Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A.
We have mapped conserved regions of enhanced DNase I accessibility within the endogenous chromosomal locus of vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger protein (ZFP) transcription factors were designed to target DNA sequences contained within the DNase I-hypersensitive regions. These ZFPs, when fused to either VP16 or p65 transcriptional activation domains, were able to activate expression of the VEGF-A gene as assayed by mRNA accumulation and VEGF-A protein secretion through a range exceeding that induced by hypoxic stress. Importantly, multiple splice variants of VEGF-A mRNA with defined physiological functions were induced by a single engineered ZFP transcription factor. We present evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for dissecting the requirements for gene regulation at a distance without altering the DNA sequence of the endogenous target locus.
Liu
P Q
PQ
Sangamo BioSciences Incorporated, Richmond, California 94804, USA.
Rebar
E J
EJ
Zhang
L
L
Liu
Q
Q
Jamieson
A C
AC
Liang
Y
Y
Qi
H
H
Li
P X
PX
Chen
B
B
Mendel
M C
MC
Zhong
X
X
Lee
Y L
YL
Eisenberg
S P
SP
Spratt
S K
SK
Case
C C
CC
Wolffe
A P
AP
D002461:Cell Line, Transformed; D002874:Chromosome Mapping; D003850:Deoxyribonuclease I / Q000235:genetics*; D016228:Endothelial Growth Factors / Q000235:genetics*; D006801:Humans; D011506:Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation; D042461:Vascular Endothelial Growth Factor A; D016335:Zinc Fingers
D016228:Endothelial Growth Factors; D011506:Proteins; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D003850:Deoxyribonuclease I