Separase prevents genomic instability by controlling replication fork speed

Proper chromosome segregation is crucial for preserving genomic integrity, and errors in this process cause chromosome mis-segregation, which may contribute to cancer development. Sister chromatid separation is triggered by Separase, an evolutionary conserved protease that cleaves the cohesin complex, allowing the dissolution of sister chromatid cohesion. Here we provide evidence that Separase participates in genomic stability maintenance by controlling replication fork speed. We found that Separase interacted with the replication licensing factors MCM2-7, and genome-wide data showed that Separase co-localized with MCM complex and cohesin. Unexpectedly, the depletion of Separase increased the fork velocity about 1.5-fold and caused a strong acetylation of cohesin's SMC3 subunit and altered checkpoint response. Notably, Separase silencing triggered genomic instability in both HeLa and human primary fibroblast cells. Our results show a novel mechanism for fork progression mediated by Separase and thus the basis for genomic instability associated with tumorigenesis

Separase prevents genomic instability by controlling replication fork speed

Proper chromosome segregation is crucial for preserving genomic integrity, and errors in this process cause chromosome mis-segregation, which may contribute to cancer development. Sister chromatid separation is triggered by Separase, an evolutionary conserved protease that cleaves the cohesin complex, allowing the dissolution of sister chromatid cohesion. Here we provide evidence that Separase participates in genomic stability maintenance by controlling replication fork speed. We found that Separase interacted with the replication licensing factors MCM2-7, and genome-wide data showed that Separase co-localized with MCM complex and cohesin. Unexpectedly, the depletion of Separase increased the fork velocity about 1.5-fold and caused a strong acetylation of cohesin's SMC3 subunit and altered checkpoint response. Notably, Separase silencing triggered genomic instability in both HeLa and human primary fibroblast cells. Our results show a novel mechanism for fork progression mediated by Separase and thus the basis for genomic instability associated with tumorigenesis

Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

ABSTRACT Loss-of-function (LOF) methods, such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing, provide unparalleled power for studying the biological function of genes of interest. When coupled with transcriptomic analyses, LOF methods allow researchers to dissect networks of transcriptional regulation. However, a major concern is nonspecific targeting, which involves depletion of transcripts other than those intended. The off-target effects of each of these common LOF methods have yet to be compared at the whole-transcriptome level. Here, we systematically and experimentally compared non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible offtarget effects in gene expression, with CRISPRi exhibiting clonal variation in the transcriptional profile. As an illustrative example, we evaluated the performance of each method for deciphering the role of a long noncoding RNA (lncRNA) with unknown function. Although all LOF methods reduced expression of the candidate lncRNA, each method yielded different sets of differentially expressed genes upon knockdown as well as a different cellular phenotype. Therefore, to definitively confirm the functional role of a transcriptional regulator, we recommend the simultaneous use of at least two different LOF methods and the inclusion of multiple, specifically designed negative controls

CCDC61/VFL3 Is a Paralog of SAS6 and Promotes Ciliary Functions.

Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively

"Evidenze del ruolo non canonico del gene SMC1β nella regolazione dell'espressione genica in cellule somatiche"

