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Nuclear proteomics to investigate multi-layered gene expression regulation

Director

Tiziana Bonaldi

HIGHLIGHTS

 

We are part of the LIBRA  Consortium (http://www.eu-libra.eu), a Coordination and Support Action project funded by the European Commission’s framework programme H2020 to boost gender equality in research.

Activities

The long-standing goal of the research carried out in the group is to investigate gene expression regulation, at different levels. Historically our research has focused on the epigenetic regulation of gene expression mediated by histone post-translational modifications (hPTMs) and variants. By using mass-spectrometry (MS) technology, we study: the combinatorial aspects of hPTMs at the level of single histone, mono- or poly-nucleosomes, up to small chromatin regions; the in vivo action of histonemodifying enzymes and the quantitative aspects related to modification dynamics. In addition, we are extending the analysis of methylations of non-histonic proteins, to explore the regulatory networks mediated by this modification in the cell. More recently, with the advent of quantitative proteomics that allows accurate and large-scale protein expression profiling, we appreciated the potential of this technology to examine the post-transcriptional events regulating gene expression, with a focus on micro-RNA mediated translational inhibition. Our research elaborates on this observation developing novel strategies to study these processes, globally. One quality of the team is the ambition to design and apply innovative and unconventional approaches to investigate various aspects of gene expression, in order to gain original perspectives and contribute new concepts to the field.

  • Research projects

    Chromatomics: proteomics of chromatin domains by ChroP approach
    Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation.
    Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. We have optimized and published a modified protocol for chromatin immunoprecipitation, combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture, to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics (ChroP) strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH (Soldi and Bonaldi, 2013).
    On-going work: we are developing the ChroP 2.0 approach, namely using ChroP to characterize enhancers and TSSs of inflammatory genes, at basal level and upon LPS stimulus, using H3K4me3 to enrich TSSs and H3K4m1 and PU.1 for enhancers. The comparison of distinct inflammatory stimuli will allow identifying stimulus- specific determinants.

    Deciphering the code of protein methylation by MS-Proteomics
    Protein methylation is a post-translational modification by which a variable number of methyl groups is transferred to Lysine and Arginine residues within proteins. Despite increased interest in this modification due to its reversible nature and its emerging role in a diverse set of biological pathways beyond chromatin, global identification of protein methylation has remained an unachieved goal. To characterise sites of Lysine and Arginine methylation globally, we employed an approach that combines heavy methyl stable isotope labelling by amino acids in cell culture (hmSILAC) with high-resolution mass spectrometry-based proteomics.
    Through a broad evaluation of immuno-affinity enrichment and the application of two classical protein separation techniques prior to mass spectrometry, to nuclear and cytosolic fractions separately, we identified a total of 501 different methylation types, on 370 distinct Lysine and Arginine sites, present on 127 unique proteins. Our results considerably extend the number of known in vivo methylation sites and indicate their significant presence on several protein complexes involved at all stages of gene expression (Bremang et al. in press in Mol. BioSystems).
    On-going work: while optimizing the analytical methods to extend the annotation of the methylome, we are investigating: the role of methylation in regulating the Microprocessor complex and the modulation of the methylome upon PRTM5 and PRMT7knock-out, in collaboration with E. Guccione at IMCB.

    Identification of miR17-92 targets in myc- B cell lymphoma by qProteomics
    The functional effect of microRNA activity depends on its molecular environment: the same miRNA may play different roles at different stages of any particular biological process, including tumorigenesis, on the basis of the changing gene expression landscape. An oncogenic role of miR-17-19b, a truncated version of the miR-17-92 cluster, has been previously associated to B-cell lymphoma. In this project, we aim at elucidating the contribution of the cluster to the maintenance of the malignant phenotype, using an established MYCdependent B-cell lymphoma as a model.
    We found that enforced expression of the cluster impaired lymphoma growth, both in vitro and in vivo, as a consequence of decreased tumor proliferation and increased apoptosis, indicating that in MYC-dependent cancer cells, miR-17-19b represses MYC function. Thus, we used a systems biology approach to dissect the gene expression regulatory network mediated by miR-17-19b: SILAC-proteomics led to the identification of over 200 novel targets of miR-17-19b. Analysis of these targets revealed that 40% are under the transcriptional control of the MYC oncogene, uncovering a widespread silencing effect operated by these miRNAs on the MYC-centered regulatory network (Mihailovic et al., in preparation).
    On-going work: among the identified miR targets we found E2F1 and Chek-2, transcriptional and translational regulator of MYC, respectively. We are assessing these two aspects of MYC regulation. In particular, MYC translational repression via suppression of Chek-2 represents a completely original mode of miR17-92 action.

