Cell-free production of transducible transcription factors for nuclear reprogramming.
ABSTRACT Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli-based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target.
[show abstract] [hide abstract]
ABSTRACT: Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by introducing Oct3/4 and Sox2 with either Klf4 and c-Myc or Nanog and Lin28 using retroviruses or lentiviruses. Patient-specific iPS cells could be useful in drug discovery and regenerative medicine. However, viral integration into the host genome increases the risk of tumorigenicity. Here, we report the generation of mouse iPS cells without viral vectors. Repeated transfection of two expression plasmids, one containing the complementary DNAs (cDNAs) of Oct3/4, Sox2, and Klf4 and the other containing the c-Myc cDNA, into mouse embryonic fibroblasts resulted in iPS cells without evidence of plasmid integration, which produced teratomas when transplanted into mice and contributed to adult chimeras. The production of virus-free iPS cells, albeit from embryonic fibroblasts, addresses a critical safety concern for potential use of iPS cells in regenerative medicine.Science 11/2008; 322(5903):949-53. · 31.20 Impact Factor
Article: Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers.[show abstract] [hide abstract]
ABSTRACT: Members of the POU and SOX transcription factor families exemplify the partnerships established between various transcriptional regulators during early embryonic development. Although functional cooperativity between key regulator proteins is pivotal for milestone decisions in mammalian development, little is known about the underlying molecular mechanisms. In this study, we focus on two transcription factors, Oct4 and Sox2, as their combination on DNA is considered to direct the establishment of the first three lineages in the mammalian embryo. Using experimental high-resolution structure determination, followed by model building and experimental validation, we found that Oct4 and Sox2 were able to dimerize onto DNA in distinct conformational arrangements. We demonstrate that the DNA enhancer region of their target genes is responsible for the correct spatial alignment of glue-like interaction domains on their surface. Interestingly, these surfaces frequently have redundant functions and are instrumental in recruiting various interacting protein partners.Genes & Development 09/2003; 17(16):2048-59. · 11.66 Impact Factor
Article: Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.[show abstract] [hide abstract]
ABSTRACT: The TAT protein transduction domain (PTD) has been used to deliver a wide variety of biologically active cargo for the treatment of multiple preclinical disease models, including cancer and stroke. However, the mechanism of transduction remains unknown. Because of the TAT PTD's strong cell-surface binding, early assumptions regarding cellular uptake suggested a direct penetration mechanism across the lipid bilayer by a temperature- and energy-independent process. Here we show, using a transducible TAT-Cre recombinase reporter assay on live cells, that after an initial ionic cell-surface interaction, TAT-fusion proteins are rapidly internalized by lipid raft-dependent macropinocytosis. Transduction was independent of interleukin-2 receptor/raft-, caveolar- and clathrin-mediated endocytosis and phagocytosis. Using this information, we developed a transducible, pH-sensitive, fusogenic dTAT-HA2 peptide that markedly enhanced TAT-Cre escape from macropinosomes. Taken together, these observations provide a scientific basis for the development of new, biologically active, transducible therapeutic molecules.Nature Medicine 04/2004; 10(3):310-5. · 22.46 Impact Factor