La coesina è un complesso proteico costituito da quattro subunità: due appartengono alla famiglia evolutivamente conservata di proteine SMC (Structural Maintenance of Chromosomes, Smc1α e Smc3) in grado di formare eterodimeri, e due sono proteine non SMC (Rad21 e Stag1/2). Mutazioni a carico dei geni codificanti le diverse subunità della coesina e dei fattori che permettono la corretta funzione del complesso sono responsabili di una serie di patologie umane, note con il termine di coesinopatie, tra cui la sindrome di Cornelia de Lange, causata da mutazioni nei geni NIPBL, SMC1α e SMC3. Il ruolo canonico della coesina è quello di garantire la coesione dei cromatidi fratelli e la loro corretta segregazione durante il ciclo cellulare. Tuttavia, recenti evidenze sperimentali suggeriscono che la coesina interviene in altri processi biologici, come la ricombinazione, la riparazione del DNA, il mantenimento della stabilità del genoma e la regolazione della trascrizione genica. Nel 2001, nei mammiferi, è stato isolato il gene Smc1β, che presenta una elevata similarità con il gene Smc1α. Nell’uomo SMC1β mappa sul cromosoma 22, mentre SMC1α è localizzato sul cromosoma X. Poiché studi successivi hanno evidenziato Smc1β come principale componente della coesina nelle cellule della linea germinale, è stata introdotta la definizione di coesina meiotica, avente una localizzazione cromosomica specifica ed un ruolo fondamentale nel garantire il corretto svolgimento della meiosi. Ad oggi non sono stati condotti studi specifici riguardanti Smc1β a livello di cellule somatiche, pertanto lo scopo di questa tesi è quello di investigare l’espressione di Smc1β sia in cellule germinali che somatiche e di analizzare il possibile ruolo di questa subunità. A questo proposito sono stati effettuati studi su differenti tessuti murini, sia a livello dell’mRNA, mediante la tecnica di Real Time PCR, sia a livello di sintesi proteica, attraverso la metodologia del Western Blotting. I risultati hanno confermato che Smc1β è presente a livello della linea germinale, ma è anche espresso in diversi tessuti somatici, tra cui cervello, cuore e milza. Attraverso la Co-Immunoprecipitazione è stato mostrato che Smc1β interagisce con Smc3 e Rad21, subunità della coesina, rafforzando l’osservazione che Smc1β non è un componente esclusivo della coesina meiotica e pertanto potrebbe dare una specificità funzionale alla coesina stessa a livello delle cellule somatiche. Per indagare il possibile ruolo della coesina in cui è presente Smc1β, sono stati effettuati studi funzionali. L’inibizione dell’espressione del gene SMC1β è stata condotta attraverso la tecnica di RNA interference (RNAi) in fibroblasti umani ed è stata saggiata mediante Western Blotting. In seguito, l’mRNA estratto dalle cellule trattate è stato analizzato tramite microarray di espressione. L’analisi dei dati ha mostrato che l’inibizione di SMC1β causa variazioni del profilo trascrizionale, suggerendo un ruolo non canonico della coesina, contenente la subunità SMC1β, nella regolazione dell’espressione genica

Towards the understanding of pericentriolar satellite biology

Pericentriolar satellites (PS) are electron dense granules surrounding the centrosome, the major microtubule-organizing centre in eukaryotic cells. In cycling cells the centrosome promotes spindle assembly and the faithful execution of mitosis. In non-cycling cells it is involved in forming the cilium, a plasma membrane-resident organelle, which mediates crucial signalling pathways in development and tissue homeostasis. PS are thought to contribute to centrosome formation, through the microtubule-dependent transport of centrosome components, and they are involved in ciliogenesis and stress response. Moreover, several proteins that localize to PS are mutated in human ciliopathies and neurodevelopmental disorders. The precise roles of PS in the various molecular pathways and diseases are however poorly understood, in part due to the limited knowledge of their composition. In the first part of my study I performed a comprehensive analysis of the pericentriolar satellite proteome. This was achieved by sucrose sedimentation of PS, combined with affinity purification of a key PS component, PCM1. To eliminate contamination by centrosomes, the PS proteome was determined from wild-type cells as well as from two cell lines genetically engineered to lack centrosomes. Mass spectrometry identified 170 PS components including most of the previously described PS proteins, confirming the validity of the approach. Having determined the proteomic composition of PS from DT40 cells, I then performed validation studies both in chicken and human cell lines. In the second part of my study, I aimed to use the list of PS proteins to uncover new biological roles for pericentriolar satellites. I devised two distinct approaches to gain functional insights. First, I generated a cell line lacking PCM1 as a tool to study the role(s) of PS and PS components. Second, I performed loss-of-function studies on a set of new PS proteins to determine their function(s) in maintaining the canonical PS distribution and in forming primary cilia.CRUK studentshi

Suppression of CHK1 by ETS Family Members Promotes DNA Damage Response Bypass and Tumorigenesis

The ETS family of transcription factors has been repeatedly implicated in tumorigenesis. In prostate cancer, ETS family members, such as ERG, ETV1, ETV4, and ETV5, are frequently overexpressed due to chromosomal translocations, but the molecular mechanisms by which they promote prostate tumorigenesis remain largely undefined. Here, we show that ETS family members, such as ERG and ETV1, directly repress the expression of the checkpoint kinase 1 (CHK1), a key DNA damage response cell-cycle regulator essential for the maintenance of genome integrity. Critically, we find that ERG expression correlates with CHK1 downregulation in human patients and demonstrate that Chk1 heterozygosity promotes the progression of high-grade prostatic intraepithelial neoplasia into prostatic invasive carcinoma in Pten(+) (/-) mice. Importantly, CHK1 downregulation sensitizes prostate tumor cells to etoposide but not to docetaxel treatment. Thus, we identify CHK1 as a key functional target of the ETS proto-oncogenic family with important therapeutic implications