3-positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1-phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.
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http://dx.doi.org/10.15252/embj.201696307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538769PMC
August 2017

Focal adhesion kinase depletion reduces human hepatocellular carcinoma growth by repressing enhancer of zeste homolog 2.

Cell Death Differ 2017 05 24;24(5):889-902. Epub 2017 Mar 24.

Liver Research Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer in humans. The focal adhesion tyrosine kinase (FAK) is often over-expressed in human HCC and FAK inhibition may reduce HCC cell invasiveness. However, the anti-oncogenic effect of FAK knockdown in HCC cells remains to be clarified. We found that FAK depletion in HCC cells reduced in vitro and in vivo tumorigenicity, by inducing G2/M arrest and apoptosis, decreasing anchorage-independent growth, and modulating the expression of several cancer-related genes. Among these genes, we showed that FAK silencing decreased transcription and nuclear localization of enhancer of zeste homolog 2 (EZH2) and its tri-methylation activity on lysine 27 of histone H3 (H3K27me3). Accordingly, FAK, EZH2 and H3K27me3 were concomitantly upregulated in human HCCs compared to non-tumor livers. In vitro experiments demonstrated that FAK affected EZH2 expression and function by modulating, at least in part, p53 and E2F2/3 transcriptional activity. Moreover, FAK silencing downregulated both EZH2 binding and histone H3K27me3 levels at the promoter of its target gene NOTCH2. Finally, we found that pharmacological inhibition of FAK activity resembled these effects although milder. In summary, we demonstrate that FAK depletion reduces HCC cell growth by affecting cancer-promoting genes including the pro-oncogene EZH2. Furthermore, we unveil a novel unprecedented FAK/EZH2 crosstalk in HCC cells, thus identifying a targetable network paving the way for new anticancer therapies.
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http://dx.doi.org/10.1038/cdd.2017.34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423113PMC
May 2017

The Dual Role of EPOP and Elongin BC in Controlling Transcriptional Activity.

Mol Cell 2016 11;64(4):637-638

Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy. Electronic address:

Two studies published in this issue of Molecular Cell (Beringer et al., 2016; Liefke et al., 2016) characterize the novel interaction of EPOP with Elongin BC in regulating gene transcription at both H3K4me3-broad active and H3K27me3 Polycomb-repressed chromatin domains.
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http://dx.doi.org/10.1016/j.molcel.2016.11.009DOI Listing
November 2016

Control of adult intestinal identity by the Polycomb repressive machinery.

Cell Cycle 2017 02 15;16(3):243-244. Epub 2016 Nov 15.

a Dpt. of Experimental Oncology , IRCCS - Istituto Europeo di Oncologia , Milan , Italy.

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http://dx.doi.org/10.1080/15384101.2016.1252582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345241PMC
February 2017

PRC2 preserves intestinal progenitors and restricts secretory lineage commitment.

EMBO J 2016 11 1;35(21):2301-2314. Epub 2016 Sep 1.

Department of Experimental Oncology, European Institute of Oncology, Milan, Italy

Chromatin modifications shape cell heterogeneity by activating and repressing defined sets of genes involved in cell proliferation, differentiation and development. Polycomb-repressive complexes (PRCs) act synergistically during development and differentiation by maintaining transcriptional repression of common genes. PRC2 exerts this activity by catalysing H3K27 trimethylation. Here, we show that in the intestinal epithelium PRC2 is required to sustain progenitor cell proliferation and the correct balance between secretory and absorptive lineage differentiation programs. Using genetic models, we show that PRC2 activity is largely dispensable for intestinal stem cell maintenance but is strictly required for radiation-induced regeneration by preventing Cdkn2a transcription. Combining these models with genomewide molecular analysis, we further demonstrate that preferential accumulation of secretory cells does not result from impaired proliferation of progenitor cells induced by Cdkn2a activation but rather from direct regulation of transcription factors responsible for secretory lineage commitment. Overall, our data uncover a dual role of PRC2 in intestinal homeostasis highlighting the importance of this repressive layer in controlling cell plasticity and lineage choices in adult tissues.
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http://dx.doi.org/10.15252/embj.201694550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090213PMC
November 2016

Maintenance of leukemic cell identity by the activity of the Polycomb complex PRC1 in mice.

