Our group previously demonstrated that when a DNA double-strand break (DSB) occurs, RNA polymerase II (RNAPII) is recruited to the exposed DNA ends and allows the by-directional synthesis of transcripts derived from exposed DNA ends that we called damage-induced long non-coding RNAs (dilncRNAs). Recently we reported that a DSB also recruits the full transcriptional machinery, in particular the preinitiation complex (PIC), just as commonly recruited at canonical RNA polymerase II-driven transcriptional promoters. This, blurs the distinction between a DSB and a transcriptional promoter. dilncRNAs were defined as non-coding as most of our genome is not coding for polypeptides and thus, DSB will most often transcribe non coding regions of DNA. However, what would be the consequences of a DSB occurring upstream of a gene unit, lacking a promoter but carrying all other features of a coding sequence, such as an open reading frame (ORF) and a poly(A) signal? Would the ensuing dilncRNA actually be a coding transcript? In other words: can a DSB trigger the synthesis of a protein? Therefore, the aim of this project is to test if a DSB appropriately positioned at the transcriptional start site (TSS) of an otherwise silent gene may assemble a functional promoter, triggering RNA synthesis which leads to protein expression. I have carried out such studies in artificial cell systems in which I have engineered promoter-less reporter genes. In addition, I have tested whether a DSB is sufficient to re-express a candidate tumour suppressor gene, HIC-1 (hyper-methylated in cancer 1), that is commonly silenced by promoter methylation. In more in detail, I tested my working hypothesis in three different systems. As a proof of concept, I first used two reporter gene-based systems: a stable clonal cell line of HeLa cells bearing an integrated lentiviral construct encoding for an enhanced green fluorescent protein (EGFP) ORF lacking its transcriptional promoter and enhancer region, and an immortalized mouse embryonic fibroblast (MEF) cell line carrying a promoter-less enhanced yellow fluorescent protein (EYFP) integrated by homologous recombination in the locus Rosa26 of the mouse genome. To induce sites-specific DSBs, I employed the CRISPR/Cas9 technology. Single guide RNAs targeting the GFP or EYFP transcriptional start site (TSS) were cloned in a lentiviral vector and cells were transduced with such a vector expressing them together with Cas9 to test the impact of DSB induction on the expression of the reporter gene. The second system takes advantage of MDA MB 231, a human breast cancer cell line, in which HIC1 endogenous gene is actively silenced by promoter DNA methylation. The goal, here, is to infect these cells with a CRISPR/Cas9 lentiviral vector to induce a DSB at the TSS of HIC1 gene and monitor the synthesis of a mature messenger RNA. These experiments suggested that a DSB, appropriately positioned at a gene TSS, triggers the transcription of RNAs that are polyadenylated, exported in the cytoplasm and translated into a functional protein. This study could be relevant to identify a novel mechanism of transcriptional regulation based on DSB at promoters. This approach could also be used to activate gene expression in genetic disease settings, or to activate the expression of tumor suppressor genes that are silenced in tumors.

CAN A PRECISELY-POSITIONED DNA DOUBLE-STRAND BREAK (DSB) ACTIVATE GENE EXPRESSION? / A. Di Lillo ; tutor: F. Nicassio, F. d’Adda di Fagagna : phd coordinator: S. Minucci. - : . Dipartimento di Oncologia ed Emato-Oncologia, 2021 Dec 13. ((32. ciclo, Anno Accademico 2020.

CAN A PRECISELY-POSITIONED DNA DOUBLE-STRAND BREAK (DSB) ACTIVATE GENE EXPRESSION?

A. DI LILLO
2021

Abstract

Our group previously demonstrated that when a DNA double-strand break (DSB) occurs, RNA polymerase II (RNAPII) is recruited to the exposed DNA ends and allows the by-directional synthesis of transcripts derived from exposed DNA ends that we called damage-induced long non-coding RNAs (dilncRNAs). Recently we reported that a DSB also recruits the full transcriptional machinery, in particular the preinitiation complex (PIC), just as commonly recruited at canonical RNA polymerase II-driven transcriptional promoters. This, blurs the distinction between a DSB and a transcriptional promoter. dilncRNAs were defined as non-coding as most of our genome is not coding for polypeptides and thus, DSB will most often transcribe non coding regions of DNA. However, what would be the consequences of a DSB occurring upstream of a gene unit, lacking a promoter but carrying all other features of a coding sequence, such as an open reading frame (ORF) and a poly(A) signal? Would the ensuing dilncRNA actually be a coding transcript? In other words: can a DSB trigger the synthesis of a protein? Therefore, the aim of this project is to test if a DSB appropriately positioned at the transcriptional start site (TSS) of an otherwise silent gene may assemble a functional promoter, triggering RNA synthesis which leads to protein expression. I have carried out such studies in artificial cell systems in which I have engineered promoter-less reporter genes. In addition, I have tested whether a DSB is sufficient to re-express a candidate tumour suppressor gene, HIC-1 (hyper-methylated in cancer 1), that is commonly silenced by promoter methylation. In more in detail, I tested my working hypothesis in three different systems. As a proof of concept, I first used two reporter gene-based systems: a stable clonal cell line of HeLa cells bearing an integrated lentiviral construct encoding for an enhanced green fluorescent protein (EGFP) ORF lacking its transcriptional promoter and enhancer region, and an immortalized mouse embryonic fibroblast (MEF) cell line carrying a promoter-less enhanced yellow fluorescent protein (EYFP) integrated by homologous recombination in the locus Rosa26 of the mouse genome. To induce sites-specific DSBs, I employed the CRISPR/Cas9 technology. Single guide RNAs targeting the GFP or EYFP transcriptional start site (TSS) were cloned in a lentiviral vector and cells were transduced with such a vector expressing them together with Cas9 to test the impact of DSB induction on the expression of the reporter gene. The second system takes advantage of MDA MB 231, a human breast cancer cell line, in which HIC1 endogenous gene is actively silenced by promoter DNA methylation. The goal, here, is to infect these cells with a CRISPR/Cas9 lentiviral vector to induce a DSB at the TSS of HIC1 gene and monitor the synthesis of a mature messenger RNA. These experiments suggested that a DSB, appropriately positioned at a gene TSS, triggers the transcription of RNAs that are polyadenylated, exported in the cytoplasm and translated into a functional protein. This study could be relevant to identify a novel mechanism of transcriptional regulation based on DSB at promoters. This approach could also be used to activate gene expression in genetic disease settings, or to activate the expression of tumor suppressor genes that are silenced in tumors.
D'ADDA DI FAGAGNA, FABRIZIO
FOIANI, MARCO
DNA damage; transcription; CRISPR/Cas9
Settore BIO/11 - Biologia Molecolare
CAN A PRECISELY-POSITIONED DNA DOUBLE-STRAND BREAK (DSB) ACTIVATE GENE EXPRESSION? / A. Di Lillo ; tutor: F. Nicassio, F. d’Adda di Fagagna : phd coordinator: S. Minucci. - : . Dipartimento di Oncologia ed Emato-Oncologia, 2021 Dec 13. ((32. ciclo, Anno Accademico 2020.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/884393
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