Synthetic design of strong promoters.
We have taken a synthetic biology approach to the generation and screening of transcription factor binding sites for activity in human cells. All possible 10-mer DNA sequences were printed on microarrays as 100-mers containing 10 repeats of the same sequence in tandem, yielding an oligonucleotide library of 52,429 unique sequences. This library of potential enhancers was introduced into a retroviral vector and screened in multiple cell lines for the ability to activate GFP transcription from a minimal CMV promoter. With this method, we isolated 100 bp synthetic enhancer elements that were as potent at activating transcription as the WT CMV immediate early enhancer. The activity of the recovered elements was strongly dependent on the cell line in which they were recovered. None of the elements were capable of achieving the same levels of transcriptional enhancement across all tested cell lines as the CMV enhancer. A second screen, for enhancers capable of synergizing with the elements from the original screen, yielded compound enhancers that were capable of twofold greater enhancement activity than the CMV enhancer, with higher levels of activity than the original synthetic enhancer across multiple cell lines. These findings suggest that the 10-mer synthetic enhancer space is sufficiently rich to allow the creation of synthetic promoters of all strengths in most, if not all, cell types.
Department of Genetics, Division of Genetics, Howard Hughes Medical Institute, Brigham and Women's Hospital, Harvard University Medical School, Boston, MA 02115, USA.
Michael R
MR
Schlabach
Jimmy K
JK
Hu
Mamie
M
Li
Stephen J
SJ
Elledge
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D046228:Microarray Analysis / Q000379:methods; D009841:Oligonucleotides / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D009841:Oligonucleotides; D014157:Transcription Factors; D049452:Green Fluorescent Proteins