Sci Adv 2016 Oct 7;2(10):e1600972. Epub 2016 Oct 7.

Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.

Leukemia is a complex heterogeneous disease often driven by the expression of oncogenic fusion proteins with different molecular and biochemical properties. Whereas several fusion proteins induce leukemogenesis by activating gene expression (Hox-activating fusions), others impinge on different pathways that do not involve the activation of genes (non-Hox-activating fusions). It has been postulated that one of the main oncogenic properties of the HOXA9 transcription factor is its ability to control the expression of the tumor suppressor locus (), thereby compensating Polycomb-mediated repression, which is dispensable for leukemias induced by Hox-activating fusions. We show, by genetically depleting the H2A ubiquitin ligase subunits of the Polycomb repressive complex 1 (PRC1), Ring1a and Ring1b, that activation cannot repress expression in the absence of PRC1 and its dependent deposition of H2AK119 monoubiquitination (H2AK119Ub). This demonstrates the essential role of PRC1 activity in supporting the oncogenic potential of Hox-activating fusion proteins. By combining genetic tools with genome-wide location and transcription analyses, we further show that PRC1 activity is required for the leukemogenic potential of both Hox-activating and non-Hox-activating fusions, thus preventing the differentiation of leukemic cells independently of the expression of the locus. Overall, our results genetically demonstrate that PRC1 activity and the deposition of H2AK119Ub are critical factors that maintain the undifferentiated identity of cancer cells, positively sustaining the progression of different types of leukemia.
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http://dx.doi.org/10.1126/sciadv.1600972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055382PMC
October 2016

EpiMINE, a computational program for mining epigenomic data.

Epigenetics Chromatin 2016 29;9:42. Epub 2016 Sep 29.

Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy.

Background: In epigenetic research, both the increasing ease of high-throughput sequencing and a greater interest in genome-wide studies have resulted in an exponential flooding of epigenetic-related data in public domain. This creates an opportunity for exploring data outside the limits of any specific query-centred study. Such data have to undergo standard primary analyses that are accessible with multiple well-stabilized programs. Further downstream analyses, such as genome-wide comparative, correlative and quantitative analyses, are critical in deciphering key biological features. However, these analyses are only accessible for computational researchers and completely lack platforms capable of handling, analysing and linking multiple interdisciplinary datasets with efficient analytical methods.

Results: Here, we present EpiMINE, a program for mining epigenomic data. It is a user-friendly, stand-alone computational program designed to support multiple datasets, for performing genome-wide correlative and quantitative analysis of ChIP-seq and RNA-seq data. Using data available from the ENCODE project, we illustrated several features of EpiMINE through different biological scenarios to show how easy some known observations can be verified. These results highlight how these approaches can be helpful in identifying novel biological features.

Conclusions: EpiMINE performs different kinds of genome-wide quantitative and correlative analyses, using ChIP-seq- and RNA-seq-related datasets. Its framework enables it to be used by both experimental and computational researchers. EpiMINE can be downloaded from https://sourceforge.net/projects/epimine/.
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http://dx.doi.org/10.1186/s13072-016-0095-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043526PMC
January 2018

Mapping the Function of Polycomb Proteins.

Authors:
Diego Pasini

Methods Mol Biol 2016 ;1480:3-6

Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139, Milan, Italy.

Polycomb group (PcG) proteins are master regulators of proliferation and development that play essential roles in human pathologies including cancers. PcGs act as gatekeepers of cellular identity, maintaining repression of a multitude of target genes. However, these properties have only been recently uncovered thanks to technological advances, first of all chromatin immunoprecipitations (ChIP), that allowed a systematic characterization of the activity of these factors in an unbiased manner at a genome-wide level. Using PcG protein as example, this chapter introduces the readers to the use of chromatin analysis (ChIP assays and replication timing) and how to move these approaches to a level of genome-wide interpretation.
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http://dx.doi.org/10.1007/978-1-4939-6380-5_1DOI Listing
January 2018

Transcription factor TLX1 controls retinoic acid signaling to ensure spleen development.

