Universitą degli studi di Pavia


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Arrigoni research activity

Role of Sequence and Chromatin Elements in Origin Firing and Gene Silencing

Project 1: “Research of the Minimal Sequence Required for Replication Firing”
DNA replication initiates by recruiting the replication machinery to thousands of sites scattered on chromosomes, termed replication initiation sites or replication origins, in a highly organized spatiotemporal manner. Origin selection is mediated by the assembly of the pre-replication complex (pre-RC), which initiates with the binding of the Origin Recognition Complex (ORC) onto replication origins, bringing its completion at the end of G1 phase of cell cycle. Hence, replication origins are “licensed” to fire DNA replication, and then DNA polymerase is recruited (pre-initiation complex, pre-IC) to start the bidirectional DNA replication in early, middle or late S phase of cell cycle. Since the metazoan pre-RC exhibits virtually no sequence-specificity, is not fully understood what determine the targeting of metazoan ORC to specific replication origins, and how replication timing in early, middle and late-S phase is regulated, yet. A combination of many genetic and epigenetic factors such base composition (AT-rich elements), DNA topology, chromatin accessibility (Rampakakis et al, 2009), and the localization near of transcriptional regulatory elements (Cadoret et al, 2008) seem to define the “oris code”. Moreover, three-dimensional organization of chromatin in the nucleus seems to be implied in replication timing. Indeed, genomic regions of similar replication timing are clustered spatially in the nucleus (De & Michor, 2011), and the active chromatin located in the nuclear interior is preferentially replicated early. Hence, genetic and epigenetic-structural factors contribute to origin selection and early or late activation in the S-phase of cell cycle.
Our interest is focused on: 1- defining the minimal sequence of a human DNA origin, namely the Lamin B2 origin (LamB2-ori), required for triggering DNA replication; 2- identifying the proteins involved in origin recognition; 3- understanding the molecular process of the timing of origin firing by studying the chromatin topology and nuclear positioning of the sequence. LamB2-ori is located on the short arm of chromosome 19 between two transcriptional units in an open chromatin environment. Previously, the bidirectional start sites of LamB2-ori was mapped within a region of about 80 to 100bp region characterized by a cell-cycle dependent footprint whose sequence is bound by component of the preRC (Abdurashidova et al, 1998; 2003; 2000). We also provided evidence supporting a genetic process of origin selection by demonstrating the ectopic origin firing activity of a 1.2kb fragment isolated from the LamB2-ori region (Paixćo et al, 2004). To avoid positional effects by genomic sequences on the activity of the ectopic LamB2-ori randomly integrated into the host genome, we applied Recombinase Mediated Cassette Exchange (RMCE) to perform an extensive mutagenesis analysis of eLamB2-ori sequence integrated in a constant genomic site. The comparison of a panel of LamB2-ori mutants will allow to define the essential and the minimal requirement of the LamB2-ori to trigger DNA synthesis. Chromatin immunoprecipitation analysis (ChIP) will be also applied to identify the pre-RC proteins bound onto LamB2-ori mutants. Moreover, biochemical and pharmacological approaches will be carried out during the cell cycle to understand the role of chromatin topology and three-dimensional structure of the nucleus on the regulation of replication timing.

Project 2: “Defining the Sequence Elements of the Human LamB2-ori Replicator Involved in the Prevention of Gene Silencing”
Gene silencing is an epigenetic phenomenon that bring to the “switching off” of the expression of both transgenes and endogenous genes. This phenomenon generates from the heterochromatinization of a selected genomic region, by DNA methylation and histone deacetylation and insertion of repressive chromatin marks on the histone tails. These modifications bring to the recruitment of specific protein complexes involved in gene expression and chromatin compaction. Gene silencing is a natural process that exerts its role in differentiation and tumor progression. Moreover, it is one of the mayor impediments in gene therapy and expression of recombinant protein in eukaryotic cell lines. Hence, there is an increasing interest in the identification of factors and/or DNA sequence that are required for the prevention of gene silencing. Scaffold/Matrix Attachment Regions (S/MARs) (Allen et al, 2000), CpG islands of housekeeping genes (Williams et al, 2005) as well as replication origins (Fu et al, 2006) were used to to prevent gene silencing of transgene including therapeutic genes.
In this project we intend to investigate the role of the LamB2-ori in preventing gene silencing by assaying a set of origin mutants for the ability to sustain the expression of a reporter gene.

Abdurashidova G, Danailov MB, Ochem A, Triolo G, Djeliova V, Radulescu S, Vindigni A, Riva S & Falaschi A (2003) Localization of proteins bound to a replication origin of human DNA along the cell cycle. EMBO J 22: 4294–4303
Abdurashidova G, Deganuto M, Klima R, Riva S, Biamonti G, Giacca M & Falaschi A (2000) Start sites of bidirectional DNA synthesis at the human lamin B2 origin. Science 287: 2023–2026
Abdurashidova G, Riva S, Biamonti G, Giacca M & Falaschi A (1998) Cell cycle modulation of protein-DNA interactions at a human replication origin. EMBO J 17: 2961–2969
Allen GC, Spiker S & Thompson WF (2000) Use of matrix attachment regions (MARs) to minimize transgene silencing. Plant Mol Biol 43: 361–376
Cadoret J-C, Meisch F, Hassan-Zadeh V, Luyten I, Guillet C, Duret L, Quesneville H & Prioleau M-N (2008) Genome-wide studies highlight indirect links between human replication origins and gene regulation. Proc Natl Acad Sci USA 105: 15837–15842
De S & Michor F (2011) DNA replication timing and long-range DNA interactions predict mutational landscapes of cancer genomes. Nat Biotechnol
Fu H, Wang L, Lin C-M, Singhania S, Bouhassira EE & Aladjem MI (2006) Preventing gene silencing with human replicators. Nat Biotechnol 24: 572–576
Paixćo S, Colaluca IN, Cubells M, Peverali FA, Destro A, Giadrossi S, Giacca M, Falaschi A, Riva S & Biamonti G (2004) Modular structure of the human lamin B2 replicator. Mol Cell Biol 24: 2958–2967
Rampakakis E, Arvanitis DN, Di Paola D & Zannis-Hadjopoulos M (2009) Metazoan origins of DNA replication: regulation through dynamic chromatin structure. J Cell Biochem 106: 512–520
Williams S, Mustoe T, Mulcahy T, Griffiths M, Simpson D, Antoniou M, Irvine A, Mountain A & Crombie R (2005) CpG-island fragments from the HNRPA2B1/CBX3 genomic locus reduce silencing and enhance transgene expression from the hCMV promoter/enhancer in mammalian cells. BMC Biotechnol 5: 17
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