associated with Dicer and Argonaute proteins, strongly suggesting a role of these fragments in RNA silencing [46,47]. On the other hand, modified nucleotides in tRNA are well known to affect their structural and metabolic stability, and thus are likely to influence directly the rate or efficiency of protein translation [42,48]. Interestingly, protein translation is hierarchi- cally controlled during stem cell self-renewal and differentiation; while parsimonious during self-renewal, it enhances during differentiation [49]. This increased protein synthesis capacity in stem cells could allow rapid elevation of translational rate in response to differentiation signals [49]. In summary, we demonstrate that the RNA methyltransferase Misu (Nsun2) stimulates a sub-population of stem cells to leave the hair bulge and become committed progenitor cells in the hair germ. Thus we identify post-transcriptional RNA modifications as a novel mechanism by which stem cells control the balance between stem cell self-renewal and differentiation. Materials and Methods Ethics statement All mouse husbandry and experiments were carried out according to the local ethics committee under the terms of a UK Home Office license. Mice Misu 2/2 mice were derived using 129S2 ES cell line carrying a Gene Trap in intron 8 of the NSUN2 gene (GGTC-clone ID: D014D11) generated by the German Gene Trap Consortium, and then mated with C57Bl6/J CBA F1 mice. F1 progeny was subsequently inbred. A PCR-based strategy was developed to distinguish the wild-type and Misu gene trap alleles. The primers are as follows: SR2, (59GCC AAA CCT ACA GGT GGG GTC TTT) and B34 (59- TGT AAA ACG ACG GGA TCC GCC) amplify a fragment 650 bp of the ß-geo cassette. The primers Misu-Int8-59 (59AGG TGG ACC TGA TCA TGG AG) and Misu-Int8-39 (59-AGGG AGG GTC TGG AAA GATG) amplify a fragment of 500 bp of the wild-type allele. Mice containing a floxed allele of the NSUN2 gene were obtained by first crossing Nsun2tm1a(EUCOMM)Wtsi mice, generated by the Wellcome Trust Sanger Institute, with transgenic mice expressing Flp recombinase to delete the LacZ-neo cassette [50]. The offspring, containing two LoxP sites flanking exon 6, were then crossed with KRT14-cre mice (The Jackson Laboratory). In KRT14-cre mice, Cre-recombinase is expressed under the control of the keratin14 (KRT14) -promoter leading to deletion of Misu in all basal, undifferentiated cells of the epidermis (K14MisuD/D). All mouse lines were bred to a mixed genetic background of CBA6C57BL/6J. Primers to identify Misuwt, Misuflox and MisuD alleles are F1 (59CCC CCA CTG CTG CTC AAC G) and R1 (59CAA TGC CAC CAC AAC CTC CTT). Hair cycle analyses Total of 97 Misu 2/2 mice with their wild-type controls and 29 K14MisuD/D with their Misuf/f control mice were genotyped and grouped by gender. Samples were taken from same regions from dorsal skin and process for H&E staining. Each mouse was classified into specific stages of the hair cycle based on established morphological guidelines [51]. The total numbers were represent- ed as percentages. tRNA bisulfite sequencing Bisulfite conversion of tRNA was carried out as previously described [21]. cDNA was PCR amplified using primers specific Misu Regulates Stem Cell Fate for the deaminated sequences of tRNA-998Leu (CAA) and tRNA- 1911Leu (CAA). tRNALeu (CAA) sequences were obtained from the Genomic tRNA Database (http://gtrnadb.ucsc.edu/). Primer sequences are as follows: Fw_Leu_De: 59GAT GGT TGA GTG GTT TAA GGT GTT, Rv_Leu998_De: 59CAC CTC CAA AAA AAA CCA AAA C and Rv_Leu1911_De: 59CAC CTC CAT TCA AAA ACC AAA AC. BrdU labelling DNA label-retaining cells (LRC) were generated by repeated BrdU (Sigma) injections of neonatal mice at P10 [25]. For LRC assays animals were chased for the times indicated. For tracing migration of bulge LRC into the hair germ, animals were sacrificed at P47 [4]. LRC were detected by BrdU immunostain- ing in tail skin whole mounts. Z-stack volumes of random areas of the slide were collected using a confocal microscope (Leica SP5). Maximum projected images were quantified using Volocity software (PerkinElmer). Images were segmented to identify and measure intensity of BrdU-positive cells constrained to specific Regions Of Interest (ROI) defining the whole hair follicle (bulge and hair germ), high and low bulge region and hair germ. Frequency distributions of BrdU intensity were calculated with Microsoft Office Excel 2007 software (Microsoft). Tissue staining Immunostainings were performed on 10 mm paraffin sections. After citrate epitope retrieval, sections were permeabilized for 5 minutes with 0.2% Triton6100 at room temperature, blocked for 1 hour with 5% FCS and incubated overnight with the appropriate antibody dilution. Immunostaining on cryosections or cultured cells was performed as for paraffin after fixation for 10 minutes in 4% paraformaldehyde at room temperature. Tail epidermal whole mounts were prepared and immunolabelled as described previously [25,36]. For LacZ staining, whole mounts of embryos or freshly obtained skin samples were fixed for 30 minutes at room temperature in buffer containing 0.1 M phosphate buffer, 5 mM EGTA, 2 mM MgCl2 and 0.2% glutaraldehyde. Samples were then washed three times for 15 minutes each in wash buffer (2 mM MgCl2 and 0.1% Nonidet P40 in 0.1 M phosphate buffer) and stained for 12 hours in a solution consisting of 1 mg/ml X-gal (Melford), 5 mM K3Fe(CN)6 and 5 mM K4Fe(CN)6 in wash buffer. The skin samples were then embedded in paraffin, sectioned at 10 mm and stained with eosin or used for stainings. Primary antibodies were used at the following dilutions: rabbit monoclonal antibody to Ki67 (1:100; SP6, Vector Labs), rabbit polyclonal anti mouse keratin 14 (1:2000; Covance), rabbit polyclonal anti mouse keratin 10 (1:500; Covance), mouse monoclonal anti Gata3 (1:50; HG3-31, Santa Cruz Biotechnolo- gy), rabbit polyclonal anti Lef1 (1:50; Cell Signaling Technology), mouse polyclonal anti Dlx3 (1:200; Abnova), guinea pig polyclonal anti hair keratins 31, 71 and 72 (1:200; Progen), rabbit polyclonal anti keratin 6 (1:5.000; Babco), rabbit polyclonal to phosphor- Smad1/5/8 (1:50; Cell Signaling Technology), rabbit polyclonal CUK-1079-A antibody to mouse Misu (1:1000; produced by Covalab), mouse monoclonal to keratin 15 (1:1000) [25]), rat monoclonal anti BrdU (1:100; Abcam), goat polyclonal anti P- cadherin (1:100; R & D Systems), rat monoclonal anti a6 integrin (1:500; GoH3, AbD Serotec), Secondary antibodies (Alexa Fluor 594- and 488-conjugated anti-rabbit, mouse, rat and guinea pig, Invitrogen) were added at a dilution of 1:500 for 1 hour at room temperature together with DAPI to label nuclei. White field images were acquired using an Olympus IX80 microscope and a DP50 camera. Confocal images were acquired PLoS Genetics | www.plosgenetics.org 10 December 2011 | Volume 7 | Issue 12 | e1002403