Publications by authors named "Giulia Della Chiara"

11 Publications

  • Page 1 of 1

Epigenomic landscape of human colorectal cancer unveils an aberrant core of pan-cancer enhancers orchestrated by YAP/TAZ.

Nat Commun 2021 04 20;12(1):2340. Epub 2021 Apr 20.

Human Organoid Models Integrative Center HOMIC, University of Milan, Milan, Italy.

Cancer is characterized by pervasive epigenetic alterations with enhancer dysfunction orchestrating the aberrant cancer transcriptional programs and transcriptional dependencies. Here, we epigenetically characterize human colorectal cancer (CRC) using de novo chromatin state discovery on a library of different patient-derived organoids. By exploring this resource, we unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic and independent of interpatient heterogeneity. We show that the transcriptional coactivators YAP/TAZ act as key regulators of the conserved CRC gained enhancers. The same YAP/TAZ-bound enhancers display active chromatin profiles across diverse human tumors, highlighting a pan-cancer epigenetic rewiring which at single-cell level distinguishes malignant from normal cell populations. YAP/TAZ inhibition in established tumor organoids causes extensive cell death unveiling their essential role in tumor maintenance. This work indicates a common layer of YAP/TAZ-fueled enhancer reprogramming that is key for the cancer cell state and can be exploited for the development of improved therapeutic avenues.
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http://dx.doi.org/10.1038/s41467-021-22544-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058065PMC
April 2021

TET2 Regulates Mast Cell Differentiation and Proliferation through Catalytic and Non-catalytic Activities.

Cell Rep 2016 05 5;15(7):1566-1579. Epub 2016 May 5.

Institute for Research in Biomedicine, Universita' della Svizzera italiana (USI), 6500 Bellinzona, Switzerland. Electronic address:

Dioxygenases of the TET family impact genome functions by converting 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC). Here, we identified TET2 as a crucial regulator of mast cell differentiation and proliferation. In the absence of TET2, mast cells showed disrupted gene expression and altered genome-wide 5hmC deposition, especially at enhancers and in the proximity of downregulated genes. Impaired differentiation of Tet2-ablated cells could be relieved or further exacerbated by modulating the activity of other TET family members, and mechanistically it could be linked to the dysregulated expression of C/EBP family transcription factors. Conversely, the marked increase in proliferation induced by the loss of TET2 could be rescued exclusively by re-expression of wild-type or catalytically inactive TET2. Our data indicate that, in the absence of TET2, mast cell differentiation is under the control of compensatory mechanisms mediated by other TET family members, while proliferation is strictly dependent on TET2 expression.
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http://dx.doi.org/10.1016/j.celrep.2016.04.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584687PMC
May 2016

Transcription of Mammalian cis-Regulatory Elements Is Restrained by Actively Enforced Early Termination.

Mol Cell 2015 Nov 22;60(3):460-74. Epub 2015 Oct 22.

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

Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adaptor protein WDR82 and its associated complexes actively limit such non-coding transcription. WDR82 targets the SET1 H3K4 methyltransferases and the nuclear protein phosphatase 1 (PP1) complexes to the initiating Pol II. WDR82 and PP1 also interact with components of the transcriptional termination and RNA processing machineries. Depletion of WDR82, SET1, or the PP1 subunit required for its nuclear import caused distinct but overlapping transcription termination defects at highly expressed genes and active enhancers and promoters, thus enabling the increased synthesis of unusually long ncRNAs. These data indicate that transcription initiated from cis-regulatory elements is tightly coordinated with termination mechanisms that impose the synthesis of short RNAs.
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http://dx.doi.org/10.1016/j.molcel.2015.09.018DOI Listing
November 2015

M1 polarization of human monocyte-derived macrophages restricts pre and postintegration steps of HIV-1 replication.

AIDS 2013 Jul;27(12):1847-56

Objective: Functional polarization of human monocyte-derived macrophages (MDMs) into M1 cells leads to inhibition of R5 HIV-1 replication and viral DNA synthesis in comparison to control, unpolarized cells together with CD4 downregulation from the cell surface and upregulation of CCR5-binding chemokine secretion. We here investigated whether a postentry restriction of virus replication is also induced by M1 polarization of MDM.

Design: MDM were first polarized to M1 cells by 18 h stimulation with interferon-[gamma] and tumor necrosis factor-[alpha]; the cytokines were then removed and the cells were infected with vesicular stomatitis virus G-protein pseudotyped enhanced green fluorescence protein HIV-1 (HIV-GFP) generating a single-round infection cycle.

Methods: HIV-1 expression was monitored in terms of eGFP expression by fluorescence activated cell sorter (FACS) analysis and real-time PCR analysis of total HIV-1 gag DNA, 2-long terminal repeat DNA, proviral DNA, and multiply spliced RNA transcripts. Expression of apolipopoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G), and APOBEC3A was tested by western blotting and FACS analysis.