    Chemical proteomics to identify targets of anticancer drugs
    A key step during drug discovery and development is the identification of drug’s targets, crucial to define drug selectivity and for the early detection of possible side effects. Target identification is conventionally achieved by screening in vitro a number of selected substrates. Chemical proteomics is an alternative strategy, where whole proteomes are screened by drug affinity chromatography to detect interactors, possibly in physiological-like conditions mimicking the cellular environment. In the last decade, quantitative proteomic strategies based on stable isotope labelling allowed the development of a more advanced version of chemical proteomics.

    - Target deconvolution for the multi-kinase inhibitor E-3810:
    different SILAC experiments have been designed to confidently identify putative targets of E-3810 in cancer cells lysates. We have identified a set of E-3810 interactors and estimated their Kd and IC50. While in vitro screening initially performed on E-3810 identified VEGRs and FGFRs as primary targets, our analysis identifies a broader set of interactors, with novel information to define the mechanism of action of the compount and to understand possible sideeffects (Colzani et al., in preparation)

    - On-going: the mPTP dissected by chemical proteomics:
    mitochondrial permeability transition is a process characterized by a sudden increase in inner mitochondrial membrane permeability, which represents a crucial step in cell death and is involved in various pathological processes, such as ischemia/ riperfusion injuries and neurodegenerative diseases. Mitochondrial permeability transition is mediated by the opening of a pore located in the inner mitochondrial membrane, known as the mitochondrial permeability transition pore (mPTP), whose structural nature remains controversial.
    Inhibitors targeting this pore may have important therapeutic applications. At the DDU a set of acrylamido derivatives is available, which inhibit the mitochondrial permeability transition, in vitro and in vivo. We are using chemical proteomics to analyze the proteins isolated from mitochondrial extracts in pull-down experiments using acrylamido derivative, coupled to beads. The IC50 and KD values calculated will identify the specific interactors of the compounds, thus leading to the biochemical characterization of the mPTP.

  • Publications

    • Soldi M, Cuomo A, Bremang M, Bonaldi T. Mass spectrometry-based proteomics for the analysis of chromatin structure and dynamics. Int J Mol Sci. 2013 Mar 6;14(3):5402-31. doi: 10.3390/ijms14035402.
    • Vella P, Scelfo A, Jammula S, Chiacchiera F, Williams K, Cuomo A, Roberto A, Christensen J, Bonaldi T, Helin K, Pasini D. Tet Proteins Connect the O-Linked N-acetylglucosamine Transferase Ogt to Chromatin in Embryonic Stem Cells. Mol Cell. 2013 Feb 21;49(4):645- 56. doi: 10.1016/j.molcel.2012.12.019. Epub 2013 Jan 24.
    • Soldi M, Bonaldi T. The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components. Mol Cell Proteomics. 2013 Mar;12(3):764-80. doi: 10.1074/mcp. M112.024307. Epub 2013 Jan 14.
    • Cuomo A, Moretti S, Minucci S, Bonaldi T. SILAC-based proteomic analysis to dissect the “histone modification signature” of human breast cancer cells. Amino Acids. 2011 Jul;41(2):387-99. doi: 10.1007/s00726-010-0668-2. Epub 2010 Jul 9.
    • Bonaldi T, Straub T, Cox J, Kumar C, Becker PB, Mann M. Combined use of RNAi and quantitative proteomics to study gene function in Drosophila. Mol Cell. 2008 Sep 5;31(5):762-72. doi: 10.1016/j.molcel.2008.07.018.

UN PICCOLO GESTO E' PER NOI UN GRANDE AIUTO

PARTNERSHIP

Università degli Studi di Milano Ecancer Medical Science IFOM-IEO Campus

CREDITS

Ministero della Salute Joint Commission International Breastcertification bollinirosa

© 2013 Istituto Europeo di Oncologia - via Ripamonti 435 Milano - P.I. 08691440153

IRCCS - ISTITUTO DI RICOVERO E CURA A CARATTERE SCIENTIFICO

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