Molecular Roadblocks for Cellular Reprogramming Thomas Vierbuchen, Marius Wernig  Molecular Cell  Volume 47, Issue 6, Pages 827-838 (September 2012) DOI: 10.1016/j.molcel.2012.09.008 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Experimental Systems for Studying Nuclear Reprogramming (A) Somatic cell nuclear transfer (SCNT). Nucleus from a donor cell is inserted into an enucleated oocyte. In mammals, the resulting cell can then be cultured in vitro to derive nuclear transfer ESCs (NT-ESCs), which can then be used to generate cloned mice via standard blastocyst injection. Alternatively, blastocysts can be derived from oocytes in vitro (at low efficiency) and implanted into pseudopregnant mice to develop. Measurements of the efficiency of NT-ESC derivation, blastocyst derivation from somatic nuclei, or the generation of live pups can serve as a measure of the efficiency of nuclear reprogramming. (B) Cell fusion. Two distinct cell types are fused together to generate chimeric cells with multiple nuclei. In order to facilitate identification of transcripts or proteins from each fusion partner, cells from different species (e.g., human and mouse) are often used. Fused cells can be purified using fluorescent-activated cell sorting or by double antibiotic selection. In heterokaryons, fused cells maintain distinct nuclei and do not undergo cell division (e.g., Bhutani et al., 2010). Cells can also be selected for stable, dividing clones in which nuclear fusion has occurred. These are referred to as synkaryons or cell hybrids (e.g., Cowan et al., 2005). (C) Transcription factor-mediated reprogramming. Reprogramming transcription factors are introduced into cells via viral vectors, DNA transfection, or RNA transfection. Numerous cell fates can be induced in addition to those shown in the figure (see Figure 2 for a complete list). Strong artificial promoters are generally used to ensure robust expression. Inducible promoters (e.g., tetracycline-inducible) can be used to shut off reprogramming factor expression to determine whether cell fate reprogramming is stable in the absence of exogenous of transcription factor expression. Molecular Cell 2012 47, 827-838DOI: (10.1016/j.molcel.2012.09.008) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Transcription Factor-Mediated Conversion of Fibroblasts into Diverse Cellular Lineages Summary of the diverse cell types generated directly from mouse and human fibroblasts by lineage reprogramming. Factors listed in parentheses are required for reprogramming human cells but not for mouse cells. References (starting from the bottom left of the figure and going counterclockwise): Ambasudhan et al., 2011; Caiazzo et al., 2011; Davis et al., 1987; Feng et al., 2008; Huang et al., 2011; Ieda et al., 2010; Kajimura et al., 2009; Lujan et al., 2012; Pang et al., 2011; Pfisterer et al., 2011; Qiang et al., 2011; Sekiya and Suzuki, 2011; Son et al., 2011; Szabo et al., 2010; Takahashi and Yamanaka, 2006; Yoo et al., 2011. Molecular Cell 2012 47, 827-838DOI: (10.1016/j.molcel.2012.09.008) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Models of Transcription Factor Binding during the Initiation of Transcriptional Reprogramming (A) Permissive enhancer model (Taberlay et al., 2011). Genes that have promoters that exhibit marks of polycomb-mediated epigenetic silencing (i.e., H3K27me3) can have enhancer elements that exist in a permissive state (i.e., H3K4me1-enriched) and allow for reprogramming factor binding and subsequent chromatin remodeling at the promoter. (B) Pioneer factor model. Reprogramming factors with pioneer activity can bind nucleosomal DNA and can thus access cis-regulatory elements that exist in a chromatin state that is thought to preclude binding of most transcription factors. Pioneer factor binding can displace nucleosomes and recruit chromatin modifying proteins or additional transcription factors, leading to activation of previously “nonpermissive” genes. (C) Spontaneous accessibility. Reprogramming transcription factors bind to cis-regulatory regions during transient unwrapping of nucleosome-bound DNA that would normally be sterically occluded. High levels of reprogramming factor expression could help to increase the likelihood of this occurring at a high enough fraction of important cis-regulatory regions that it is relevant for reprogramming. (D) Accessibility during cell division. Reprogramming transcription factors gain access to occluded cis-regulatory regions during cell division. Copying the genome requires pre-existing nucleosomes to be temporarily displaced and the insertion of initially unmodified histones into newly copied DNA strands. These processes are likely to interrupt the stable epigenetic silencing of these loci and could thus allow reprogramming factors (especially when expressed at high levels) to bind to these temporarily available sites and cause chromatin remodeling, leading to stable transcriptional reprogramming. Molecular Cell 2012 47, 827-838DOI: (10.1016/j.molcel.2012.09.008) Copyright © 2012 Elsevier Inc. Terms and Conditions