B1 SINE-binding ZFP266 impedes mouse iPSC generation through suppression of chromatin opening mediated by reprogramming factors

Induced pluripotent stem cell (iPSC) reprogramming is inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 24 reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhances murine iPSC generation in several reprogramming settings, emerging as the most robust roadblock. We show that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2, and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with co-activator domains converts ZFP266 from an inhibitor to a potent facilitator of iPSC reprogramming. We propose that the SINE-KRAB-ZFP interaction is a critical regulator of chromatin accessibility at regulatory elements required for efficient cellular identity changes. In addition, this work serves as a resource to further illuminate molecular mechanisms hindering reprogramming.Induced pluripotent stem cell (iPSC) reprogramming is inherently inefficient. Here the authors identify 24 reprogramming roadblock genes through a CRISPR/Cas9-mediated genome-wide knockout screen including a KRAB-ZFP Zfp266, knockout of which consistently enhances murine iPSC generation.Peer reviewe

Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence

Expression of the transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) reprograms somatic cells into induced pluripotent stem cells (iPSCs). Reprogramming is a slow and inefficient process, suggesting the presence of safeguarding mechanisms that counteract cell fate conversion. One such mechanism is senescence. To identify modulators of reprogramming-induced senescence, we performed a genome-wide shRNA screen in primary human fibroblasts expressing OSKM. In the screen, we identified novel mediators of OSKM-induced senescence and validated previously implicated genes such as CDKN1A. We developed an innovative approach that integrates single-cell RNA sequencing (scRNA-seq) with the shRNA screen to investigate the mechanism of action of the identified candidates. Our data unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin (mTOR) influences reprogramming. On one hand, mTOR inhibition blunts the induction of cyclin-dependent kinase (CDK) inhibitors (CDKIs), including p16INK4a, p21CIP1, and p15INK4b, preventing OSKM-induced senescence. On the other hand, inhibition of mTOR blunts the senescence-associated secretory phenotype (SASP), which itself favors reprogramming. These contrasting actions contribute to explain the complex effect that mTOR has on reprogramming. Overall, our study highlights the advantage of combining functional screens with scRNA-seq to accelerate the discovery of pathways controlling complex phenotypes

Constitutively active Smad2/3 are broad scope potentiators of transcription factor-mediated cellular reprogramming

Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering. Ruetz et al. show that constitutively active SMAD2/3 has a surprising ability to boost the efficiency of cell reprogramming both to iPSCs and across lineages and may therefore be a general factor that can enhance transcription-factor-mediated reprogramming in a variety of contexts

Mutation of a conserved residue enhances sensitivity of analogue sensitized kinases to generate a novel approach for mitotic studies in fission yeast

The chemical genetic strategy in which mutational enlargement of the ATP-binding site sensitises of a protein kinase to bulky ATP analogues has proved to be an elegant tool for the generation of conditional analogue-sensitive kinase alleles in a variety of model organisms. Here, we describe a novel substitution mutation in the kinase domain that can enhance the sensitivity of analogue-sensitive kinases. Substitution of a methionine residue to phenylalanine in the +2 position after HRDLKxxN motif of the subdomain VIb within the kinase domain markedly increased the sensitivities of the analogue-sensitive kinases to ATP analogues in three out of five S. pombe kinases (i.e. Plo1, Orb5 and Wee1) that harbor this conserved methionine residue. Kinome alignment established that a methionine residue is found at this site in 5–9% of kinases in key model organisms, suggesting that a broader application of this structural modification may enhance ATP analogue sensitivity of analogue-sensitive kinases in future studies. We also show that the enhanced sensitivity of the wee1.as8 allele in a cdc25.22 background can be exploited to generate highly synchronised mitotic and S phase progression at 36°C. Proof-of-principle experiments show how this novel synchronisation technique will prove of great use in the interrogation of the mitotic or S-phase functions through temperature sensitivity mutation of molecules of interest in fission yeast

Effect of training and methodology development on the effectiveness of discussion groups on grassland innovation

Within the framework of the Horizon 2020 thematic network Inno4Grass (www.inno4grass.eu), 55 ‘practice & science meetings’ have been carried out using a multi-stakeholder, participatory approach, in order to bridge the gap between science and practice and deliver a comprehensive analysis of grassland innovations across eight European countries. Three project phases can be distinguished: (1) common guidelines under discussion, (2) introduction and preliminary test of the guidelines, (3) additional training on the discussion process. A structured feedback on the meetings held was provided by the organisers by means of a questionnaire, including information about duration, preparatory material, participants, discussion methods and evaluation of the meetings by the organisers. This information allows computing of mean values and distribution of the most relevant variables. Only a few of themwere affected by the project phase. In particular, there was an increase in the use of farm portraits and of scientific literature over the project phases. All aspects describing the success of meetings were positively assessed. This suggests that the methodology for the conduction of the practice & science meetings within Inno4Grass was already relatively well established at the beginning of the project

Effect of training and methodology development on the effectiveness of discussion groups on grassland innovation

Within the framework of the Horizon 2020 thematic network Inno4Grass (www.inno4grass.eu), 55 ‘practice & science meetings’ have been carried out using a multi-stakeholder, participatory approach, in order to bridge the gap between science and practice and deliver a comprehensive analysis of grassland innovations across eight European countries. Three project phases can be distinguished: (1) common guidelines under discussion, (2) introduction and preliminary test of the guidelines, (3) additional training on the discussion process. A structured feedback on the meetings held was provided by the organisers by means of a questionnaire, including information about duration, preparatory material, participants, discussion methods and evaluation of the meetings by the organisers. This information allows computing of mean values and distribution of the most relevant variables. Only a few of them were affected by the project phase. In particular, there was an increase in the use of farm portraits and of scientific literature over the project phases. All aspects describing the success of meetings were positively assessed. This suggests that the methodology for the conduction of the practice & science meetings within Inno4Grass was already relatively well established at the beginning of the project

Constitutively active SMAD2/3 are broad-scope potentiators of transcription-factor-mediated cellular reprogramming

Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering.This work was supported by the ERC (grants ROADTOIPS 261075 to K.K. and BRAINCELL 261063 to S.L. and iN-Brain 309712 to M.P.), the BBSRC (project grant BB/L023474/1 to K.K.), the Anne Rowling Regenerative Neurology Clinic (K.K.), the Swedish Research Council (grant STARGET to S.L. and grants 521-2012-5624 and 521-2013-3347 to M.P.), and the Wellcome Trust (grant WT098051 to K.Y.). T.R., L.T., J.A., and D.F.K. are funded by a Darwin Trust scholarship, a CMVM scholarship, and Principal's Career Development scholarship from the University of Edinburgh, respectively. D.F.K. is supported by the BBSRC (EASTBIO doctoral training partnership). T.V.T. is supported by a Juan de la Cierva postdoctoral fellowship (MINECO, FJCI-2014-22946). B.D.S. was supported by an EMBO long-term fellowship (ALTF 1143-2015). K.K. is an MRC senior non-clinical fellow (MR/N008715/1). M.P. is a New York Stem Cell Foundation Robertson Investigator