J Clin Invest 2016 07 23;126(7):2452-64. Epub 2016 May 23.

The molecular mechanisms that underlie spleen development and congenital asplenia, a condition linked to increased risk of overwhelming infections, remain largely unknown. The transcription factor TLX1 controls cell fate specification and organ expansion during spleen development, and Tlx1 deletion causes asplenia in mice. Deregulation of TLX1 expression has recently been proposed in the pathogenesis of congenital asplenia in patients carrying mutations of the gene-encoding transcription factor SF-1. Herein, we have shown that TLX1-dependent regulation of retinoic acid (RA) metabolism is critical for spleen organogenesis. In a murine model, loss of Tlx1 during formation of the splenic anlage increased RA signaling by regulating several genes involved in RA metabolism. Uncontrolled RA activity resulted in premature differentiation of mesenchymal cells and reduced vasculogenesis of the splenic primordium. Pharmacological inhibition of RA signaling in Tlx1-deficient animals partially rescued the spleen defect. Finally, spleen growth was impaired in mice lacking either cytochrome P450 26B1 (Cyp26b1), which results in excess RA, or retinol dehydrogenase 10 (Rdh10), which results in RA deficiency. Together, these findings establish TLX1 as a critical regulator of RA metabolism and provide mechanistic insights into the molecular determinants of human congenital asplenia.
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http://dx.doi.org/10.1172/JCI82956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922703PMC
July 2016

Emerging roles for Polycomb proteins in cancer.

Curr Opin Genet Dev 2016 02 2;36:50-8. Epub 2016 May 2.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain; Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain; Institucio Catalana de Recerca i Estudis Avancats, Pg Lluis Companys 23, 08010 Barcelona, Spain. Electronic address:

The activities of the heterogeneous Polycomb (PcG) group of proteins ensure that the developmental processes of proliferation and cellular identity establishment are carried out correctly. PcG proteins assemble stable multiprotein complexes that, together with additional factors, maintain their target genes in a transcriptionally repressive state. The biochemical and functional features of PcG proteins have been extensively investigated over the years. Here we analyse the biochemical and mechanistic proprieties of PcG proteins with respect to recent advances that link the genetic alterations of PcG activity to cancer development.
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http://dx.doi.org/10.1016/j.gde.2016.03.013DOI Listing
February 2016

Polycomb Complex PRC1 Preserves Intestinal Stem Cell Identity by Sustaining Wnt/β-Catenin Transcriptional Activity.

Cell Stem Cell 2016 Jan 29;18(1):91-103. Epub 2015 Oct 29.

Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy. Electronic address:

Polycomb repressive complexes (PRCs) are among the most important gatekeepers of establishing and maintaining cell identity in metazoans. PRC1, which plays a dominant role in this context, executes its functions via multiple subcomplexes, which all contribute to H2AK119 mono-ubiquitination (H2Aubq). Despite our comprehensive knowledge of PRC1-dependent H2Aubq in embryonic stem cells and during early development, its role in adult stem cells still remains poorly characterized. Here we show that PRC1 activity is required for the integrity of the intestinal epithelium, regulating stem cell self-renewal via a cell-autonomous mechanism that is independent from Cdkn2a expression. By dissecting the PRC1-dependent transcription program in intestinal stem cells, we demonstrate that PRC1 represses a large number of non-lineage-specific transcription factors that directly affect β-catenin/Tcf transcriptional activity. Our data reveal that PRC1 preserves Wnt/β-catenin activity in adult stem cells to maintain intestinal homeostasis and supports tumor formation induced by the constitutive activation of this pathway.
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http://dx.doi.org/10.1016/j.stem.2015.09.019DOI Listing
January 2016
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