Boiani

Project Title: Identification of oocytic reprogramming factors and gain-of-function in mouse induced pluripotent stem cells

The swift reprogramming of somatic nuclei afforded by oocytes (Boiani et al., Genes Dev 16:1209-1219, 2002) and pluripotent stem cells (Han et al., Stem Cells 26:445-454, 2008) hints to an active reprogramming machinery operating in these two types of cells, possibly coupled to DNA repair (Wossidlo et al., EMBO J 29:1877-1888, 2010). In contrast, transcription factor-based reprogramming seems to involve a passive mechanism, as transcription factors themselves lack enzymatic activity and require several cell cycles to yield an effect (Hanna et al., Nature 462:595-601, 2009). The Boiani group aims to shed light on the active reprogramming factors that are hosted in the mouse oocyte. Our practical goal is to identify the protein components of the oocyte’s reprogramming machinery. This information will not only benefit direct reprogramming by adding oocytic factors to it, but will also extend and refine our molecular understanding of female germ cell biology. Using liquid chromatography coupled to mass spectrometry (LC-MS), work in the Boiani group led to the identification of 3699 proteins in metaphase II mouse oocytes, the largest oocyte proteome at the time of writing  (Pfeiffer et al., 10:2140-2153, 2011). Based on the shared reprogramming ability of oocytes and ES cells, proteins present in both cell types were selected. A further selection was based on the assumption that reprogramming factors would include nuclear enzymes that act on chromatin. Therefore, the shared genes were filtered for the gene ontology (GO) terms ‘nucleus’, ‘chromatin modification’ and ‘catalytic activity’. Twenty-eight proteins fulfill these selection criteria and are considered as candidate reprogramming factors. It is reassuring to see that chromatin remodelling ATPase Brg1 (Hansis et al., Curr Biol 14:1475-1480, 2004; Singhal et al., Cell 141:943-955, 2010) and protein arginine methyltransferase Prmt5 (Nagamatsu et al., The Journal of biological chemistry 286:10641-10648, 2011), which are proteins known to increase nuclear reprogramming activity, are present in our list of 28.

In our ongoing work, six of the 28 candidate proteins have been tested for their reprogramming potential in mouse embryonic fibroblasts. Brcc3, Carm1, Hat1, Prmt1, Rnf2 and Ruvbl2 were superimposed to an inducible secondary iPS system (courtesy of Marius Wernig), as follows. Using a retroviral vector system, each factor was overexpressed in MEFs that already carried the OSKM factors under the control of a doxycycline (dox) inducible promoter (Wernig et al., Nat Biotechnol 26:916-924, 2008). Speed and efficiency of reprogramming were measured by the number of alkaline phosphatase (AP), Nanog positive colonies and by the expression of early pluripotency marker SSEA1 via FACS analyses. Our preliminary results show that Carm1 has a positive effect (about 2 fold) on reprogramming efficiency as measured by SSEA1 positive cell proportion, whereas Prmt1, Rnf2 and Ruvbl2 may increase reprogramming speed, but not overall reprogramming efficiency. Additionally, using Oct4-GFP transgenic MEFs, we are currently testing Carm1 for its potential to substitute for other reprogramming factors. The necessary vectors for the 22 remaining factors are currently being cloned and will be streamlined into our reprogramming assays in the near future. Pending ultimate confirmation of the beneficial effect of Carm1 on reprogramming, it will be interesting to clarify its mode of action in further experiments. We plan to manipulate levels of Carm1 in oocytes and ES cells prior to nuclear transfer and cell fusion via mRNA injection or mRNA interference, respectively. We also plan to introduce specific point mutations in the mRNA, to test the role of specific Carm1 protein domains in reprogramming.