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Synergistic and tunable human gene activation by combinations of synthetic transcription factors
npg 2013 Nature America, Inc. All rights reserved.
Pablo Perez-Pinera1, David G Ousterout1, Jonathan M Brunger1,2, Alicia M Farin3, Katherine A Glass1,2, Farshid Guilak1,2,
Gregory E Crawford4,5, Alexander J Hartemink4,6 & Charles A Gersbach1,4
Mammalian genes are regulated by the cooperative and synergistic actions of many transcription factors. In thisstudy we recapitulate this complex regulation in humancells by targeting endogenous gene promoters, including regions of closed chromatin upstream of silenced genes,with combinations of engineered transcription activatorlike effectors (TALEs). These combinations of TALE transcription factors induced substantial gene activation and allowed tuning of gene expression levels that will broadly enable synthetic biology, gene therapy and biotechnology.
Synthetic biology aims to study the control of gene expression by constructing gene regulation systems from the bottom up to better understand natural biological systems and develop useful tools for biotechnology1. Despite many substantial accomplishments, this field has been limited primarily to studying artificial promoter-transgene systems with one or two transactivators, typically in microorganisms16. In contrast, the natural regulation of mammalian gene expression is extraordinarily complex and typically achieved through the combinatorial control of each gene by many regulatory factors. This level of complexity has not yet been achieved in synthetic gene regulation systems and has not been possible for the regulation of endogenous genes. However, the recent emergence of technologies for engineering transcription activatorlike effectors (TALEs) targeted to almost any DNA sequence714 provides a unique opportunity for recapitulating this natural complexity. In the current study, we achieved the combinatorial regulation of endogenous mammalian genes in their natural chromosomal context by engineering several TALE transcription factors (TALE-TFs) to bind nearby sites upstream of the
transcription start site (TSS) for a target gene. The composition of these combinations of independent TALE-TFs can be manipulated to control gene activation. Synergistic regulation of gene expression by multiple transcriptional activators is known to occur through simultaneous binding and stabilization of components of the preinitiation complex15,16. Building on this model, we activated endogenous genes with combinations of engineered transcription factors and were able to tune gene expression levels by systematically varying these combinations.
Each TALE-TF has two distinct protein domains that carry out individual molecular functions: (i) the repeat variable diresidue...