Results: Inhibition of HIV-GFP expression was observed in M1-MDM along with impaired viral DNA synthesis, delayed proviral integration, and reduced proviral transcription. Although APOBEC3G levels were similar in M1 and unpolarized MDM, APOBEC 3A was selectively expressed only by M1 cells.

Conclusion: M1 polarization of in-vitro differentiated primary MDM determines a transient, but profound restriction of HIV-1 replication affecting multiple (entry and postentry) steps in the virus life cycle likely involving the upregulated expression of APOBEC3A.
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http://dx.doi.org/10.1097/QAD.0b013e328361d059DOI Listing
July 2013

Passport control for foreign integrated DNAs: An unexpected checkpoint by class II HDAC4 revealed by amino acid starvation.

Mob Genet Elements 2012 Sep;2(5):233-238

Center for Translational Genomics and Bioinformatics; San Raffaele Scientific Institute; Milan, Italy.

The endless battle between mammalian host cells and microbes has evolved mechanisms to shut down the expression of exogenous transcriptional units integrated into the genome with the goal of limiting their spreading. Recently, we observed that deprivation of essential amino acids leads to a selective, reversible upregulation of expression of exogenous transgenes, either carried by integrated plasmids or retroviral vectors, but not of their endogenous counterparts. This effect was dependent on epigenetic modifications and was mediated by the downregulation of the class II histone deacetylase-4 (HDAC4). Indeed, HDAC4 expression inversely correlated with that of the transgene and its inhibition or downregulation enhanced transgene expression. Could this be true also for "naturally" integrated proviruses? We investigated this question in the case of HIV-1, the etiological agent of AIDS and we observed that both amino acid starvation and HDAC4 inhibition triggered HIV-1 reactivation in chronically infected ACH-2 T lymphocytic cells (HDAC4), but not in similarly infected U1 promonocytic cells (HDAC4-negative). Thus, an HDAC4-dependent pathway may contribute to unleash virus expression by latently infected cells, which represent nowadays a major obstacle to HIV eradication. We discuss here the implications and open questions of these novel findings, as well as their serendipitous prelude.
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http://dx.doi.org/10.4161/mge.22610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575431PMC
September 2012

Amino acid starvation induces reactivation of silenced transgenes and latent HIV-1 provirus via down-regulation of histone deacetylase 4 (HDAC4).

Proc Natl Acad Sci U S A 2012 Aug 23;109(34):E2284-93. Epub 2012 Jul 23.

Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Ospedale San Raffaele, 20132 Milan, Italy.

The epigenetic silencing of exogenous transcriptional units integrated into the genome represents a critical problem both for long-term gene therapy efficacy and for the eradication of latent viral infections. We report here that limitation of essential amino acids, such as methionine and cysteine, causes selective up-regulation of exogenous transgene expression in mammalian cells. Prolonged amino acid deprivation led to significant and reversible increase in the expression levels of stably integrated transgenes transcribed by means of viral or human promoters in HeLa cells. This phenomenon was mediated by epigenetic chromatin modifications, because histone deacetylase (HDAC) inhibitors reproduced starvation-induced transgene up-regulation, and transcriptome analysis, ChIP, and pharmacological and RNAi approaches revealed that a specific class II HDAC, namely HDAC4, plays a critical role in maintaining the silencing of exogenous transgenes. This mechanism was also operational in cells chronically infected with HIV-1, the etiological agent of AIDS, in a latency state. Indeed, both amino acid starvation and pharmacological inhibition of HDAC4 promoted reactivation of HIV-1 transcription and reverse transcriptase activity production in HDAC4(+) ACH-2 T-lymphocytic cells but not in HDAC4(-) U1 promonocytic cells. Thus, amino acid deprivation leads to transcriptional derepression of silenced transgenes, including integrated plasmids and retroviruses, by a process involving inactivation or down-regulation of HDAC4. These findings suggest that selective targeting of HDAC4 might represent a unique strategy for modulating the expression of therapeutic viral vectors, as well as that of integrated HIV-1 proviruses in latent reservoirs without significant cytotoxicity.
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http://dx.doi.org/10.1073/pnas.1202174109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427085PMC
August 2012

Negative regulation of HIV-1 transcription by a heterodimeric NF-κB1/p50 and C-terminally truncated STAT5 complex.

J Mol Biol 2011 Jul;410(5):933-43

AIDS Immunopathogenesis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, 20132 Milano, Italy.

