An ambitious EC-funded research initiative on epigenetics advancing towards systems biology

ETH Zurich

Description

Long interspersed elements type 1 (LINE-1s, or L1s) have impacted mammalian genomes at multiple levels. In particular, L1 continue to affect our genome, and their movement can lead to sporadic cases of disease, including Hemophilia A, muscular dystrophy, and colon cancer. Approximately 45% of the human genome and 40% of the mouse genome are derived from transposable elements and L1 elements represented 17% and 20% respectively (Beck et al., 2011). In plants, fungi and metazoans, silencing small (s)RNAs suppress TEs at both transcriptional and post-transcriptional levels (Zaratiegui et al., 2007). In mice, germline-specific, 26-31-nt PIWI-associated RNAs (piRNAs) derived from TE-enriched clusters are loaded into ARGONAUTE-like PIWI proteins directing de-novo cytosine methylation and RNA degradation of active TEs, including L1 (Aravin et al., 2008). In most healthy somatic tissues, L1s are silenced via 5’-UTR promoter methylation, established from 7.5 days of embryogenesis (Smith et al., 2012). In pre-implantation embryos, by contrast, L1 methylation progressively decreases, to reach 13-23% in blastocysts (Howlett and Reik, 1991), which accumulate full-length L1 transcripts and undergo mosaic retrotransposition, a potential source of heritable and non-heritable mutations (Packer et al., 1993; Kano et al., 2009). Pre-implantation embryogenesis thus defines a critical window during which L1s should be tightly controlled despite their hypo-methylated status and the lack of piRNAs. Being isolated from the blastocyst’s inner mass, cultured mESCs are thus potentially suited to study the mechanism(s) that might restrict L1 retrotransposition during pre-implantation, including, possibly, RNAi.
We previously demonstrated “Young” active L1 elements are transcriptionally up-regulated in RNAi background ESCs (Dicer, Argonaute) (Ciaudo et al., 2013 under review). We also shown that RNAi-deficient diploid ESCs allow the retrotransposition of endogenous L1 and engineered eGFP-L1 elements, (Yang and Kazazian, 2006). Furthermore, tracking L1 retrotransposition have been already achieved in diploid mouse ESCs using L1-GFP elements (Prak et al., 2003). In order to identify new regulators of L1 transcription and/or retrotransposition, the PhD student in charge of the project will conduct screens in haploid and diploid WT and Dicer deficient mESCs carrying an engineered L1-GFP reporter transgene. Moreover, the candidate genes from the screens will be finally validated by generating stable interfered mutant mESC lines using shRNAs (Ciaudo et al., 2006) or CRISPR/CAS9 system and by assessing L1 retrotransposition and expression in this “pure” background. The small-RNA profiling of these new mutant cell lines will be assess be deep-sequencing and the involvement of the candidate gene to regulate RNAi pathway will be investigated. We expect to find new regulators of L1 retrotransposition acting directly on the L1 element or through the regulation of the RNAi pathway. Finally, in the future, the most interesting mutant mESC lines will be further directly reintroduce into mouse blastocyst to generate mutant mice. This last experiment will finally allow us to study the L1 regulation in vivo.

Qualifications

The candidate will contribute to the implementation of a brand new laboratory in the new HPL building at ETH.

The successful applicant should have the following qualifications:

• Strong experience in molecular biology: cloning procedures (design of primers, digestion, amplification, bacteria transfection…) as well as DNA, RNA and protein extractions are required.
• Experience in mammalian cell culture is highly recommended;
• Experience in FACS facility would be also desirable, but not mandatory.

The applicant will also be expected to:

• Participate to group meetings;
• Participate to collaborative tasks, duties of the lab;

Salary

A salary for a PhD student (according to ETH salary):
First year kCHF 47.7/ second year kCHF 51.1 third year kCHF 54.5 = 153 300 CHF

Application details

Please apply by sending your CV and publication list together with a motivation letter to:
Ciaudo - This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Starting date: 2nd September 2013
Closing date: 2nd September 2016