Analysis of Argonaute-Chromatin Interactions in Human Somatic Cells

A main regulator of mRNA abunance in the cell are Argonaute (Ago) proteins. Agos are guided by an associating small RNA molecule to complementary mRNAs in the cytoplasm resulting in their translational repression and degradation. Apart from their cytoplasmic function which is conserved across many species, some Ago proteins were also found to influence mRNA abundance at the step of transcription in the nucleus of cells. There, Ago proteins bound to a small RNA recognize nascent transcripts of mRNAs and transposable elements by complementary base pairing leading to their transcriptional silencing. This is often linked to the recruitment of chromatin modifying factors. The nuclear function of Ago proteins, which was studied especially in yeast, plants and worms, was found to be inducible also in human cells by targeting Ago to chromatin by promoter complementary small RNA species [Meister, 2013]. Therefore, the question after endogenous triggers and the genomic target sites of this potential function emerged. In order to identify potential target gene loci in human somatic cells and mouse embryonic stem cells, we examined Ago chromatin interaction sites by applying two complementary techniques, Chromatin Immunoprecipitation (ChIP) and DNA Adenine Methyltransferase Identification (DAMID) combined with Next-Generation Sequencing analysis. The ChiP experiments using three different antibodies for Ago1 and Ago2, yielded only low numbers of overlapping peaks and bad signal-to-noise levels. This suggested either lack of strong chromatin interaction sites of Ago proteins or experimental failure. Next, we applied the antibody-independent DAMID approach. This method deduces chromatin association sites from DNA-methylation events caused by a recombinant fusion protein consisting of a DNA methyltransferase from E. coli and the target protein [Vogel et al., 2006]. Despite successful establishment of the method, which was shown by the results for a positive control, the validation of the predicted nuclear Ago-binding sites by ChIP experiments failed for the human cell line. For mESC only few significant peaks were detected which are currently validated by our collaboration partner. Excitingly, both DAMID experiments showed high enrichment of mitochondrial DNA sequences in Ago samples compared to controls. Therefore, we tested a potential mitochondrial localisation of Ago proteins, which was also suggested by hints in the current literature but was not thoroughly assessed [Bian et al., 2010; Bandiera et al., 2011; Sripada et al., 2012; Zhang et al., 2014]. Assisted by the expertise of the lab of Prof. Dr. P. Rehling in mitochondrial biology, we analysed import and localisation of Ago in mitochondria. While some of the experiments supported a potential inter membrane space localisation, no evidence for matrix-import required for specific interaction of Ago proteins with mitochondrial DNA could be found. When the results from mitochondrial localisation were combined with in depth analysis of the sequencing reads form the DAMID experiments, it appeared that a high fraction of the reads were highly amplified non-specific mitochondrial fragments reducing the complexity of DNA library. As implied in the literature [Casbon et al., 2011], this low complexity has a negative influence on quantification of the lower abundant DNA fragments in the library. In conclusion, we can assume that the DAMID approach is also feasible for Ago proteins but needs further improvements like nuclear targeting and depletion of mitochondrial sequences.