Signal transducers and activator of transcription (STAT) proteins are often constitutively activated in leukocytes of HIV-1(+) individuals, which frequently show a dominant expression of a C-terminally truncated isoform of STAT5 (STAT5Δ). STAT5Δ can act as a negative regulator of human immunodeficiency virus type 1 (HIV-1) expression in both CD8-depleted primary leukocytes and chronically infected promonocytic U1 cells stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF). Activated STAT5Δ can directly bind to two consensus sequences in the HIV-1 long terminal repeat (LTR) promoter; binding impairs recruitment of RNA polymerase II (Crotti, A., Lusic, M., Lupo, R., Lievens, P. M., Liboi, E., Della Chiara, G., et al. (2007). Naturally occurring C-terminally truncated STAT5 is a negative regulator of HIV-1 expression. Blood, 109, 5380-5389). One of the STAT consensus sequences overlaps with one nuclear factor κB (NF-κB) binding site; interestingly, NF-κB1/p50 homodimers, frequently detected in monocytic cells, are negative regulators of HIV transcription. Here, we show that GM-CSF stimulation of U1 cells, while not inducing NF-κB activation, leads to STAT5Δ phosphorylation and binding to the NF-κB/STAT target sequence in the HIV LTR promoter, which already associates with p50 under unstimulated conditions. STAT5Δ was found to associate with p50, but not with RelA/p65, in both U1 cells expressing endogenous proteins and 293T cells overexpressing these factors. Furthermore, GM-CSF stimulation promoted concurrent binding of STAT5Δ and p50 at the HIV LTR promoter in U1 cells. Immunoprecipitation of chromatin from GM-CSF-stimulated U1 cells confirmed in vivo binding of p50 to the viral promoter together with STAT5Δ. Thus, cytokine-activated STAT5Δ/p50 complexes can contribute to the maintenance of HIV-1 latency in monocytic cells.
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http://dx.doi.org/10.1016/j.jmb.2011.03.044DOI Listing
July 2011

The rise and fall of intermittent interleukin-2 therapy in HIV infection.

Eur Cytokine Netw 2010 Sep 23;21(3):197-201. Epub 2010 Aug 23.

AIDS Immunopathogenesis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy.

In 1995, a breakthrough paper showed that intermittent cycles of interleukin-2 (IL-2), together with suboptimal ART, caused an unprecedented, stable increase in CD4+ T cell counts, without altering the steady state levels of viremia. At the time, this was somewhat obscured by the first successes of combination antiretroviral therapy (cART). However, since then, numerous studies have confirmed this basic finding, opening up a new perspective in the long-term management of chronic HIV infection. One of the benchmarks of this experimental treatment is the expansion of CD4+CD25+ T lymphocytes probably including T regulatory cells (Tregs). Based on these encouraging findings, two major phase III clinical trials, ESPRIT and SILCAAT, involving thousands of patients worldwide, were launched and continued over several years. Unfortunately, they both resulted in the highly unexpected, yet unequivocal, outcome of a lack of a protective effect of IL-2-expanded CD4+ T cells on HIV disease progression towards the acquired immunodeficiency syndrome (AIDS) or death. In addition, there was the suggestion of an increase in certain deleterious effects on treated patients in terms of cardiovascular and inflammatory events. While IL-2 therapy is unlikely to be studied any further in the context of HIV infection, other cytokines, such as IL-7, are still being tested in the hope of more promising results.
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http://dx.doi.org/10.1684/ecn.2010.0199DOI Listing
September 2010

Naturally occurring C-terminally truncated STAT5 is a negative regulator of HIV-1 expression.

Blood 2007 Jun 1;109(12):5380-9. Epub 2007 Mar 1.

AIDS Immunopathogenesis Unit and the Division of Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy.

CD4(+) cells of most individuals infected with HIV-1 harbor a C-terminally truncated and constitutively activated form of signal transducer and activator of transcription-5 (STAT5 Delta). We report that the chronically HIV-infected U1 cell line expresses STAT5 Delta but not full-length STAT5. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of U1 cells promoted early activation of STAT5 Delta and of extracellular signal regulated kinases (ERKs), followed by later activation of activator protein 1 (AP-1) and HIV expression. Inhibition of ERK/AP-1 by PD98,059 abolished, whereas either tyrphostin AG490 or a STAT5 small interfering RNA (siRNA) enhanced, virion production in GM-CSF-stimulated U1 cells. Chromatin immunoprecipitation demonstrated the induction of STAT5 Delta binding to STAT consensus sequences in the HIV-1 promoter together with a decreased recruitment of RNA polymerase II after 1 hour of GM-CSF stimulation of U1 cells. Down-regulation of STAT5 Delta by siRNA resulted in the up-regulation of both HIV-1 gag-pol RNA and p24 Gag antigen expression in CD8-depleted leukocytes of several HIV-positive individuals cultivated ex vivo in the presence of interleukin-2 but not of interleukin-7. Thus, the constitutively activated STAT5 Delta present in the leukocytes of most HIV-positive individuals acts as a negative regulator of HIV expression.
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http://dx.doi.org/10.1182/blood-2006-08-042556DOI Listing
June 2007

In vitro inhibition of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) expression and leukemogenic activity by DNA/LNA chimeric antisense oligos.

Oncol Res 2005 ;16(3):157-66

Centre of Biotechnology, University of Urbino, Fano, Italy.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.
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http://dx.doi.org/10.3727/000000006783981152DOI Listing
December 2006